Research Article Detection of Extended-Spectrum...

Research ArticleDetection of Extended-Spectrum Beta-Lactamase-ProducingEscherichia coli in Market-Ready Chickens in Zambia

K Chishimba12 B M Hangrsquoombe2 K Muzandu2 S E Mshana3 M I Matee4

C Nakajima56 and Y Suzuki56

1Lusaka City Council Public Health Department PO Box 30789 Lusaka Zambia2Microbiology Unit Paraclinical Studies University of Zambia PO Box 32379 Lusaka Zambia3Weill Bugando School of Medicine Catholic University of Health and Allied Sciences PO Box 1464 Mwanza Tanzania4Department of Microbiology and Immunology School of Medicine Muhimbili University of Health and Allied SciencesPO Box 65001 Dar es Salaam Tanzania5Division of Bioresources Hokkaido University Research Center for Zoonosis Control Kita-20 Nishi-10 Kita-kuSapporo 001-0020 Japan6The Global Station for Zoonosis Control Hokkaido University Sapporo Kita-20 Nishi-10 Kita-ku Sapporo 001-0020 Japan

Correspondence should be addressed to B M Hangrsquoombe mudenda68yahoocom

Received 27 November 2015 Revised 2 March 2016 Accepted 29 March 2016

Academic Editor Karl Drlica

Copyright copy 2016 K Chishimba et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains The objective of this study was to detect the presence of extended-spectrum 120573-lactamase- (ESBL-) producingEscherichia coli in poultry in Zambia A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichiacoli The cultured E coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detectionof 119887119897119886CTX-M 119887119897119886SHV and 119887119897119886TEM genes Overall 201 77384 (95 CI 432ndash655) of total samples analyzed contained ESBL-producing Escherichia coli The antimicrobial sensitivity test revealed that 857 (6677 CI 757ndash92) of ESBL-producing E coliisolates conferred resistance to beta-lactam and other antimicrobial agentsThese results indicate that poultry is a potential reservoirfor ESBL-producing Escherichia coli The presence of ESBL-producing Escherichia coli in poultry destined for human consumptionrequires strengthening of the antibiotic administering policy This is important as antibiotic administration in food animals isgaining momentum for improved animal productivity in developing countries such as Zambia

1 Introduction

Extended-spectrum beta-lactamase (ESBL) producers areGram-negative bacteria that produce enzymes that bestowresistance to most beta-lactam antibiotics like penicillinscephalosporins and the monobactam aztreonam [1] TheseESBL producers have been noticed mainly in the Enterobac-teriaceae family of bacteriawhichmay harbour several antibi-otic resistance determinants making treatment of infectionscaused by these pathogens more difficult [2]

Extended-spectrum beta-lactamase producers have acomplex epidemiology the most prominent bacteria in-volved include E coli and K pneumoniae whose reservoirscomprise the environment (soil and water) wild animals

farm animals food and pets [3] In some communities back-yard poultry houses at residential premises may disseminateantimicrobial-resistant ESBL producers in the environmentas previously observed [4] A study conducted in Spainidentified the presence of ESBL-producing bacteria in poultrywith E coli strains comprising ESBL CTX-M-14 CTX-M-9 and SHV-12 [5] In another study conducted in Britain545 of CTX-M-carrying E coli was isolated from broilerabattoirs and 36 from individual broiler caecal samples [6]Food animals colonized with ESBL-producing bacteria canenhance the spread of bacteria at the community level [7]Antimicrobial-resistant E coli asymptomatically colonizesthe intestinal flora of food animals with a likelihood ofbecoming infectious to humans if consumed through the

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 5275724 5 pageshttpdxdoiorg10115520165275724

2 International Journal of Microbiology

food chain [8] Furthermore the predominant presence ofCefotaxime-Munich genes in healthy broiler chickens can inthe long run spread the resistant ESBL-genes from the rearingenvironment into the food chain [3] Contamination of meatproducts during slaughtering dressing and evisceration ofinternal contents is another risk factor that can result infurther spread of resistant ESBLs genes within the humanpopulation [3 6]

