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Background Escherichia coli and Klebsiella species are most common ESBL producers and associated with UTI in both community and hospital setting, often limiting the treatment therapy of UTI. Aim of present study was Isolation and Identication of ESBL producing E. coli and Klebsiella Species in urine sample from cases of UTI and their antibiotic susceptibility pattern. Materials and Methods This descriptive cross-sectional study was conducted at Nobel Medical College Teaching Hospital with effect from October 2018 to June 2019 after approval from the Institutional Review Committee (IRC) of the college as per clinical laboratory standards institute of (CLSI) guidelines. Results Out of total 2567 urine samples, 631(24.5%) samples showed microbial growth. Among which E. coli was 288 (45.6%), Klebsiella species were 20(31.5%), other gram-negative bacteria were 158(25%), gram positive bacteria were 114(18%) and fungus 51 (8%) were recovered. Out of 631 culture positive urine samples, 308 urine samples were positive for E. coli/K. species 288 (93.5%)/20 (42.5%) respectively. Among which ESBL isolates were 213 (69%) and non ESBL isolates were 95 (31%). All ESBL producing E. coli and K. species were found (100%) sensitive to meropenem. Similarly, among other antibiotics also showed increased susceptibility towards the piperacillin/tazobactum (97.2%), cefoperazone/sulbactum (94.4%) and amikacin (93.0%). Conclusion The highest numbers of ESBL producers E. coli and K. species from urine sample are increasing day by day and creating serious problem in treating patients in Nepal. So it should be mandatory and very essential to have routine monitoring system to detect the ESBL producing isolates in clinical laboratories. Keyword: Antibiotic, Escherichia coli, Urinary Tract Infections Kumari Ragani Yadav et.al. 32 Nepal Journals Online: www.nepjol.info Ofcial website: www.jonmc.info Journal of Nobel Medical College Vol. 8, No. 2, Issue 15, July-December 2019 Introduction The organisms producing Extended Spectrum BetaLactamases (ESBL) enzymes are resistant st nd rd to all penicillins and 1 , 2 and 3 generation cephalosporins and monobactum, however remains sensitive to carbapenems and cephamycin [1]. ESBL are commonly detected in the members of the Enterobacteriaceae like Klebsiella pneumoniae and E. coli. E. coli and Klebsiella pneumoniae are most two predominant and important bacteria isolated from urine samples in cases of UTI in both in hospital setting and community [2]. The multidrug resistant gram negative bacteria are serious global problem at present [3]. The easy transmission of the ESBL codzing plasmids between the species has become a major threat in hospital setting and outbreaks occurring in hospital are often due to infection caused by organisms producing ESBL [4]. This issue is of great challenge and inviting rst priority to every laboratory to detect ESBL isolates. Aim of Present study was Isolation and Identication of ESBL producing E. coli and Klebsiella species in urine sample from cases of UTI and their antibiotic susceptibility pattern in Nobel Medical College Teaching Hospital, Biratnagar, Nepal. Materials and Methods This descriptive cross-sectional study was conducted at Nobel Medical College Teaching Hospital, Biratnagar, Nepal with effect from October 2018 to June 2019 after approval from the Institutional Review Committee (IRC) of the college. The studies consider 95% condence interval and 80% power to evaluate the sample size. According to literature review the rate of ESBL producing E. coli and Klebsiella species in urine sample from cases of UTI is estimated approximately 50%. So by using formula n = 2 2 z pq/l where Z= 1.96 at 95% condence interval, p=50%, q= 50% and l= 20% of p i.e. 10. So now n= 4x50x50/100 =100. But load of patient attending to the microbiology laboratory was high so total 2567 cases of UTI included during the study. Study populations were cases of urinary tract infection patients attending to Hospital. Inclusion criteria for this study was all the age groups from cases of UTI and the samples that were received unlabeled, collecting tubes and bottles which were cracked or broken and urine sample from nonUTI cases was excluded. Data collection was done by lling self-structured Proforma designed and endorsed by the department for the study. Data was collected throughout the study to meet the sample size for the study. A total 2567 clean catch mid-stream urine (MSU) samples was collected in wide mouth sterile container from cases of UTI attending in hospital. All the mid-stream urine samples were inoculated on cysteine lactose electrolyte-decient agar (CLED) and culture plates were incubated into the 0 incubator at 37 C for overnight. The colonies from signicant bacterial growth plate were processed and organisms were identied by standard microbiological techniques by using various biochemical tests and other conrmatory tests [5]. Antibiotic susceptibility testing was performed by Kirby-Bauer's disc diffusion method as per CLSI guidelines [6]. Different antimicrobial disc used were: nitrofurantoin (NIT) 300μg, noroxacin (NX) 10μg, gentamycin (GEN) 10μg, levooxacin (LE) 5μg, ampicillin (AMP) 10μg, cefuroxime (CXM) 30μg, ceftazidime (CAZ) 30μg, cefotaxime (CTX) 30μg, meropenem (MRP) 10μg, piperacillin /tazobactum (PTZ)100/10μg, ampicillin/salbactum (A/S) 10/10μg, amikacin (AK) 30μg, co-trimozole (COT) 25μg, ciprooxacin(CIP) 5μg and cefoperazone /salbctum (CFS) 5/30μg. According to CLSI guidelines, [7] the organisms (Escherichia coli and Klebsiella species) were screened for ESBL production using ceftazidime (30μg), cefotaxime (30μg) and ceftriaxone (30μg). The Escherichia coli and Klebsiella species showing reduced susceptibility to at least one of these antibiotic with zone of inhibition on Mueller Hinton Agar for ceftazidime (30μg) ≤22mm, ≤25mm with ceftriaxone (30μg) and ≤27mm with cefotaxime (30μg) were considered as potential ESBL producing organisms and selected for phenotypic conrmatory test for ESBL. Phenotypic conrmatory test The antibiotic susceptibility test were performed on Mueller Hinton Agar by using disc of third generation cephalosporins alone and disc of third generation cephalosporins along with clavulanic acid by Kirby-Bauer's disc diffusion method following the CLSI guidelines [6]. The suspected ESBL producing Escherichia coli and Klebsiella species were inoculated as lawn culture on Mueller Hinton Agar. Then the discs of ceftazidime (30μg) alone and ceftazidime + clavulanic acid (30μg +10μg) discs along with cefotaxime (30μg) discs alone and cefotaxime + clavulanic acid (30μg +10μg) discs were placed with 25 mm apart on lawn culture of organism on Mueller Hinton Agar. After overnight of aerobic incubation into the 0 incubator at 37 C the difference in zone diameter of cephalosporin alone and cephalosporins + clavulanic acid were measured by using verniercaliper scale. An increase of ≥5 mm of zone diameter around cephalosporins + clavulanic acid disc compared to cephalosporins alone was conrmed as ESBL producer. Original Article

Study of Extended Spectrum Beta-Lactamases Producing Escherichia Coli and Klebsiella Species in a Tertiary Care Hospital, Biratnagar, Nepal