Objective: To determine frequency, distribution and sensitivity pattern of Extended-Spectrum ß Lactamase (EBSL) producing organism at a tertiary care hospital in Pakistan. Methods: All members of enterobacteriacae isolated between April and August 2002 were studied. Isolates were speciated according to standard biochemical tests. Susceptibility testing was performed by Kirby-Bauer method. ESBL was detected using double disc method using cefotaxime versus cefotaxime plus clavulanate according to NCCLS. Statistical analysis was performed by SPSS version 10. Test of significance were calculated using chi-square test. Results: During the study period, 1137/2840 (40%) of the isolates tested were found to be ESBL producing. ESBL positivity was detected in 50% Enterobacter sp., 41% E.coli and 36% K.pneumoniae. ESBL production was noted in 52% of nosocomial isolates tested (415/799). ESBL was more frequent in patients at the extremes of ages (under 5 years and more than 60 years). Cross-resistance to non-beta lactam antibiotics (flouoroquinolones, aminoglycosides and co-trimoxazole) was also more frequent in ESBL producing organisms Conclusion: A high frequency of ESBL positivity amongst our isolates is documented which is alarming in low-income settings where expensive second line agents are unavailable. Our data supports urgent need for regular screening and surveillance for these organisms (JPMA 55:436;2005)