Extended-spectrum beta-lactamase-producing bacteriaare frequently resistant to many antimicrobial agents usuallyrecommended for the treatment of infections such as gentam-icin fluoroquinolones and trimethoprim-sulfamethoxazole[9] This leads to serious challenges in the treatment ofESBL-Enterobacteriaceae infections because the bacterialplasmid may harbour several antibiotic resistance determi-nants [2] Heavy usage of antibiotics has been reportedto be a risk factor in the acquisition of ESBL-producingorganisms An increased trend of resistance to commonlyused antibiotics namely ampicillin cotrimoxazole gen-tamicin erythromycin tetracycline and third-generationcephalosporins has been observed [10 11]

In the present study we found the presence of ESBL-producing E coli bacteria in market-ready chickens Theseorganisms have been established to confer resistance tomany antimicrobial agents recommended for the treatmentof infections such as gentamicin fluoroquinolones andtrimethoprim-sulfamethoxazole [2] This leads to seriouschallenges in the treatment of ESBL-Enterobacteriaceaeinfections [10 11] A baseline study was therefore initiated todetermine the carriage of ESBL in food of animal origin inmarket-ready broiler chickens in Zambia

2 Materials and Methods

21 Study Area and Sampling The study was conducted inLusaka the capital city of Zambia Lusaka covers an estimatedarea of 360 km2 and is located at 15∘301015840 latitude south and28∘171015840 longitude east The city lies on a plateau 1280m abovesea level [12] A poultry abattoir was considered for thepurpose of poultry swab sampling at a point where broilerchickens were to enter the market A total of 384 poultrysamples were collected at the slaughterhouse before the car-casses were chilled

The swabs were labelled appropriately and collectionof the swab samples was aseptically done This involvedthe use of sterile swab sticks (Oxoid Basingstoke UK)which were placed in tubes containing a Cary-Blair transportmedium (Oxoid Basingstoke UK)The poultry samples wereinoculated on MacConkey agar (Oxoid Basingstoke UK)containing 2mgL of cefotaxime (Sigma-Aldrich MunichGermany) for preliminary screening of ESBL-producing bac-teria [13]The plates were later incubated at 37∘C for 24 hoursThe colonies that grew onMacConkey agar were identified aslactose fermenters or nonlactose fermenters Identification ofE coli lactose-fermenting positive colonies was done usingphenotypic characteristics and confirmed by the Triple SugarIron (TSI) and IMViC tests as described by Rayamajhi et al[13] and Batchelor et al [14] For genetic detection E coliisolates were cultured on brain-heart-infusion broth (Nissui

Tokyo Japan) at 37∘C for 24 hours After incubation DNAwas extracted by boiling methods [15] The E coli isolateswere subjected to PCR for confirmation of resistance genesTEM (Temoniera) SHV (Sulphydryl Variable) and CTX-M (Cefotaxime-Munich) using primers previously used byother workers [14 16] The PCR (Finnzymes Oy Finland)was performed in a total reaction volume of 10 120583L consistingof 5 120583L Phusion master mix 2 120583L sterile distilled water2 120583L primers (forward and reverse) and 1120583L bacterial DNAtemplateThe PCRwas performed using the rapid cycle DNAamplificationmethod comprising an initial denaturation stepat 98∘C for 30 seconds followed by 35 cycles of templatedenaturation at 98∘C for 1 second and primer annealing at60∘C for 5 seconds and 72∘C for 1 second with final extensionat 72∘C for 10 seconds The PCR products were later viewedwith ethidium bromide after electrophoresis through 15agarose gel [17]

22 Antibiotic Sensitivity Testing The antimicrobial suscep-tibility testing was done using the Kirby-Bauer disc dif-fusion method on Mueller Hinton Agar (Becton Dick-inson and Company MD USA) based on the Clini-cal Laboratory Standard Institute (CLSI) guidelines [18]The antibiotic discs (Becton Dickinson and CompanyMD USA) used included ampicillin (10 120583g) sulfamethox-azoletrimethoprim (1252375120583g) streptomycin (300 120583g)ciprofloxacin (5120583g) tetracycline (30 120583g) gentamicin (10 120583g)nalidixic acid (30 120583g) chloramphenicol (30120583g) ceftazidime(30 120583g) norfloxacin (10 120583g) and cefotaxime (30 120583g) Thephenotypic confirmation of ESBL isolates was done by thecombination of disc approximation method using eitherceftazidime (30 120583g) or cefotaxime (30 120583g) alone followed byovernight incubation at 37∘C for 18ndash24 hrs Interpretationof susceptibility patterns on other antimicrobial discs wasdone using guidelines laid down in the CLSI which providesbreak points corresponding to zone of inhibition diameterAn increase in antibiotic zone diameter (5ndash12mm) for eitherceftazidime or cefotaxime indicated ESBL production [10 18]Quality control standard laboratory procedures were strictlyadhered to to avoid contamination Escherichia coli ATCC25922 was used as a quality control organism

