Research Article Antimicrobial Resistance of Salmonella ...
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Research Article Antimicrobial Resistance of Salmonella enterica Isolates from Tonsil and Jejunum with Lymph Node Tissues of Slaughtered Swine in Metro Manila, Philippines Kamela Charmaine S. Ng 1 and Windell L. Rivera 1,2 1 Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines 2 Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines Correspondence should be addressed to Windell L. Rivera; [email protected] Received 1 January 2014; Accepted 27 January 2014; Published 4 March 2014 Academic Editors: C. M. A. Khan, T. Krishnan, and V. L. Traina-Dorge Copyright © 2014 K. C. S. Ng and W. L. Rivera. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Due to frequent antibiotic exposure, swine is now recognized as potential risk in disseminating drug-resistant Salmonella enterica strains. is study thus subjected 20 randomly selected S. enterica isolates from tonsil and jejunum with lymph node (JLN) tissues of swine slaughtered in Metro Manila, Philippines, to VITEK 2 antimicrobial susceptibility testing (AST). e test revealed all 20 isolates had resistance to at least one antimicrobial agent, in which highest occurrence of resistance was to amikacin (100%), cefazolin (100%), cefuroxime (100%), cefuroxime axetil (100%), cefoxitin (100%), and gentamicin (100%), followed by ampicillin (50%), and then by sulfamethoxazole trimethoprim (30%). ree multidrug-resistant (MDR) isolates were detected. e sole S. enterica serotype Enteritidis isolate showed resistance to 12 different antibiotics including ceſtazidime, ceſtriaxone, amikacin, gentamicin, and tigecycline. is study is the first to report worldwide on the novel resistance to tigecycline of MDR S. enterica serotype Enteritidis isolated from swine tonsil tissues. is finding poses huge therapeutic challenge since MDR S. enterica infections are associated with increased rate of hospitalization or death. us, continual regulation of antimicrobial use in food animals and prediction of resistant serotypes are crucial to limit the spread of MDR S. enterica isolates among hogs and humans. 1. Introduction Salmonella is a rod-shaped, Gram-negative, oxidase negative, nonspore forming, predominantly peritrichous enterobac- terium [1]. It has been reported and recognized as one of the leading causes of food borne illness, causing diarrheal diseases and enteric fever that may be complicated by extraintestinal infections, such as bacteremia, meningitis, and osteomyelitis, leading to millions of cases of hospitalizations and deaths worldwide each year [2, 3]. It has been isolated from a wide variety of animals, of which swine are the most commonly recognized carriers [4]. e demand for the production of quality livestock meat is increasing. However, the hog livestock production system, despite being the top livestock industry in the Philippines, is constantly challenged with various microbial diseases such as salmonellosis that lead to morbidity-linked reduction in productivity and increased cost of disease treatment [5]. e threat and prevalence of this disease in the country continue to be high [6]. Food poisoning outbreaks and livestock infection caused by Salmonella spp. are widespread in the Philippines as evidenced by cases of food poisoning reported in Benguet, Tondo, Manila, and Bulacan and cases of hog morbidity and mortality in Tacloban and Leyte [6–9]. e widespread use of antibiotics has resulted in the emergence of drug-resistant Salmonella strains. Since antibi- otics are widely used for growth promotion and disease treatment in commercial swine production systems, swine is now recognized as a potential risk in disseminating multidrug-resistant (MDR) Salmonella spp. strains [4, 10, 11]. e VITEK 2 system (bioMerieux) has revolutionized antimi- crobial susceptibility testing through its rapid and automated fluorescence-based technology. Livermore and coworkers [12] have commended the accuracy of identification and antimicrobial susceptibility testing (AST) of the VITEK 2 Hindawi Publishing Corporation ISRN Microbiology Volume 2014, Article ID 364265, 9 pages http://dx.doi.org/10.1155/2014/364265
Transcript of Research Article Antimicrobial Resistance of Salmonella ...
Research ArticleAntimicrobial Resistance of Salmonella entericaIsolates from Tonsil and Jejunum with Lymph Node Tissues ofSlaughtered Swine in Metro Manila Philippines
Kamela Charmaine S Ng1 and Windell L Rivera12
1 Institute of Biology College of Science University of the Philippines Diliman Quezon City 1101 Philippines2 Natural Sciences Research Institute University of the Philippines Diliman Quezon City 1101 Philippines
Correspondence should be addressed to Windell L Rivera wlriverascienceupdeduph
Received 1 January 2014 Accepted 27 January 2014 Published 4 March 2014
Academic Editors C M A Khan T Krishnan and V L Traina-Dorge
Copyright copy 2014 K C S Ng and W L RiveraThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Due to frequent antibiotic exposure swine is now recognized as potential risk in disseminating drug-resistant Salmonella entericastrains This study thus subjected 20 randomly selected S enterica isolates from tonsil and jejunum with lymph node (JLN) tissuesof swine slaughtered in Metro Manila Philippines to VITEK 2 antimicrobial susceptibility testing (AST) The test revealed all20 isolates had resistance to at least one antimicrobial agent in which highest occurrence of resistance was to amikacin (100)cefazolin (100) cefuroxime (100) cefuroxime axetil (100) cefoxitin (100) and gentamicin (100) followed by ampicillin(50) and then by sulfamethoxazole trimethoprim (30) Three multidrug-resistant (MDR) isolates were detected The soleS enterica serotype Enteritidis isolate showed resistance to 12 different antibiotics including ceftazidime ceftriaxone amikacingentamicin and tigecycline This study is the first to report worldwide on the novel resistance to tigecycline of MDR S entericaserotype Enteritidis isolated from swine tonsil tissues This finding poses huge therapeutic challenge since MDR S entericainfections are associated with increased rate of hospitalization or death Thus continual regulation of antimicrobial use in foodanimals and prediction of resistant serotypes are crucial to limit the spread of MDR S enterica isolates among hogs and humans
1 Introduction
Salmonella is a rod-shaped Gram-negative oxidase negativenonspore forming predominantly peritrichous enterobac-terium [1] It has been reported and recognized as one ofthe leading causes of food borne illness causing diarrhealdiseases and enteric fever that may be complicated byextraintestinal infections such as bacteremiameningitis andosteomyelitis leading to millions of cases of hospitalizationsand deaths worldwide each year [2 3] It has been isolatedfrom a wide variety of animals of which swine are the mostcommonly recognized carriers [4]
The demand for the production of quality livestock meatis increasing However the hog livestock production systemdespite being the top livestock industry in the Philippines isconstantly challenged with various microbial diseases suchas salmonellosis that lead to morbidity-linked reduction inproductivity and increased cost of disease treatment [5] The
threat and prevalence of this disease in the country continueto be high [6] Food poisoning outbreaks and livestockinfection caused by Salmonella spp are widespread in thePhilippines as evidenced by cases of food poisoning reportedin Benguet Tondo Manila and Bulacan and cases of hogmorbidity and mortality in Tacloban and Leyte [6ndash9]
The widespread use of antibiotics has resulted in theemergence of drug-resistant Salmonella strains Since antibi-otics are widely used for growth promotion and diseasetreatment in commercial swine production systems swineis now recognized as a potential risk in disseminatingmultidrug-resistant (MDR) Salmonella spp strains [4 10 11]TheVITEK2 system (bioMerieux) has revolutionized antimi-crobial susceptibility testing through its rapid and automatedfluorescence-based technology Livermore and coworkers[12] have commended the accuracy of identification andantimicrobial susceptibility testing (AST) of the VITEK 2
Hindawi Publishing CorporationISRN MicrobiologyVolume 2014 Article ID 364265 9 pageshttpdxdoiorg1011552014364265
2 ISRNMicrobiology
system and the significantly reduced handling time thatenhances the work flow of clinical microbiology laboratory
This study aimed to characterize S enterica isolates fromtonsil and jejunum with lymph node (JLN) tissues of swineat slaughter in selected accredited and non-accredited meatestablishments in Metro Manila In order to detect MDRstrains and shed light on the appropriate treatment againstthe pathogen VITEK 2 AST was performed in this study
2 Materials and Methods
21 Sample Collection Tonsils and their corresponding JLNtissues were collected from 30 hogs in each of the four non-accredited meat establishments in Quezon City and fouraccredited slaughterhouses in Malabon Makati Pasig andQuezon City in Metro Manila Philippines A 15 cm longsegment of JLNwas secured with sterile threads on both endsand excised with the flame sterilized knife of the butcher Itwas immediately transferred to a sterile bag that was cooledduring transport to the laboratory Afterwards 25 g of JLNwas weighed in a sterile foil and pre-enriched with 225mL ofbuffered peptone water in a sterile bottle agitated for 2minand incubated for 18ndash24 h at 37∘C The tonsil tissues werecollected using flame sterilized forceps and butcherrsquos knifeandwere pre-enriched the sameway as the intestinal samples
