Antibiotic Profile of Extended Spectrum Beta Lactamase Escherichia Coli from Clinical Samples

Background: Extended spectrum β-lactamases have addressed the serious challenges worldwide due to the emergence of ESBL producing genes which possess a serious threat for the treatment of infections both in community and hospitals since it is found to be increasing trends of multidrug resistance. This study was focused to find out the antibiotic profile of multidrug resistant Escherichia .coli and status of ESBLs producing E.coli. Methods: This was a cross-sectional study conducted over a period of 2 years (September 2017 to April 2019) at microbiology laboratory of Nepal Mediciti Hospital. A total of 16542 samples were processed. Various clinical samples were collected from both inpatients and outpatients aseptically and without contaminating skin commensals. Standard microbiological techniques were used for isolation and identification of pathogens. Extended spectrum beta-lactamases were phenotypically confirmed by combined disc method. Results: Out of 1449 E.coli isolates, 323(22.29%) were found to be MDR E.coli. Isolation rate of ESBL producing E.coli (66.56%) were found to be high among MDR E.coli isolates. Conclusion: There was increasing prevalence of ESBL producing E.coli and was essential to monitor antibiotic susceptibility pattern and formulate antibiotic policy to prevent the spread of MDR and ESBL producers.


INTRODUCTION:
Extended-spectrum beta-lactamases (ESBLs) are the group of beta-lactamase enzymes, which hydrolyze and cause resistance to the oxyimino-cephalosporins (cefotaxime,ceftazidime,ceftriaxone,cefuroxime and cefepime) and monobactams(aztreonam), but not the cephamycins (cefoxitin and cefotetan) or carbapenems (imipenem, meropenem, and ertapenem), produced by Escherichea coli and Klebsiella pneumoniae. 1 Beta-Lactam antibiotics include (penicillins, cephalosporin, carbapenems & monobactams) are common to treat bacterial infections. The emergence of β-Lactamases mediated bacterial resistance, are due to the irrational use of antibiotics, particularly the third generation cephalosporins, which subsequently led to the development of ESBL producing bacteria. 2 Emergence of resistant bacteria worldwide possess a great threat to treatment outcomes in community and hospital settings. E. coli is one of the commonest pathogen to exhibit multidrug resistance. Important risk factors for infection with MDR and ESBL Escherichia coli are prolonged antibiotic exposure, overstay in hospital, irrational use of 3 rd generation cephalosporins, severe illness,frequent use of IV devices and catheters. 3 Extended spectrum beta lactamases are plasmid mediated enzymes which confer bacterial resistance to the penicillins, first, second and third generation cephalosporins and aztreonam. 4 The origin of Extended spectrum beta lactamases from mutant forms of broad spectrum beta lactamases such as the TEM-1,TEM-2 and SHV-1 enzymes coded by genes located on transferrable plasmids, which can easily spread form one organism to another. 5 The epidemiology of ESBL-producing bacteria has been increasing day by day with crossing boundaries between hospitals and the community. The acquisition of efficient mobile elements has accelerated the transfer of various antibiotic resistance genes. Continuous surveillance system to monitor multidrug-resistant bacteria is urgently required worldwide. 6 Current knowledge of prevalence of ESBL producing strains and antibiotic status of MDR, ESBL strains will help global awareness for the formulation of antibiotic policy and antibiotic stewardship program to optimize antibiotic use for improving patient outcomes, reducing adverse consequences and provide cost-effective therapy. Therefore, the present study was conducted with an objectives of studying the antibiotic profile of MDR and ESBL Escherichia coli producing strains which helps to formulate antibiotic policy and antibiotic stewardship program for the prevention and control of infections.

MATERIAL AND METHODS
This study was a cross sectional study conducted in Microbiology Laboratory of Nepal Mediciti Hospital, Bhaisepati; Nepal from September 2017 to April 2019.This study was approved by Institutional Review Committee of Nepal Mediciti Hospital. A total of 16542 samples were processed. Various clinical samples were collected from both inpatients and outpatients aseptically and without contaminating skin commensals. Urine samples were inoculated on CLED agar whereas pus, sputum, swab ,biopsy, fluid , foley'stip, vaginal swab, catheter tip, CVP tip, BAL, suction tube, bile were inoculated aseptically on blood agar, chocolate agar and Mac-Conkey agar and incubated aerobically at 37±°C for 24-48 hours. About 8-10 ml of blood from adult patients and 1 -3 ml of blood from pediatric cases were inoculated in BACTEC TM plus Aerobic/F vial and BACTEC TM PEDS PLUS/F vial respectively. The culture bottles were incubated at 37 o c overnight (at least 5 days) inside BACTEC TM FX and checked for the turbidity or any visual change daily. The specimens were sub cultured on Blood agar, chocolate agar and MacConkey agar plates and incubated aerobically at 37±°C for 24 hours using a standard operative procedures. Significant bacterial count (10 5 CFU/ ml) was recorded from urine culture isolates. The identification of bacterial isolates were carried out by cultural, morphological characters, Gram stain and appropriate biochemical tests (triple sugar iron, indole, citrate, urease and motility) following standard procedures.

