EXTENDED SPECTRUM BETA LACTAMASES DETECTION AND MULTIPLE ANTIBIOTIC RESISTANCE INDEXING OF ESCHERICHIA COLI FROM URINE SAMPLES OF PATIENTS FROM A REFERRAL HOSPITAL OF EASTERN NEPAL

Background: Escherichia coli is the most common causative agent of urinary tract infection. Antibiotic resistance among uropathogens has become a prominent public health problem. Multidrug resistance bacteria have limited the therapeutic possibilities by producing Extended Spectrum Beta Lactamases (ESBL). Objective: Since routine monitoring of ESBL producers are not conducted in clinical laboratories their true prevalence is still unknown. So the objective of this research was to assess multiple antibiotic resistance (MAR) indices and determine ESBL production among Escherichia coli isolated from urine samples. Methods: Standard microbiological techniques and antibiotic sensitivity test were performed by Kirby Bauer disc diffusion method to identify E. coli. ESBL screening was done by using Ceftriaxone, Aztreonam, Cefotaxime, Ceftazidime and Cefpodoxime whereas confirmation by combined disc assay. SPSS 16 software was used to analyze data. Results: 86.95% E. coli isolates were MDR strains. 27 isolates had multiple antibiotic resistance (MAR) index of 0.2 and 5 isolates had MAR index of 0.7. E. coli isolates showed higher degree of resistance towards Amoxicillin (100%) while 100% were sensitive towards Gentamicin followed by Nitrofurantoin (62.31%). The reliable screening agent for ESBL detection with sensitivity 100% and positive predictive value of 80% was Cefotaxime. Combined disc assay detected 12/69 (17.31%) of E. coli isolates as confirmed ESBL producers. Conclusion: The ubiquity of ESBL-producing E. coli was observed emphasizing the necessity of regular surveillance of ESBL producing clinical isolates in clinical samples to minimize multi-drug resistance strains and avert the ineffectiveness of antimicrobial agent for good health practices.


Introduction
Urinary tract infection (UTI) is a common bacterial disease prevalent in community. E. coli accounts for 75.0-90.0% of all UTIs (Dromigney et al., 2005). UTI is a common disease prevalent among Nepalese population (Kattel et al., 2008).
Antimicrobial therapy of UTI caused by E. coli has been continually weakened due to the resistance against beta lactam antibiotics. β-lactamases are the major defense of Gram negative bacteria against β-lactam antibiotics (Jacoby et al., 2005). Extended spectrum β-lactamases (ESBLs) are defined as the plasmid-mediated bacterial enzymes granting resistance to the penicillins (except temocillin), first, second and third-generation cephalosporins, and aztreonam (except cephamycins or carbapenems) but inhibited by β-lactamase inhibitors such as clavulanic acid (Paterson et al., 2005). MAR index helps to assess the spread of bacterial resistance to more than three antibiotics (Krumperman, 1983).
The emergence of MDR and ESBL E. coli pose antibiotic management problems (Lim et al., 2009). Multiple antibiotic resistances in bacteria and production of ESBL is most commonly associated with the presence of plasmids which contain one or more resistance genes, each coding a single antibiotic resistance phenotype (Paterson et al., 2005;Daini et al., 2008).
This study was designed to estimate the current prevalence and antimicrobial resistance patterns along with ESBL producing urinary isolates of E. coli among urinary tract infection patients visiting Bijayapur Hopsital, Dharan, Nepal.