3 Results

Of the 384 E coli isolates analyzed for the presence ofESBL-producing isolates on PCR a total of 201 77384(95 CI 432ndash655) were confirmed to be ESBL-producingisolates The ESBL-producing E coli isolates carried the 120573-lactamase genes of either 119887119897119886CTX-M 119887119897119886SHV and 119887119897119886TEM or acombination The major gene detected on PCR was 119887119897119886CTX-Mcluster followed by 119887119897119886SHV cluster and 119887119897119886TEM cluster Acombination of 119887119897119886CTX-M + 119887119897119886TEM cluster showed 44 (17isolates) This was followed by a combination of 119887119897119886CTX-M +119887119897119886SHV cluster and 119887119897119886CTX-M + 119887119897119886SHV + 119887119897119886TEM cluster with052 (2 isolates) and the lowest was a combination of 119887119897119886TEM+ 119887119897119886SHV cluster with 03 (1 isolate) (Table 1)

The seventy-seven (77) E coli isolates preliminarily iden-tified as ESBL-producing E coli were subjected to antibioticsusceptibility testing Of 77 E coli isolates analyzed 857

International Journal of Microbiology 3

Table 1 Confirmed ESBL-producing E coli (using PCR and genedetection) isolates

Detected gene (s) Number of Ecoli isolates

of E coliisolates

(119899 = 384)a

SHV 2 052CTX-M 50 13TEM 3 08CTX-M and TEM 17 44CTX-M and SHV 2 052TEM and SHV 1 03CTX-M SHV and TEM 2 052Noneb 307 799Proven ESBL producers 77 201a384 E coli isolates suspected of being ESBL producers were examinedbNegative in all PCRs

Table 2 Antibiotic susceptibilities of the isolates 119899 = 77

Antibiotic Resistant number ()Cefotaximeceftazidime 77 (100)Ampicillin 77 (100)Chloramphenicol 44 (571)Ciprofloxacin 37 (481)Gentamicin 29 (377)Nalidixic acid 37 (481)Norfloxacin 42 (545)Streptomycin 15 (208)Sulfamethoxazoletrimethoprim 32 (416)Tetracycline 46 (597)

(6677 CI 757ndash92) were resistant to one or several antimi-crobial compounds The diversity of the antibiotic resistantand susceptible E coli isolates was clearly observed with66 isolates being resistant to one or more antibiotics usedand 11 isolates being susceptible to all antibiotics testedInterestingly high resistance rates were noticed in ampicillin(100) cefotaximeceftazidime (100) tetracycline (597)chloramphenicol (571) and norfloxacin (545) (Table 2)It was further observed that multidrug resistance (MDR)of E coli isolates to six or more drugs was most frequent(455 3577) followed by resistance to five drugs (117977) and four drugs (117 977) Based on the antimicrobialsusceptibility test results E coli frompoultry in this study canbe grouped in six phenotypes (Table 3)

4 Discussion

Bacteria in food-producing animals are spread throughthe food chain In Africa the desire to optimize animalproduction has led to indiscriminate use of antibiotics Theheavy usage of antibiotics has been reported to be a riskfactor for acquisition of ESBL-producing organisms resulting

in an increased trend of resistance to commonly used antibi-otics namely ampicillin cotrimoxazole gentamicin ery-thromycin tetracycline and third generation cephalosporins[16] In Zambia drugstores which stock human and livestockantimicrobial agents are coming up without proper guidanceon the dispensation of drugs The inadequate monitoring ofthe drugstores in the nation has led people to easily access thelivestock antibiotics without following the recommended laiddown clinical prescription procedures thereby increasing therisk of ESBL in food animals Detection of ESBL-producingE coli isolates in poultry has not been fully conducted inZambia This cross sectional study revealed that an overallof 20 ESBL-producing E coli isolates were detected inpoultryThehigh prevalence observedmay be due to frequentadministration of antimicrobials to poultry which in turnincreases the risk of higher antimicrobial-resistant E colistrains in the normal intestinal flora as observed in a studyconducted by Carattoli [3] In this study it was establishedthat food animals were possible reservoirs for resistant faecalflora particularly E coli Furthermore the high colonizationrate could be attributed to cross contamination of poultryin abattoirs particularly during slaughtering and dressingThe processes of slaughtering and dressing are potential riskfactors that may exacerbate the transmission rate of ESBL-producing E coli resistant genes as described by Lavilla et al[8] and Reich et al [16]