22 Single-Enrichment Broth Culture Method One hundredmicroliters of pre-enriched tonsil tissue and JLN sampleswere inoculated into Rappaport-Vassiliadis Broth (10mL)while onemLof the pre-enriched sampleswas inoculated intoTetrathionate Broth (10mL) and was incubated at 37∘C for24 h After incubation broth cultures were streak-plated ontoselective chromogenic medium Rainbow Agar Salmonella(RAS)
23 DNA Extraction Three colonies of Salmonella spp cellsfrom RAS were suspended in 150 120583L of sterile distilled waterThe suspension was heated at 100∘C for 10min and cooledto room temperature afterwards The cell debris was pelletedby centrifugation at 13000 rpm for 2min The clear super-natant obtained was used as DNA template in PCR [14]Concentration of DNA extracts was then measured usingNanoDrop 2000 following the manufacturerrsquos instructions
24 PCR-Based Identification of Salmonella spp Isolates InvAprimers invA-F and invA-R which amplify a 244 bp frag-ment of the invA gene specific for Salmonella spp wereused for initial detection and confirmation of suspectedSalmonella spp isolates [15] Promega GoTaq Green MasterMix consisting of GoTaq DNA polymerase 2X Green GoTaqReaction Buffer 3mM MgCl
2and 04mM dNTPs was used
for PCR amplification of invA region DNA amplificationwas performed in a reaction volume of 25120583L PCR wasperformed under the following cycling conditions an initialdenaturation at 95∘C for 2min followed by 35 cycles ofdenaturation at 95∘C for 30 s annealing at 56∘C for 30 s andextension at 72∘C for 2min Final extension was done at72∘C for 5min For each run DNA from S enterica serotype
Typhimuriumwas used as positive control while sterile wateras template was included as negative control
Amplicons were checked by separating PCR productsthrough agarose gel electrophoresis in 1x TAE buffer at 100volts for 30 to 40min All PCR products were analyzed in a15 agarose gel stainedwith 05 120583gmL ethidiumbromide for20min and visualized on a UV transilluminator The sizes ofthe bands were estimated using Vivantis 1000 bp DNA ladderas molecular weight marker
25 DNA Sequencing of Selected Amplicons PCR productsobtained with the primers representing each serogroup weresent to Macrogen Inc (Seoul South Korea) for purifica-tion and DNA sequencing for validation of their identitiesNucleotide sequence data obtained were checked in BioEditv 7090 sequence alignment program [16] and compared toavailable sequences of Salmonella spp in GenBank using theBasic Local Alignment Search Tool (BLAST) algorithm avail-able in the National Center for Biotechnology Informationwebsite (httpwwwncbinlmnihgovBLAST)
26 Antimicrobial Susceptibility Testing VITEK 2 AST of 20randomly selected S enterica isolates from slaughtered swinein both accredited and non-accredited meat establishmentswas performed to generate the antibiograms and detectMDRstrains (Table 1) The stock culture strains were subculturedonto Salmonella-Shigella agar plates to confirm their purityThe turbidity of the bacterial suspensions was adjustedwith a densitometer (DENSICHEK) to match that of aMcFarland 04ndash06 standard in 045 sterile sodium chloridesolution The time interval between suspension preparationand card filling was less than 30min to avoid changes inturbidity Afterwards the VITEK 2 AST N091 antimicrobialsusceptibility cards and bacterial suspension in tubes bothcontained in a cassette weremanually loaded into the VITEK2 system Each test card was automatically filled with abacterial suspension sealed incubated and read by kineticfluorescence measurement The reporting time for the directtesting of susceptibility against the 17 antibiotics for 20 swinetissue culture isolates by the VITEK 2 system ranged from 85to 105 hours
3 Results
VITEK 2 AST of 20 randomly selected S enterica iso-lates from slaughtered swine in Metro Manila Philippines(Table 1) revealed that all had resistance to at least oneantimicrobial agent in which highest occurrence of resis-tance was to amikacin (100) cefazolin (100) cefuroxime(100) cefuroxime axetil (100) cefoxitin (100) and gen-tamicin (100) followed by ampicillin (50) and thenby sulfamethoxazole trimethoprim (30) Tables 2(a) and2(b) show the complete antibiogram of S enterica isolatesgenerated through VITEK 2 AST while Tables 3 and 4reflect the distribution of in vitro and in vivo antimicrobialresistance respectively of S enterica serotypes detectedAs seen in Tables 2(a) and 2(b) four S enterica serotypeTyphimurium isolates the sole serotype Heidelberg one
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Table 1 Sequence similarities () of isolates and reference Salmonella enterica sequences obtained from GenBank
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Lt16 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 677 97 00 99
Lt21 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 671 98 00 99
Lt24 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 653 99 00 99
Lt30 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 686 95 00 99
Lai1 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 674 98 00 99
Lai27 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 672 95 00 99
Lat23 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 678 97 00 99
Lat27 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 665 97 00 99
Lbt30 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 684 97 00 99
Lct47 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 674 93 00 100
Nt4 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 684 94 00 99
Pt26 rfbJ BSalmonella enterica subsp
enterica serotypeHeidelberg str B182
CP0011201 662 98 00 99
Lat20 wzxC1 C1Salmonella enterica subsp
enterica serotypeCholeraesuis str SC-B67
AE0172201 490 99 00 99
Lbt18 wzxC1 C1Salmonella enterica subsp
enterica serotypeCholeraesuis str SC-B67
AE0172201 487 99 00 99
Lt25 tyv DSalmonella enterica subsp
enterica serotypeEnteritidis str P125109
AM9331721 619 99 00 99
Li16 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 344 1002119890 minus 176
99
Lt3 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 357 694119890 minus 115
98
4 ISRNMicrobiology
Table 1 Continued
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Mbi8 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 354 993119890 minus 171
98
Mbi25 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 348 993119890 minus 180
100
Pt12 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 253 963119890 minus 119
99
serotypeCholeraesuis the sole serotype Enteritidis and threeserotype Weltevreden isolates were resistant to ampicillinand two S enterica serotype Typhimurium isolates andone each from serotypes Heidelberg Choleraesuis Enteri-tidis and Weltevreden were resistant to sulfamethoxazoletrimethoprim In addition the sole serotype Enteritidis wasfound to be resistant to ceftriaxone ertapenem tigecycline(Table 3) and ceftazidime (Table 4) Out of the 20 randomlyselected S enterica isolates three (15) MDR serotypes weredetected in the study namely Choleraesuis and Enteritidisfrom non-accredited meat establishments and Weltevredenfrom an accredited meat establishment (Tables 2(a) and2(b)) Among the three MDR isolates S enterica serotypeEnteritidis was found to be resistant to 12 antibiotics ofvarious antimicrobial classes including third generationcephalosporins ceftazidime and ceftriaxone third generationaminoglycosides amikacin and gentamicin as well as to theglycylcycline tigecycline (Tables 3 and 4) VITEK 2 AST alsoshowed that all 20 S enterica isolates tested were susceptibleto ceftazidime cefepime imipenem meropenem amikacinand levofloxacin (Tables 2(a) and 2(b)) Moreover VITEK 2ASTdemonstrated that S enterica isolates classified under thesame serogroup had varying antibiograms as shown in Tables2(a) and 2(b) for isolates Lt3 and Mbi8
4 Discussion
AST is traditionally performed through the Kirby-Bauer discdiffusion assay However this method is laborious and proneto inconsistencies subjectivity and human errorThe VITEK2 system (bioMerieux) has revolutionized AST through itsrapid and automated fluorescence-based technology thatallows determination of minimum inhibitory concentration(MIC) by the analysis of growth kinetics of bacteria withantibiotics in test cards [12]
Among the antibiotics included in the VITEK 2 Gramnegative susceptibility card used in the study piperacillintazobactam was suppressed from analysis while ESBL (extraspectrum 120573-lactamase) was not claimed by the machinesince the latter is only considered relevant to Escherichia andKlebsiella species [13] However ESBL should not be anymore
excluded from AST of S enterica since an ESBL-producing Senterica serotype Typhi has been isolated in the Philippines[17] and many of the isolates tested were found to be resistantto cephalosporins (Table 2(a)) the subsequent characteristicof possessing the enzyme ESBL
VITEK 2 MIC interpretation guideline is based on Clini-cal and Laboratory Standards Institute [13] Referring toTables 2(a) and 2(b) MIC values and interpretations ofalmost all of the isolates to cefazolin cefuroxime cefuroximeaxetil cefoxitin amikacin and gentamicin and of S entericaserotype Enteritidis to ceftazidime were edited to resistant bythe machine for the reason that these antibiotics may onlyappear active in vitro against S enterica but are not effectivein vivo (clinically) and should not be reported as susceptibleThis editing of antibiograms based on inferredmechanisms isin agreement with theNational Committee for Clinical Labo-ratory Standards [12]
