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

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 Identication 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 Ofcial 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 Identication 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% condence 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% condence 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-decient agar (CLED) and culture plates were incubated into the 0 incubator at 37 C for overnight. The colonies from signicant bacterial growth plate were processed and organisms were identied by standard microbiological techniques by using various biochemical tests and other conrmatory 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, noroxacin (NX) 10μg, gentamycin (GEN) 10μg, levooxacin (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, ciprooxacin(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 conrmatory test for ESBL. Phenotypic conrmatory 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 conrmed as ESBL producer. Original Article


Introduction
The organisms producing Extended Spectrum Beta-Lactamases (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 Ident-ication 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. 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-decient agar (CLED) and culture plates were incubated into the 0 incubator at 37 C for overnight. The colonies from signicant bacterial growth plate were processed and organisms were identied by standard microbiological techniques by using various biochemical tests and other conrmatory 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, noroxacin (NX) 10µg, gentamycin (GEN) 10µg, levooxacin (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, ciprooxa-cin(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 conrmatory test for ESBL.

Phenotypic conrmatory 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 conrmed as ESBL producer.

Statistical analysis
The collected data were analyses using statistical package for the social science for windows (SPSS) version 20. Parametric variables were assessed using chi-squared test, as appropriate. A difference was considered statistically signicant if the p-value <0.05. This difference is statistically signicant with p value 0.005, which is shown in gure 1. Figure 2 shows that most of the patients were from OPD 177(57.0%) and 131 (43.0%) were from IPD. Cases of UTI were higher in OPD as compare to IPD.  Table 3 shows that both ESBL/non ESBL isolates were recovered with wide range from the age group between 19-35 years and least with 0-1 years of age. Figure 3 shows that total cases of UTI were 631. Out of 631 UTI cases 308 cases were caused by E. coli and K. species. Among which ESBL producing bacteria were 213(69.0%) and non ESBL producing bacteria were 95 (31.0%). ESBL isolates were more as compare to non ESBL isolates recovered from cases of UTI.     This is due to not proper using the antibiotics rd including 3 generation cephalosporins and not follow the standard infection control practice in hospital as well as in community. In the present study, total numbers of positive urine samples were 631 processed from the OPDs and IPDs. Among which, 308 urine sample showed growth of Escherichia coli species 288(93.5%) and Klebsiella species 20(42.5%) were recovered from cases of UTI which was very similar to study conducted by Nepal K et al [11]. In our study, the cases of UTI were higher in female 246(79.9%) as compared to male 62(20.1%).Male: female ratio was 1:3.9. Female is more prone for UTI as compared to male due to the anatomical structure of female urogenital system [8]. Females showed a higher rate of ESBL producing among the E. coli (70.5%) and K. species (50.0%), which was similar to that of Ahmed SM et al [12]. This study revealed a higher occurrence of ESBL producing uropathogens in the adult age group of 19-35 years, which was similar to study done by Chander A et al [13].

Discussion
In hospital setting and community based UTI are second most common bacterial infectious diseases in human being. Misuse and overuse of antimicrobials in community and in hospital set up will not only be expensive to the patients but also increase the drug resistance in future. Mostly in hospital, ICU patients are more susceptible to infection and chances of bacterial colonization are high by various pathogens due to weak immune system [8]. ESBL producing organisms have emerged as major global problem and affect the treatment with broad spectrum antibiotics which may cause drug resistance as a serious problem in future [9]. The ESBL producers from urinary isolates in our study were (69.2%) but it was half less, (31.6%) in a study conducted by Chatterjee M et al [10].   The studies by Spanu T et al [14] showed ESBL production was higher in K. pneumonia strains, as compared to E. coli isolates. Which was contrast to our study has shown increased number of ESBL producing E. coli was (70.5%) as compared to K. species (50.0%).Higher numbers of isolates were from outpatient department (57.5%) as compared to inpatient department (42.5%) and which was similar to the study conducted by Nepal K et al [11]. In present study numbers of ESBL/non ESBL producing isolates were recovered more from the OPD 114(64.4%)/63 (35.6%) as compared to IPD 99(75.6%)/32(24.4%). This is uncommon, very alarming situation and shows the trend of dissemination of ESBL producing bacteria to the community. This correlates well with study conducted by Bonnet R et al showed that higher number of ESBL-producing isolates from outpatients was E. coli (57.8%) [15]. Escherichia coli accounts for a large number of urinary isolates as well as higher numbers of ESBL production than K. pneumoniae [16]. This is similar to our study. The increase in ESBL production among the clinical isolates is found to be ranging from <1 to 74.0% worldwide [11]. The probable reason for gradual increase in ESBLs production in various parts of world due to the rd random and inappropriate use of 3 generation cephalosporins, which contribute to the evolution of ESBLs, not using of standard infection control practices in healthcare facilities and lack of national antibiotic policy. We found in our study ESBLs producing isolates were 100.0% susceptible to meropenem which conrms clearly with the CDC (1999) ESBLs denition and study conducted by Raut S et al [17]. All ESBL producing E. coli and K. species were found (100.0%) susceptibility to meropenem. Similarly, other antibiotic also showed increased susceptibility towards the piperacillin/tazobactum (97.2%), cefoperazone/ sulbactum (94.4%) and amikacin (93.0%), and which was similar to study conducted by Singh N et al [18] and Iroha IR et al [19].The ESBL producing organisms are increasing rapidly and becoming a major problem in the area of infectious diseases which includes multi drug resistance, difculty in detection and treatment and increase in mortality of patients. As compared to other available antimicrobial agents, carbapen-emes are the drug of choice and are reliable for treatment for infections caused by ESBL isolates. However, overuse and misuse of carbapenemes may also lead to drugs resistance among the other gramnegative bacteria. Therefore, knowledge of drug susceptibility, rational of drugs use and proper use of antibiotics including third-generation cephalosporins, must take effective measures to control infection in hospital environ-ment as well as in a community. These methods are the most effective means of controlling and preventing the spread of ESBL producing microorganisms.

Conclusion
ESBL are clinically signicant and when detected, indicate the need for the use of appropriate antibacterial agents. Infection with strains expressing ESBLs is a great challenge for both microbiologists and clinicians as they are having less therapeutic options. In this study infection with ESBL production among the E. coli and K. species isolated from urine sample are high and creating serious problem in country like Nepal. Study also observed that high numbers of ESBL