The predominant ESBL-producing E coli gene identifiedin this study was 119887119897119886CTX-M cluster with 13 which wasrelatively low in relation to 545 of 119887119897119886CTX-M carryingE coli isolated from poultry chickens in Britain [6] InZambia the economic situation has led many residents torearing broiler chickens as an income generating ventureThis brings humans into close contact with poultry whichcould be a possible route of shedding ESBL producers intothe environment However no detailed studies have yet beenconducted in Zambia to specifically describe the types ofESBL-producing Enterobacteriaceae present in apparentlyhealthy chickens and other food animals

In addition the study also showed that ESBL-producingE coli isolates carried multiple types of beta-lactamase genesand that the combination of 119887119897119886CTX-M + 119887119897119886TEM cluster waspredominant followed by 119887119897119886SHV cluster and 119887119897119886TEM clusterThis therefore confirms that ESBL-mediated plasmids arecapable of carrying more than one beta-lactamase gene andthus result in high-level presence of beta-lactam resistantphenotypes as described by Rottier et al [2] Previous studiesin Spain identified E coli strains comprising 119887119897119886CTX-M-14119887119897119886CTX-M-9 and 119887119897119886SHV-12 as the predominant ESBL-producingsubtypes isolated among poultry [12]

Antimicrobial susceptibility testing revealed interestingpatterns with resistance rates observed in the majority ofantimicrobial agents tested From the antimicrobial sus-ceptibility results 857 (6677 CI 757ndash92) of isolateswere resistant to chloramphenicol ciprofloxacin gentam-icin nalidixic acid norfloxacin streptomycin sulfamethoxa-zoletrimethoprim and tetracycline The findings are similarto studies conducted by Mshana et al [10] For instancea study on published literature for Democratic Republicof Congo Mozambique Tanzania and Zambia revealed an


Table 3 Antibiotic resistance patterns of E coli isolates

Antibiotic combination Number of resistant isolates Observation

Ampicillin and tetracycline 1 13 Resistant to two antibiotics

Ampicillin tetracycline chloramphenicol andceftazidime

3 39 Resistant to four antibiotics

Ampicillin ceftazidime chloramphenicol andnorfloxacin


Ampicillin chloramphenicol cefotaxime andsulfamethoxazole


Ampicillin streptomycin gentamicin tetracycline andcefotaxime

9 117 Resistant to five antibiotics

Ampicillin streptomycin tetracycline cefotaximenalidixic acid ceftazidime norfloxacin andciprofloxacin

35 455 Resistant to six antibiotics

increased trend of resistance to ampicillin cotrimoxazolegentamicin erythromycin tetracycline and third-generationcephalosporins [11] In this study high resistance rates tobeta-lactam drugs namely ampicillin (100) cefotaximeand ceftazidime (100) were observed among the isolatesinvestigated At least six phenotypes were observed from thisstudy indicating a wide diversity of resistance Unfortunatelythe study had a limitation as it was not able to establish thetype of antimicrobials frequently used for poultry feed andgrowth promotion

5 Conclusion

This is the first study to be conducted in Zambia on detectionof ESBL-producing E coli isolates in poultry This thereforefurther confirms that poultry can be one of the major andpotential reservoirs for the antimicrobial ESBL resistant geneswhich could spread into the food chain The study hasalso shown a widespread occurrence of multidrug resistancepatterns of E coli isolates in poultry

Abbreviations

ESBL Extended-spectrum beta-lactamasesPCR Polymerase chain reaction

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

K Chishimba B M Hangrsquoombe K Muzandu S EMshana M I Matee C Nakajima and Y Suzuki conceivedand designed the experiments K Chishimba and B MHangrsquoombe performed the experiments and analyzed andinterpreted the data S E Mshana M I Matee and Y Suzukihelped in study design coordinated the study and reviewedthe paper K Chishimba and B M Hangrsquoombe wrote thepaper All authors have read and approved the final paper

