Phenotypic Characterization of Multidrug-resistant Escherichia Coli with Special Reference to Extended-spectrum-beta-lactamases and Metallo-beta-lactamases in a Tertiary Care

Introduction: The increasing reports on extended-spectrum-beta-lactamase and metallo-betalactamase producing Escherichia coli have addressed a potential threat to global health since it is found to be highly resistance to most of the currently available antibiotics including carbapenems. The present study was aimed to determine the antibiogram of extended-spectrum-beta-lactamase and metallo-beta-lactamase producing MDR E. coli isolates from various clinical samples.


INtrODUctION
The production of beta-lactamases (β-lactamases) is the most common mechanism responsible for resistance to β-lactams among clinical isolates of Enterobacteriaceae family. 1 The β-lactamases receiving the most attention are the extended-spectrum-beta-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases and carbapenemases because of rapid global dissemination of these enzymes. 1,2ESBLs confer bacterial resistance to all β-lactams except carbapenems and cephamycins, which are inhibited by β-lactamase inhibitors such as clavulanic acid. 3Carbapenemases consist of serineβ-lactamases (KPC, OXA, GES, etc.) and Metallo-βlactamases (MBLs) which are associated with resistance to aminoglycosides and fluoroquinolones. 4enotypic Characterization of Multidrug-resistant Escherichia Coli with Special Reference to Extended-spectrum-beta-lactamases and Metallo-beta-lactamases in a Tertiary Care Center OPEN ACCESS E.coli has been reported as opportunistic, worrisome, nosocomial and community-associated pathogen and the most frequent isolate in various clinical specimens. 5.coli is a major concern in medical community because of worldwide emergence of MDR strains mediated by ESBL and MBL enzymes.3,6,7 Little information is currently available regarding ESBL and MBL producing E.coli in Nepal.Keeping in view the above background, this cross-sectional study was conducted to provide information on antibiotic susceptibility with special reference to ESBL and MBL in MDR E.coli isolates from hospitalized patients.

MEtHODs
A cross-sectional study was conducted at the bacteriology laboratory of

Antimicrobial susceptibility testing
Antibiotic susceptibilities were determined by Kirby-Bauer disk diffusion method and the results were interpreted according to the guidelines of the Clinical Laboratory Standard Institute (CLSI). 9The antibiotic discs used were amikacin (30 μg), amoxycillin (

Tests for ESBL-production
All of the 250 isolates were screened for ESBL production by CLSI phenotypic confirmatory test of combined disc assay method. 9One disc of ceftazidime (30 μg) alone and one in combination with clavulanic acid (30 μg /10 μg) were placed at a distance of 20 mm on a Muller Hinton agar plate inoculated with a bacterial suspension of 0.5 McFarland turbidity standards, and incubated overnight at 37°C.The ESBL-producing strains showed a variation greater than 5mm between the inhibition zones around cefotaxime or ceftazidime discs alone in comparison with the inhibition zone around cefotaxime/clavulanic acid or ceftazidime/ clavulanic acid discs.Klebsiella pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative control strains respectively.

Tests for MBL-production
screening test: The isolates were subjected for MBL detection when the zone of inhibition (ZOI) for ceftazidime (CAZ) (30 μg) was <18 mm.The sensitivity or resistivity pattern to imipenem (IPM) (10mg) and or meropenem (MEM) (10 μg) were not considered for MBL detection as bacteria might harbour "hidden MBL" and if only the carbapenem resistant phenotypes were considered, then such hidden MBL carrying isolates would be missed.

MBL confirmation by combination disk method:
All 250 isolates were phenotypically confirmed for metalloβ -lactamase production as described by Franklin et al. 11 Briefly, two imipenem (IPM) disks (10 µg), one containing 10 μl of 0.1 M (292 μg) anhydrous EDTA (Sigma Chemicals, St. Louis, MO), were placed 25 mm apart (center to center).An increase in zone diameter of >4 mm around the IPM/EDTA disk compared to that of the IPM disk alone was considered positive for an MBL.For MBL test standardization, Pseudomonas aeruginosa ATCC 27853 was used as a negative control strain and Pseudomonas aeruginosa PA 105663 was used as a positive control.

Distribution of ESBL and MBL in MDR E .coli
Out of 250 E. coli strains studied, (n=239, 95.6%) strains produced any of the 2 types of β -lactamases i.e.ESBL and MBL, either alone or co-producer (Figure 2).In our study, (n=15, 6.2%) E. coli strains were positive for both types of β-lactamases i.e.ESBL and MBL in combination.

Distribution of ESBL and MBL-producers in different wards
ESBL and MBL producing MDR E. coli were the most common in surgical wards.Out of 250 MDR E. coli isolates, (n=80, 53.3%) ESBL-producers and (n=26, 60.4%) MBL-producers were isolated from surgical wards (Table 4).

Antibiogram of ESBL and MBL co-producer MDR E. coli
All the ESBL and MBL co-producer isolates were highly sensitive (n=15, 100.0%) to polymyxins and tigecycline but completely resistant to all penicillins and cephalosporins (including inhibitor combinations), aminoglycosides, cephamycins, fluoroquinolones, folate pathway inhibitors and nitrofurantoin.

