Antibiotic Susceptibility Pattern and Biofilm Formation in...

7
Research Article Antibiotic Susceptibility Pattern and Biofilm Formation in Clinical Isolates of Enterococcus spp. Sridevi Shridhar and Biranthabail Dhanashree Department of Microbiology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India Correspondence should be addressed to Biranthabail Dhanashree; [email protected] Received 15 January 2019; Accepted 5 February 2019; Published 3 March 2019 Academic Editor: Subhada Prasad Pani Copyright © 2019 Sridevi Shridhar and Biranthabail Dhanashree. 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. Enterococcus is a commensal in the intestine and is now emerging as a drug-resistant pathogen. It produces different virulence factors. Enterococcus surface protein (esp) is a virulence factor that helps in the adhesion, but its role in biofilm formation is still contradictory. Moreover, in many bacterial species, strong biofilm producer exhibits multidrug resistance. Hence, this study is done to know the antimicrobial susceptibility pattern of Enterococcus spp. and to correlate the drug resistance with biofilm production and esp gene. Enterococcal isolates were collected from various clinical specimens. Antibiotic susceptibility testing was done by disc diffusion, and biofilm production was performed by microtiter plate method. PCR was performed for detection of esp gene. Two E. faecium strains resistant to vancomycin and high-level aminoglycoside (HLAR) were non-biofilm-producers and did not harbor esp gene. However, other biofilm-producing E. faecium harbored esp gene, and this association was found to be statistically significant (p=0.024). It was observed that there was no significant association between biofilm formation and presence of esp gene in E. faecalis. Moreover, a significant correlation was not found between drug resistance and biofilm production in both Enterococcus species. us, biofilm formation is not always associated with the presence or absence of esp gene and or drug resistance in Enterococcus spp. 1. Introduction Enterococci are normal flora of oral cavity, gut, and female genital tract of humans and are known to cause nosocomial infections [1–4]. E. faecalis is responsible for 80-90 percent and E. faecium 5-10 percent of the human enterococcal infec- tions [5, 6]. Most frequent infections caused by Enterococcus spp. are urinary tract infections followed by intra-abdominal abscesses and bloodstream infections [7]. A high mortality rate of enterococcal infections is due to increasing resistance of the organism to -lactam antibiotics, aminoglycosides, and glycopeptides and inadequate response to the treatment [5, 8]. Pandemic spread of vancomycin- resistant Enterococci (VRE) and acquisition of resistance to newer antimicrobials warrant continued surveillance and early detection of VRE along with Minimum Inhibitory Concentrations (MIC) [9]. Biofilm protects Enterococci from host immune response and antibiotics. Biofilm-producing Enterococci cause recur- rent, chronic, and antibiotic-resistant infections [10–12]. According to the National Institute of Health, 80% of infec- tions are related to biofilm-forming microbes [13, 14]. Apart from biofilm-forming ability, Enterococcus spp. are known to produce various virulence factors [15]. Moreover, clinical isolates have been reported to harbor gene coding for esp virulence factor rather than the commensal strains [16]. Hence, the study was done to know the prevalence of drug resistance in clinical isolates of Enterococcus spp. and to find the association of drug resistance with biofilm formation and esp genes in this part of the country. 2. Materials and Methods 2.1. Isolation and Identification of Enterococcus spp. Entero- cocci isolated from clinical samples like pus, sputum, vaginal swab, and aspirates (n=150) received for routine culture at the Department of Microbiology, Kasturba Medical College (KMC) Mangalore, from December 2016 to June 2017 were included in the study. Institutional Ethics Committee, KMC Mangalore, India, has approved this study. All the media, Hindawi Interdisciplinary Perspectives on Infectious Diseases Volume 2019, Article ID 7854968, 6 pages https://doi.org/10.1155/2019/7854968

Transcript of Antibiotic Susceptibility Pattern and Biofilm Formation in...

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Research ArticleAntibiotic Susceptibility Pattern and Biofilm Formation inClinical Isolates of Enterococcus spp

Sridevi Shridhar and Biranthabail Dhanashree

Department of Microbiology Kasturba Medical College Mangalore Manipal Academy of Higher Education Manipal India

Correspondence should be addressed to Biranthabail Dhanashree dbiranthabailyahoocoin

Received 15 January 2019 Accepted 5 February 2019 Published 3 March 2019

Academic Editor Subhada Prasad Pani

Copyright copy 2019 Sridevi Shridhar and Biranthabail Dhanashree This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Enterococcus is a commensal in the intestine and is now emerging as a drug-resistant pathogen It produces different virulencefactors Enterococcus surface protein (esp) is a virulence factor that helps in the adhesion but its role in biofilm formation is stillcontradictoryMoreover in many bacterial species strong biofilm producer exhibits multidrug resistance Hence this study is doneto know the antimicrobial susceptibility pattern of Enterococcus spp and to correlate the drug resistance with biofilm productionand esp gene Enterococcal isolates were collected from various clinical specimens Antibiotic susceptibility testing was done by discdiffusion and biofilm production was performed by microtiter plate method PCR was performed for detection of esp gene Two Efaecium strains resistant to vancomycin and high-level aminoglycoside (HLAR) were non-biofilm-producers and did not harbor espgene However other biofilm-producing E faecium harbored esp gene and this association was found to be statistically significant(p=0024) It was observed that therewas no significant association between biofilm formation andpresence of esp gene inE faecalisMoreover a significant correlation was not found between drug resistance and biofilm production in both Enterococcus speciesThus biofilm formation is not always associated with the presence or absence of esp gene and or drug resistance in Enterococcusspp

1 Introduction

Enterococci are normal flora of oral cavity gut and femalegenital tract of humans and are known to cause nosocomialinfections [1ndash4] E faecalis is responsible for 80-90 percentand E faecium 5-10 percent of the human enterococcal infec-tions [5 6] Most frequent infections caused by Enterococcusspp are urinary tract infections followed by intra-abdominalabscesses and bloodstream infections [7]

A high mortality rate of enterococcal infections is due toincreasing resistance of the organism to 120573-lactam antibioticsaminoglycosides and glycopeptides and inadequate responseto the treatment [5 8] Pandemic spread of vancomycin-resistant Enterococci (VRE) and acquisition of resistance tonewer antimicrobials warrant continued surveillance andearly detection of VRE along with Minimum InhibitoryConcentrations (MIC) [9]

Biofilm protects Enterococci from host immune responseand antibiotics Biofilm-producing Enterococci cause recur-rent chronic and antibiotic-resistant infections [10ndash12]

According to the National Institute of Health 80 of infec-tions are related to biofilm-forming microbes [13 14] Apartfrom biofilm-forming ability Enterococcus spp are knownto produce various virulence factors [15] Moreover clinicalisolates have been reported to harbor gene coding for espvirulence factor rather than the commensal strains [16]Hence the study was done to know the prevalence of drugresistance in clinical isolates of Enterococcus spp and to findthe association of drug resistance with biofilm formation andesp genes in this part of the country

2 Materials and Methods

21 Isolation and Identification of Enterococcus spp Entero-cocci isolated from clinical samples like pus sputum vaginalswab and aspirates (n=150) received for routine culture atthe Department of Microbiology Kasturba Medical College(KMC) Mangalore from December 2016 to June 2017 wereincluded in the study Institutional Ethics Committee KMCMangalore India has approved this study All the media

HindawiInterdisciplinary Perspectives on Infectious DiseasesVolume 2019 Article ID 7854968 6 pageshttpsdoiorg10115520197854968

2 Interdisciplinary Perspectives on Infectious Diseases

antibiotic discs and chemicals used in the study were pro-cured from Hi-Media Laboratories Pvt Ltd Mumbai IndiaEnterococcal isolateswere identified by colony characteristicsand common biochemical reactions [17] and confirmed byVITEK-2 automated system (bioMerieux USA)

22 Antibiotic Susceptibility Test Antimicrobial susceptibil-ity to ampicillin (10120583g) penicillin (10 units) tetracycline(30120583g) erythromycin (15120583g) chloramphenicol (30120583g) van-comycin (30120583g) teicoplanin (30120583g) ciprofloxacin (5120583g) andnitrofurantoin (300120583g) was determined by Kirby-Bauer diskdiffusion [18] and interpreted as per CLSI guidelines [19]Thequality control strain used was ATCC E faecalis 29212

23 High-Level Aminoglycoside Resistance (HLAR) DetectionDetection of HLAR was performed with disks containinggentamicin (120120583g) and streptomycin (300 120583g) by disk dif-fusion method Results were read after incubation at 35∘Cfor 24h and after 48h for streptomycin A zone diameterof 6mm indicates resistance 7-9mm shows that the resultsare inconclusive and more than 10mm suggests that theisolates are sensitive to aminoglycosides Resistance by discdiffusion to gentamicin corresponds to MIC of gt500 120583gmland susceptibility corresponds to MIC of lt 500 120583gmlHowever for high-level streptomycin MIC of gt1000 120583gmlby broth dilution and gt2000 120583gml by agar dilution methodcorresponds to a zone diameter of 6mm by disk diffusionMIC of le500 120583gml by broth and le1000 120583gml by agardilution corresponds to 10 mm diameter by disk diffusionmethod [19]

24 Detection of MIC for Vancomycin Enterococcal isolateswere inoculated onto Muller Hinton Agar supplementedwith 5 defibrinated sheep blood Vancomycin E-strips (EzyMIC) were placed on the inoculated plates and incubatedat 37∘C in 5 CO

2for 24 h The MIC was read where

the ellipse intersects the MIC scale on the strip E faecalisATCC 29212 and ATCC 51299 were used as negative andpositive controls respectively The results were interpretedas sensitive (MIC le4120583gml) intermediate (MIC 8-16 120583gml)and resistant (MIC ge32 120583gml) based on the CLSI guidelines[19]

25 Biofilm Formation All the clinical isolates were checkedfor biofilm production by the procedure used by Kafil andMobarez (2013) and Triveda and Gomathi (2016) [20 21]Briefly freshly subcultured strains of Enterococcus on bloodagar plates were inoculated in 1ml of Brain Heart Infusion(BHI) broth with 1 glucose and incubated at 37∘C for 24 hTo 180120583l of fresh BHI medium 20120583l of 24-hour-old bacterialgrowth was added which corresponded to a turbidity of 05McFarland standard 200120583l of the suspension of the clinicalisolates and the control strain (E faecalis ATCC 29212) wereinoculated into flat bottommicrotiter plates in duplicates andincubated at 37∘C in 5 CO

2for 24h After incubation the

contents of the plate were removed tapped and washed threetimes with phosphate buffer saline The biofilm was fixed byadding 150120583l of methanol for 20 min It was air-dried for

about 30 min in an inverted position and later stained with01 crystal violet for 15min Excess stain was removed andplates were washed with distilled water 150120583l of 33 aceticacid was added in each well and kept for 30 min withoutshaking The optical density (OD) was measured at 570nmBased on the OD values the isolates were categorized asstrong biofilm formers (OD

570gt 2) medium (OD

570gt 1

but lt2) weak (OD570gt 05 but lt1) and non-biofilm-formers

(OD570le 05) [13]

26 Detection of the esp Gene by Polymerase Chain Reac-tion (PCR) All the isolates of E faecalis and E faeciumwere subjected to PCR for the detection of the esp geneDNA extraction was done by boiling method Brieflythree to four colonies of enterococcal isolates were sus-pended in 100120583l of distilled water The bacterial cells werelysed by boiling for 10 minutes in a dry bath The lysatewas centrifuged briefly and 2120583l of the supernatant wasused as the DNA PCR was done by using primers esp11 (51015840- TTGCTAATGCTAGTCCACGACC-31015840) and esp 12(51015840-GCGTCAACACTTGCATTGCCGAA-31015840 ) Nuclease-freewater and E faecalis ATCC 29212 were used as esp negativeand positive controls respectively PCR reaction mixtureconsisted of 200120583M of dNTP mixture and 25 U Taq poly-merase with 1X buffer and 25mMMgCl

2 02120583M of primers

and 1120583l of DNA The PCR tubes containing master mixprimer and DNA were amplified in a thermocycler (Bio RadInc USA) PCR reaction conditionswere initial denaturationat 95∘C for 2 min followed by 30 cycles of 94∘C for 45 secs63∘C for 45 secs and 72∘C for 1 min Final extension wascarried out at 72∘C for 10min The amplified product wasresolved by agarose gel electrophoresis using 15 agarose in1X Tris-acetate EDTA (TAE) buffer The gel was stained with05 mgml ethidium bromide Gels were visualized under UVtransilluminator and gel pictures were photographed usinggel documentation system (Alpha View 130 Alpha InnotechCorporation Multi Image Light Cabinet) [15 22]

27 Statistical Analysis The data were tabulated and ana-lyzed by statistical package SPSS ver110 (Chicago IL USA)to compare antibiotic resistance between different clinicalisolates and biofilm production in the presenceabsence ofesp gene among Enterococcus spp Chi-square test was usedfor categorical variables and P value lt 005 was considered assignificant