Acknowledgments

This work was supported by the Wellcome Trust Grantno WT 087546MA for one medicine Africa-UK researchcapacity development partnership for infectious diseases inSouthern Africa under the Southern African Center forinfectious disease surveillance and in part by the grant fromthe Ministry of Education Culture Sports Science andTechnology of Japan for the Joint Research Program of theResearch Center for Zoonosis Control Hokkaido UniversityIt was also supported by the Global COE Program on theEstablishment of International Collaboration Centers forZoonosis Control Hokkaido University and J-GRID and theJapan Initiative for Global Research Network on InfectiousDiseases from the Ministry of Education Culture SportsScience and Technology of Japan The authors thank MrM Mubanga Mr L Moonga and Mr E Mulenga for thetechnical assistance

References

[1] L M Silvia and A G Jacoby Extended Spectrum Beta-Lac-tamases Wolters Kluwer Health 2014

[2] WC RottierH SMAmmerlaan andM JM Bonten ldquoEffectsof confounders and intermediates on the association of bac-teraemia caused by extended-spectrum 120573-lactamase-produc-ing Enterobacteriaceae and patient outcome a meta-analysisrdquoJournal of Antimicrobial Chemotherapy vol 67 no 6 pp 1311ndash1320 2012

[3] A Carattoli ldquoAnimal reservoirs for extended spectrum 120573-lac-tamase producersrdquo Clinical Microbiology and Infection vol 14no 1 pp 117ndash123 2008

[4] W D Nafarnda I E Ajayi J C Shawulu et al ldquoBacteriologicalquality of abattoir effluents discharged into water bodies inAbuja Nigeriardquo ISRN Veterinary Science vol 2012 Article ID515689 5 pages 2012

[5] L BrinasMAMorenoM Zarazaga et al ldquoDetection of CMY-2 CTX-M-14 and SHV-12120573-lactamases inEscherichia coli fecal-sample isolates from healthy chickensrdquo Antimicrobial Agentsand Chemotherapy vol 47 no 6 pp 2056ndash2058 2003


[6] L P Randall C Clouting R A Horton et al ldquoPrevalenceof Escherichia coli carrying extended-spectrum 120573-lactamases(CTX-M and TEM-52) from broiler chickens and turkeys inGreat Britain between 2006 and 2009rdquo Journal of AntimicrobialChemotherapy vol 66 no 1 pp 86ndash95 2011

[7] V Bortolaia L Guardabassi M Bisgaard J Larsen and A MBojesen ldquoEscherichia coli producing CTX-M-1 -2 and -9 group120573-lactamases in organic chicken egg productionrdquoAntimicrobialAgents and Chemotherapy vol 54 no 8 pp 3527ndash3528 2010

[8] S Lavilla J J Gonzalez-Lopez E Miro et al ldquoDissemination ofextended-spectrum 120573-lactamase-producing bacteria the food-borne outbreak lessonrdquo Journal of Antimicrobial Chemotherapyvol 61 no 6 pp 1244ndash1251 2008

[9] J Rodrıguez-Bano J C Alcala J M Cisneros et al ldquoCommu-nity infections caused by extended-spectrum 120573-lactamase-pro-ducing Escherichia colirdquo Archives of Internal Medicine vol 168no 17 pp 1897ndash1902 2008

[10] S E Mshana E Kamugisha M Mirambo T Chakraborty andE F Lyamuya ldquoPrevalence of multiresistant gram-negative org-anisms in a tertiary hospital in Mwanza Tanzaniardquo BMC Re-search Notes vol 2 article 49 2009

[11] S E Mshana M Matee and M Rweyemamu ldquoAntimicrobialresistance in human and animal pathogens in Zambia Demo-cratic Republic of CongoMozambique and Tanzania an urgentneed of a sustainable surveillance systemrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 12 no 1 article 28 2013

[12] MFNP Lusaka District Draft Development Plan 2006 to 2011Ministry of Finance and National Planning Planning andEconomic Management Department Lusaka Zambia 2005