DIscUssION
ESBL and MBL enzymes are of increasing clinical concern.
ESBLs are most commonly produced by Escherichia coli and Klebsiella spp.but may also be present in other gram negative bacteria.Many MDR bacteria produce multiple β-lactamases including combinations of these different enzymes.With the increasing number of MBL and KPC producing bacteria and ESBL and AmpC producing bacteria associated with porin loss and efflux mechanisms, there has been an increasing resistance to carbapenems. 4Prolonged antibiotic exposure, overstay in hospitals, severe illness, unprecedented use of third generation cephalosporin, and increased use of intravenous devices or catheters are important risk factors for infection with MDR E. coli. 12e current study demonstrated that (n=127, 50.8%) of MDR E. coli were isolated from urine samples and (n=141, 56.4%) from surgical wards.4][15] In the present study, most of the patients in surgical wards have indwelling urinary catheter.The indwelling urinary catheter as an invasive device has a significant association with hospital acquired urinary tract infections for it provides either a portal of entry for microorganism or a place for colonization of microorganisms. 16or all of MDR E.coli isolates, colistin, polymyxin B and tigecycline had the excellent activity followed by chloramphenicol, Piperacillin/tazobactam, amikacin and carbapenems.The resistance of E. coli isolates towards the third generation and fourth generation cephalosporins-cefotaxime and cefepime could be attributed to ESBL or some other relevant underlying mechanisms.Results of our study have shown that infection with ESBL producing MDR E. coli was (n=150, 60.0%) in our setting which was alarmingly high.Others have reported 50-70% prevalence of ESBL producing among MDR E. coli. 17,18ESBL production varies from hospital to hospital because of variation in selection of type of antibiotics.The selective pressures which are generated by the indiscriminate use of the beta-lactam antibiotics have led to the selection of a variety of mutated forms of β-lactamases such as ESBLs. 19 this study (n=43, 17.0%) isolates were MBL producers.1][22] Our findings were in concordance with the studies which were done by Bora et al 23 who reported 18.9% and Bandekar et al, 24 who reported 15.7% MBL producers.MBL producing organisms were isolated mainly from surgical wards (n=26, 60.4%).Indwelling medical devices are commonly used in these wards, which play a key role in the spread of infective agents.In any hospital setting, carbapenems are used as the last resort for treatment of MDR gram-negative bacterial infection.Antibiotic overuse is an important contributor for the emergence and spread of resistance; association between carbapenem consumption and resistance has been previously documented. 25However, since last 15 years, acquired resistance which is mainly mediated by MBLs to these life saving antimicrobials has been increasingly reported worldwide including Nepal not only in Pseudomonas and Acinetobacter spp, but also among members of Enterobacteriaceae. 26,27alysis of antimicrobial susceptibility pattern of ESBL producing E. coli isolates demonstrated high susceptibility rates towards imipenem(n=135, 90.0%) and meropenem (n=120, 80.0%) followed by piperacillin/tazobactam (n=119, 79.0%), amikacin (n=117, 78.0%) and chloramphenicol (n=46, 78.0%).9][30] MBL producing bacterial isolates can confer resistance to carbapenems and all beta-lactam agents except aztreonam although Shrestha et al.Phenotypic Characterization of Multidrug-Resistant Escherichia Coli with Special Reference... coexistence of other resistance mechanisms such as AmpC type beta-lactamases or ESBLs render them resistant to aztreonam. 31We observed all MBL producer E.coli were resistant to imipenem and meropenem.These isolates also demonstrated a high level of resistance to amoxycillin, the third and fourth generation cephalosporins, amikacin and gentamicin as well as to the beta-lactam/beta-lactamase inhibitor combination tested in the study.These findings are similar with other reports. 23,27Present study identified ESBL and MBL co-producers in (n=15, 6.2%) isolates.An increased rate of occurrence of ESBL and MBL co-producers (8.7 %) was also observed among nosocomial isolates of E.coli in a recent report from India. 32This study has demonstrated a very high level of resistance to the most of antibiotics tested in ESBL and MBL co-producer E.coli.Only polymyxins and tigecycline have potent activity against these isolates.Although the isolates were uniformly susceptible to polymyxin B and tigecycline in vitro, outcomes for infected patients treated with these agents remain unknown. 33The coexistence of different classes of β-lactamases in a single bacterial isolate may pose diagnostic and treatment challenges because the treatment options are fast running out.They are of significant concern because they create therapeutic dilemma, cause treatment failures and are increasing in occurrence worldwide.It might be undertaken that in the absence of novel agents in the near future, the spread of ESBL and MBL co-producers may lead to therapeutic dead ends.

CONCLUSIONS
Of particular concern are our results showing frequent carbapenem resistance among E. coli isolates, as well as the high rates of resistance to non-beta-lactam agents.This report underlined a real threat from the emergence of extreme drug-resistant and pan drug-resistant bacteria in near future.The spread of ESBL and MBL producing bacteria has been noticeably rapid worldwide including Nepal, indicating that continuous monitoring systems and effective infection control measures are absolutely required.Therapeutic options for infections due to ESBL and MBL producers have also become increasingly limited.Therefore, a better understanding of β-lactamase mediated resistance mechanisms is critical for optimizing therapy.In view of the exhaustion of available therapeutic options, investment in infection control resources and optimal antibiotic use, along with harmonized efforts from all concerned authorities is urgently required.

table 1 .
Prevalence of MDR E. coli in