Hasan, Rumina

Jabeen, Kauser

Zafar, Afia

English

eCommons@AKU

eCommons@AKUDepartment of Pathology and Laboratory Medicine Medical College, PakistanJanuary 2005Frequency and sensitivity pattern of extendedspectrum beta lactamase producing isolates in atertiary care hospital laboratory of PakistanKauser JabeenAga Khan University, kausar.jabeen@aku.eduAfia ZafarAga Khan University, afia.zafar@aku.eduRumina HasanAga Khan University, rumina.hasan@aku.eduFollow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiolPart of the Pathology CommonsRecommended CitationJabeen, K., Zafar, A., Hasan, R. (2005). Frequency and sensitivity pattern of extended spectrum beta lactamase producing isolates in atertiary care hospital laboratory of Pakistan. Journal of Pakistan Medical Association, 55(10), 436-439.Available at: http://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiol/621436 J Pak Med AssocOriginal ArticleFrequency and sensitivity pattern of Extended Spectrum Beta Lactamase produc-ing isolates in a tertiary care hospital laboratory of PakistanKausar Jabeen, Afia Zafar, Rumina HasanDepartment of Pathology, The Aga Khan University Hospital, Karachi, Pakistan.AbstractObjective: To determine frequency, distribution and sensitivity pattern of Extended-Spectrum ß Lactamase(EBSL) producing organism at a tertiary care hospital in Pakistan.Methods: All members of enterobacteriacae isolated between April and August 2002 were studied. Isolates were spe-ciated according to standard biochemical tests. Susceptibility testing was performed by Kirby-Bauer method. ESBLwas detected using double disc method using cefotaxime versus cefotaxime plus clavulanate according to NCCLS.Statistical analysis was performed by SPSS version 10. Test of significance were calculated using chi-square test.Results: During the study period, 1137/2840 (40%) of the isolates tested were found to be ESBL producing. ESBLpositivity was detected in 50% Enterobacter sp., 41% E.coli and 36% K.pneumoniae. ESBL production was noted in52% of nosocomial isolates tested (415/799). ESBL was more frequent in patients at the extremes of ages (under 5years and more than 60 years). Cross-resistance to non-beta lactam antibiotics (flouoroquinolones, aminoglycosidesand co-trimoxazole) was also more frequent in ESBL producing organisms (p<0.05). Conclusion: A high frequency of ESBL positivity amongst our isolates is documented which is alarming in low-income settings where expensive second line agents are unavailable. Our data supports urgent need for regu-lar screening and surveillance for these organisms (JPMA 55:436;2005).IntroductionExtended-spectrum ß-lactamase (ESBL) producingorganisms are a major problem in the area of infectious dis-ease1 conferring resistance to all ß-lactam antibiotics exceptcephamycins and carbapenems.2 In addition, ESBL-produc-ing organisms frequently show cross-resistance to manyother classes of antibiotics; including aminoglycosides andfluoroquinolones3 thus treatment of these infections is oftena therapeutic challenge.The frequency of ESBL-producing organisms dif-fers significantly in accordance with geographic location.4-6 The ESBL positivity rate amongst K.pneumoniae isreported at 45% in Latin America and 7% in the UnitedStates. In New York, on the other hand, surveillance of 15hospitals in Brooklyn report, 34% ESBL positivity wasfound in K. pneumoniae.5 Similarly, although frequency ofESBL producing E.coli in Europe, North, Latin Americaand Western Pacific is reported at 1-8%4, its prevalence inthe Asia Pacific region and South Africa is reported atmore than 20%.7 Mathur et al from India recently reported68% ESBL positivity rate in their enterobacteriacae iso-lates8 while Shah et al and Zaman et al have reported a fre-quency of 48% and 35% respectively from Pakistan(Table).9,10Detection of ESBL is a major challenge for the clin-ical microbiology laboratory.11 Its presence in bacterialcells does not always produce phenotypic resistance result-ing in some ESBL isolates appearing susceptible to third-generation cephalosporin in vitro. However treatment ofthese isolates with third-generation cephalosporins is fre-quently ineffective.12,13A proficiency testing project for clinical laborato-ries participating in the National Nosocomial InfectionsSurveillance System indicated that as many as 58% oflaboratories failed to detect and report ESBL isolates cor-rectly.14 In some studies, 37% of ESBL producing organ-isms were misreported15 whereas in another study only 7out of 38 laboratories correctly identified and reportedthese organisms.16 These data suggest that improvementin the ability of clinical laboratories to detect ESBLs isneeded. National Committee for Clinical LaboratoryStandards (NCCLS) and British Society for Antimicrobialand Chemotherapy (BSAC) have published standardizedcriteria for screening, confirmatory testing and reportingof these organisms.17,18 All ESBL producing organismsTable. Reported ESBL positivity rates from Asia.Year Country % (n) (ESBL) Reference  2002 Pakistan 48 (400) 9 2002 China, Japan, Taiwan, Singapore, Philippine  >20 (2193) 7 2002 India 68 (678) 8 2000 Hong Kong 11-13 (1174) 25 1999 Pakistan 35 (200) 10 Vol. 55, No. 10, October 2005 437should be reported as being resistant to all peni-cillins, cephalosporins and aztreonam.In developing countries, many laboratories do notroutinely detect ESBL production, a practice which islikely to result in misreporting and hence treatment fail-ures. On the other hand, in areas with low ESBL levels itmay not be cost effective to test for