This study is first to report ampicillin-resistant serotypesof S enterica namely Typhimurium Heidelberg Cholerae-suis Enteritidis and Weltevreden isolated from tonsil andJLN tissues of slaughtered swine in the Philippines usingVITEK 2 AST The routine administration of ampicillin forgastroenteritis in bothman and swine and its commonuse forsensitivity testing in diagnostic laboratories led to the occur-rence of ampicillin-resistant S enteric strains [18]
Amoxicillinclavulanic acid-resistant serotypes of Senterica namely Enteritidis and Weltevreden from tonsiland JLN tissues of slaughtered swine in Metro ManilaPhilippines were also first detected in this study usingVITEK 2 AST (Table 2(a)) Resistance to cefazolin cefurox-ime cefuroxime axetil and cefoxitin was revealed by VITEK2 AST in all 20 isolates tested in this study (Table 2(a))This is the first report on resistance to the aforementionedcephalosporins of S enterica serotypes Agona CholeraesuisEnteritidis Heidelberg Typhimurium and Weltevredenobtained from tonsil and JLN tissues of slaughtered swinein the Philippines using VITEK 2 AST (Tables 3 and 4) Itis remarkable to note that the said cephalosporins cefa-zolin cefuroxime cefuroxime axetil and cefoxitin arenot commonly employed as therapeutic agents or growthpromoters in livestock in the Philippines [19]The emergence
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6 ISRNMicrobiology
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Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLt3
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sle20
SMbi8
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sge320
RMbi25
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S2
Sle20
SPt12
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
Sa ETP
ertapenem
IPM
imipenem
MEM
merop
enem
ANamikacinG
MgentamicinLEV
levofl
oxacinT
GC
tigecyclin
eSX
Ttrim
etho
prim
sulfametho
xazoleM
minim
uminhibitory
concentration(M
IC)
Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
2 ISRNMicrobiology
system and the significantly reduced handling time thatenhances the work flow of clinical microbiology laboratory
This study aimed to characterize S enterica isolates fromtonsil and jejunum with lymph node (JLN) tissues of swineat slaughter in selected accredited and non-accredited meatestablishments in Metro Manila In order to detect MDRstrains and shed light on the appropriate treatment againstthe pathogen VITEK 2 AST was performed in this study
2 Materials and Methods
21 Sample Collection Tonsils and their corresponding JLNtissues were collected from 30 hogs in each of the four non-accredited meat establishments in Quezon City and fouraccredited slaughterhouses in Malabon Makati Pasig andQuezon City in Metro Manila Philippines A 15 cm longsegment of JLNwas secured with sterile threads on both endsand excised with the flame sterilized knife of the butcher Itwas immediately transferred to a sterile bag that was cooledduring transport to the laboratory Afterwards 25 g of JLNwas weighed in a sterile foil and pre-enriched with 225mL ofbuffered peptone water in a sterile bottle agitated for 2minand incubated for 18ndash24 h at 37∘C The tonsil tissues werecollected using flame sterilized forceps and butcherrsquos knifeandwere pre-enriched the sameway as the intestinal samples
22 Single-Enrichment Broth Culture Method One hundredmicroliters of pre-enriched tonsil tissue and JLN sampleswere inoculated into Rappaport-Vassiliadis Broth (10mL)while onemLof the pre-enriched sampleswas inoculated intoTetrathionate Broth (10mL) and was incubated at 37∘C for24 h After incubation broth cultures were streak-plated ontoselective chromogenic medium Rainbow Agar Salmonella(RAS)
23 DNA Extraction Three colonies of Salmonella spp cellsfrom RAS were suspended in 150 120583L of sterile distilled waterThe suspension was heated at 100∘C for 10min and cooledto room temperature afterwards The cell debris was pelletedby centrifugation at 13000 rpm for 2min The clear super-natant obtained was used as DNA template in PCR [14]Concentration of DNA extracts was then measured usingNanoDrop 2000 following the manufacturerrsquos instructions
24 PCR-Based Identification of Salmonella spp Isolates InvAprimers invA-F and invA-R which amplify a 244 bp frag-ment of the invA gene specific for Salmonella spp wereused for initial detection and confirmation of suspectedSalmonella spp isolates [15] Promega GoTaq Green MasterMix consisting of GoTaq DNA polymerase 2X Green GoTaqReaction Buffer 3mM MgCl
2and 04mM dNTPs was used
for PCR amplification of invA region DNA amplificationwas performed in a reaction volume of 25120583L PCR wasperformed under the following cycling conditions an initialdenaturation at 95∘C for 2min followed by 35 cycles ofdenaturation at 95∘C for 30 s annealing at 56∘C for 30 s andextension at 72∘C for 2min Final extension was done at72∘C for 5min For each run DNA from S enterica serotype
Typhimuriumwas used as positive control while sterile wateras template was included as negative control
Amplicons were checked by separating PCR productsthrough agarose gel electrophoresis in 1x TAE buffer at 100volts for 30 to 40min All PCR products were analyzed in a15 agarose gel stainedwith 05 120583gmL ethidiumbromide for20min and visualized on a UV transilluminator The sizes ofthe bands were estimated using Vivantis 1000 bp DNA ladderas molecular weight marker
25 DNA Sequencing of Selected Amplicons PCR productsobtained with the primers representing each serogroup weresent to Macrogen Inc (Seoul South Korea) for purifica-tion and DNA sequencing for validation of their identitiesNucleotide sequence data obtained were checked in BioEditv 7090 sequence alignment program [16] and compared toavailable sequences of Salmonella spp in GenBank using theBasic Local Alignment Search Tool (BLAST) algorithm avail-able in the National Center for Biotechnology Informationwebsite (httpwwwncbinlmnihgovBLAST)
26 Antimicrobial Susceptibility Testing VITEK 2 AST of 20randomly selected S enterica isolates from slaughtered swinein both accredited and non-accredited meat establishmentswas performed to generate the antibiograms and detectMDRstrains (Table 1) The stock culture strains were subculturedonto Salmonella-Shigella agar plates to confirm their purityThe turbidity of the bacterial suspensions was adjustedwith a densitometer (DENSICHEK) to match that of aMcFarland 04ndash06 standard in 045 sterile sodium chloridesolution The time interval between suspension preparationand card filling was less than 30min to avoid changes inturbidity Afterwards the VITEK 2 AST N091 antimicrobialsusceptibility cards and bacterial suspension in tubes bothcontained in a cassette weremanually loaded into the VITEK2 system Each test card was automatically filled with abacterial suspension sealed incubated and read by kineticfluorescence measurement The reporting time for the directtesting of susceptibility against the 17 antibiotics for 20 swinetissue culture isolates by the VITEK 2 system ranged from 85to 105 hours
3 Results
VITEK 2 AST of 20 randomly selected S enterica iso-lates from slaughtered swine in Metro Manila Philippines(Table 1) revealed that all had resistance to at least oneantimicrobial agent in which highest occurrence of resis-tance was to amikacin (100) cefazolin (100) cefuroxime(100) cefuroxime axetil (100) cefoxitin (100) and gen-tamicin (100) followed by ampicillin (50) and thenby sulfamethoxazole trimethoprim (30) Tables 2(a) and2(b) show the complete antibiogram of S enterica isolatesgenerated through VITEK 2 AST while Tables 3 and 4reflect the distribution of in vitro and in vivo antimicrobialresistance respectively of S enterica serotypes detectedAs seen in Tables 2(a) and 2(b) four S enterica serotypeTyphimurium isolates the sole serotype Heidelberg one
ISRNMicrobiology 3
Table 1 Sequence similarities () of isolates and reference Salmonella enterica sequences obtained from GenBank
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Lt16 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 677 97 00 99
Lt21 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 671 98 00 99
Lt24 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 653 99 00 99
Lt30 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 686 95 00 99
Lai1 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 674 98 00 99
Lai27 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 672 95 00 99
Lat23 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 678 97 00 99
Lat27 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 665 97 00 99
Lbt30 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 684 97 00 99
Lct47 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 674 93 00 100
Nt4 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 684 94 00 99
Pt26 rfbJ BSalmonella enterica subsp
enterica serotypeHeidelberg str B182
CP0011201 662 98 00 99
Lat20 wzxC1 C1Salmonella enterica subsp
enterica serotypeCholeraesuis str SC-B67
AE0172201 490 99 00 99
Lbt18 wzxC1 C1Salmonella enterica subsp
enterica serotypeCholeraesuis str SC-B67
AE0172201 487 99 00 99
Lt25 tyv DSalmonella enterica subsp
enterica serotypeEnteritidis str P125109
AM9331721 619 99 00 99
Li16 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 344 1002119890 minus 176
99