3 Results

31 Distribution of Enterococcus spp A total of 150 Enterococ-cus isolateswere included in the study Among these 82 (58)were E faecalis and 63 (42) were E faecium 563 of the Efaecalis and 699 of E faeciumwere isolated from the maleswhereas 437 of E faecalis and 301 of E faecium wereisolated from female patients Enterococcus spp isolated fromdifferent clinical samples are shown in Table 1 Antibioticsusceptibility was performed on all 150 isolates Howeverbiofilm and eae gene detection was done on 137 isolates as13 stored isolates were lost during recovery which included

Interdisciplinary Perspectives on Infectious Diseases 3

Table 1 Enterococcus spp isolated from different clinical samples

Clinical specimen E faecalisN ()

E faeciumN ()

TotalEnterococcus spp

N ()Tissue and pus 25 (568) 19 (431) 44 (293)High vaginal swabs (HVS) 07 (636) 04 (363) 11(73)Bile 03 (60) 02 (40) 05 (33)Urine 41 (273) 29 (414) 70 (466)Blood and body fluids 09 (529) 08 (47) 17 (113)Respiratory specimens 02 (666) 01 (333) 03 (2)Total 87 (58) 63 (42) 150

8 (4 E faecalis and 4 E faecium) urinary isolates and 5 (4 Efaecalis and 1 E faecium) isolates from pus

32 Antibiotic Susceptibility of Enterococcus spp All the Efaecalis isolates were sensitive to vancomycin Three strainsof E faecium were resistant to vancomycin by disk dif-fusion method Among these two isolates had a MIC ofge32120583gml and one MIC of ge8120583gml Thus based on MIConly two strains were vancomycin-resistant One each ofvancomycin-resistant E faecium isolates was from pus andtissue Resistance pattern of Enterococcus spp to variousantibiotics tested is shown in Table 2 It was observed thatsignificantly higher number of Enterococcus spp isolatedfrom tissue and pus samples showed resistance to amikacin(p=0009) amoxiclav (p=0002) ampicillin (ple0001) high-level gentamicin (p=0004) erythromycin (ple0001) peni-cillin (p=0006) piperacillin-tazobactam (p=0005) and van-comycin (p=004) when compared to enterococcal isolatesfrom other samples Moreover a significant number ofurinary enterococcal isolates showed resistance to ampicillin(p=0014)when compared to isolates fromother samplesTheresistance of enterococcal isolates from blood and body fluidto imipenemwas found to be statistically significant (p=003)It was observed that significant number of E faecalis from allthe clinical samples showed resistance to HLG (ple0001) anderythromycin (p=0009)

33 Biofilm Production Among the 137 Enterococcus testedfor biofilm production five (2 E faecium and 3 E fae-calis) urinary isolates and one E faecium from pus andone E faecalis from bronchoalveolar lavage (BAL) werestrong biofilm producers Among the three strong biofilm-producing urinary E faecalis isolates one was resistant tocotrimoxazole and one was resistant to HLG Among thetwo strong biofilm-producing urinary E faecium isolates onewas resistant to cotrimoxazole and HLS and another strainto ampicillin Strong biofilm-producing E faecalis from BALwas sensitive to all the antibiotics tested Strong biofilm-producing E faecium from pus was found to be resistant toHLSHowever 107 (781) isolates were non-biofilm-formerswhich included both sensitive and resistant strains of Efaecalis (n=65) and E faecium (n=42)

M 1 2 3 4 5 6 7 8 9 10 11 12 PC NC

950bp esp gene

Figure 1 Agarose gel picture of PCR reaction showing Enterococcusspp positive for esp gene Lanes M molecular weight marker 1isolate negative for esp 2 to 8 isolates positive for esp 9 isolatenegative for esp 10 to 12 isolates positive for esp PC positive controlfor esp NC negative control for esp

34 Detection of espGene by PCR PCRwas performed on 137isolates for the detection of esp genes and 40 isolates (22 Efaecalis and 18 E faecium) were positive for esp gene Agarosegel picture of PCR showing Enterococcus spp positive for espgene is shown in Figure 1 The relation between biofilm andpresence of esp gene among Enterococcus spp is shown inTable 3 The occurrence of esp gene and biofilm productionwas not statistically significant in case of E faecalis (p =0117)while it was statistically significant in case of E faecium(p=0024)

4 Discussion

Enterococcus is one of the significant pathogens affecting allage groups E faecium is more resistant than E faecalisHence speciation and antibiotic susceptibility testing arenecessary to detect the emergence and changing patternof drug resistance Vancomycin-resistant Enterococcus is asignificant cause of concern as this might share its resistancegene with other bacterial strains causing crossover of generendering others resistant to vancomycin

In the present study out of 150 Enterococcus isolates 58were E faecalis and 42 E faecium The rate of isolation ofEnterococcus spp was higher from urine (466) and pus(293) followed by blood and body fluids (113) as shown

4 Interdisciplinary Perspectives on Infectious Diseases

Table 2 Antibiotic resistance pattern of E faecalis and E faecium

Antibiotics tested Percentage resistanceE faecalis E faecium Enterococcus spp

Amikacin 622 885 732Amoxiclav 152 525 295Ampicillin 207 587 366Erythromycin 407 667 806High-level gentamicin 483 651 566High-level streptomycin 483 714 566Imipenem 36 595 459Meropenem 42 595 494Nitrofurantoin 73 241 142Piperacillin 39 655 50Piperacillin-tazobactam 25 488 367Teicoplanin 8 127 10Vancomycin 0 317 13

Table 3 Relationship between biofilm and presence of esp gene among Enterococcus spp

Type of biofilm (OD570

)

Enterococcus sppwith esp gene

Enterococcus sppwithout esp gene Total Enterococcus

spp N ()E faecalisN ()

E faeciumN ()

E faecalisN ()

E faeciumN ()

Strong (gt2) 3 (136) 3 (166) 1(17) 0 (0) 7 (51)Medium (1 to 2) 2 (90) 2 (111) 3 (52) 1 (25) 8 (58)Weak (05 to 1) 2 (90) 2 (111) 3 (52) 8 (20) 15 (109)Non-biofilm-formers(le05) 15 (681) 11 (611) 50 (877) 31 (775) 107 (781)

Total 22 (161) 18 (131) 57 (4160) 40 (423) 137 (100)

in Table 1 Earlier studies from India and abroad reportdifferent rates of isolation of Enterococcus spp from clinicalsamples which ranged from 10 to 80 from urine from 16 to43 from pus and from 3 to 36 from blood [4 6 23ndash25]Isolation rate of Enterococcus spp in the current study is atpar with few of the earlier studies [6 26] Thus our reportand reports from earlier workers from India and abroadclearly indicate that variation in isolation rate depends onthe geographical area and the clinical samples chosen in thestudy

In our study 731 and 53 of E faecalis isolated fromurine were resistant to HLG and HLS respectively Mean-while the rate of resistance of urinary E faeciumwas found tobe 482 to amoxiclav 655 each to HLG and piperacillinand 689 to HLS (Table 2) A similar earlier study onurinary Enterococcus isolates from India reported resistancefor HLG (40) piperacillin (54) nitrofurantoin (115)and vancomycin (85) [25] However in our study two(31) ofE faecium strainswere resistant and all theE faecalisstrains were sensitive to vancomycin One each vancomycin-resistant E faecium strain from pus and tissue were resistantto HLAR and sensitive to teicoplanin In an Iranian study byTalebi et al the resistance pattern was different from that ofour research where isolates were resistant to teicoplanin (3)and vancomycin (9) along with few other drugs [27] This

shows that resistance varies from region to region or frominstitution to institution in the same areaHence it is essentialto know the antibiogram of the enterococcal isolates in anarea to formulate antibiotic policy

Resistance to erythromycin was shown by a higher num-ber of E faecium strains (p=0002) as they are intrinsicallyresistant to macrolides lincosamides and streptogramin B(MLSB phenotype) Cross-resistance to all macrolides arisesfrom modification of the 23S rRNA target (except linezolidresistance) by a variety of methylase genes commonly ermBHence macrolides and lincosamides are not used to treatenterococcal infections even if E faecalis and E faecium aresusceptible to quinupristin-dalfopristin in vitro [28] In thepresent study erythromycin was tested for its susceptibilityjust to know the resistance pattern and not to use fortreatment

In this study 219 of enterococcal isolates producedbiofilm which included 275 E faecium and 177 E faecalisA study from Tamil Nadu [29] showed 68 isolates to bebiofilm formers The study used isolates from diverse clinicalsamples and detected biofilm formation by three differentmethods microtiter plate method tube method and Congored method In our study we have used only microtiter platemethod Thus the method used for the detection of biofilmand origin of the isolate will influence the biofilm formation

Interdisciplinary Perspectives on Infectious Diseases 5

Among the biofilm-producing E faecalis (n=14) isolates13 were resistant to HLAR two were resistant to teicoplaninand one was sensitive to all the antibiotics except amikacinAmong the biofilm-producing E faecium (n=16) isolates 14isolates were resistant to HLAR However all the biofilm-producing E faecalis and E faecium were susceptible tovancomycinThus vancomycin-resistantE faeciumwas non-biofilm-producer In the present study 292 of the isolatescarried esp gene (Figure 1) while the rest did not In a studydone by Kafil et al [20 30] 75 of the Enterococcus isolatesproducing biofilm carried the esp gene A survey by Toledo-Arana et al [15] reported biofilm production by 465 of espgene carrying E faecalis However there are no reports fromIndia to show a clear relation between presence of esp geneand biofilm production

In our study an association of biofilm and presence ofesp gene as depicted in Table 3 was not significant amongE faecalis (p=0117) while for E faecium it was statisticallysignificant (p=0024) A study by Kafil et al [24] showedno significant association between biofilm formation and thepresence or absence of esp gene (pgt005) However studiesfrom Iran byKafil et al [30] and Toledo-Arana [15] shows thatpresence of esp gene in urinary drug-resistant Enterococcusisolates led to strong biofilm formation and a firm adherenceto host cells However in our study only ten urinary Efaecalis and nine E faecium were biofilm-producers andfive each had esp gene Of the 19 biofilm-producing urinaryEnterococcus spp 14 showed HLAR resistance and two wereresistant to teicoplanin Moreover vancomycin-resistant Efaecium did not harbor esp gene and did not produce biofilmThus biofilm production presence of esp genes and drugresistance were not interrelated in the present study

A study by Dale et al (2015) had shown evidence of Efaecalis genetic determinants mediating antibiotic resistancewithin biofilms [30] The same research also suggests thatE faecalis employs biofilm-specific mechanisms and notthe simple extracellular matrix diffusion barriers to keepantibiotics away from their targets Since our study periodwas only of four monthsrsquo duration we targeted for esp genealone However further research is required to study morevirulence factors and correlation of the same with biofilmproduction and antibiotic resistance In conclusion biofilmformation is not always associated with the presence of espgene or drug resistance Emergence of VRE HLAR andresistance to teicoplanin has left us with very few therapeuticoptions for enterococcal infection

Data Availability

The datasets generated andor analyzed during the currentstudy are available from the corresponding author uponreasonable request

Ethical Approval

All procedures performed in this study were in accordancewith the ethical standards of the Kasturba Medical CollegeEthical committee

Consent

For this type of study formal consent was not required

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors acknowledge the Department of MicrobiologyKasturba Medical College Mangalore Manipal Academy ofHigher Education Manipal for giving them the opportunityto conduct the study

References

[1] W C Winn Jr S D Allen W H Jande and P C KonemanKonmanrsquos Color Atlas and Textbook of Diagnostic Microbiologypp 1294-1298 2006

[2] B E Murray and G M Weinstock Enterococci New Aspects ofAn Old Organism Blackwell Science Inc 1999

[3] M J Richards J R Edwards D H Culver and R P GaynesldquoNosocomial infections in combinedmedical-surgical intensivecare units in the United Statesrdquo Infection Control and HospitalEpidemiology vol 21 no 8 pp 510ndash515 2000

[4] K Mukherjee D Bhattacharjee G Chakraborti and S SChatterjee ldquoPrevalence and antibiotic susceptibility pattern ofenterococcus species from various clinical samples in a tertiarycare hospital in Kolkatardquo International Journal of ContemporaryMedical Research vol 3 pp 1565ndash1567 2016

[5] R C Moellering ldquoEmergence of enterococcus as a significantpathogenrdquo Clinical Infectious Diseases vol 14 no 6 pp 1173ndash1178 1992

[6] S Sreeja P R Sreenivasa Babu and A G Prathab ldquoTheprevalence and the characterization of the enterococcus speciesfrom various clinical samples in a tertiary care hospitalrdquo Journalof Clinical and Diagnostic Research vol 6 pp 1486ndash1488 2012

[7] D Low N Keller A Barth and R Jones ldquoClinical prevalenceantimicrobial susceptibility and geographic resistance patternsof enterococci results from the Sentry antimicrobial surveil-lance program 1997ndash1999rdquo Clinical Infectious Diseases vol 32no s2 pp S133ndashS145 2001

