Incidence of blaIMP and blaVIM Genes among Carbapenemase Producing Escherichia coli in Lahore, Pakistan

This study was conducted between Sept 2017 and April 2018. A total of one hundred non duplicate E. coli isolates, which were found resistant to at least one carbapenem drug were obtained from collection of a tertiary care hospital at Lahore, Punjab Pakistan.. E. coli was reconfirmed on the basis of colonial characteristics and morphology and biochemically using an API 20E Test System for Enterobacteriaceae (bioMérieux, France). Antimicrobial susceptibility of isolates was tested by the Kirby-Bauer disc diffusion method using mueller-hinton agar (Oxoid, UK), according to Clinical Laboratory Standards Institute (CLSI). The antibiotic discs contained imipenem (10 µg) and meropenem (10 µg). Carbapenemase-producing E. coli were identified by the modified hodge test (Genc et al., 2016) and phenotypic detection of MBL was done by a combined disc diffusion method as per guidelines of Clinical laboratory Standard Institute (CLSI, 2016). In the combined disc test, if the increase in inhibition zone and imipenem-EDTA disc was ≥7 mm than the imipenem alone, the test was considered as MBL-positive.

Isolates found positive for carbapenemase production were subjected to PCR based identification for blaIMP and blaVIM using primers described in Table I. Plasmid DNA was extracted through plasmid isoaltion kit (TIANGEN Biotech Beijing, Co., Ltd.). The quality and quantity of extracted DNA was determined through nanodrop as well gel electrophoresis. Primers for PCR were synthesized by GeneLink, while master mix was purchased from Thermo Scientific. PCR reaction was performed in a total of 25µl reaction mixture. PCR was performed at a range of temperatures of 50-56°C and the optimum temperature was identified as 55°C.

All E. coli isolates were picked up from carbapenem-resistant culture collection and were tested again against meropenem and imipenem antibiotics to ensure its resistance pattern against carbapenem drugs. Results showed that all of these 100 isolates of E. coli were carbapenem non-susceptible, all being resistant to imipenem (100%) and meropenem (100%). Out of these 100 isolates, 81 were found positive for carbapenemase production based on Modified Hodge Test, whilst only 28 isolates were phenotypically positive for MBL production. Our current observed incidence rate of carbapenemase production among imipenem-resistant E. coli is higher as compared to previous report of 37.1% from Lahore in 2015 (Ain et al., 2018). Furthermore, in the same study, Ain et al. (2018) observed an overall 63.38% frequency of occurrence of MBL among all clinical isolates as compared to 28% observed in our study (Ain et al., 2018). This suggests that in the current study, MBL incidence rate was found much lower as compared to previous study. This could be the strain difference, as we have only focused on E. coli while, Ain et al. (2018) studied overall prevalence among different strains including E. coli. Our results showed that 25 isolates (30.8%) of

carbapenemase-producing E. coli were found to harbor bla-VIM gene, while 13 isolates (16.04%) were found to carry blaIMP gene (Fig. 1). All PCR amplicon of the targeted genes were sequenced and confirmed through BLAST. A recent study from district Lahore concluded that of 100 carbapenem resistant isolates, 93 and 89 isolates were positive for carbapenemase and β-lactamase production, respectively, while, 3.3% of these isolates were found positive for bla-IMP and 32.5% were found positive for bla-VIM (Akhtar et al., 2018). Our results are corroborates with the result of studies reported in India, Madagascar and Brazil where 94.4%, 89.8% and 88.2% carbapenemase positivity was described among gram-negative bacteria, respectively (Amudhan et al., 2012; Andriamanantena et al., 2010; Franco et al., 2010). Other studies suggest widespread presence of carbapenemase encoding genes in other parts of Paksitan suggesting to initiate steps to discourage dissemination of these features. This suggest that incidence of carbapenemase producers are quite high with considerably proportion of strains carrying bla-IMP gene. The higher incidence rate of MBL and subsequent higher occurrence of bla-IMP and bla-VIM genes in our clinical isolates is possibly due to the ability of E. coli to capture novel resistance genes through horizontal recombination located on the conjugative plasmids. The absence of bla-IMP and bla-VIM genes in the rest of carbapenem resistant strains does reflect that other types of carbapenemase encoding genes may also be present in the vicinity. It is speculated that other types of carbapenemase encoding genes such blaNDM-1, blaOXA-48 etc. may also be widespread in the area and should be investigated. In conclusion, the results describe here indicate high prevalence of carbapenemase-producing E. coli which, if not properly managed, will present a devastating challenge for the health care system of Pakistan.

It is concluded that E. coli isolated from different patients at a tertiary care hospital at Lahore indicate high prevalence of carbapenemase production stains comprising MBL. Majority of these isolates were encoding bla-IMP and bla-VIM genes, however, possibility of existence of other genes cannot be rule out. Notably, bla-VIM gene was found more prevalent (30.8%) than the bla-IMP gene (16.04%). This suggests to restrict the use of carbapenem drugs in order to restrict further dissemination of resistance.

There was no funding available for the work.

The authors have no conflicts of interest to report.