Lt3 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 357 694119890 minus 115
98
4 ISRNMicrobiology
Table 1 Continued
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Mbi8 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 354 993119890 minus 171
98
Mbi25 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 348 993119890 minus 180
100
Pt12 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 253 963119890 minus 119
99
serotypeCholeraesuis the sole serotype Enteritidis and threeserotype Weltevreden isolates were resistant to ampicillinand two S enterica serotype Typhimurium isolates andone each from serotypes Heidelberg Choleraesuis Enteri-tidis and Weltevreden were resistant to sulfamethoxazoletrimethoprim In addition the sole serotype Enteritidis wasfound to be resistant to ceftriaxone ertapenem tigecycline(Table 3) and ceftazidime (Table 4) Out of the 20 randomlyselected S enterica isolates three (15) MDR serotypes weredetected in the study namely Choleraesuis and Enteritidisfrom non-accredited meat establishments and Weltevredenfrom an accredited meat establishment (Tables 2(a) and2(b)) Among the three MDR isolates S enterica serotypeEnteritidis was found to be resistant to 12 antibiotics ofvarious antimicrobial classes including third generationcephalosporins ceftazidime and ceftriaxone third generationaminoglycosides amikacin and gentamicin as well as to theglycylcycline tigecycline (Tables 3 and 4) VITEK 2 AST alsoshowed that all 20 S enterica isolates tested were susceptibleto ceftazidime cefepime imipenem meropenem amikacinand levofloxacin (Tables 2(a) and 2(b)) Moreover VITEK 2ASTdemonstrated that S enterica isolates classified under thesame serogroup had varying antibiograms as shown in Tables2(a) and 2(b) for isolates Lt3 and Mbi8
4 Discussion
AST is traditionally performed through the Kirby-Bauer discdiffusion assay However this method is laborious and proneto inconsistencies subjectivity and human errorThe VITEK2 system (bioMerieux) has revolutionized AST through itsrapid and automated fluorescence-based technology thatallows determination of minimum inhibitory concentration(MIC) by the analysis of growth kinetics of bacteria withantibiotics in test cards [12]
Among the antibiotics included in the VITEK 2 Gramnegative susceptibility card used in the study piperacillintazobactam was suppressed from analysis while ESBL (extraspectrum 120573-lactamase) was not claimed by the machinesince the latter is only considered relevant to Escherichia andKlebsiella species [13] However ESBL should not be anymore
excluded from AST of S enterica since an ESBL-producing Senterica serotype Typhi has been isolated in the Philippines[17] and many of the isolates tested were found to be resistantto cephalosporins (Table 2(a)) the subsequent characteristicof possessing the enzyme ESBL
VITEK 2 MIC interpretation guideline is based on Clini-cal and Laboratory Standards Institute [13] Referring toTables 2(a) and 2(b) MIC values and interpretations ofalmost all of the isolates to cefazolin cefuroxime cefuroximeaxetil cefoxitin amikacin and gentamicin and of S entericaserotype Enteritidis to ceftazidime were edited to resistant bythe machine for the reason that these antibiotics may onlyappear active in vitro against S enterica but are not effectivein vivo (clinically) and should not be reported as susceptibleThis editing of antibiograms based on inferredmechanisms isin agreement with theNational Committee for Clinical Labo-ratory Standards [12]
This study is first to report ampicillin-resistant serotypesof S enterica namely Typhimurium Heidelberg Cholerae-suis Enteritidis and Weltevreden isolated from tonsil andJLN tissues of slaughtered swine in the Philippines usingVITEK 2 AST The routine administration of ampicillin forgastroenteritis in bothman and swine and its commonuse forsensitivity testing in diagnostic laboratories led to the occur-rence of ampicillin-resistant S enteric strains [18]
Amoxicillinclavulanic acid-resistant serotypes of Senterica namely Enteritidis and Weltevreden from tonsiland JLN tissues of slaughtered swine in Metro ManilaPhilippines were also first detected in this study usingVITEK 2 AST (Table 2(a)) Resistance to cefazolin cefurox-ime cefuroxime axetil and cefoxitin was revealed by VITEK2 AST in all 20 isolates tested in this study (Table 2(a))This is the first report on resistance to the aforementionedcephalosporins of S enterica serotypes Agona CholeraesuisEnteritidis Heidelberg Typhimurium and Weltevredenobtained from tonsil and JLN tissues of slaughtered swinein the Philippines using VITEK 2 AST (Tables 3 and 4) Itis remarkable to note that the said cephalosporins cefa-zolin cefuroxime cefuroxime axetil and cefoxitin arenot commonly employed as therapeutic agents or growthpromoters in livestock in the Philippines [19]The emergence
ISRNMicrobiology 5Ta
ble2Antibiogram
ofSalm
onellaenteric
aiso
latesg
enerated
throug
hVITEK
2antim
icrobialsusceptib
ilitytesting
(a)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
AMP
AMC
CZCX
MCX
MAX
FOX
CAZ
CRO
FEP
MI
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt21
BTy
phim
urium
ge32
Rle2
Sge64
Rge64
Rge64
Rle4
lowast
Rle1
Sle1
Sle1
SLt24
BTy
phim
urium
ge32
R16
Ile4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt30
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai1
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai27
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat23
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat27
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t30
BTy
phim
urium
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLct47
BTy
phim
urium
ge32
R8
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SNt4
BTy
phim
urium
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SPt26
BHeidelberg
ge32
R4
Sle4
lowast
R8
lowast
R8
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat20
C1Ch
oleraesuis
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t18C1
Choleraesuis
ge32
R16
Ige64
R8
lowast
R8
lowast
Rge64lowast
Rle1
Sle1
Sle1
SLt25
DEn
teritidis
ge32
Rge32
Rge64
Rge64
Rge64
Rge64
R4lowast
lowast
Rge64
R2
SLi16
EWeltevreden
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt3
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi8
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi25
EWeltevreden
ge32
Rge32
Rge64
R16
lowast
R16
lowast
R16
lowast
Rle1
Sle1
Sle1
SPt12
EWeltevreden
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
Sa A
MP
ampicillin
AMC
amoxicillinclav
ulanicacidC
Zcefazolin
CXM
cefuroxim
eCX
MAX
cefuroximea
xetilF
OX
cefoxitin
CAZ
cefta
zidimeCR
Oceft
riaxoneF
EPcefepim
eMm
inim
uminhibitory
concentration(M
IC)Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
(b)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt21
BTy
phim
urium
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sle20
SLt24
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLai1
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLai27
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLat23
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLat27
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLb
t30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLct47
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S1
Sge320
RNt4
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RPt26
BHeidelberg
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RLat20
C1Ch
oleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLb
t18C1
Choleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sge320
RLt25
DEn
teritidis
ge8
R4
S1
Sle2
lowast
Rle1
lowast
R1
Sge8
Rge320
R
6 ISRNMicrobiology
(b)Con
tinued
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Li16
EWeltevreden
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLt3
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sle20
SMbi8
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sge320
RMbi25
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S2
Sle20
SPt12
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
Sa ETP
ertapenem
IPM
imipenem
MEM
merop
enem
ANamikacinG
MgentamicinLEV
levofl
oxacinT
GC
tigecyclin
eSX
Ttrim
etho
prim
sulfametho
xazoleM
minim
uminhibitory
concentration(M
IC)
Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
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International Journal of
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ISRNMicrobiology 3
Table 1 Sequence similarities () of isolates and reference Salmonella enterica sequences obtained from GenBank
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Lt16 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 677 97 00 99
Lt21 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 671 98 00 99
Lt24 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 653 99 00 99
Lt30 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 686 95 00 99
Lai1 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 674 98 00 99
Lai27 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 672 95 00 99
Lat23 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 678 97 00 99
Lat27 rfbJ BSalmonella enterica subspenterica serotype Agona str
SL483CP0011381 665 97 00 99
Lbt30 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 684 97 00 99
Lct47 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 674 93 00 100
Nt4 rfbJ BSalmonella enterica subsp
enterica serotypeTyphimurium str U288
CP0038361 684 94 00 99
Pt26 rfbJ BSalmonella enterica subsp
enterica serotypeHeidelberg str B182
CP0011201 662 98 00 99