[8] L Shah S Mulla G P Patel and S Rewadiwala ldquoPrevalenceof enterococci with higher resistance level in a tertiary carehospital a matter of concernrdquo National Journal of MedicalResearch vol 2 pp 25ndash27 2012

[9] A Tripathi S K Shukla A Singh and K Prasad ldquoA newapproach of real time polymerase chain reaction in detectionof vancomycin-resistant enterococci and its comparison withother methodsrdquo Indian Journal of Medical Microbiology vol 31no 1 pp 47ndash52 2013

[10] J Costerton ldquoCystic fibrosis pathogenesis and the role ofbiofilms in persistent infectionrdquo Trends in Microbiology vol 9no 2 pp 50ndash52 2001

[11] R Moniri A Ghasemi S G Moosavi K Dastehgoli and MRezaei ldquoVirulence genersquos relationship with biofilm formationand detection of aac (6rsquo)aph (210158401015840) in enterococcus faecalisisolated from patients with urinary tract infectionrdquo JundishapurJournal of Microbiology vol 6 p e6244 2013

6 Interdisciplinary Perspectives on Infectious Diseases

[12] P Watnick and R Kolter ldquoBiofilm city of microbesrdquo Journal ofBacteriology vol 182 no 10 pp 2675ndash2679 2000

[13] J AMohamedWHuang S R Nallapareddy F Teng and B EMurray ldquoInfluence of origin of isolates especially endocarditisisolates and various genes on biofilm formation by enterococ-cus faecalisrdquo Infection and Immunity vol 72 no 6 pp 3658ndash3663 2004

[14] J A Mohamed and D B Huang ldquoBiofilm formation byenterococcirdquo Journal of MedicalMicrobiology vol 56 no 12 pp1581ndash1588 2007

[15] A Toledo-Arana J Valle C Solano et al ldquoThe enterococcalsurface protein esp is involved in enterococcus faecalis biofilmformationrdquoApplied and EnvironmentalMicrobiology vol 67 no10 pp 4538ndash4545 2001

[16] G P M Upadhyaya K L Ravikumar and B L UmapathyldquoReview of virulence factors of enterococcus an emergingnosocomial pathogenrdquo Indian Journal of Medical Microbiologyvol 27 no 4 pp 301ndash305 2009

[17] J G Collee T J Mackie and J E McCartney Mackie ampMcCartney Practical MedicalMicrobiology Elsevier NewDelhiIndia 14th edition 1996

[18] A W Bauer W M Kirby J C Sherris and M TurchldquoSusceptibility testing by a standardized single disc methodrdquoAmerican Journal of Clinical Pathology vol 45 pp 493ndash4961966

[19] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 27thinformational supplement CLSI documentM100-S27rdquo Clinicaland Laboratory Standards Institute In M100-S27 2017

[20] A Mobarez M Moghadam and H Kafil ldquoAdhesion andvirulence factor properties of Enterococci isolated from clinicalsamples in Iranrdquo Indian Journal of Pathology and Microbiologyvol 56 no 3 pp 238ndash242 2013

[21] L Triveda and S Gomathi ldquoDetection of biofilm formationamong the clinical isolates of Enterococci An evaluation ofthree different screening methodsrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 5 no 3 pp 643ndash650 2016

[22] G P M Upadhyaya U B Lingadevaru and R K LingegowdaldquoComparative study among clinical and commensal isolatesof Enterococcus faecalis for presence of esp gene and biofilmproductionrdquo The Journal of Infection in Developing Countriesvol 5 no 5 pp 365ndash369 2011

[23] M Salem-Bekhit M Muharram H Hefni I Moussa and FAlanazy ldquoPrevalence and antimicrobial resistance pattern ofmultidrug-resistant enterococci isolated from clinical speci-mensrdquo Indian Journal of Medical Microbiology vol 30 no 1 pp44ndash51 2012

[24] H S Kafil andAMMobarez ldquoAssessment of biofilm formationby enterococci isolates from urinary tract infections withdifferent virulence profilesrdquo Journal of King Saud University -Science vol 27 no 4 pp 312ndash317 2015

[25] P Srivastava R Mehta and P Nirwan ldquoPrevalence and antimi-crobial susceptibility of enterococcus species isolated fromdifferent clinical samples in a tertiary care hospital of NorthIndiardquo National Journal of Medical Research pp 389-91 2011

[26] S Mohanty S Jose R Singhal et al ldquoSpecies prevalence andantimicrobial susceptibility of enterococci isolated in a tertiarycare hospital of North Indiardquo Southeast Asian Journal of TropicalMedicine and Public Health vol 36 no 4 pp 962ndash965 2005

[27] M Talebi N Asghari Moghadam Z Mamooii M Enayati MSaifi and M R Pourshafie ldquoAntibiotic resistance and biofilm

formation of enterococcus faecalis in patient and environmen-tal samplesrdquo Jundishapur Journal of Microbiology vol 8 pe23349 2015

[28] W R Miller J M Munita and C A Arias ldquoMechanisms ofantibiotic resistance in enterococcirdquo Expert Review of Anti-infective Therapy vol 12 no 10 pp 1221ndash1236 2014

[29] V Sindhanai S S Avanthiga and V C S Chander ldquoAntibioticsusceptibility pattern of biofilm forming and biofilm non form-ing enterococci speciesrdquo IOSR Journal of Dental and MedicalSciences vol 15 no 4 pp 33ndash37 2016

[30] H S Kafil and A M Mobarez ldquoSpread of enterococcalsurface protein in antibiotic resistant entero-coccus faeciumand enterococcus faecalis isolates fromurinary tract infectionsrdquoThe Open Microbiology Journal vol 9 no 1 pp 14ndash17 2015

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Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Page 2: Antibiotic Susceptibility Pattern and Biofilm Formation in ...downloads.hindawi.com/journals/ipid/2019/7854968.pdf · ResearchArticle Antibiotic Susceptibility Pattern and Biofilm

2 Interdisciplinary Perspectives on Infectious Diseases

antibiotic discs and chemicals used in the study were pro-cured from Hi-Media Laboratories Pvt Ltd Mumbai IndiaEnterococcal isolateswere identified by colony characteristicsand common biochemical reactions [17] and confirmed byVITEK-2 automated system (bioMerieux USA)

22 Antibiotic Susceptibility Test Antimicrobial susceptibil-ity to ampicillin (10120583g) penicillin (10 units) tetracycline(30120583g) erythromycin (15120583g) chloramphenicol (30120583g) van-comycin (30120583g) teicoplanin (30120583g) ciprofloxacin (5120583g) andnitrofurantoin (300120583g) was determined by Kirby-Bauer diskdiffusion [18] and interpreted as per CLSI guidelines [19]Thequality control strain used was ATCC E faecalis 29212

23 High-Level Aminoglycoside Resistance (HLAR) DetectionDetection of HLAR was performed with disks containinggentamicin (120120583g) and streptomycin (300 120583g) by disk dif-fusion method Results were read after incubation at 35∘Cfor 24h and after 48h for streptomycin A zone diameterof 6mm indicates resistance 7-9mm shows that the resultsare inconclusive and more than 10mm suggests that theisolates are sensitive to aminoglycosides Resistance by discdiffusion to gentamicin corresponds to MIC of gt500 120583gmland susceptibility corresponds to MIC of lt 500 120583gmlHowever for high-level streptomycin MIC of gt1000 120583gmlby broth dilution and gt2000 120583gml by agar dilution methodcorresponds to a zone diameter of 6mm by disk diffusionMIC of le500 120583gml by broth and le1000 120583gml by agardilution corresponds to 10 mm diameter by disk diffusionmethod [19]

24 Detection of MIC for Vancomycin Enterococcal isolateswere inoculated onto Muller Hinton Agar supplementedwith 5 defibrinated sheep blood Vancomycin E-strips (EzyMIC) were placed on the inoculated plates and incubatedat 37∘C in 5 CO

2for 24 h The MIC was read where

the ellipse intersects the MIC scale on the strip E faecalisATCC 29212 and ATCC 51299 were used as negative andpositive controls respectively The results were interpretedas sensitive (MIC le4120583gml) intermediate (MIC 8-16 120583gml)and resistant (MIC ge32 120583gml) based on the CLSI guidelines[19]

25 Biofilm Formation All the clinical isolates were checkedfor biofilm production by the procedure used by Kafil andMobarez (2013) and Triveda and Gomathi (2016) [20 21]Briefly freshly subcultured strains of Enterococcus on bloodagar plates were inoculated in 1ml of Brain Heart Infusion(BHI) broth with 1 glucose and incubated at 37∘C for 24 hTo 180120583l of fresh BHI medium 20120583l of 24-hour-old bacterialgrowth was added which corresponded to a turbidity of 05McFarland standard 200120583l of the suspension of the clinicalisolates and the control strain (E faecalis ATCC 29212) wereinoculated into flat bottommicrotiter plates in duplicates andincubated at 37∘C in 5 CO

2for 24h After incubation the

contents of the plate were removed tapped and washed threetimes with phosphate buffer saline The biofilm was fixed byadding 150120583l of methanol for 20 min It was air-dried for

about 30 min in an inverted position and later stained with01 crystal violet for 15min Excess stain was removed andplates were washed with distilled water 150120583l of 33 aceticacid was added in each well and kept for 30 min withoutshaking The optical density (OD) was measured at 570nmBased on the OD values the isolates were categorized asstrong biofilm formers (OD

570gt 2) medium (OD

570gt 1

but lt2) weak (OD570gt 05 but lt1) and non-biofilm-formers

(OD570le 05) [13]

26 Detection of the esp Gene by Polymerase Chain Reac-tion (PCR) All the isolates of E faecalis and E faeciumwere subjected to PCR for the detection of the esp geneDNA extraction was done by boiling method Brieflythree to four colonies of enterococcal isolates were sus-pended in 100120583l of distilled water The bacterial cells werelysed by boiling for 10 minutes in a dry bath The lysatewas centrifuged briefly and 2120583l of the supernatant wasused as the DNA PCR was done by using primers esp11 (51015840- TTGCTAATGCTAGTCCACGACC-31015840) and esp 12(51015840-GCGTCAACACTTGCATTGCCGAA-31015840 ) Nuclease-freewater and E faecalis ATCC 29212 were used as esp negativeand positive controls respectively PCR reaction mixtureconsisted of 200120583M of dNTP mixture and 25 U Taq poly-merase with 1X buffer and 25mMMgCl

2 02120583M of primers

and 1120583l of DNA The PCR tubes containing master mixprimer and DNA were amplified in a thermocycler (Bio RadInc USA) PCR reaction conditionswere initial denaturationat 95∘C for 2 min followed by 30 cycles of 94∘C for 45 secs63∘C for 45 secs and 72∘C for 1 min Final extension wascarried out at 72∘C for 10min The amplified product wasresolved by agarose gel electrophoresis using 15 agarose in1X Tris-acetate EDTA (TAE) buffer The gel was stained with05 mgml ethidium bromide Gels were visualized under UVtransilluminator and gel pictures were photographed usinggel documentation system (Alpha View 130 Alpha InnotechCorporation Multi Image Light Cabinet) [15 22]

27 Statistical Analysis The data were tabulated and ana-lyzed by statistical package SPSS ver110 (Chicago IL USA)to compare antibiotic resistance between different clinicalisolates and biofilm production in the presenceabsence ofesp gene among Enterococcus spp Chi-square test was usedfor categorical variables and P value lt 005 was considered assignificant

3 Results

31 Distribution of Enterococcus spp A total of 150 Enterococ-cus isolateswere included in the study Among these 82 (58)were E faecalis and 63 (42) were E faecium 563 of the Efaecalis and 699 of E faeciumwere isolated from the maleswhereas 437 of E faecalis and 301 of E faecium wereisolated from female patients Enterococcus spp isolated fromdifferent clinical samples are shown in Table 1 Antibioticsusceptibility was performed on all 150 isolates Howeverbiofilm and eae gene detection was done on 137 isolates as13 stored isolates were lost during recovery which included

Interdisciplinary Perspectives on Infectious Diseases 3

Table 1 Enterococcus spp isolated from different clinical samples

Clinical specimen E faecalisN ()

E faeciumN ()

TotalEnterococcus spp

N ()Tissue and pus 25 (568) 19 (431) 44 (293)High vaginal swabs (HVS) 07 (636) 04 (363) 11(73)Bile 03 (60) 02 (40) 05 (33)Urine 41 (273) 29 (414) 70 (466)Blood and body fluids 09 (529) 08 (47) 17 (113)Respiratory specimens 02 (666) 01 (333) 03 (2)Total 87 (58) 63 (42) 150

8 (4 E faecalis and 4 E faecium) urinary isolates and 5 (4 Efaecalis and 1 E faecium) isolates from pus