[13] N Rayamajhi S G Kang D Y Lee et al ldquoCharacterization ofTEM- SHV- and AmpC-type 120573-lactamases from cephalospo-rin-resistant Enterobacteriaceae isolated from swinerdquo Interna-tional Journal of Food Microbiology vol 124 no 2 pp 183ndash1872008

[14] M Batchelor K Hopkins E J Threlfall et al ldquo119887119897119886CTX-Mgenes in clinical Salmonella isolates recovered from humans inEngland and Wales from 1992 to 2003rdquo Antimicrobial Agentsand Chemotherapy vol 49 no 4 pp 1319ndash1322 2005

[15] K Changkaew A Intarapuk F Utrarachkij C Nakajima OSuthienkul and Y Suzuki ldquoAntimicrobial resistance extended-spectrum b-Lactamase productivity and class 1 integrons inEscherichia coli from healthy swinerdquo Journal of Food Protectionvol 78 no 8 pp 1443ndash1450 2015

[16] F Reich V Atanassova and G Klein ldquoExtended-spectrum120573-lactamase- and ampc-producing Enterobacteria in healthybroiler chickens Germanyrdquo Emerging Infectious Diseases vol19 no 8 pp 1253ndash1259 2013

[17] R Ranjbar C Mammina M R Pourshafie and M M Soltan-Dallal ldquoCharacterization of endemic Shigella boydii strainsisolated in Iran by serotyping antimicrobial resistance plasmidprofile ribotyping and pulsed-field gel electrophoresisrdquo BMCResearch Notes vol 1 article 74 2008

[18] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing 19th InformationalSupplementM100-S19 Clinical and Laboratory Standards Insti-tute Wayne Pa USA 2009

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International Journal of

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Zoology


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BioinformaticsAdvances in

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Evolutionary BiologyInternational Journal of



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Advances in

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


food chain [8] Furthermore the predominant presence ofCefotaxime-Munich genes in healthy broiler chickens can inthe long run spread the resistant ESBL-genes from the rearingenvironment into the food chain [3] Contamination of meatproducts during slaughtering dressing and evisceration ofinternal contents is another risk factor that can result infurther spread of resistant ESBLs genes within the humanpopulation [3 6]

Extended-spectrum beta-lactamase-producing bacteriaare frequently resistant to many antimicrobial agents usuallyrecommended for the treatment of infections such as gentam-icin fluoroquinolones and trimethoprim-sulfamethoxazole[9] This leads to serious challenges in the treatment ofESBL-Enterobacteriaceae infections because the bacterialplasmid may harbour several antibiotic resistance determi-nants [2] Heavy usage of antibiotics has been reportedto be a risk factor in the acquisition of ESBL-producingorganisms An increased trend of resistance to commonlyused antibiotics namely ampicillin cotrimoxazole gen-tamicin erythromycin tetracycline and third-generationcephalosporins has been observed [10 11]

In the present study we found the presence of ESBL-producing E coli bacteria in market-ready chickens Theseorganisms have been established to confer resistance tomany antimicrobial agents recommended for the treatmentof infections such as gentamicin fluoroquinolones andtrimethoprim-sulfamethoxazole [2] This leads to seriouschallenges in the treatment of ESBL-Enterobacteriaceaeinfections [10 11] A baseline study was therefore initiated todetermine the carriage of ESBL in food of animal origin inmarket-ready broiler chickens in Zambia

2 Materials and Methods

21 Study Area and Sampling The study was conducted inLusaka the capital city of Zambia Lusaka covers an estimatedarea of 360 km2 and is located at 15∘301015840 latitude south and28∘171015840 longitude east The city lies on a plateau 1280m abovesea level [12] A poultry abattoir was considered for thepurpose of poultry swab sampling at a point where broilerchickens were to enter the market A total of 384 poultrysamples were collected at the slaughterhouse before the car-casses were chilled

The swabs were labelled appropriately and collectionof the swab samples was aseptically done This involvedthe use of sterile swab sticks (Oxoid Basingstoke UK)which were placed in tubes containing a Cary-Blair transportmedium (Oxoid Basingstoke UK)The poultry samples wereinoculated on MacConkey agar (Oxoid Basingstoke UK)containing 2mgL of cefotaxime (Sigma-Aldrich MunichGermany) for preliminary screening of ESBL-producing bac-teria [13]The plates were later incubated at 37∘C for 24 hoursThe colonies that grew onMacConkey agar were identified aslactose fermenters or nonlactose fermenters Identification ofE coli lactose-fermenting positive colonies was done usingphenotypic characteristics and confirmed by the Triple SugarIron (TSI) and IMViC tests as described by Rayamajhi et al[13] and Batchelor et al [14] For genetic detection E coliisolates were cultured on brain-heart-infusion broth (Nissui