Lat20 wzxC1 C1Salmonella enterica subsp
enterica serotypeCholeraesuis str SC-B67
AE0172201 490 99 00 99
Lbt18 wzxC1 C1Salmonella enterica subsp
enterica serotypeCholeraesuis str SC-B67
AE0172201 487 99 00 99
Lt25 tyv DSalmonella enterica subsp
enterica serotypeEnteritidis str P125109
AM9331721 619 99 00 99
Li16 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 344 1002119890 minus 176
99
Lt3 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 357 694119890 minus 115
98
4 ISRNMicrobiology
Table 1 Continued
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Mbi8 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 354 993119890 minus 171
98
Mbi25 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 348 993119890 minus 180
100
Pt12 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 253 963119890 minus 119
99
serotypeCholeraesuis the sole serotype Enteritidis and threeserotype Weltevreden isolates were resistant to ampicillinand two S enterica serotype Typhimurium isolates andone each from serotypes Heidelberg Choleraesuis Enteri-tidis and Weltevreden were resistant to sulfamethoxazoletrimethoprim In addition the sole serotype Enteritidis wasfound to be resistant to ceftriaxone ertapenem tigecycline(Table 3) and ceftazidime (Table 4) Out of the 20 randomlyselected S enterica isolates three (15) MDR serotypes weredetected in the study namely Choleraesuis and Enteritidisfrom non-accredited meat establishments and Weltevredenfrom an accredited meat establishment (Tables 2(a) and2(b)) Among the three MDR isolates S enterica serotypeEnteritidis was found to be resistant to 12 antibiotics ofvarious antimicrobial classes including third generationcephalosporins ceftazidime and ceftriaxone third generationaminoglycosides amikacin and gentamicin as well as to theglycylcycline tigecycline (Tables 3 and 4) VITEK 2 AST alsoshowed that all 20 S enterica isolates tested were susceptibleto ceftazidime cefepime imipenem meropenem amikacinand levofloxacin (Tables 2(a) and 2(b)) Moreover VITEK 2ASTdemonstrated that S enterica isolates classified under thesame serogroup had varying antibiograms as shown in Tables2(a) and 2(b) for isolates Lt3 and Mbi8
4 Discussion
AST is traditionally performed through the Kirby-Bauer discdiffusion assay However this method is laborious and proneto inconsistencies subjectivity and human errorThe VITEK2 system (bioMerieux) has revolutionized AST through itsrapid and automated fluorescence-based technology thatallows determination of minimum inhibitory concentration(MIC) by the analysis of growth kinetics of bacteria withantibiotics in test cards [12]
Among the antibiotics included in the VITEK 2 Gramnegative susceptibility card used in the study piperacillintazobactam was suppressed from analysis while ESBL (extraspectrum 120573-lactamase) was not claimed by the machinesince the latter is only considered relevant to Escherichia andKlebsiella species [13] However ESBL should not be anymore
excluded from AST of S enterica since an ESBL-producing Senterica serotype Typhi has been isolated in the Philippines[17] and many of the isolates tested were found to be resistantto cephalosporins (Table 2(a)) the subsequent characteristicof possessing the enzyme ESBL
VITEK 2 MIC interpretation guideline is based on Clini-cal and Laboratory Standards Institute [13] Referring toTables 2(a) and 2(b) MIC values and interpretations ofalmost all of the isolates to cefazolin cefuroxime cefuroximeaxetil cefoxitin amikacin and gentamicin and of S entericaserotype Enteritidis to ceftazidime were edited to resistant bythe machine for the reason that these antibiotics may onlyappear active in vitro against S enterica but are not effectivein vivo (clinically) and should not be reported as susceptibleThis editing of antibiograms based on inferredmechanisms isin agreement with theNational Committee for Clinical Labo-ratory Standards [12]
This study is first to report ampicillin-resistant serotypesof S enterica namely Typhimurium Heidelberg Cholerae-suis Enteritidis and Weltevreden isolated from tonsil andJLN tissues of slaughtered swine in the Philippines usingVITEK 2 AST The routine administration of ampicillin forgastroenteritis in bothman and swine and its commonuse forsensitivity testing in diagnostic laboratories led to the occur-rence of ampicillin-resistant S enteric strains [18]
Amoxicillinclavulanic acid-resistant serotypes of Senterica namely Enteritidis and Weltevreden from tonsiland JLN tissues of slaughtered swine in Metro ManilaPhilippines were also first detected in this study usingVITEK 2 AST (Table 2(a)) Resistance to cefazolin cefurox-ime cefuroxime axetil and cefoxitin was revealed by VITEK2 AST in all 20 isolates tested in this study (Table 2(a))This is the first report on resistance to the aforementionedcephalosporins of S enterica serotypes Agona CholeraesuisEnteritidis Heidelberg Typhimurium and Weltevredenobtained from tonsil and JLN tissues of slaughtered swinein the Philippines using VITEK 2 AST (Tables 3 and 4) Itis remarkable to note that the said cephalosporins cefa-zolin cefuroxime cefuroxime axetil and cefoxitin arenot commonly employed as therapeutic agents or growthpromoters in livestock in the Philippines [19]The emergence
ISRNMicrobiology 5Ta
ble2Antibiogram
ofSalm
onellaenteric
aiso
latesg
enerated
throug
hVITEK
2antim
icrobialsusceptib
ilitytesting
(a)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
AMP
AMC
CZCX
MCX
MAX
FOX
CAZ
CRO
FEP
MI
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt21
BTy
phim
urium
ge32
Rle2
Sge64
Rge64
Rge64
Rle4
lowast
Rle1
Sle1
Sle1
SLt24
BTy
phim
urium
ge32
R16
Ile4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt30
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai1
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai27
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat23
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat27
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t30
BTy
phim
urium
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLct47
BTy
phim
urium
ge32
R8
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SNt4
BTy
phim
urium
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SPt26
BHeidelberg
ge32
R4
Sle4
lowast
R8
lowast
R8
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat20
C1Ch
oleraesuis
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t18C1
Choleraesuis
ge32
R16
Ige64
R8
lowast
R8
lowast
Rge64lowast
Rle1
Sle1
Sle1
SLt25
DEn
teritidis
ge32
Rge32
Rge64
Rge64
Rge64
Rge64
R4lowast
lowast
Rge64
R2
SLi16
EWeltevreden
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt3
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi8
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi25
EWeltevreden
ge32
Rge32
Rge64
R16
lowast
R16
lowast
R16
lowast
Rle1
Sle1
Sle1
SPt12
EWeltevreden
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
Sa A
MP
ampicillin
AMC
amoxicillinclav
ulanicacidC
Zcefazolin
CXM
cefuroxim
eCX
MAX
cefuroximea
xetilF
OX
cefoxitin
CAZ
cefta
zidimeCR
Oceft
riaxoneF
EPcefepim
eMm
inim
uminhibitory
concentration(M
IC)Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
(b)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt21
BTy
phim
urium
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sle20
SLt24
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLai1
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLai27
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLat23
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLat27
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLb
t30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLct47
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S1
Sge320
RNt4
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RPt26
BHeidelberg
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RLat20
C1Ch
oleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLb
t18C1
Choleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sge320
RLt25
DEn
teritidis
ge8
R4
S1
Sle2
lowast
Rle1
lowast
R1
Sge8
Rge320
R
6 ISRNMicrobiology
(b)Con
tinued
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Li16
EWeltevreden
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLt3
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sle20
SMbi8
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sge320
RMbi25
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S2
Sle20
SPt12
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
Sa ETP
ertapenem
IPM
imipenem
MEM
merop
enem
ANamikacinG
MgentamicinLEV
levofl
oxacinT
GC
tigecyclin
eSX
Ttrim
etho
prim
sulfametho
xazoleM
minim
uminhibitory
concentration(M
IC)
Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 ISRNMicrobiology
Table 1 Continued
Isolate Region Serogroup Strain Accession numberQuery lengthand cover 119864
value
Maximumidentity
Mbi8 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 354 993119890 minus 171
98
Mbi25 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 348 993119890 minus 180
100
Pt12 wzxE1 ESalmonella enterica subsp
enterica serotypeWeltevreden str2007-60-3289-1
FR7752241 253 963119890 minus 119
99
