Distribution of Integrons and Phylogenetic Groups among Enteropathogenic Escherichia coli Isolates from Children

Taru Singh,V G Ramachandran,Dheeraj Shah,Sayim Wani,Khan A Maroof,Shukla Das,Aditi Sharma

doi:10.3389/fmicb.2017.00561

Abstract

Integrons by means of horizontal gene transfer carry multidrug resistance genes (MDR) among bacteria, including E. coli. The aim of this study was to determine the antibiotic resistance profiles and the genes associated with them, to gain insights in the distribution of phylogroups, prevalence, and characterization of class 1, 2 and 3 integrons among Enteropathogenic E. coli (EPEC) isolates, from children upto 5 years of age from Delhi and National Capital Region (NCR), India. A total of 120 E. coli isolates, including 80 from diarrheagenic E. coli (cases) and 40 from healthy isolates (controls) were recruited in this study. After isolation of E. coli, screening for EPEC was done by conventional multiplex PCR. Antibiotic suseptibility test was performed using disk diffusion method and further confirmed by minimum inhibitory concentration (MICs) by E-test. The presence and characterization of integrons and antimicrobial resistance genes were performed by PCR and DNA sequencing. Phylogeny determination was carried out by quadruplex PCR. EPEC strains were found in 64 of the 80 diarrheagenic cases, out of which 38 were MDR. In the 40 healthy controls, 23 were found to be EPEC strain, out of which only 2 were MDR. Amongst 80 diarrheagenic cases, class 1 integron were observed in 43 isolates, class 2 integron in 12 isolates and 9 isolates were found with co-existence of both. Similarly, in healthy controls; class 1 integron in 9 and class 2 integron in 7 isolates were observed with co-existence in 3 isolates. None of the isolates included class 3 integron. The dfr was the most commonly identified gene cassette within the integron-positive isolates. Phylogenetic studies showed considerable representation of phylogroup B2 in both diarrheagenic cases and healthy controls. This study reiterates the importance of class 1 integron predominantly for acquisition of antibiotic resistance genes among EPEC isolates. Furthermore, it also ascertains the possible association between multidrug resistance and presence of integrons. Approximately 91% of isolates were easily assigned to their respective phylogroups. Assessment of the relationship between antibiotic resistance and dominant phylogroups detected was also attempted. This study also highlights the increased burden of antimicrobial resistance in healthy controls.

Highlights

Childhood diarrhea is a predominant cause of child mortality worldwide, with an estimated of 2.2 million children getting affected in the developing countries alone (Ramana and Tamanna, 2012)
The amplified PCR products of Enteropathogenic Escherichia coli (EPEC) virulence for eae, bfpA and eaf genes on 1.5% agarose gel represents the uniplex PCR (Figure 1, Supplementary File), while Figure 1 shows that of multiplex PCR for all the three genes
Typical EPEC [eae+bfpA(eaf)] was found in 18.75% and 2.5% isolates (P = 0.001), atypical EPEC was found in 25% and 37.5% isolates (P = 0.673) while the total EPEC was found in 80% and 57.5% isolates (P = 0.012) of diarrheagenic cases and healthy controls respectively. 50/80 (62.5%) diarrheagenic cases (38 EPEC and 12 non-EPEC isolates) and 5/40 (12.5%) healthy controls (2 EPEC and 3 non-EPEC isolates) were multi drug resistant

Summary

Introduction

Childhood diarrhea is a predominant cause of child mortality worldwide, with an estimated of 2.2 million children getting affected in the developing countries alone (Ramana and Tamanna, 2012). EPEC adherence factor plasmid (pEAF) encodes bundle-forming pili which is essential for EPEC virulence, antigenicity, auto-aggregation, and localized adherence to epithelial cells (Trabulsi et al, 2002). The genetic determinants that produce A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that contains the genes eae encoding intimin (McDaniel et al, 1995). Atypical EPEC strains (lacking pEAF) are considered less virulent, as the genes encoding virulence factors are located on transmissible plasmids and on transposons or bacteriophages (Trabulsi et al, 2002); these have not been proven to be less pathogenic (Levine et al, 1985)

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