Screening of ESBL
3 rd generation cephalosporins (ceftazidime, cefotaxime and ceftriaxone) were used to screen ESBL producers. Isolates resistant to more than one of these agents were identified as possible ESBL producers. 7, 8 Confirmation of ESBL by confirmatory test following CLSI guidelines.

Confirmatory Test for ESBL
ESBL detection was confirmed by two phenotypic methods.

Double disc synergy test (DDST)
Microbial inoculums matched with 0.5 McFarland Standard turbidity were inoculated on dried MHA plates. Antibiotic disc of amoxicillinclavulanate(20µg/10µg was placed at the center and ceftazidime(30µg) and cefotaxime(30µg) were placed on either side of 30mm apart from center to center. Plates were incubated at 37°C for 18-24 hours. Those inoculums which exhibited an enhanced zone of inhibition in between amoxicillin/clavulanic acid and ceftazidime and cefotaxime were identified as confirmed ESBL producers. 9, 10 Combined disc method Ceftazidime (30µg) alone and ceftazidime with clavulanic acid(30µg/10µg)and cefotaxime (30µg) and cefotaxime with clavulanic acid (30µg/10µg) were placed at an appropriate distance from each other on inoculated MHA plate. The plate was incubated at 37°C for 18-24 hours aerobically. Inhibition zone differences by ≥5mm in either ceftazidime clavulanic acid with ceftazidime alone or cefotaxime clavulanic acid with cefotaxime alone was interpreted as confirmed ESBL. 11 The quality control was done using Escherichia coli ATCC 25922 as negative control.

Statistical Analysis
The data was entered and percentage calculation were analyzed by using Statistical Package for Social Sciences (SPSS) version 21 software.

RESULTS
Total of 16542 samples were collected in which 1449 Escherichia coli isolates were recovered from various clinical samples of inpatients and outpatients of the hospital. The highest number of Escherichia coli was isolated from urine followed by sputum, swab,pus,blood,fluid,foley's tip,vaginal swab, catheter tip,BAL,biopsy,bile suction tube,CVP tip,ET tube. Distribution of Escherichia coli isolates on the basis of source. (Fig.1)  In the present study, it is observed that Escherichia coli isolates from urine culture is 96.54% to nitrofurantoin followed by amikacin (80.78%).
Most of E.coli from urine were resistant to third generation cephalosporins indicates possible ESBL producers.
Similarly, Escherichia coli isolates from sputum, swab, pus and blood were found to be 100% sensitive to tigecycline followed by colistin and polymyxin b. Resistance to third generation cephalosporins is between 26% and 93%, which is highly variable. Whereas, resistant pattern of carbapenem is between 4% and 20% which is quite low. Isolates resistant to third generation cephalosporins were considered as possible ESBL producers  Antibiotic susceptibility pattern of ESBL E.coli producers showed similar pattern of sensitivity to tigecycline (100%) followed by polymyxin b, colistin and meropenem as non-ESBL producing E.coli. But the resistant to third generation cephalosporins showed increasing trends as compared to non-ESBL producers (table 4).   18 .This may be due to the rational use of these drugs in UTIs cases since it is reserved drug for UTIs.
In this study, analysis of antibiotic susceptibility of E.coli isolated from sputum, blood, swab, pus demonstrated a significant degree of susceptibility towards tigecycline (100%) followed by colistin (98% to 100%), polymyxinb (97% to 100%), meropenem (91% to 96%) and imipenem (79% to 90%).Similar results were shown in other studies .14, 19. It was found to be higher resistant pattern of cephalosporins (22% to 93%), fluoroquinolones (26% to 85%),aminoglycosides (8% to 59%) as compared to urine isolates. Several studies conducted in Nepal showed similar results. 12, 19,20 In contrast to our study, Bamford et al.noted higher susceptibility pattern towards cephalosporins, fluoroquinolones, aminoglycosides. 21 The increased level of drug resistance is a major concern worldwide since these are the first line drugs recommended internationally 22,23 and are irrational used in public and private sectors. 24