32 Antibiotic Susceptibility of Enterococcus spp All the Efaecalis isolates were sensitive to vancomycin Three strainsof E faecium were resistant to vancomycin by disk dif-fusion method Among these two isolates had a MIC ofge32120583gml and one MIC of ge8120583gml Thus based on MIConly two strains were vancomycin-resistant One each ofvancomycin-resistant E faecium isolates was from pus andtissue Resistance pattern of Enterococcus spp to variousantibiotics tested is shown in Table 2 It was observed thatsignificantly higher number of Enterococcus spp isolatedfrom tissue and pus samples showed resistance to amikacin(p=0009) amoxiclav (p=0002) ampicillin (ple0001) high-level gentamicin (p=0004) erythromycin (ple0001) peni-cillin (p=0006) piperacillin-tazobactam (p=0005) and van-comycin (p=004) when compared to enterococcal isolatesfrom other samples Moreover a significant number ofurinary enterococcal isolates showed resistance to ampicillin(p=0014)when compared to isolates fromother samplesTheresistance of enterococcal isolates from blood and body fluidto imipenemwas found to be statistically significant (p=003)It was observed that significant number of E faecalis from allthe clinical samples showed resistance to HLG (ple0001) anderythromycin (p=0009)

33 Biofilm Production Among the 137 Enterococcus testedfor biofilm production five (2 E faecium and 3 E fae-calis) urinary isolates and one E faecium from pus andone E faecalis from bronchoalveolar lavage (BAL) werestrong biofilm producers Among the three strong biofilm-producing urinary E faecalis isolates one was resistant tocotrimoxazole and one was resistant to HLG Among thetwo strong biofilm-producing urinary E faecium isolates onewas resistant to cotrimoxazole and HLS and another strainto ampicillin Strong biofilm-producing E faecalis from BALwas sensitive to all the antibiotics tested Strong biofilm-producing E faecium from pus was found to be resistant toHLSHowever 107 (781) isolates were non-biofilm-formerswhich included both sensitive and resistant strains of Efaecalis (n=65) and E faecium (n=42)

M 1 2 3 4 5 6 7 8 9 10 11 12 PC NC

950bp esp gene

Figure 1 Agarose gel picture of PCR reaction showing Enterococcusspp positive for esp gene Lanes M molecular weight marker 1isolate negative for esp 2 to 8 isolates positive for esp 9 isolatenegative for esp 10 to 12 isolates positive for esp PC positive controlfor esp NC negative control for esp

34 Detection of espGene by PCR PCRwas performed on 137isolates for the detection of esp genes and 40 isolates (22 Efaecalis and 18 E faecium) were positive for esp gene Agarosegel picture of PCR showing Enterococcus spp positive for espgene is shown in Figure 1 The relation between biofilm andpresence of esp gene among Enterococcus spp is shown inTable 3 The occurrence of esp gene and biofilm productionwas not statistically significant in case of E faecalis (p =0117)while it was statistically significant in case of E faecium(p=0024)

4 Discussion

Enterococcus is one of the significant pathogens affecting allage groups E faecium is more resistant than E faecalisHence speciation and antibiotic susceptibility testing arenecessary to detect the emergence and changing patternof drug resistance Vancomycin-resistant Enterococcus is asignificant cause of concern as this might share its resistancegene with other bacterial strains causing crossover of generendering others resistant to vancomycin

In the present study out of 150 Enterococcus isolates 58were E faecalis and 42 E faecium The rate of isolation ofEnterococcus spp was higher from urine (466) and pus(293) followed by blood and body fluids (113) as shown

4 Interdisciplinary Perspectives on Infectious Diseases

Table 2 Antibiotic resistance pattern of E faecalis and E faecium

Antibiotics tested Percentage resistanceE faecalis E faecium Enterococcus spp

Amikacin 622 885 732Amoxiclav 152 525 295Ampicillin 207 587 366Erythromycin 407 667 806High-level gentamicin 483 651 566High-level streptomycin 483 714 566Imipenem 36 595 459Meropenem 42 595 494Nitrofurantoin 73 241 142Piperacillin 39 655 50Piperacillin-tazobactam 25 488 367Teicoplanin 8 127 10Vancomycin 0 317 13

Table 3 Relationship between biofilm and presence of esp gene among Enterococcus spp

Type of biofilm (OD570

)

Enterococcus sppwith esp gene

Enterococcus sppwithout esp gene Total Enterococcus

spp N ()E faecalisN ()

E faeciumN ()

E faecalisN ()

E faeciumN ()

Strong (gt2) 3 (136) 3 (166) 1(17) 0 (0) 7 (51)Medium (1 to 2) 2 (90) 2 (111) 3 (52) 1 (25) 8 (58)Weak (05 to 1) 2 (90) 2 (111) 3 (52) 8 (20) 15 (109)Non-biofilm-formers(le05) 15 (681) 11 (611) 50 (877) 31 (775) 107 (781)

Total 22 (161) 18 (131) 57 (4160) 40 (423) 137 (100)

in Table 1 Earlier studies from India and abroad reportdifferent rates of isolation of Enterococcus spp from clinicalsamples which ranged from 10 to 80 from urine from 16 to43 from pus and from 3 to 36 from blood [4 6 23ndash25]Isolation rate of Enterococcus spp in the current study is atpar with few of the earlier studies [6 26] Thus our reportand reports from earlier workers from India and abroadclearly indicate that variation in isolation rate depends onthe geographical area and the clinical samples chosen in thestudy

In our study 731 and 53 of E faecalis isolated fromurine were resistant to HLG and HLS respectively Mean-while the rate of resistance of urinary E faeciumwas found tobe 482 to amoxiclav 655 each to HLG and piperacillinand 689 to HLS (Table 2) A similar earlier study onurinary Enterococcus isolates from India reported resistancefor HLG (40) piperacillin (54) nitrofurantoin (115)and vancomycin (85) [25] However in our study two(31) ofE faecium strainswere resistant and all theE faecalisstrains were sensitive to vancomycin One each vancomycin-resistant E faecium strain from pus and tissue were resistantto HLAR and sensitive to teicoplanin In an Iranian study byTalebi et al the resistance pattern was different from that ofour research where isolates were resistant to teicoplanin (3)and vancomycin (9) along with few other drugs [27] This

shows that resistance varies from region to region or frominstitution to institution in the same areaHence it is essentialto know the antibiogram of the enterococcal isolates in anarea to formulate antibiotic policy

Resistance to erythromycin was shown by a higher num-ber of E faecium strains (p=0002) as they are intrinsicallyresistant to macrolides lincosamides and streptogramin B(MLSB phenotype) Cross-resistance to all macrolides arisesfrom modification of the 23S rRNA target (except linezolidresistance) by a variety of methylase genes commonly ermBHence macrolides and lincosamides are not used to treatenterococcal infections even if E faecalis and E faecium aresusceptible to quinupristin-dalfopristin in vitro [28] In thepresent study erythromycin was tested for its susceptibilityjust to know the resistance pattern and not to use fortreatment

In this study 219 of enterococcal isolates producedbiofilm which included 275 E faecium and 177 E faecalisA study from Tamil Nadu [29] showed 68 isolates to bebiofilm formers The study used isolates from diverse clinicalsamples and detected biofilm formation by three differentmethods microtiter plate method tube method and Congored method In our study we have used only microtiter platemethod Thus the method used for the detection of biofilmand origin of the isolate will influence the biofilm formation

Interdisciplinary Perspectives on Infectious Diseases 5

Among the biofilm-producing E faecalis (n=14) isolates13 were resistant to HLAR two were resistant to teicoplaninand one was sensitive to all the antibiotics except amikacinAmong the biofilm-producing E faecium (n=16) isolates 14isolates were resistant to HLAR However all the biofilm-producing E faecalis and E faecium were susceptible tovancomycinThus vancomycin-resistantE faeciumwas non-biofilm-producer In the present study 292 of the isolatescarried esp gene (Figure 1) while the rest did not In a studydone by Kafil et al [20 30] 75 of the Enterococcus isolatesproducing biofilm carried the esp gene A survey by Toledo-Arana et al [15] reported biofilm production by 465 of espgene carrying E faecalis However there are no reports fromIndia to show a clear relation between presence of esp geneand biofilm production

In our study an association of biofilm and presence ofesp gene as depicted in Table 3 was not significant amongE faecalis (p=0117) while for E faecium it was statisticallysignificant (p=0024) A study by Kafil et al [24] showedno significant association between biofilm formation and thepresence or absence of esp gene (pgt005) However studiesfrom Iran byKafil et al [30] and Toledo-Arana [15] shows thatpresence of esp gene in urinary drug-resistant Enterococcusisolates led to strong biofilm formation and a firm adherenceto host cells However in our study only ten urinary Efaecalis and nine E faecium were biofilm-producers andfive each had esp gene Of the 19 biofilm-producing urinaryEnterococcus spp 14 showed HLAR resistance and two wereresistant to teicoplanin Moreover vancomycin-resistant Efaecium did not harbor esp gene and did not produce biofilmThus biofilm production presence of esp genes and drugresistance were not interrelated in the present study

A study by Dale et al (2015) had shown evidence of Efaecalis genetic determinants mediating antibiotic resistancewithin biofilms [30] The same research also suggests thatE faecalis employs biofilm-specific mechanisms and notthe simple extracellular matrix diffusion barriers to keepantibiotics away from their targets Since our study periodwas only of four monthsrsquo duration we targeted for esp genealone However further research is required to study morevirulence factors and correlation of the same with biofilmproduction and antibiotic resistance In conclusion biofilmformation is not always associated with the presence of espgene or drug resistance Emergence of VRE HLAR andresistance to teicoplanin has left us with very few therapeuticoptions for enterococcal infection

Data Availability

The datasets generated andor analyzed during the currentstudy are available from the corresponding author uponreasonable request

Ethical Approval

All procedures performed in this study were in accordancewith the ethical standards of the Kasturba Medical CollegeEthical committee

Consent

For this type of study formal consent was not required

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors acknowledge the Department of MicrobiologyKasturba Medical College Mangalore Manipal Academy ofHigher Education Manipal for giving them the opportunityto conduct the study

References

[1] W C Winn Jr S D Allen W H Jande and P C KonemanKonmanrsquos Color Atlas and Textbook of Diagnostic Microbiologypp 1294-1298 2006

[2] B E Murray and G M Weinstock Enterococci New Aspects ofAn Old Organism Blackwell Science Inc 1999

[3] M J Richards J R Edwards D H Culver and R P GaynesldquoNosocomial infections in combinedmedical-surgical intensivecare units in the United Statesrdquo Infection Control and HospitalEpidemiology vol 21 no 8 pp 510ndash515 2000

[4] K Mukherjee D Bhattacharjee G Chakraborti and S SChatterjee ldquoPrevalence and antibiotic susceptibility pattern ofenterococcus species from various clinical samples in a tertiarycare hospital in Kolkatardquo International Journal of ContemporaryMedical Research vol 3 pp 1565ndash1567 2016

[5] R C Moellering ldquoEmergence of enterococcus as a significantpathogenrdquo Clinical Infectious Diseases vol 14 no 6 pp 1173ndash1178 1992

[6] S Sreeja P R Sreenivasa Babu and A G Prathab ldquoTheprevalence and the characterization of the enterococcus speciesfrom various clinical samples in a tertiary care hospitalrdquo Journalof Clinical and Diagnostic Research vol 6 pp 1486ndash1488 2012

[7] D Low N Keller A Barth and R Jones ldquoClinical prevalenceantimicrobial susceptibility and geographic resistance patternsof enterococci results from the Sentry antimicrobial surveil-lance program 1997ndash1999rdquo Clinical Infectious Diseases vol 32no s2 pp S133ndashS145 2001

[8] L Shah S Mulla G P Patel and S Rewadiwala ldquoPrevalenceof enterococci with higher resistance level in a tertiary carehospital a matter of concernrdquo National Journal of MedicalResearch vol 2 pp 25ndash27 2012

[9] A Tripathi S K Shukla A Singh and K Prasad ldquoA newapproach of real time polymerase chain reaction in detectionof vancomycin-resistant enterococci and its comparison withother methodsrdquo Indian Journal of Medical Microbiology vol 31no 1 pp 47ndash52 2013

[10] J Costerton ldquoCystic fibrosis pathogenesis and the role ofbiofilms in persistent infectionrdquo Trends in Microbiology vol 9no 2 pp 50ndash52 2001

[11] R Moniri A Ghasemi S G Moosavi K Dastehgoli and MRezaei ldquoVirulence genersquos relationship with biofilm formationand detection of aac (6rsquo)aph (210158401015840) in enterococcus faecalisisolated from patients with urinary tract infectionrdquo JundishapurJournal of Microbiology vol 6 p e6244 2013

6 Interdisciplinary Perspectives on Infectious Diseases

[12] P Watnick and R Kolter ldquoBiofilm city of microbesrdquo Journal ofBacteriology vol 182 no 10 pp 2675ndash2679 2000