serotypeCholeraesuis the sole serotype Enteritidis and threeserotype Weltevreden isolates were resistant to ampicillinand two S enterica serotype Typhimurium isolates andone each from serotypes Heidelberg Choleraesuis Enteri-tidis and Weltevreden were resistant to sulfamethoxazoletrimethoprim In addition the sole serotype Enteritidis wasfound to be resistant to ceftriaxone ertapenem tigecycline(Table 3) and ceftazidime (Table 4) Out of the 20 randomlyselected S enterica isolates three (15) MDR serotypes weredetected in the study namely Choleraesuis and Enteritidisfrom non-accredited meat establishments and Weltevredenfrom an accredited meat establishment (Tables 2(a) and2(b)) Among the three MDR isolates S enterica serotypeEnteritidis was found to be resistant to 12 antibiotics ofvarious antimicrobial classes including third generationcephalosporins ceftazidime and ceftriaxone third generationaminoglycosides amikacin and gentamicin as well as to theglycylcycline tigecycline (Tables 3 and 4) VITEK 2 AST alsoshowed that all 20 S enterica isolates tested were susceptibleto ceftazidime cefepime imipenem meropenem amikacinand levofloxacin (Tables 2(a) and 2(b)) Moreover VITEK 2ASTdemonstrated that S enterica isolates classified under thesame serogroup had varying antibiograms as shown in Tables2(a) and 2(b) for isolates Lt3 and Mbi8
4 Discussion
AST is traditionally performed through the Kirby-Bauer discdiffusion assay However this method is laborious and proneto inconsistencies subjectivity and human errorThe VITEK2 system (bioMerieux) has revolutionized AST through itsrapid and automated fluorescence-based technology thatallows determination of minimum inhibitory concentration(MIC) by the analysis of growth kinetics of bacteria withantibiotics in test cards [12]
Among the antibiotics included in the VITEK 2 Gramnegative susceptibility card used in the study piperacillintazobactam was suppressed from analysis while ESBL (extraspectrum 120573-lactamase) was not claimed by the machinesince the latter is only considered relevant to Escherichia andKlebsiella species [13] However ESBL should not be anymore
excluded from AST of S enterica since an ESBL-producing Senterica serotype Typhi has been isolated in the Philippines[17] and many of the isolates tested were found to be resistantto cephalosporins (Table 2(a)) the subsequent characteristicof possessing the enzyme ESBL
VITEK 2 MIC interpretation guideline is based on Clini-cal and Laboratory Standards Institute [13] Referring toTables 2(a) and 2(b) MIC values and interpretations ofalmost all of the isolates to cefazolin cefuroxime cefuroximeaxetil cefoxitin amikacin and gentamicin and of S entericaserotype Enteritidis to ceftazidime were edited to resistant bythe machine for the reason that these antibiotics may onlyappear active in vitro against S enterica but are not effectivein vivo (clinically) and should not be reported as susceptibleThis editing of antibiograms based on inferredmechanisms isin agreement with theNational Committee for Clinical Labo-ratory Standards [12]
This study is first to report ampicillin-resistant serotypesof S enterica namely Typhimurium Heidelberg Cholerae-suis Enteritidis and Weltevreden isolated from tonsil andJLN tissues of slaughtered swine in the Philippines usingVITEK 2 AST The routine administration of ampicillin forgastroenteritis in bothman and swine and its commonuse forsensitivity testing in diagnostic laboratories led to the occur-rence of ampicillin-resistant S enteric strains [18]
Amoxicillinclavulanic acid-resistant serotypes of Senterica namely Enteritidis and Weltevreden from tonsiland JLN tissues of slaughtered swine in Metro ManilaPhilippines were also first detected in this study usingVITEK 2 AST (Table 2(a)) Resistance to cefazolin cefurox-ime cefuroxime axetil and cefoxitin was revealed by VITEK2 AST in all 20 isolates tested in this study (Table 2(a))This is the first report on resistance to the aforementionedcephalosporins of S enterica serotypes Agona CholeraesuisEnteritidis Heidelberg Typhimurium and Weltevredenobtained from tonsil and JLN tissues of slaughtered swinein the Philippines using VITEK 2 AST (Tables 3 and 4) Itis remarkable to note that the said cephalosporins cefa-zolin cefuroxime cefuroxime axetil and cefoxitin arenot commonly employed as therapeutic agents or growthpromoters in livestock in the Philippines [19]The emergence
ISRNMicrobiology 5Ta
ble2Antibiogram
ofSalm
onellaenteric
aiso
latesg
enerated
throug
hVITEK
2antim
icrobialsusceptib
ilitytesting
(a)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
AMP
AMC
CZCX
MCX
MAX
FOX
CAZ
CRO
FEP
MI
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt21
BTy
phim
urium
ge32
Rle2
Sge64
Rge64
Rge64
Rle4
lowast
Rle1
Sle1
Sle1
SLt24
BTy
phim
urium
ge32
R16
Ile4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt30
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai1
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai27
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat23
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat27
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t30
BTy
phim
urium
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLct47
BTy
phim
urium
ge32
R8
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SNt4
BTy
phim
urium
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SPt26
BHeidelberg
ge32
R4
Sle4
lowast
R8
lowast
R8
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat20
C1Ch
oleraesuis
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t18C1
Choleraesuis
ge32
R16
Ige64
R8
lowast
R8
lowast
Rge64lowast
Rle1
Sle1
Sle1
SLt25
DEn
teritidis
ge32
Rge32
Rge64
Rge64
Rge64
Rge64
R4lowast
lowast
Rge64
R2
SLi16
EWeltevreden
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt3
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi8
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi25
EWeltevreden
ge32
Rge32
Rge64
R16
lowast
R16
lowast
R16
lowast
Rle1
Sle1
Sle1
SPt12
EWeltevreden
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
Sa A
MP
ampicillin
AMC
amoxicillinclav
ulanicacidC
Zcefazolin
CXM
cefuroxim
eCX
MAX
cefuroximea
xetilF
OX
cefoxitin
CAZ
cefta
zidimeCR
Oceft
riaxoneF
EPcefepim
eMm
inim
uminhibitory
concentration(M
IC)Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
(b)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt21
BTy
phim
urium
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sle20
SLt24
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLai1
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLai27
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLat23
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLat27
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLb
t30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLct47
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S1
Sge320
RNt4
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RPt26
BHeidelberg
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RLat20
C1Ch
oleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLb
t18C1
Choleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sge320
RLt25
DEn
teritidis
ge8
R4
S1
Sle2
lowast
Rle1
lowast
R1
Sge8
Rge320
R
6 ISRNMicrobiology
(b)Con
tinued
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Li16
EWeltevreden
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLt3
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sle20
SMbi8
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sge320
RMbi25
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S2
Sle20
SPt12
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
Sa ETP
ertapenem
IPM
imipenem
MEM
merop
enem
ANamikacinG
MgentamicinLEV
levofl
oxacinT
GC
tigecyclin
eSX
Ttrim
etho
prim
sulfametho
xazoleM
minim
uminhibitory
concentration(M
IC)
Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
ISRNMicrobiology 5Ta
ble2Antibiogram
ofSalm
onellaenteric
aiso
latesg
enerated
throug
hVITEK
2antim
icrobialsusceptib
ilitytesting
(a)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
AMP
AMC
CZCX
MCX
MAX
FOX
CAZ
CRO
FEP
MI
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt21
BTy
phim
urium
ge32
Rle2
Sge64
Rge64
Rge64
Rle4
lowast
Rle1
Sle1
Sle1
SLt24
BTy
phim
urium
ge32
R16
Ile4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt30
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai1
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLai27
BTy
phim
urium
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat23
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat27
BAgona
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t30
BTy
phim
urium
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLct47
BTy
phim
urium
ge32
R8
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SNt4
BTy
phim
urium
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SPt26
BHeidelberg
ge32
R4
Sle4
lowast
R8
lowast
R8
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLat20
C1Ch
oleraesuis
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLb
t18C1
Choleraesuis
ge32
R16
Ige64
R8
lowast
R8
lowast
Rge64lowast
Rle1
Sle1
Sle1
SLt25
DEn
teritidis
ge32
Rge32
Rge64
Rge64
Rge64
Rge64
R4lowast
lowast
Rge64
R2
SLi16
EWeltevreden
le2
Sle2
Sle4
lowast
Rle1
lowast
Rle1
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SLt3
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi8
EWeltevreden
ge32
R4
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
SMbi25
EWeltevreden
ge32
Rge32
Rge64
R16
lowast
R16
lowast
R16
lowast
Rle1
Sle1
Sle1
SPt12
EWeltevreden
le2
Sle2
Sle4
lowast
R4
lowast
R4
lowast
Rle4
lowast
Rle1
Sle1
Sle1
Sa A
MP
ampicillin
AMC
amoxicillinclav
ulanicacidC
Zcefazolin
CXM
cefuroxim
eCX
MAX
cefuroximea
xetilF
OX
cefoxitin
CAZ
cefta
zidimeCR
Oceft
riaxoneF
EPcefepim
eMm
inim
uminhibitory
concentration(M
IC)Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
(b)
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Lt16
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt21