Methodology
In a descriptive cross-sectional study conducted from March to August 2014, a total of 752 urine samples from suspected UTI patients visiting Bijayapur Hospital, Dharan were processed in the laboratory of Sunsari Technical College, Dharan for the isolation of E. coli. Informed consent was obtained for each sample used in the study from Research Article the patients. Each sample was mixed well and aseptically inoculated on MacConkey agar plates and incubated at 37 o C for 24 hours aerobically. Significant UTI was defined as the presence of >10 5 colony forming unit (CFU)/ml in the culture.
Further identification of E. coli was done by their cultural characteristics, Gram stain and different biochemical reactions. The antimicrobial susceptibility testing (AST) of E. coli isolates was done by Kirby Bauer Disc Diffusion Method as per CLSI guideline (CLSI, 2013). E. coli ATCC 25922 was used as a reference strain.
Multiple antibiotic resistance (MAR) index was determined using the formula MAR=a/b, where "a" denotes the number of antibiotics to which test isolate showed resistance and "b" is the total number of antibiotics employed for sensitivity (Akinjogunla et al., 2010). An isolate was considered to be Multidrug Resistant (MDR) when it showed resistance to two or more drugs of different structural classes.
All the processed E. coli isolates were subjected to phenotypic confirmatory test using Combined Disks Assay consisting Ceftazidime (30µg) and Ceftazidime (30µg) plus Clavulanic acid (10µg) and Cefotaxime (30µg) and Cefotaxime (30µg) plus Clavulanic acid (10µg). An increase in zone diameter of ≥5mm in the presence of Clavulanate from either of the combination discs confirmed ESBL isolates (CLSI 2013). Data collected was analysed by using SPSS. P-value ≤0.05 was considered to be statistically significant.

Results
Out of 105 positive isolates, the overall prevalence of E. coli was found 69.6% in total 99 Gram negative isolates. Out of 69 E. coli isolates, 60 (86.95%) were multiple drug resistance and 12 (17.3%) isolates were found to be ESBL producers. The isolates were highly sensitive to Gentamicin and Tobramycin (100%) followed by Nitrofurantoin (62.31%). All the 69 isolates of E. coli were resistance towards Amoxicillin (Table 1).
Nine multidrug resistance patterns were observed in E. coli for the seven antimicrobial agents tested. Resistance to Amx-Cz was the most frequent pattern observed in 41.7% of E. coli isolates, 8.3% of E. coli isolates showed Amx-E-Cz-NA-Nit resistant pattern ( Table 2). The MAR index ranges from 0.14 to 0.71. Out of 69 E. coli isolates, only 9 showed MAR index of 0.1 (< 0.2). 5 isolates showed MAR index of 0.7 i.e. these isolates were resistance to five antibiotics used in the testing (Table 3).

Discussion
In this study, the overall prevalence of E. coli was found to be 69 (65.7%) in total 105 isolates. Similar result was reported by Sharma et al.(2011) who found 67.5% E. coli.
Most of the E. coli isolates showed the multidrug resistant (86.95%) in agreement with other studies (Bashar et al., 2009;Moyo et al., 2010;Hassan et al., 2011;Sharma et al., 2013). This study demonstrated 100% resistant of E. coli isolates to Amoxicillin which was similar to previously reported finding (Khadgi et al., 2013). Resistance to penicillins may be determined by the organisms due to the production of penicillin destroying enzymes such as betalactamase (Forbes et al., 2007).
With the highest sensitivity and PPV cefotaxime was found the most reliable agent for ESBL screening test. This result matches with other findings (Ho et al., 2000;Poudyal et al., 2011). TEM-1, TEM-2, and SHV-1 β-lactamases are the primary causes for resistance towards β-lactam antimicrobial agents among gram negative rods (Livermore, 1995). Two isolates screened as ESBL screen negatives by Ceftazidime, however, were found ESBL producers on confirmatory test. This suggests the possible production of CTX-M type ESBL by these isolates (Bush, 2008). CTX-M ESBLs differ from TEM and SHV types as they hydrolyse Cefotaxime and Ceftriaxone better compared to Ceftazidime (Lewis et al., 2007). Out of 69 E. coli isolates, 12 (17.39%) confirmed to be ESBL positive. This result is in harmony with previous study (Chander et al., 2013).

Conclusion
This study reveals that E. coli is the most predominant pathogen in urinary tract infection accounting for 65.7 % in total positive isolates. ESBL producers were found higher in females 11 (91.6%) than males 1 (8.3%). The prevalence of MDR E. coli isolates was high i.e. 86.95%. Likewise, MAR index data revealed that isolates with lowest and highest MAR index are present in our surrounding that can pose health hazards.
The prevalence of ESBL producers was 17.39% among total E. coli. Ceftazidime had the lowest sensitivity detecting ESBL producers and can miss CTX-M producing bacteria thus signifying use of more than one screening agents and combination disk assay for more reliable detection of ESBL. Hence this kind of study aids in evaluating the exact cause and mechanism of rapid development of antibiotic resistance by bacteria.