[13] J AMohamedWHuang S R Nallapareddy F Teng and B EMurray ldquoInfluence of origin of isolates especially endocarditisisolates and various genes on biofilm formation by enterococ-cus faecalisrdquo Infection and Immunity vol 72 no 6 pp 3658ndash3663 2004

[14] J A Mohamed and D B Huang ldquoBiofilm formation byenterococcirdquo Journal of MedicalMicrobiology vol 56 no 12 pp1581ndash1588 2007

[15] A Toledo-Arana J Valle C Solano et al ldquoThe enterococcalsurface protein esp is involved in enterococcus faecalis biofilmformationrdquoApplied and EnvironmentalMicrobiology vol 67 no10 pp 4538ndash4545 2001

[16] G P M Upadhyaya K L Ravikumar and B L UmapathyldquoReview of virulence factors of enterococcus an emergingnosocomial pathogenrdquo Indian Journal of Medical Microbiologyvol 27 no 4 pp 301ndash305 2009

[17] J G Collee T J Mackie and J E McCartney Mackie ampMcCartney Practical MedicalMicrobiology Elsevier NewDelhiIndia 14th edition 1996

[18] A W Bauer W M Kirby J C Sherris and M TurchldquoSusceptibility testing by a standardized single disc methodrdquoAmerican Journal of Clinical Pathology vol 45 pp 493ndash4961966

[19] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 27thinformational supplement CLSI documentM100-S27rdquo Clinicaland Laboratory Standards Institute In M100-S27 2017

[20] A Mobarez M Moghadam and H Kafil ldquoAdhesion andvirulence factor properties of Enterococci isolated from clinicalsamples in Iranrdquo Indian Journal of Pathology and Microbiologyvol 56 no 3 pp 238ndash242 2013

[21] L Triveda and S Gomathi ldquoDetection of biofilm formationamong the clinical isolates of Enterococci An evaluation ofthree different screening methodsrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 5 no 3 pp 643ndash650 2016

[22] G P M Upadhyaya U B Lingadevaru and R K LingegowdaldquoComparative study among clinical and commensal isolatesof Enterococcus faecalis for presence of esp gene and biofilmproductionrdquo The Journal of Infection in Developing Countriesvol 5 no 5 pp 365ndash369 2011

[23] M Salem-Bekhit M Muharram H Hefni I Moussa and FAlanazy ldquoPrevalence and antimicrobial resistance pattern ofmultidrug-resistant enterococci isolated from clinical speci-mensrdquo Indian Journal of Medical Microbiology vol 30 no 1 pp44ndash51 2012

[24] H S Kafil andAMMobarez ldquoAssessment of biofilm formationby enterococci isolates from urinary tract infections withdifferent virulence profilesrdquo Journal of King Saud University -Science vol 27 no 4 pp 312ndash317 2015

[25] P Srivastava R Mehta and P Nirwan ldquoPrevalence and antimi-crobial susceptibility of enterococcus species isolated fromdifferent clinical samples in a tertiary care hospital of NorthIndiardquo National Journal of Medical Research pp 389-91 2011

[26] S Mohanty S Jose R Singhal et al ldquoSpecies prevalence andantimicrobial susceptibility of enterococci isolated in a tertiarycare hospital of North Indiardquo Southeast Asian Journal of TropicalMedicine and Public Health vol 36 no 4 pp 962ndash965 2005

[27] M Talebi N Asghari Moghadam Z Mamooii M Enayati MSaifi and M R Pourshafie ldquoAntibiotic resistance and biofilm

formation of enterococcus faecalis in patient and environmen-tal samplesrdquo Jundishapur Journal of Microbiology vol 8 pe23349 2015

[28] W R Miller J M Munita and C A Arias ldquoMechanisms ofantibiotic resistance in enterococcirdquo Expert Review of Anti-infective Therapy vol 12 no 10 pp 1221ndash1236 2014

[29] V Sindhanai S S Avanthiga and V C S Chander ldquoAntibioticsusceptibility pattern of biofilm forming and biofilm non form-ing enterococci speciesrdquo IOSR Journal of Dental and MedicalSciences vol 15 no 4 pp 33ndash37 2016

[30] H S Kafil and A M Mobarez ldquoSpread of enterococcalsurface protein in antibiotic resistant entero-coccus faeciumand enterococcus faecalis isolates fromurinary tract infectionsrdquoThe Open Microbiology Journal vol 9 no 1 pp 14ndash17 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Page 3: Antibiotic Susceptibility Pattern and Biofilm Formation in ...downloads.hindawi.com/journals/ipid/2019/7854968.pdf · ResearchArticle Antibiotic Susceptibility Pattern and Biofilm

Interdisciplinary Perspectives on Infectious Diseases 3

Table 1 Enterococcus spp isolated from different clinical samples

Clinical specimen E faecalisN ()

E faeciumN ()

TotalEnterococcus spp

N ()Tissue and pus 25 (568) 19 (431) 44 (293)High vaginal swabs (HVS) 07 (636) 04 (363) 11(73)Bile 03 (60) 02 (40) 05 (33)Urine 41 (273) 29 (414) 70 (466)Blood and body fluids 09 (529) 08 (47) 17 (113)Respiratory specimens 02 (666) 01 (333) 03 (2)Total 87 (58) 63 (42) 150

8 (4 E faecalis and 4 E faecium) urinary isolates and 5 (4 Efaecalis and 1 E faecium) isolates from pus

32 Antibiotic Susceptibility of Enterococcus spp All the Efaecalis isolates were sensitive to vancomycin Three strainsof E faecium were resistant to vancomycin by disk dif-fusion method Among these two isolates had a MIC ofge32120583gml and one MIC of ge8120583gml Thus based on MIConly two strains were vancomycin-resistant One each ofvancomycin-resistant E faecium isolates was from pus andtissue Resistance pattern of Enterococcus spp to variousantibiotics tested is shown in Table 2 It was observed thatsignificantly higher number of Enterococcus spp isolatedfrom tissue and pus samples showed resistance to amikacin(p=0009) amoxiclav (p=0002) ampicillin (ple0001) high-level gentamicin (p=0004) erythromycin (ple0001) peni-cillin (p=0006) piperacillin-tazobactam (p=0005) and van-comycin (p=004) when compared to enterococcal isolatesfrom other samples Moreover a significant number ofurinary enterococcal isolates showed resistance to ampicillin(p=0014)when compared to isolates fromother samplesTheresistance of enterococcal isolates from blood and body fluidto imipenemwas found to be statistically significant (p=003)It was observed that significant number of E faecalis from allthe clinical samples showed resistance to HLG (ple0001) anderythromycin (p=0009)

33 Biofilm Production Among the 137 Enterococcus testedfor biofilm production five (2 E faecium and 3 E fae-calis) urinary isolates and one E faecium from pus andone E faecalis from bronchoalveolar lavage (BAL) werestrong biofilm producers Among the three strong biofilm-producing urinary E faecalis isolates one was resistant tocotrimoxazole and one was resistant to HLG Among thetwo strong biofilm-producing urinary E faecium isolates onewas resistant to cotrimoxazole and HLS and another strainto ampicillin Strong biofilm-producing E faecalis from BALwas sensitive to all the antibiotics tested Strong biofilm-producing E faecium from pus was found to be resistant toHLSHowever 107 (781) isolates were non-biofilm-formerswhich included both sensitive and resistant strains of Efaecalis (n=65) and E faecium (n=42)

M 1 2 3 4 5 6 7 8 9 10 11 12 PC NC

950bp esp gene

Figure 1 Agarose gel picture of PCR reaction showing Enterococcusspp positive for esp gene Lanes M molecular weight marker 1isolate negative for esp 2 to 8 isolates positive for esp 9 isolatenegative for esp 10 to 12 isolates positive for esp PC positive controlfor esp NC negative control for esp

34 Detection of espGene by PCR PCRwas performed on 137isolates for the detection of esp genes and 40 isolates (22 Efaecalis and 18 E faecium) were positive for esp gene Agarosegel picture of PCR showing Enterococcus spp positive for espgene is shown in Figure 1 The relation between biofilm andpresence of esp gene among Enterococcus spp is shown inTable 3 The occurrence of esp gene and biofilm productionwas not statistically significant in case of E faecalis (p =0117)while it was statistically significant in case of E faecium(p=0024)

4 Discussion

Enterococcus is one of the significant pathogens affecting allage groups E faecium is more resistant than E faecalisHence speciation and antibiotic susceptibility testing arenecessary to detect the emergence and changing patternof drug resistance Vancomycin-resistant Enterococcus is asignificant cause of concern as this might share its resistancegene with other bacterial strains causing crossover of generendering others resistant to vancomycin

In the present study out of 150 Enterococcus isolates 58were E faecalis and 42 E faecium The rate of isolation ofEnterococcus spp was higher from urine (466) and pus(293) followed by blood and body fluids (113) as shown

4 Interdisciplinary Perspectives on Infectious Diseases

Table 2 Antibiotic resistance pattern of E faecalis and E faecium

Antibiotics tested Percentage resistanceE faecalis E faecium Enterococcus spp

Amikacin 622 885 732Amoxiclav 152 525 295Ampicillin 207 587 366Erythromycin 407 667 806High-level gentamicin 483 651 566High-level streptomycin 483 714 566Imipenem 36 595 459Meropenem 42 595 494Nitrofurantoin 73 241 142Piperacillin 39 655 50Piperacillin-tazobactam 25 488 367Teicoplanin 8 127 10Vancomycin 0 317 13

Table 3 Relationship between biofilm and presence of esp gene among Enterococcus spp

Type of biofilm (OD570

)

Enterococcus sppwith esp gene

Enterococcus sppwithout esp gene Total Enterococcus

spp N ()E faecalisN ()

E faeciumN ()

E faecalisN ()

E faeciumN ()

Strong (gt2) 3 (136) 3 (166) 1(17) 0 (0) 7 (51)Medium (1 to 2) 2 (90) 2 (111) 3 (52) 1 (25) 8 (58)Weak (05 to 1) 2 (90) 2 (111) 3 (52) 8 (20) 15 (109)Non-biofilm-formers(le05) 15 (681) 11 (611) 50 (877) 31 (775) 107 (781)

Total 22 (161) 18 (131) 57 (4160) 40 (423) 137 (100)

in Table 1 Earlier studies from India and abroad reportdifferent rates of isolation of Enterococcus spp from clinicalsamples which ranged from 10 to 80 from urine from 16 to43 from pus and from 3 to 36 from blood [4 6 23ndash25]Isolation rate of Enterococcus spp in the current study is atpar with few of the earlier studies [6 26] Thus our reportand reports from earlier workers from India and abroadclearly indicate that variation in isolation rate depends onthe geographical area and the clinical samples chosen in thestudy

In our study 731 and 53 of E faecalis isolated fromurine were resistant to HLG and HLS respectively Mean-while the rate of resistance of urinary E faeciumwas found tobe 482 to amoxiclav 655 each to HLG and piperacillinand 689 to HLS (Table 2) A similar earlier study onurinary Enterococcus isolates from India reported resistancefor HLG (40) piperacillin (54) nitrofurantoin (115)and vancomycin (85) [25] However in our study two(31) ofE faecium strainswere resistant and all theE faecalisstrains were sensitive to vancomycin One each vancomycin-resistant E faecium strain from pus and tissue were resistantto HLAR and sensitive to teicoplanin In an Iranian study byTalebi et al the resistance pattern was different from that ofour research where isolates were resistant to teicoplanin (3)and vancomycin (9) along with few other drugs [27] This

shows that resistance varies from region to region or frominstitution to institution in the same areaHence it is essentialto know the antibiogram of the enterococcal isolates in anarea to formulate antibiotic policy

Resistance to erythromycin was shown by a higher num-ber of E faecium strains (p=0002) as they are intrinsicallyresistant to macrolides lincosamides and streptogramin B(MLSB phenotype) Cross-resistance to all macrolides arisesfrom modification of the 23S rRNA target (except linezolidresistance) by a variety of methylase genes commonly ermBHence macrolides and lincosamides are not used to treatenterococcal infections even if E faecalis and E faecium aresusceptible to quinupristin-dalfopristin in vitro [28] In thepresent study erythromycin was tested for its susceptibilityjust to know the resistance pattern and not to use fortreatment

In this study 219 of enterococcal isolates producedbiofilm which included 275 E faecium and 177 E faecalisA study from Tamil Nadu [29] showed 68 isolates to bebiofilm formers The study used isolates from diverse clinicalsamples and detected biofilm formation by three differentmethods microtiter plate method tube method and Congored method In our study we have used only microtiter platemethod Thus the method used for the detection of biofilmand origin of the isolate will influence the biofilm formation

Interdisciplinary Perspectives on Infectious Diseases 5

Among the biofilm-producing E faecalis (n=14) isolates13 were resistant to HLAR two were resistant to teicoplaninand one was sensitive to all the antibiotics except amikacinAmong the biofilm-producing E faecium (n=16) isolates 14isolates were resistant to HLAR However all the biofilm-producing E faecalis and E faecium were susceptible tovancomycinThus vancomycin-resistantE faeciumwas non-biofilm-producer In the present study 292 of the isolatescarried esp gene (Figure 1) while the rest did not In a studydone by Kafil et al [20 30] 75 of the Enterococcus isolatesproducing biofilm carried the esp gene A survey by Toledo-Arana et al [15] reported biofilm production by 465 of espgene carrying E faecalis However there are no reports fromIndia to show a clear relation between presence of esp geneand biofilm production