BTy
phim
urium
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sle20
SLt24
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLt30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLai1
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLai27
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLat23
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLat27
BAgona
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sle20
SLb
t30
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLct47
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S1
Sge320
RNt4
BTy
phim
urium
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RPt26
BHeidelberg
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S2
Sge320
RLat20
C1Ch
oleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
SLb
t18C1
Choleraesuis
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
Sle05
Sge320
RLt25
DEn
teritidis
ge8
R4
S1
Sle2
lowast
Rle1
lowast
R1
Sge8
Rge320
R
6 ISRNMicrobiology
(b)Con
tinued
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Li16
EWeltevreden
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLt3
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sle20
SMbi8
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sge320
RMbi25
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S2
Sle20
SPt12
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
Sa ETP
ertapenem
IPM
imipenem
MEM
merop
enem
ANamikacinG
MgentamicinLEV
levofl
oxacinT
GC
tigecyclin
eSX
Ttrim
etho
prim
sulfametho
xazoleM
minim
uminhibitory
concentration(M
IC)
Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 ISRNMicrobiology
(b)Con
tinued
Isolate
Serogrou
pSenteric
aserotype
Antim
icrobial
a
ETP
IPM
MEM
AN
GM
LEV
TGC
SXT
MI
MI
MI
MI
MI
MI
MI
MI
Li16
EWeltevreden
le05
S4lowast
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S4
Ile20
SLt3
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sle20
SMbi8
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rge16
Rle012
Sle05
Sge320
RMbi25
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
R1
S2
Sle20
SPt12
EWeltevreden
le05
Sle1
Sle025
Sle2
lowast
Rle1
lowast
Rle012
S1
Sle20
Sa ETP
ertapenem
IPM
imipenem
MEM
merop
enem
ANamikacinG
MgentamicinLEV
levofl
oxacinT
GC
tigecyclin
eSX
Ttrim
etho
prim
sulfametho
xazoleM
minim
uminhibitory
concentration(M
IC)
Iinterpretatio
nlowast
mod
ified
bymachine
(Clin
icalandLabo
ratory
StandardsInstitute2012
[13])
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
ISRNMicrobiology 7
Table 3 Distribution of in vitro antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
AMP AMC CZ CXM CXM AX FOX CRO ETP GM TGC SXTCholeraesuis (2) 1 1 1 1Enteritidis (1) 1 1 1 1 1 1 1 1 1 1Heidelberg (1) 1 1Typhimurium(8) 4 1 1 1 2
Weltevreden (5) 3 1 1 2 1aAMP ampicillin AMC amoxicillinclavulanic acid CZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CRO ceftriaxone ETPertapenem GM gentamicin TGC tigecycline SXT trimethoprimsulfamethoxazole (Clinical and Laboratory Standards Institute 2012 [13])
Table 4 Distribution of in vivo antimicrobial resistance of Salmonella enterica serotypes isolated from swine at slaughter in Metro Manila
Serotype(number tested)
Number of isolates resistant to the antimicrobial agentsa
CZ CXM CXM AX FOX CAZ AN GMAgona (3) 3 3 3 3 3 3Choleraesuis (2) 1 2 2 1 2 2Enteritidis (1) 1 1 1Heidelberg (1) 1 1 1 1 1 1Typhimurium(8) 7 7 7 8 8 8
Weltevreden (5) 4 5 5 5 5 3aCZ cefazolin CXM cefuroxime CXM AX cefuroxime axetil FOX cefoxitin CAZ ceftazidime AN amikacin GM gentamicin (Clinical and LaboratoryStandards Institute 2012 [13])
of resistance to cephalosporins has been attributed toplasmid-mediated resistance to AmpC (CMY-2) 120573-lactamase[20]
The sole S enterica serotype Enteritidis detected in thisstudy exhibited resistance to third generation cephalosporinsceftazidime and ceftriaxone (Table 2(a)) In view of the highrate of resistance to fluoroquinolone ciprofloxacin third gen-eration cephalosporins such as ceftazidime and ceftriaxoneare suggested as drugs of choice in the treatment of inva-sive nontyphoid Salmonella infections Resistance to theseantibiotics has been emerging due to production of variousclass A ESBLs and class C cephalosporinases in S entericastrains [21] Lee and coworkers [10] have detected ceftriaxoneresistance only in S enterica isolates under serogroups B andC1 from TaiwanThey did not detect ceftriaxone resistance inS enterica isolates from the PhilippinesThis study is the firstto report on in vitro resistance to ceftriaxone (Table 3) and invivo resistance to ceftazidime (Table 4) of S enterica serotypeEnteritidis isolated from tonsil and JLN tissues of slaughteredswine in the Philippines using VITEK 2 AST
Ampicillin amoxicillinclavulanic acid and third gener-ation cephalosporins are commonly used to treat complexsalmonellosis [21] The augmenting emergence of resistanceto these antibiotics worldwide has brought about huge thera-peutic challenge to animal and human medicine Resistanceto ertapenemwas observed in isolate Lt25 (serogroupD sero-type Enteritidis) (Table 2(b)) According to the study of Liv-ermore and coworkers [22] ertapenem was the most active
agent tested against members of the family Enterobacteri-aceae including Salmonella spp as compared to imipenemcefepime ceftriaxone and piperacillin-tazobactam Basedon their broth microdilution experiments Salmonella sppisolates from Europe and Australia were susceptible toertapenem In the study of Su and coworkers [23] theydetected a ceftriaxone and ciprofloxacin-resistant S entericaserotype Typhimurium strain which developed carbapenemresistance during ertapenem treatment which they attributedto a single gene mutation in the organism This is the firstreport on ertapenem resistance of S enterica serotype Enteri-tidis from tonsil and JLN tissues of slaughtered swine in thePhilippines using VITEK 2 AST
Amikacin and gentamicin are aminoglycosides that bindto the bacterial 30S ribosome and interfere with proteinsynthesis These aminoglycosides have broader spectra ofactivity than streptomycin and kanamycin [24] In 1992Arboleda and coworkers [25] isolated a gentamicin-sensitiveSalmonella spp from a piglet in Laguna Philippines whereasMaluping in 2005 [26] isolated gentamicin-resistant S enter-ica serotype Choleraesuis also from the same animal inBulacan PhilippinesThe result obtained in the present studyreflects the increasing resistance of S enterica to gentamicinas two gentamicin-resistant S enterica serotype Weltevredenisolates were detected for the first time from tonsil and JLNtissues of slaughtered swine in the Philippines using VITEK 2AST (Table 3) In vivo resistance against amikacin was foundin all 20 S enterica isolates from tonsil and JLN tissues of
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 ISRNMicrobiology
slaughtered swine specifically in serotypes Agona Cholerae-suis Enteritidis Heidelberg Typhimurium andWeltevredenfor the first time in the Philippines using VITEK 2 AST(Table 4) This finding is novel and needs further study sinceaminoglycoside phosphotransferase APH(31015840)-I detected in Senterica generates resistance only to kanamycin neomycinlividomycin paromomycin and ribostamycin [24]
The most alarming resistance of multidrug isolates foundwas to tigecycline a broad-spectrum derivative of minocy-cline and a member of the novel class glycylcyclines [27]It is considered a promising drug for treating complexinfections since it has a bacteriostatic mode of action againsta broad spectrum of aerobic and anaerobic atypical Gram-positive and Gram-negative organisms and even MDRESBL-expressing Enterobacteriaceae and carbapenem-resist-ant strains [27ndash30] It is said to circumvent efflux and riboso-mal protection the twomost frequent genetic mechanisms oftetracycline resistance It is also unaffected by the presence ofcoresistance to unrelated antimicrobials such as 120573-lactamsaminoglycosides and quinolones [30]
Fritsche and coworkers [31] found that 957 of all Enter-obacteriaceae strains tested including Salmonella spp weresusceptible to tigecycline In 2010 Hentschke and coworkers[27] isolated tigecycline-resistant S enterica serotype Hadarwith MIC of 16 120583gmL from a human patient in GermanyResults obtained in the present study are remarkably incontrast to the findings of Fritsche and coworkers [31] butin agreement with that of Hentschke and coworkers [27]implicating the augmenting emergence of S enterica strainsReferring to Table 2(b) VITEK 2 AST revealed resistance ofisolate Lt25 (serogroupD serotype Enteritidis) to tigecyclineThis is the first report in the world on tigecycline-resistantS enterica serotype Enteritidis with MIC of ge8120583gmL fromanimal source using VITEK 2 AST This finding is animportant contribution to the global data bank of MDRtigecycline-resistant S enterica serotypes Further character-ization of this novel tigecycline-resistant S enterica strainshould be consequently done as this may reveal genetic basisof resistance and factors involved in it