ESBL on a routinebasis.19 It is therefore essential that ESBL positivity ratesare monitored and that decision regarding appropriate lab-oratory practices made in light of local/regional ESBLdata. Moreover correct reporting would limit inappropri-ate antimicrobial usage and hence decrease emergenceand extension of antimicrobial resistance worldwide. Inthis paper, ESBL positivity rates among isolates fromKarachi, Pakistan are discussed along with implicationswith regard to laboratory practices in developing coun-tries. Therefore frequency, distribution and sensitivitypattern of EBSL producing organism in samples submit-ted to a tertiary care referral hospital laboratory wasdetermined. Material and MethodsThis descriptive study was performed at a 550 bedtertiary care hospital located in Karachi, Pakistan. Clinicalmicrobiology laboratory of the hospital receives samplesfrom in and outpatients presenting to the tertiary care centreas well as from referrals other hospitals, clinics and generalpractitioners across the city.All enterobacteriacae isolated between April toOctober 2002 (2840 isolates) were studied for ESBL pro-duction. These included 1248 isolates from patients present-ing to our hospital (including inpatients, and patients fromemergency room, consulting clinics as well as from ourcommunity health centre). While 1590 isolates were fromreferrals outside (i.e. from other hospitals, clinics and gen-eral practitioners across the city). Enterobacteriaceae growing in clinical speci-mens were identified using routine biochemical tests.20Kirby Bauer was performed in accordance with NCCLSguidelines17 using Mueller Hinton agar (Oxoid). ESBLdetection method used was double disc method usingcefotaxime (30 µg) in comparison to cefotaxime plusclavulanate (30+10 µg) (Oxoid) according to NCCLScriteria.17SPSS version 10 was used to enter and analyze data.Descriptive analysis was carried out and test of significancewas calculated using chi-square test.Results During the study period 2840 isolates of enter-obacteriaceae were identified. Of these, 2016 (71%) wereE.coli, 429 (15%) K.pneumonae and 256 (9%)Enterobacter sp. Overall 1137/2840 (40%) were ESBLproducing. The sources of ESBL positive isolates includ-ed urine (n=784), blood (n=119), sterile body fluids (CSF,pleural and peritoneal fluids) (n= 86), respiratory speci-m e n s(n= 62) and central lines (n=20). Frequency of ESBL pos-itivity was highest amongst Enterobacter species 50%(n=256) followed by E.coli 41% (n=2016), Klebsillaspecies 36% (n=429), Morganella species 27% (n=111),Proteus species 20% (n=51) with Citrobacter sp. showingthe lowest positivity rate 14% (n=22).Out of 2840 enterobacteriaceae isolates, 1248 iso-lates from patients presenting to the tertiary care centreitself were further analyzed in terms of location and ESBLpositivity. Unfortunately, the remaining isolates from out-side referrals could not be analyzed due to incomplete clin-ical information. While ESBL positivity was more than30% in all study areas, highest positivity rate 52% (n=799)was significantly noted amongst inpatient isolates. Thepositivity rate was also high in isolates from the emergencyroom 45% (n=269) and consulting clinics 39% (n=140).Whereas ESBL isolation rate was significantly lower, 30%(n= 40) in community health centre patients as compared toinpatients. (p>0.01)Age wise break up of ESBL positivity rate isshown in Figure 1. Mean age of patients with ESBL pro-ducing organisms was 47 (Range: 1-95) versus 43(Range 1-100) for non ESBL producers (p<0.01).Furthermore ESBL production is significantly more inpatients less than 5 years and more than 60 years of age(p<0.01) (Figure 1). Figure 1. Age wise distribution of ESBL producing organisms exhibiting increase frequency ofESBL isolates at extremes of ages. % ESBL isolates represents ESBL positive isolates divided bytotal number of isolates obtained from a particular age group. An analysis of cross resistance to other antibioticsamongst ESBL producing isolates showed that ESBLpositive isolates had significantly higher resistance toother classes of antibiotics; aminoglycosides, quinolonesAge wise distribution of ESBL producing organisms452734404505101520253035404550<5 n=340 5-15 n=157 15-45 n=805 45-60 n=569 >60 n=994% ESBL isolates438 J Pak Med Assocand cotrimoxazole (p<0.01) (Figure 2). No resist-ance was seen to carbapenems and only 23 (1%) isolateswere resistant to piperacillin/tazobactam. DiscussionESBL producing organisms are among the fastestgrowing problems in the area of infectious diseases. Clinicalmicrobiological laboratories can no longer rely on simple invitro susceptibility data in the absence of the proper detec-tion of ESBL. Two other studies from Pakistan9,10 have alsoreported high frequencies of ESBL positivity rate. Shah et alfrom Pakistan reported that 48% of their ESBL positive iso-lates were from patients between 50-60 years of age.9 Otherstudies report a mean ages of 8321 and 5822 years for ESBLas compared to 47 years in this study. The increasing preva-lence in the lower age group is likely to be related to theoverall increase in ESBL load in our setting, a hypothesissupported by other surveillance studies from Asia and AsianPacific region reporting an alarming increase7 in ESBL pos-itivity.  