In our study an association of biofilm and presence ofesp gene as depicted in Table 3 was not significant amongE faecalis (p=0117) while for E faecium it was statisticallysignificant (p=0024) A study by Kafil et al [24] showedno significant association between biofilm formation and thepresence or absence of esp gene (pgt005) However studiesfrom Iran byKafil et al [30] and Toledo-Arana [15] shows thatpresence of esp gene in urinary drug-resistant Enterococcusisolates led to strong biofilm formation and a firm adherenceto host cells However in our study only ten urinary Efaecalis and nine E faecium were biofilm-producers andfive each had esp gene Of the 19 biofilm-producing urinaryEnterococcus spp 14 showed HLAR resistance and two wereresistant to teicoplanin Moreover vancomycin-resistant Efaecium did not harbor esp gene and did not produce biofilmThus biofilm production presence of esp genes and drugresistance were not interrelated in the present study

A study by Dale et al (2015) had shown evidence of Efaecalis genetic determinants mediating antibiotic resistancewithin biofilms [30] The same research also suggests thatE faecalis employs biofilm-specific mechanisms and notthe simple extracellular matrix diffusion barriers to keepantibiotics away from their targets Since our study periodwas only of four monthsrsquo duration we targeted for esp genealone However further research is required to study morevirulence factors and correlation of the same with biofilmproduction and antibiotic resistance In conclusion biofilmformation is not always associated with the presence of espgene or drug resistance Emergence of VRE HLAR andresistance to teicoplanin has left us with very few therapeuticoptions for enterococcal infection

Data Availability

The datasets generated andor analyzed during the currentstudy are available from the corresponding author uponreasonable request

Ethical Approval

All procedures performed in this study were in accordancewith the ethical standards of the Kasturba Medical CollegeEthical committee

Consent

For this type of study formal consent was not required

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors acknowledge the Department of MicrobiologyKasturba Medical College Mangalore Manipal Academy ofHigher Education Manipal for giving them the opportunityto conduct the study

References

[1] W C Winn Jr S D Allen W H Jande and P C KonemanKonmanrsquos Color Atlas and Textbook of Diagnostic Microbiologypp 1294-1298 2006

[2] B E Murray and G M Weinstock Enterococci New Aspects ofAn Old Organism Blackwell Science Inc 1999

[3] M J Richards J R Edwards D H Culver and R P GaynesldquoNosocomial infections in combinedmedical-surgical intensivecare units in the United Statesrdquo Infection Control and HospitalEpidemiology vol 21 no 8 pp 510ndash515 2000

[4] K Mukherjee D Bhattacharjee G Chakraborti and S SChatterjee ldquoPrevalence and antibiotic susceptibility pattern ofenterococcus species from various clinical samples in a tertiarycare hospital in Kolkatardquo International Journal of ContemporaryMedical Research vol 3 pp 1565ndash1567 2016

[5] R C Moellering ldquoEmergence of enterococcus as a significantpathogenrdquo Clinical Infectious Diseases vol 14 no 6 pp 1173ndash1178 1992

[6] S Sreeja P R Sreenivasa Babu and A G Prathab ldquoTheprevalence and the characterization of the enterococcus speciesfrom various clinical samples in a tertiary care hospitalrdquo Journalof Clinical and Diagnostic Research vol 6 pp 1486ndash1488 2012

[7] D Low N Keller A Barth and R Jones ldquoClinical prevalenceantimicrobial susceptibility and geographic resistance patternsof enterococci results from the Sentry antimicrobial surveil-lance program 1997ndash1999rdquo Clinical Infectious Diseases vol 32no s2 pp S133ndashS145 2001

[8] L Shah S Mulla G P Patel and S Rewadiwala ldquoPrevalenceof enterococci with higher resistance level in a tertiary carehospital a matter of concernrdquo National Journal of MedicalResearch vol 2 pp 25ndash27 2012

[9] A Tripathi S K Shukla A Singh and K Prasad ldquoA newapproach of real time polymerase chain reaction in detectionof vancomycin-resistant enterococci and its comparison withother methodsrdquo Indian Journal of Medical Microbiology vol 31no 1 pp 47ndash52 2013

[10] J Costerton ldquoCystic fibrosis pathogenesis and the role ofbiofilms in persistent infectionrdquo Trends in Microbiology vol 9no 2 pp 50ndash52 2001

[11] R Moniri A Ghasemi S G Moosavi K Dastehgoli and MRezaei ldquoVirulence genersquos relationship with biofilm formationand detection of aac (6rsquo)aph (210158401015840) in enterococcus faecalisisolated from patients with urinary tract infectionrdquo JundishapurJournal of Microbiology vol 6 p e6244 2013

6 Interdisciplinary Perspectives on Infectious Diseases

[12] P Watnick and R Kolter ldquoBiofilm city of microbesrdquo Journal ofBacteriology vol 182 no 10 pp 2675ndash2679 2000

[13] J AMohamedWHuang S R Nallapareddy F Teng and B EMurray ldquoInfluence of origin of isolates especially endocarditisisolates and various genes on biofilm formation by enterococ-cus faecalisrdquo Infection and Immunity vol 72 no 6 pp 3658ndash3663 2004

[14] J A Mohamed and D B Huang ldquoBiofilm formation byenterococcirdquo Journal of MedicalMicrobiology vol 56 no 12 pp1581ndash1588 2007

[15] A Toledo-Arana J Valle C Solano et al ldquoThe enterococcalsurface protein esp is involved in enterococcus faecalis biofilmformationrdquoApplied and EnvironmentalMicrobiology vol 67 no10 pp 4538ndash4545 2001

[16] G P M Upadhyaya K L Ravikumar and B L UmapathyldquoReview of virulence factors of enterococcus an emergingnosocomial pathogenrdquo Indian Journal of Medical Microbiologyvol 27 no 4 pp 301ndash305 2009

[17] J G Collee T J Mackie and J E McCartney Mackie ampMcCartney Practical MedicalMicrobiology Elsevier NewDelhiIndia 14th edition 1996

[18] A W Bauer W M Kirby J C Sherris and M TurchldquoSusceptibility testing by a standardized single disc methodrdquoAmerican Journal of Clinical Pathology vol 45 pp 493ndash4961966

[19] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 27thinformational supplement CLSI documentM100-S27rdquo Clinicaland Laboratory Standards Institute In M100-S27 2017

[20] A Mobarez M Moghadam and H Kafil ldquoAdhesion andvirulence factor properties of Enterococci isolated from clinicalsamples in Iranrdquo Indian Journal of Pathology and Microbiologyvol 56 no 3 pp 238ndash242 2013

[21] L Triveda and S Gomathi ldquoDetection of biofilm formationamong the clinical isolates of Enterococci An evaluation ofthree different screening methodsrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 5 no 3 pp 643ndash650 2016

[22] G P M Upadhyaya U B Lingadevaru and R K LingegowdaldquoComparative study among clinical and commensal isolatesof Enterococcus faecalis for presence of esp gene and biofilmproductionrdquo The Journal of Infection in Developing Countriesvol 5 no 5 pp 365ndash369 2011

[23] M Salem-Bekhit M Muharram H Hefni I Moussa and FAlanazy ldquoPrevalence and antimicrobial resistance pattern ofmultidrug-resistant enterococci isolated from clinical speci-mensrdquo Indian Journal of Medical Microbiology vol 30 no 1 pp44ndash51 2012

[24] H S Kafil andAMMobarez ldquoAssessment of biofilm formationby enterococci isolates from urinary tract infections withdifferent virulence profilesrdquo Journal of King Saud University -Science vol 27 no 4 pp 312ndash317 2015

[25] P Srivastava R Mehta and P Nirwan ldquoPrevalence and antimi-crobial susceptibility of enterococcus species isolated fromdifferent clinical samples in a tertiary care hospital of NorthIndiardquo National Journal of Medical Research pp 389-91 2011

[26] S Mohanty S Jose R Singhal et al ldquoSpecies prevalence andantimicrobial susceptibility of enterococci isolated in a tertiarycare hospital of North Indiardquo Southeast Asian Journal of TropicalMedicine and Public Health vol 36 no 4 pp 962ndash965 2005

[27] M Talebi N Asghari Moghadam Z Mamooii M Enayati MSaifi and M R Pourshafie ldquoAntibiotic resistance and biofilm

formation of enterococcus faecalis in patient and environmen-tal samplesrdquo Jundishapur Journal of Microbiology vol 8 pe23349 2015

[28] W R Miller J M Munita and C A Arias ldquoMechanisms ofantibiotic resistance in enterococcirdquo Expert Review of Anti-infective Therapy vol 12 no 10 pp 1221ndash1236 2014

[29] V Sindhanai S S Avanthiga and V C S Chander ldquoAntibioticsusceptibility pattern of biofilm forming and biofilm non form-ing enterococci speciesrdquo IOSR Journal of Dental and MedicalSciences vol 15 no 4 pp 33ndash37 2016

[30] H S Kafil and A M Mobarez ldquoSpread of enterococcalsurface protein in antibiotic resistant entero-coccus faeciumand enterococcus faecalis isolates fromurinary tract infectionsrdquoThe Open Microbiology Journal vol 9 no 1 pp 14ndash17 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Page 4: Antibiotic Susceptibility Pattern and Biofilm Formation in ...downloads.hindawi.com/journals/ipid/2019/7854968.pdf · ResearchArticle Antibiotic Susceptibility Pattern and Biofilm

4 Interdisciplinary Perspectives on Infectious Diseases

Table 2 Antibiotic resistance pattern of E faecalis and E faecium

Antibiotics tested Percentage resistanceE faecalis E faecium Enterococcus spp

Amikacin 622 885 732Amoxiclav 152 525 295Ampicillin 207 587 366Erythromycin 407 667 806High-level gentamicin 483 651 566High-level streptomycin 483 714 566Imipenem 36 595 459Meropenem 42 595 494Nitrofurantoin 73 241 142Piperacillin 39 655 50Piperacillin-tazobactam 25 488 367Teicoplanin 8 127 10Vancomycin 0 317 13

Table 3 Relationship between biofilm and presence of esp gene among Enterococcus spp

Type of biofilm (OD570

)

Enterococcus sppwith esp gene

Enterococcus sppwithout esp gene Total Enterococcus

spp N ()E faecalisN ()

E faeciumN ()

E faecalisN ()

E faeciumN ()

Strong (gt2) 3 (136) 3 (166) 1(17) 0 (0) 7 (51)Medium (1 to 2) 2 (90) 2 (111) 3 (52) 1 (25) 8 (58)Weak (05 to 1) 2 (90) 2 (111) 3 (52) 8 (20) 15 (109)Non-biofilm-formers(le05) 15 (681) 11 (611) 50 (877) 31 (775) 107 (781)

Total 22 (161) 18 (131) 57 (4160) 40 (423) 137 (100)

in Table 1 Earlier studies from India and abroad reportdifferent rates of isolation of Enterococcus spp from clinicalsamples which ranged from 10 to 80 from urine from 16 to43 from pus and from 3 to 36 from blood [4 6 23ndash25]Isolation rate of Enterococcus spp in the current study is atpar with few of the earlier studies [6 26] Thus our reportand reports from earlier workers from India and abroadclearly indicate that variation in isolation rate depends onthe geographical area and the clinical samples chosen in thestudy

In our study 731 and 53 of E faecalis isolated fromurine were resistant to HLG and HLS respectively Mean-while the rate of resistance of urinary E faeciumwas found tobe 482 to amoxiclav 655 each to HLG and piperacillinand 689 to HLS (Table 2) A similar earlier study onurinary Enterococcus isolates from India reported resistancefor HLG (40) piperacillin (54) nitrofurantoin (115)and vancomycin (85) [25] However in our study two(31) ofE faecium strainswere resistant and all theE faecalisstrains were sensitive to vancomycin One each vancomycin-resistant E faecium strain from pus and tissue were resistantto HLAR and sensitive to teicoplanin In an Iranian study byTalebi et al the resistance pattern was different from that ofour research where isolates were resistant to teicoplanin (3)and vancomycin (9) along with few other drugs [27] This

shows that resistance varies from region to region or frominstitution to institution in the same areaHence it is essentialto know the antibiogram of the enterococcal isolates in anarea to formulate antibiotic policy

Resistance to erythromycin was shown by a higher num-ber of E faecium strains (p=0002) as they are intrinsicallyresistant to macrolides lincosamides and streptogramin B(MLSB phenotype) Cross-resistance to all macrolides arisesfrom modification of the 23S rRNA target (except linezolidresistance) by a variety of methylase genes commonly ermBHence macrolides and lincosamides are not used to treatenterococcal infections even if E faecalis and E faecium aresusceptible to quinupristin-dalfopristin in vitro [28] In thepresent study erythromycin was tested for its susceptibilityjust to know the resistance pattern and not to use fortreatment