Chu and coworkers [32] isolated trimethoprim resistant-S enterica serotype Virchow from human in Taiwan VITEK2 AST performed in this study revealed sulfamethoxazoletrimethoprim-resistant S enterica serotypes TyphimuriumCholeraesuis Enteritidis and Weltevreden isolates (Tables2(b) and 3) This result is of huge relevance since this antimi-crobial is commonly administered to treat salmonellosis inthe Philippines In 2005Maluping [26] isolated sulfamethox-azole trimethoprim-resistant S enterica serotype Cholerae-suis from swine in Bulacan Philippines The present study isconsistent with the latter as sulfamethoxazole trimethoprim-resistant S enterica serotype Choleraesuis was isolated fromtonsil tissues of slaughtered swine in Metro Manila Philip-pines Additionally this study is the first to report on detec-tion of sulfamethoxazole trimethoprim-resistant S entericaserotypes namely Enteritidis Heidelberg Typhimuriumand Weltevreden from tonsil and JLN tissues of slaugh-tered swine in the Philippines using VITEK 2 AST Due toincreasing resistance of nontyphoid S enterica serotypes to
sulfamethoxazole trimethoprim it is not anymore consideredas appropriate treatment against invasive salmonellosis [33]
Multiple drug resistance is defined as resistance to threeor more classes of antimicrobials [31] MDR Salmonella sppisolates have been reported since the 1960s [11 30 32 33]Thiscould be attributed to the use of high levels of combinations ofantibiotics without adequate supervision or veterinary advicethat is common in small-scale hog-raising in the country [19]Detection of MDR Salmonella spp isolates from Philippinehogs thus deserves great attention
Cephalosporins and fluoroquinolones are recommendedfor treatment against strains resistant to ampicillin and sul-famethoxazole trimethoprim [33] Although isolates resistantto cephalosporins have been detected in the present study100 susceptibility of all isolates tested to levofloxacin a thirdgeneration fluoroquinolone was noted (Table 2(b))
5 Conclusion
The results obtained from this study confirm the role ofswine as reservoir of MDR S enterica Moreover this studydetected for the first time in the world MDR tigecycline-resistant S enterica serotype Enteritidis from animal sourceusing VITEK 2 AST that poses huge therapeutic challengeto animal and human medicine Hence there is a need forcontinuing regulation of antimicrobial use and mandatoryantimicrobial susceptibility testing in food animals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by Grants from the Office ofthe Vice Chancellor for Research and Development of theUniversity of the Philippines Diliman Office of the VicePresident for Academic Affairs of the University of thePhilippines System and the Department of the Agriculture-Biotechnology Program Implementation Unit
References
[1] R S Hendriksen M Mikoleit C Kornschober et al ldquoEmer-gence of multidrug-resistant Salmonella concord infections inEurope and the United States in children adopted from Ethio-pia 2003ndash2007rdquoThePediatric Infectious Disease Journal vol 28no 9 pp 814ndash818 2009
[2] E L Hohmann ldquoNontyphoidal salmonellosisrdquo Clinical Infec-tious Diseases vol 32 no 2 pp 263ndash269 2001
[3] CDC ldquoGeneral information on Salmonellardquo Atlanta Geor-gia USA 2012 httpwwwcdcgovSalmonellageneralindexhtml
[4] W A Gebreyes P R Davies P K Turkson et al ldquoCharac-terization of antimicrobial-resistant phenotypes and genotypesamong Salmonella enterica recovered from pigs on farms fromtransport trucks and from pigs after slaughterrdquo Journal of FoodProtection vol 67 no 4 pp 698ndash705 2004
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
ISRNMicrobiology 9
[5] B Nowak T von Muffling S Chaunchom and J HartungldquoSalmonella contamination in pigs at slaughter and on the farma field study using an antibody ELISA test and a PCR techniquerdquoInternational Journal of Food Microbiology vol 115 no 3 pp259ndash267 2007
[6] V Labro ldquoSalmonella infection in pigs risesrdquo Metro ManilaPhilippines 2009 httpservicesinquirernetprintprintphparticle id=20090201-186729
[7] J B Zabala D A Mendoza F Caput M C Roces and MWhite ldquoSalmonella food poisoning in Benguetrdquo Philippine Jour-nal of Microbiology Infectious Diseases vol 22 pp 5ndash7 1993
[8] A Lopez ldquoDoH eyes Salmonella in Tondo food poisoningrdquoMetro Manila Philippines 2008 httpnewsinfoinquirernetbreakingnewsmetroview20080925-162897DoH-eyes-Salmo-nella-in-Tondo-food-poisoning
[9] C Reyes-Estrope ldquoSalmonella found cause of food poisoningrdquoMetro Manila Philippines 2011 httpnewsinfoinquirernet17168Salmonella-found-cause-of-bulacan-food-poisoning
[10] H Y Lee L H Su M H Tsai et al ldquoHigh rate of reduced sus-ceptibility to ciprofloxacin and ceftriaxone among nontyphoidSalmonella clinical isolates in Asiardquo Antimicrobial Agents andChemotherapy vol 53 no 6 pp 2696ndash2699 2009
[11] H J Yoon S H Cho and SH Kim ldquoA case ofmultidrug-resist-ant Salmonella enterica serovar typhi treated with a bench tobedside approachrdquoYonseiMedical Journal vol 50 no 1 pp 147ndash151 2009
[12] D M Livermore M Struelens J Amorim et al ldquoMulticen-tre evaluation of the VITEK 2 advanced expert system forinterpretive reading of antimicrobial resistance testsrdquo Journal ofAntimicrobial Chemotherapy vol 49 no 2 pp 289ndash300 2002
[13] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-SecondInformational Supplement Clinical Laboratory Standards Insti-tute Wayne Pa USA 2012
[14] M E E Nori and K L Thong ldquoDifferentiation of Salmonellaenterica based on PCR detection of selected somatic andflagellar antigensrdquoAfrican Journal ofMicrobiology Research vol4 no 9 pp 871ndash876 2010
[15] C H Chiu and J T Ou ldquoRapid identification of Salmonellaserovars in feces by specific detection of virulence genes invAand spvC by an enrichment broth culture- multiplex PCRcombination assayrdquo Journal of Clinical Microbiology vol 34 no10 pp 2619ndash2622 1996
[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999
[17] N Al Naiemi B Zwart M C Rijnsburger et al ldquoExtended-spectrum-beta-lactamase production in a Salmonella entericaserotype typhi strain from the Philippinesrdquo Journal of ClinicalMicrobiology vol 46 no 8 pp 2794ndash2795 2008
[18] L R Baldrias and A K Raymundo ldquoAntimicrobial resistanceprofile of local campylobacter jejuni recovered from ceca ofdressed chickens of commercial and backyard raisers in LagunaPhilippinesrdquo Philippine Journal of Veterinary Medicine vol 46no 2 pp 87ndash94 2009
[19] T T Huynh A J Aarnink A Drucker and M W VerstegenldquoPig production in Cambodia Laos Philippines and Vietnama reviewrdquo Asian Journal of Agriculture and Development vol 4pp 69ndash90 2007
[20] P D Fey T J Safranek M E Rupp et al ldquoCeftriaxone-resistantSalmonella infection acquired by a child from cattlerdquo The New
England Journal of Medicine vol 342 no 17 pp 1242ndash12492000
[21] M de Toro Y Saenz E Cercenado et al ldquoGenetic characteriza-tion of the mechanisms of resistance to amoxicillinclavulanateand third-generation cephalosporins in Salmonella entericafrom three Spanish hospitalsrdquo International Microbiology vol14 no 3 pp 173ndash181 2011
[22] D M Livermore M W Carter S Bagel et al ldquoIn vitro activ-ities of ertapenem (MK-0826) against recent clinical bacteriacollected in Europe and Australiardquo Antimicrobial Agents andChemotherapy vol 45 no 6 pp 1860ndash1867 2001
[23] L H Su T L Wu and C H Chiu ldquoDevelopment of carba-penem resistance during therapy for non-typhoid Salmonellainfectionrdquo Clinical Microbiology and Infection vol 18 no 4 ppE91ndashE94 2012
[24] S B Vakulenko and S Mobashery ldquoVersatility of aminogly-cosides and prospects for their futurerdquo Clinical MicrobiologyReviews vol 16 no 3 pp 430ndash450 2003
[25] N P Arboleda M F Vizmanos E M Valera and E B BranaldquoSepticemic salmonellosis in a pigletrdquo Philippine Journal ofVeterinary Medicine vol 29 no 2 pp 57ndash58 1992
[26] R PMaluping ldquoPneumonia associatedwith SalmonellaCholer-aesuis infection in a pigletrdquo Philippine Journal of VeterinaryMedicine vol 42 no 1 pp 53ndash55 2005
[27] M Hentschke M Christner I Sobottka M Aepfelbacher andHRohde ldquoCombined ramRmutation and presence of aTn1721-associated tet(A) variant in a clinical isolate of Salmonellaenterica serovar hadar resistant to tigecyclinerdquo AntimicrobialAgents and Chemotherapy vol 54 no 3 pp 1319ndash1322 2010
[28] A C Fluit A Florijn J Verhoef and D Milatovic ldquoPresence oftetracycline resistance determinants and susceptibility to tige-cycline andminocyclinerdquoAntimicrobial Agents and Chemother-apy vol 49 no 4 pp 1636ndash1638 2005
[29] D J Hoban S K Bouchillon B M Johnson J L Johnsonand M J Dowzicky ldquoIn vitro activity of tigecycline against6792 Gram-negative and Gram-positive clinical isolates fromthe global Tigecycline Evaluation and Surveillance Trial (TESTProgram 2004)rdquo Diagnostic Microbiology and Infectious Dis-ease vol 52 no 3 pp 215ndash227 2005
[30] T Horiyama E Nikaido A Yamaguchi and K Nishino ldquoRolesof Salmonellamultidrug efflux pumps in tigecycline resistancerdquoJournal of Antimicrobial Chemotherapy vol 66 no 1 pp 105ndash110 2011
[31] T R Fritsche P A Strabala H S Sader M J Dowzicky andR N Jones ldquoActivity of tigecycline tested against a global col-lection of Enterobacteriaceae including tetracycline-resistantisolatesrdquoDiagnostic Microbiology and Infectious Disease vol 52no 3 pp 209ndash213 2005
[32] C Chu B Doublet Y L Lee et al ldquoSalmonella genomic island1-J variants associated with change in the antibiotic resistancegene cluster in multidrug-resistant Salmonella enterica serovarVirchow isolated from humans Taiwan 2004ndash2006rdquo ClinicalMicrobiology and Infection vol 18 no 1 pp 47ndash53 2012
[33] L H Su C H Chiu C Chu and J T Ou ldquoAntimicrobial resis-tance in nontyphoid Salmonella serotypes a global challengerdquoClinical Infectious Diseases vol 39 no 4 pp 546ndash551 2004
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