Tokyo Japan) at 37∘C for 24 hours After incubation DNAwas extracted by boiling methods [15] The E coli isolateswere subjected to PCR for confirmation of resistance genesTEM (Temoniera) SHV (Sulphydryl Variable) and CTX-M (Cefotaxime-Munich) using primers previously used byother workers [14 16] The PCR (Finnzymes Oy Finland)was performed in a total reaction volume of 10 120583L consistingof 5 120583L Phusion master mix 2 120583L sterile distilled water2 120583L primers (forward and reverse) and 1120583L bacterial DNAtemplateThe PCRwas performed using the rapid cycle DNAamplificationmethod comprising an initial denaturation stepat 98∘C for 30 seconds followed by 35 cycles of templatedenaturation at 98∘C for 1 second and primer annealing at60∘C for 5 seconds and 72∘C for 1 second with final extensionat 72∘C for 10 seconds The PCR products were later viewedwith ethidium bromide after electrophoresis through 15agarose gel [17]

22 Antibiotic Sensitivity Testing The antimicrobial suscep-tibility testing was done using the Kirby-Bauer disc dif-fusion method on Mueller Hinton Agar (Becton Dick-inson and Company MD USA) based on the Clini-cal Laboratory Standard Institute (CLSI) guidelines [18]The antibiotic discs (Becton Dickinson and CompanyMD USA) used included ampicillin (10 120583g) sulfamethox-azoletrimethoprim (1252375120583g) streptomycin (300 120583g)ciprofloxacin (5120583g) tetracycline (30 120583g) gentamicin (10 120583g)nalidixic acid (30 120583g) chloramphenicol (30120583g) ceftazidime(30 120583g) norfloxacin (10 120583g) and cefotaxime (30 120583g) Thephenotypic confirmation of ESBL isolates was done by thecombination of disc approximation method using eitherceftazidime (30 120583g) or cefotaxime (30 120583g) alone followed byovernight incubation at 37∘C for 18ndash24 hrs Interpretationof susceptibility patterns on other antimicrobial discs wasdone using guidelines laid down in the CLSI which providesbreak points corresponding to zone of inhibition diameterAn increase in antibiotic zone diameter (5ndash12mm) for eitherceftazidime or cefotaxime indicated ESBL production [10 18]Quality control standard laboratory procedures were strictlyadhered to to avoid contamination Escherichia coli ATCC25922 was used as a quality control organism

3 Results

Of the 384 E coli isolates analyzed for the presence ofESBL-producing isolates on PCR a total of 201 77384(95 CI 432ndash655) were confirmed to be ESBL-producingisolates The ESBL-producing E coli isolates carried the 120573-lactamase genes of either 119887119897119886CTX-M 119887119897119886SHV and 119887119897119886TEM or acombination The major gene detected on PCR was 119887119897119886CTX-Mcluster followed by 119887119897119886SHV cluster and 119887119897119886TEM cluster Acombination of 119887119897119886CTX-M + 119887119897119886TEM cluster showed 44 (17isolates) This was followed by a combination of 119887119897119886CTX-M +119887119897119886SHV cluster and 119887119897119886CTX-M + 119887119897119886SHV + 119887119897119886TEM cluster with052 (2 isolates) and the lowest was a combination of 119887119897119886TEM+ 119887119897119886SHV cluster with 03 (1 isolate) (Table 1)

The seventy-seven (77) E coli isolates preliminarily iden-tified as ESBL-producing E coli were subjected to antibioticsusceptibility testing Of 77 E coli isolates analyzed 857




of E coliisolates

(119899 = 384)a





4 Discussion





















5 Conclusion


Abbreviations


Competing Interests




Acknowledgments


References



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




of E coliisolates

(119899 = 384)a





4 Discussion





















5 Conclusion


Abbreviations


Competing Interests




Acknowledgments


References



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















5 Conclusion


Abbreviations


Competing Interests




Acknowledgments


References



























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Detection of Extended-Spectrum...

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