In this study 219 of enterococcal isolates producedbiofilm which included 275 E faecium and 177 E faecalisA study from Tamil Nadu [29] showed 68 isolates to bebiofilm formers The study used isolates from diverse clinicalsamples and detected biofilm formation by three differentmethods microtiter plate method tube method and Congored method In our study we have used only microtiter platemethod Thus the method used for the detection of biofilmand origin of the isolate will influence the biofilm formation

Interdisciplinary Perspectives on Infectious Diseases 5

Among the biofilm-producing E faecalis (n=14) isolates13 were resistant to HLAR two were resistant to teicoplaninand one was sensitive to all the antibiotics except amikacinAmong the biofilm-producing E faecium (n=16) isolates 14isolates were resistant to HLAR However all the biofilm-producing E faecalis and E faecium were susceptible tovancomycinThus vancomycin-resistantE faeciumwas non-biofilm-producer In the present study 292 of the isolatescarried esp gene (Figure 1) while the rest did not In a studydone by Kafil et al [20 30] 75 of the Enterococcus isolatesproducing biofilm carried the esp gene A survey by Toledo-Arana et al [15] reported biofilm production by 465 of espgene carrying E faecalis However there are no reports fromIndia to show a clear relation between presence of esp geneand biofilm production

In our study an association of biofilm and presence ofesp gene as depicted in Table 3 was not significant amongE faecalis (p=0117) while for E faecium it was statisticallysignificant (p=0024) A study by Kafil et al [24] showedno significant association between biofilm formation and thepresence or absence of esp gene (pgt005) However studiesfrom Iran byKafil et al [30] and Toledo-Arana [15] shows thatpresence of esp gene in urinary drug-resistant Enterococcusisolates led to strong biofilm formation and a firm adherenceto host cells However in our study only ten urinary Efaecalis and nine E faecium were biofilm-producers andfive each had esp gene Of the 19 biofilm-producing urinaryEnterococcus spp 14 showed HLAR resistance and two wereresistant to teicoplanin Moreover vancomycin-resistant Efaecium did not harbor esp gene and did not produce biofilmThus biofilm production presence of esp genes and drugresistance were not interrelated in the present study

A study by Dale et al (2015) had shown evidence of Efaecalis genetic determinants mediating antibiotic resistancewithin biofilms [30] The same research also suggests thatE faecalis employs biofilm-specific mechanisms and notthe simple extracellular matrix diffusion barriers to keepantibiotics away from their targets Since our study periodwas only of four monthsrsquo duration we targeted for esp genealone However further research is required to study morevirulence factors and correlation of the same with biofilmproduction and antibiotic resistance In conclusion biofilmformation is not always associated with the presence of espgene or drug resistance Emergence of VRE HLAR andresistance to teicoplanin has left us with very few therapeuticoptions for enterococcal infection

Data Availability

The datasets generated andor analyzed during the currentstudy are available from the corresponding author uponreasonable request

Ethical Approval

All procedures performed in this study were in accordancewith the ethical standards of the Kasturba Medical CollegeEthical committee

Consent

For this type of study formal consent was not required

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors acknowledge the Department of MicrobiologyKasturba Medical College Mangalore Manipal Academy ofHigher Education Manipal for giving them the opportunityto conduct the study

References

[1] W C Winn Jr S D Allen W H Jande and P C KonemanKonmanrsquos Color Atlas and Textbook of Diagnostic Microbiologypp 1294-1298 2006

[2] B E Murray and G M Weinstock Enterococci New Aspects ofAn Old Organism Blackwell Science Inc 1999

[3] M J Richards J R Edwards D H Culver and R P GaynesldquoNosocomial infections in combinedmedical-surgical intensivecare units in the United Statesrdquo Infection Control and HospitalEpidemiology vol 21 no 8 pp 510ndash515 2000

[4] K Mukherjee D Bhattacharjee G Chakraborti and S SChatterjee ldquoPrevalence and antibiotic susceptibility pattern ofenterococcus species from various clinical samples in a tertiarycare hospital in Kolkatardquo International Journal of ContemporaryMedical Research vol 3 pp 1565ndash1567 2016

[5] R C Moellering ldquoEmergence of enterococcus as a significantpathogenrdquo Clinical Infectious Diseases vol 14 no 6 pp 1173ndash1178 1992

[6] S Sreeja P R Sreenivasa Babu and A G Prathab ldquoTheprevalence and the characterization of the enterococcus speciesfrom various clinical samples in a tertiary care hospitalrdquo Journalof Clinical and Diagnostic Research vol 6 pp 1486ndash1488 2012

[7] D Low N Keller A Barth and R Jones ldquoClinical prevalenceantimicrobial susceptibility and geographic resistance patternsof enterococci results from the Sentry antimicrobial surveil-lance program 1997ndash1999rdquo Clinical Infectious Diseases vol 32no s2 pp S133ndashS145 2001

[8] L Shah S Mulla G P Patel and S Rewadiwala ldquoPrevalenceof enterococci with higher resistance level in a tertiary carehospital a matter of concernrdquo National Journal of MedicalResearch vol 2 pp 25ndash27 2012

[9] A Tripathi S K Shukla A Singh and K Prasad ldquoA newapproach of real time polymerase chain reaction in detectionof vancomycin-resistant enterococci and its comparison withother methodsrdquo Indian Journal of Medical Microbiology vol 31no 1 pp 47ndash52 2013

[10] J Costerton ldquoCystic fibrosis pathogenesis and the role ofbiofilms in persistent infectionrdquo Trends in Microbiology vol 9no 2 pp 50ndash52 2001

[11] R Moniri A Ghasemi S G Moosavi K Dastehgoli and MRezaei ldquoVirulence genersquos relationship with biofilm formationand detection of aac (6rsquo)aph (210158401015840) in enterococcus faecalisisolated from patients with urinary tract infectionrdquo JundishapurJournal of Microbiology vol 6 p e6244 2013

6 Interdisciplinary Perspectives on Infectious Diseases

[12] P Watnick and R Kolter ldquoBiofilm city of microbesrdquo Journal ofBacteriology vol 182 no 10 pp 2675ndash2679 2000

[13] J AMohamedWHuang S R Nallapareddy F Teng and B EMurray ldquoInfluence of origin of isolates especially endocarditisisolates and various genes on biofilm formation by enterococ-cus faecalisrdquo Infection and Immunity vol 72 no 6 pp 3658ndash3663 2004

[14] J A Mohamed and D B Huang ldquoBiofilm formation byenterococcirdquo Journal of MedicalMicrobiology vol 56 no 12 pp1581ndash1588 2007

[15] A Toledo-Arana J Valle C Solano et al ldquoThe enterococcalsurface protein esp is involved in enterococcus faecalis biofilmformationrdquoApplied and EnvironmentalMicrobiology vol 67 no10 pp 4538ndash4545 2001

[16] G P M Upadhyaya K L Ravikumar and B L UmapathyldquoReview of virulence factors of enterococcus an emergingnosocomial pathogenrdquo Indian Journal of Medical Microbiologyvol 27 no 4 pp 301ndash305 2009

[17] J G Collee T J Mackie and J E McCartney Mackie ampMcCartney Practical MedicalMicrobiology Elsevier NewDelhiIndia 14th edition 1996

[18] A W Bauer W M Kirby J C Sherris and M TurchldquoSusceptibility testing by a standardized single disc methodrdquoAmerican Journal of Clinical Pathology vol 45 pp 493ndash4961966

[19] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 27thinformational supplement CLSI documentM100-S27rdquo Clinicaland Laboratory Standards Institute In M100-S27 2017

[20] A Mobarez M Moghadam and H Kafil ldquoAdhesion andvirulence factor properties of Enterococci isolated from clinicalsamples in Iranrdquo Indian Journal of Pathology and Microbiologyvol 56 no 3 pp 238ndash242 2013

[21] L Triveda and S Gomathi ldquoDetection of biofilm formationamong the clinical isolates of Enterococci An evaluation ofthree different screening methodsrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 5 no 3 pp 643ndash650 2016

[22] G P M Upadhyaya U B Lingadevaru and R K LingegowdaldquoComparative study among clinical and commensal isolatesof Enterococcus faecalis for presence of esp gene and biofilmproductionrdquo The Journal of Infection in Developing Countriesvol 5 no 5 pp 365ndash369 2011

[23] M Salem-Bekhit M Muharram H Hefni I Moussa and FAlanazy ldquoPrevalence and antimicrobial resistance pattern ofmultidrug-resistant enterococci isolated from clinical speci-mensrdquo Indian Journal of Medical Microbiology vol 30 no 1 pp44ndash51 2012

[24] H S Kafil andAMMobarez ldquoAssessment of biofilm formationby enterococci isolates from urinary tract infections withdifferent virulence profilesrdquo Journal of King Saud University -Science vol 27 no 4 pp 312ndash317 2015

[25] P Srivastava R Mehta and P Nirwan ldquoPrevalence and antimi-crobial susceptibility of enterococcus species isolated fromdifferent clinical samples in a tertiary care hospital of NorthIndiardquo National Journal of Medical Research pp 389-91 2011

[26] S Mohanty S Jose R Singhal et al ldquoSpecies prevalence andantimicrobial susceptibility of enterococci isolated in a tertiarycare hospital of North Indiardquo Southeast Asian Journal of TropicalMedicine and Public Health vol 36 no 4 pp 962ndash965 2005

[27] M Talebi N Asghari Moghadam Z Mamooii M Enayati MSaifi and M R Pourshafie ldquoAntibiotic resistance and biofilm

formation of enterococcus faecalis in patient and environmen-tal samplesrdquo Jundishapur Journal of Microbiology vol 8 pe23349 2015

[28] W R Miller J M Munita and C A Arias ldquoMechanisms ofantibiotic resistance in enterococcirdquo Expert Review of Anti-infective Therapy vol 12 no 10 pp 1221ndash1236 2014

[29] V Sindhanai S S Avanthiga and V C S Chander ldquoAntibioticsusceptibility pattern of biofilm forming and biofilm non form-ing enterococci speciesrdquo IOSR Journal of Dental and MedicalSciences vol 15 no 4 pp 33ndash37 2016

[30] H S Kafil and A M Mobarez ldquoSpread of enterococcalsurface protein in antibiotic resistant entero-coccus faeciumand enterococcus faecalis isolates fromurinary tract infectionsrdquoThe Open Microbiology Journal vol 9 no 1 pp 14ndash17 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Page 5: Antibiotic Susceptibility Pattern and Biofilm Formation in ...downloads.hindawi.com/journals/ipid/2019/7854968.pdf · ResearchArticle Antibiotic Susceptibility Pattern and Biofilm

Interdisciplinary Perspectives on Infectious Diseases 5

Among the biofilm-producing E faecalis (n=14) isolates13 were resistant to HLAR two were resistant to teicoplaninand one was sensitive to all the antibiotics except amikacinAmong the biofilm-producing E faecium (n=16) isolates 14isolates were resistant to HLAR However all the biofilm-producing E faecalis and E faecium were susceptible tovancomycinThus vancomycin-resistantE faeciumwas non-biofilm-producer In the present study 292 of the isolatescarried esp gene (Figure 1) while the rest did not In a studydone by Kafil et al [20 30] 75 of the Enterococcus isolatesproducing biofilm carried the esp gene A survey by Toledo-Arana et al [15] reported biofilm production by 465 of espgene carrying E faecalis However there are no reports fromIndia to show a clear relation between presence of esp geneand biofilm production

In our study an association of biofilm and presence ofesp gene as depicted in Table 3 was not significant amongE faecalis (p=0117) while for E faecium it was statisticallysignificant (p=0024) A study by Kafil et al [24] showedno significant association between biofilm formation and thepresence or absence of esp gene (pgt005) However studiesfrom Iran byKafil et al [30] and Toledo-Arana [15] shows thatpresence of esp gene in urinary drug-resistant Enterococcusisolates led to strong biofilm formation and a firm adherenceto host cells However in our study only ten urinary Efaecalis and nine E faecium were biofilm-producers andfive each had esp gene Of the 19 biofilm-producing urinaryEnterococcus spp 14 showed HLAR resistance and two wereresistant to teicoplanin Moreover vancomycin-resistant Efaecium did not harbor esp gene and did not produce biofilmThus biofilm production presence of esp genes and drugresistance were not interrelated in the present study

A study by Dale et al (2015) had shown evidence of Efaecalis genetic determinants mediating antibiotic resistancewithin biofilms [30] The same research also suggests thatE faecalis employs biofilm-specific mechanisms and notthe simple extracellular matrix diffusion barriers to keepantibiotics away from their targets Since our study periodwas only of four monthsrsquo duration we targeted for esp genealone However further research is required to study morevirulence factors and correlation of the same with biofilmproduction and antibiotic resistance In conclusion biofilmformation is not always associated with the presence of espgene or drug resistance Emergence of VRE HLAR andresistance to teicoplanin has left us with very few therapeuticoptions for enterococcal infection