This study observed highest positivity for ESBLproduction in Enterobacter sp. followed by E.coli.Zaman et al from Pakistan reported highest frequency ofESBL production in Klebsiella sp. followed by E.coli.10The SENTRY surveillance programme form Asia Pacificand South Africa reports that most common ESBL pro-ducer was Klebsiella sp.23 Mathur et al8 from India havealso reported Klebsiella sp. as the top ESBL producingorganism.Antibiotic pressure is reported to result in a muta-tion in beta lactamase gene with production of ESBL.3Risk factors responsible for this high frequency in our set-ting have not been determined. However, the higher posi-tivity rate in inpatient isolates as compared to those frompatients presenting to the community health centre is like-ly to reflect greater antibiotic pressure amongst the inpa-tients. ESBL positivity was also high in patients fromemergency room and consulting clinics which is likely tobe a reflection of tertiary referrals and discharged inpa-tients being seen in these areas respectively. The impor-tance of antibiotic pressure is further supported by thesignificantly higher ESBL positivity in isolates frompatients at the extremes of ages where antimicrobial usageis likely to be higher. One of the dilemmas of ESBL producing organ-ism is that they are frequently resistant to antibioticsother than beta lactams as they contain plasmids withgenes that encode resistance to aminoglycosides,quinolones and trimethoprim sulfmethoxazole.24 Variousstudies22,23 have documented that ESBL producingorganisms showed reduced susceptibility to all antibi-otics except amikacin and carbapenems. We note a sim-ilar phenomenon with increased resistance to all antibi-otics in ESBL producing organisms in comparison withnon-ESBLs. Thus treatment options in these infectionsare very limited.In conclusion, we document a high prevalence ofESBL positivity amongst our isolates. Due to limitedresources, several laboratories in developing countries donot routinely detect ESBL production. However, our datasupports an urgent need for regular screening and sur-veillance for these organisms in this region. IncreasedESBL positivity in isolates from patients at the extremesof ages most likely reflects high antimicrobial usage inthis population. Moreover, cross-resistance in ESBL pos-itive isolates to non-beta lactam agents severely limitstherapeutic choices and is alarming particularly in low-income settings where expensive second line agents areunavailable. References1. Nathisuwan S, Burgess S, Lewis JS 2nd . Extended-Spectrum ß-Lactamases:Epidemiology, Detection, and Treatment. Pharmacotherapy 2001;21:920-8.2. Jacoby GA, Mederios AA. More Extended Spectrum Beta Lactamases.Antimicrob Agents Chemother 1991;35:1697-704.3. Jacoby GA. Genetics of extended-spectrum ß-Lactamases. Eur J Clin InfectDis 1994;13(suppl 1):2-11. 4. Winokur P, Jones RN, Pfaller MA (2000). Characterization of strains fromEurope, North and Latin America, and Western Pacific that express an extend-ed-spectrum ß-lactamase phenotype: report from the SENTRY antimicrobialsurveillance program (1997-1999). Presented at the 40th interscience confer-ence on antimicrobial agents and chemotherapy. Toronto, Canada, September17-20, 2000.5. Adedeji A, Vangala K, Saurina G (2000). Molecular epidemiology ofKlebsiella pneumoniae with extended-spectrum ß-lactamase (ESBLs) inBrooklyn, NY. Presented at the 38th annual meeting of the Infectious DiseaseSociety of America, New Orleans, LA, September 7-10, 2000. 6. Lautenbach E, Patel JB, Bilker WB, Edelstein PH, Fishman NO. Extended-spectrum ß-lactamases-producing Escherichia coli and Klebsiella pneumoni-ae: risk factors for infection and impact on resistance of outcomes. Clin InfectDis 2001;32:1162-71.7. Bell JM, Turnidge JD, Gales AC, Pfaller MA, Jones RN; the Sentry APACStudy    Group. Prevalence of extended spectrum beta-lactamase (ESBL)-pro-ducing clinical isolates in the Asia-Pacific region and South Africa: regionalresults from SENTRY Antimicrobial Surveillance Program (1998-99). DiagnMicrobiol Infect Dis 2002;42:193-8.8. Mathur P, Kapil A, Das B, Dhawan B. Prevalece of extended spectrum betalactamase producing gram negative bacteria in a tertiary care hospital. Ind JMed Res 2002;115:153-7.9. Shah AA, Hasan F, Ahmed S, Hameed A. Extended spectrum beta lactamasesin Enterobacteriaceae: related to age and gender. New Microbiol2002;25:363-6.1566776811555270102030405060708090Amikacin Gentamicin Cotrimoxazole Ofloxacin% ResistanceESBL+ven=1137ESBL-ven=1703Figure 2. Comparison of sensitivity pattern of ESBL versus non ESBL to amikacin, gentamicin,cotrimoxazole and ofloxacin showing cross resistance to these antibiotics as well.Vol. 55, No. 10, October 2005 43910. Zaman G, Karamat AK, Abbasi AS, Rafi S, Ikram A . Prevalence of extendedspectrum beta lactamase producing Enterobacteriaceae in nosocomial isolates.Pak Armed Forces Med J 1999;49:91-6.11. Paterson DL, Yu LV. Extended spectrum beta lactamases: A call for improveddetection and control Clin Inf Dis 1999;29:1419-22.12. Karas JA, Pillay DG, Muckart D, Sterm AW. Treatment failure due to extend-ed spectrum beta lactamases. J Antimicrob Chemother 1996;37:203-4.13. Paterson DL, Ko W, Von Gottberg, Mohapatra S, Casellas J, Molazimglu L.In vitro susceptibility and clinical outcomes of bacterimia due to extendedspectrum beta lactamase producing Klebsiella pneumoniae. Clin Inf Dis1998;27:956.14. 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Frequency and sensitivity pattern of extended spectrum beta lactamase producing isolates in a tertiary care hospital laboratory of Pakistan

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Frequency and sensitivity pattern of extended spectrum beta lactamase producing isolates in a tertiary care hospital laboratory of Pakistan

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