Data Availability

The datasets generated andor analyzed during the currentstudy are available from the corresponding author uponreasonable request

Ethical Approval

All procedures performed in this study were in accordancewith the ethical standards of the Kasturba Medical CollegeEthical committee

Consent

For this type of study formal consent was not required

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors acknowledge the Department of MicrobiologyKasturba Medical College Mangalore Manipal Academy ofHigher Education Manipal for giving them the opportunityto conduct the study

References

[1] W C Winn Jr S D Allen W H Jande and P C KonemanKonmanrsquos Color Atlas and Textbook of Diagnostic Microbiologypp 1294-1298 2006

[2] B E Murray and G M Weinstock Enterococci New Aspects ofAn Old Organism Blackwell Science Inc 1999

[3] M J Richards J R Edwards D H Culver and R P GaynesldquoNosocomial infections in combinedmedical-surgical intensivecare units in the United Statesrdquo Infection Control and HospitalEpidemiology vol 21 no 8 pp 510ndash515 2000

[4] K Mukherjee D Bhattacharjee G Chakraborti and S SChatterjee ldquoPrevalence and antibiotic susceptibility pattern ofenterococcus species from various clinical samples in a tertiarycare hospital in Kolkatardquo International Journal of ContemporaryMedical Research vol 3 pp 1565ndash1567 2016

[5] R C Moellering ldquoEmergence of enterococcus as a significantpathogenrdquo Clinical Infectious Diseases vol 14 no 6 pp 1173ndash1178 1992

[6] S Sreeja P R Sreenivasa Babu and A G Prathab ldquoTheprevalence and the characterization of the enterococcus speciesfrom various clinical samples in a tertiary care hospitalrdquo Journalof Clinical and Diagnostic Research vol 6 pp 1486ndash1488 2012

[7] D Low N Keller A Barth and R Jones ldquoClinical prevalenceantimicrobial susceptibility and geographic resistance patternsof enterococci results from the Sentry antimicrobial surveil-lance program 1997ndash1999rdquo Clinical Infectious Diseases vol 32no s2 pp S133ndashS145 2001

[8] L Shah S Mulla G P Patel and S Rewadiwala ldquoPrevalenceof enterococci with higher resistance level in a tertiary carehospital a matter of concernrdquo National Journal of MedicalResearch vol 2 pp 25ndash27 2012

[9] A Tripathi S K Shukla A Singh and K Prasad ldquoA newapproach of real time polymerase chain reaction in detectionof vancomycin-resistant enterococci and its comparison withother methodsrdquo Indian Journal of Medical Microbiology vol 31no 1 pp 47ndash52 2013

[10] J Costerton ldquoCystic fibrosis pathogenesis and the role ofbiofilms in persistent infectionrdquo Trends in Microbiology vol 9no 2 pp 50ndash52 2001

[11] R Moniri A Ghasemi S G Moosavi K Dastehgoli and MRezaei ldquoVirulence genersquos relationship with biofilm formationand detection of aac (6rsquo)aph (210158401015840) in enterococcus faecalisisolated from patients with urinary tract infectionrdquo JundishapurJournal of Microbiology vol 6 p e6244 2013

6 Interdisciplinary Perspectives on Infectious Diseases

[12] P Watnick and R Kolter ldquoBiofilm city of microbesrdquo Journal ofBacteriology vol 182 no 10 pp 2675ndash2679 2000

[13] J AMohamedWHuang S R Nallapareddy F Teng and B EMurray ldquoInfluence of origin of isolates especially endocarditisisolates and various genes on biofilm formation by enterococ-cus faecalisrdquo Infection and Immunity vol 72 no 6 pp 3658ndash3663 2004

[14] J A Mohamed and D B Huang ldquoBiofilm formation byenterococcirdquo Journal of MedicalMicrobiology vol 56 no 12 pp1581ndash1588 2007

[15] A Toledo-Arana J Valle C Solano et al ldquoThe enterococcalsurface protein esp is involved in enterococcus faecalis biofilmformationrdquoApplied and EnvironmentalMicrobiology vol 67 no10 pp 4538ndash4545 2001

[16] G P M Upadhyaya K L Ravikumar and B L UmapathyldquoReview of virulence factors of enterococcus an emergingnosocomial pathogenrdquo Indian Journal of Medical Microbiologyvol 27 no 4 pp 301ndash305 2009

[17] J G Collee T J Mackie and J E McCartney Mackie ampMcCartney Practical MedicalMicrobiology Elsevier NewDelhiIndia 14th edition 1996

[18] A W Bauer W M Kirby J C Sherris and M TurchldquoSusceptibility testing by a standardized single disc methodrdquoAmerican Journal of Clinical Pathology vol 45 pp 493ndash4961966

[19] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 27thinformational supplement CLSI documentM100-S27rdquo Clinicaland Laboratory Standards Institute In M100-S27 2017

[20] A Mobarez M Moghadam and H Kafil ldquoAdhesion andvirulence factor properties of Enterococci isolated from clinicalsamples in Iranrdquo Indian Journal of Pathology and Microbiologyvol 56 no 3 pp 238ndash242 2013

[21] L Triveda and S Gomathi ldquoDetection of biofilm formationamong the clinical isolates of Enterococci An evaluation ofthree different screening methodsrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 5 no 3 pp 643ndash650 2016

[22] G P M Upadhyaya U B Lingadevaru and R K LingegowdaldquoComparative study among clinical and commensal isolatesof Enterococcus faecalis for presence of esp gene and biofilmproductionrdquo The Journal of Infection in Developing Countriesvol 5 no 5 pp 365ndash369 2011

[23] M Salem-Bekhit M Muharram H Hefni I Moussa and FAlanazy ldquoPrevalence and antimicrobial resistance pattern ofmultidrug-resistant enterococci isolated from clinical speci-mensrdquo Indian Journal of Medical Microbiology vol 30 no 1 pp44ndash51 2012

[24] H S Kafil andAMMobarez ldquoAssessment of biofilm formationby enterococci isolates from urinary tract infections withdifferent virulence profilesrdquo Journal of King Saud University -Science vol 27 no 4 pp 312ndash317 2015

[25] P Srivastava R Mehta and P Nirwan ldquoPrevalence and antimi-crobial susceptibility of enterococcus species isolated fromdifferent clinical samples in a tertiary care hospital of NorthIndiardquo National Journal of Medical Research pp 389-91 2011

[26] S Mohanty S Jose R Singhal et al ldquoSpecies prevalence andantimicrobial susceptibility of enterococci isolated in a tertiarycare hospital of North Indiardquo Southeast Asian Journal of TropicalMedicine and Public Health vol 36 no 4 pp 962ndash965 2005

[27] M Talebi N Asghari Moghadam Z Mamooii M Enayati MSaifi and M R Pourshafie ldquoAntibiotic resistance and biofilm

formation of enterococcus faecalis in patient and environmen-tal samplesrdquo Jundishapur Journal of Microbiology vol 8 pe23349 2015

[28] W R Miller J M Munita and C A Arias ldquoMechanisms ofantibiotic resistance in enterococcirdquo Expert Review of Anti-infective Therapy vol 12 no 10 pp 1221ndash1236 2014

[29] V Sindhanai S S Avanthiga and V C S Chander ldquoAntibioticsusceptibility pattern of biofilm forming and biofilm non form-ing enterococci speciesrdquo IOSR Journal of Dental and MedicalSciences vol 15 no 4 pp 33ndash37 2016

[30] H S Kafil and A M Mobarez ldquoSpread of enterococcalsurface protein in antibiotic resistant entero-coccus faeciumand enterococcus faecalis isolates fromurinary tract infectionsrdquoThe Open Microbiology Journal vol 9 no 1 pp 14ndash17 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Page 6: Antibiotic Susceptibility Pattern and Biofilm Formation in ...downloads.hindawi.com/journals/ipid/2019/7854968.pdf · ResearchArticle Antibiotic Susceptibility Pattern and Biofilm

6 Interdisciplinary Perspectives on Infectious Diseases

[12] P Watnick and R Kolter ldquoBiofilm city of microbesrdquo Journal ofBacteriology vol 182 no 10 pp 2675ndash2679 2000

[13] J AMohamedWHuang S R Nallapareddy F Teng and B EMurray ldquoInfluence of origin of isolates especially endocarditisisolates and various genes on biofilm formation by enterococ-cus faecalisrdquo Infection and Immunity vol 72 no 6 pp 3658ndash3663 2004

[14] J A Mohamed and D B Huang ldquoBiofilm formation byenterococcirdquo Journal of MedicalMicrobiology vol 56 no 12 pp1581ndash1588 2007

[15] A Toledo-Arana J Valle C Solano et al ldquoThe enterococcalsurface protein esp is involved in enterococcus faecalis biofilmformationrdquoApplied and EnvironmentalMicrobiology vol 67 no10 pp 4538ndash4545 2001

[16] G P M Upadhyaya K L Ravikumar and B L UmapathyldquoReview of virulence factors of enterococcus an emergingnosocomial pathogenrdquo Indian Journal of Medical Microbiologyvol 27 no 4 pp 301ndash305 2009

[17] J G Collee T J Mackie and J E McCartney Mackie ampMcCartney Practical MedicalMicrobiology Elsevier NewDelhiIndia 14th edition 1996

[18] A W Bauer W M Kirby J C Sherris and M TurchldquoSusceptibility testing by a standardized single disc methodrdquoAmerican Journal of Clinical Pathology vol 45 pp 493ndash4961966

[19] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testing 27thinformational supplement CLSI documentM100-S27rdquo Clinicaland Laboratory Standards Institute In M100-S27 2017

[20] A Mobarez M Moghadam and H Kafil ldquoAdhesion andvirulence factor properties of Enterococci isolated from clinicalsamples in Iranrdquo Indian Journal of Pathology and Microbiologyvol 56 no 3 pp 238ndash242 2013

[21] L Triveda and S Gomathi ldquoDetection of biofilm formationamong the clinical isolates of Enterococci An evaluation ofthree different screening methodsrdquo International Journal ofCurrentMicrobiology andApplied Sciences vol 5 no 3 pp 643ndash650 2016

[22] G P M Upadhyaya U B Lingadevaru and R K LingegowdaldquoComparative study among clinical and commensal isolatesof Enterococcus faecalis for presence of esp gene and biofilmproductionrdquo The Journal of Infection in Developing Countriesvol 5 no 5 pp 365ndash369 2011

[23] M Salem-Bekhit M Muharram H Hefni I Moussa and FAlanazy ldquoPrevalence and antimicrobial resistance pattern ofmultidrug-resistant enterococci isolated from clinical speci-mensrdquo Indian Journal of Medical Microbiology vol 30 no 1 pp44ndash51 2012

[24] H S Kafil andAMMobarez ldquoAssessment of biofilm formationby enterococci isolates from urinary tract infections withdifferent virulence profilesrdquo Journal of King Saud University -Science vol 27 no 4 pp 312ndash317 2015

[25] P Srivastava R Mehta and P Nirwan ldquoPrevalence and antimi-crobial susceptibility of enterococcus species isolated fromdifferent clinical samples in a tertiary care hospital of NorthIndiardquo National Journal of Medical Research pp 389-91 2011

[26] S Mohanty S Jose R Singhal et al ldquoSpecies prevalence andantimicrobial susceptibility of enterococci isolated in a tertiarycare hospital of North Indiardquo Southeast Asian Journal of TropicalMedicine and Public Health vol 36 no 4 pp 962ndash965 2005

[27] M Talebi N Asghari Moghadam Z Mamooii M Enayati MSaifi and M R Pourshafie ldquoAntibiotic resistance and biofilm

formation of enterococcus faecalis in patient and environmen-tal samplesrdquo Jundishapur Journal of Microbiology vol 8 pe23349 2015

[28] W R Miller J M Munita and C A Arias ldquoMechanisms ofantibiotic resistance in enterococcirdquo Expert Review of Anti-infective Therapy vol 12 no 10 pp 1221ndash1236 2014

[29] V Sindhanai S S Avanthiga and V C S Chander ldquoAntibioticsusceptibility pattern of biofilm forming and biofilm non form-ing enterococci speciesrdquo IOSR Journal of Dental and MedicalSciences vol 15 no 4 pp 33ndash37 2016

[30] H S Kafil and A M Mobarez ldquoSpread of enterococcalsurface protein in antibiotic resistant entero-coccus faeciumand enterococcus faecalis isolates fromurinary tract infectionsrdquoThe Open Microbiology Journal vol 9 no 1 pp 14ndash17 2015

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Page 7: Antibiotic Susceptibility Pattern and Biofilm Formation in ...downloads.hindawi.com/journals/ipid/2019/7854968.pdf · ResearchArticle Antibiotic Susceptibility Pattern and Biofilm

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom