Abstract

Twenty-five diarrheal fecal samples from Kolkata were examined to determine the relative abundance of antimicrobial resistance genes (ARGs) against eight common classes of antibiotics with polymerase chain reaction (PCR) and Sanger sequencing. Relative abundance of an ARG was calculated as the percentage of fecal samples showing the presence of that particular ARG. The frequency of occurrence of resistance marker against each class of antibiotic was calculated as the percentage of fecal samples carrying at least one resistance marker for that particular class of antimicrobials. Antibiogram of Vibrio cholerae (V. cholerae) O1 strains isolated from four of these samples was obtained by disc diffusion method and was compared with the ARG profile of corresponding fecal samples from which the strains were isolated. A 464 bp amplicon of the V3-V4 region of bacterial 16S rDNA was obtained by PCR from 9 of these 25 samples using the primer pair S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 and sequenced to determine the major operational taxonomic unit (OTU). These 9 samples represented diarrhea due to diverse etiology and also unresolved etiology as determined by culture method. We conclude that the diarrheal intestinal microbiome has a common gene pool of ARGs against the major classes of antibiotics and may be serving as a reservoir of ARG dissemination. ARG profile of cholera stool showed that ARGs present in the gut of cholera patients may be transferred to the V. cholerae genome and pose a serious threat to the treatment of cholera by triggering resistance against potential drugs to which contemporary strains of V. cholerae were found to be sensitive in the present study. Fecal samples which were culture negative for diarrheal pathogens we tested also carried ARGs and OTU. Abundance of resistance markers against macrolides, tetracyclines, and aminoglycosides was the highest. Phylum Proteobacteria was the most abundant OTU suggesting proteobacterial blooms characteristic of disturbed gut microflora. Our study is the first comparative study of ARG profile of diarrheal samples with varying etiologic agent revealing the presence of ARGs against the most important classes of antibiotics in the gut of diarrheal patients by common, robust molecular methods, which are easily accessible by molecular epidemiological laboratories worldwide.

Highlights

  • Antimicrobial resistance (AMR) among diarrheal pathogens has emerged as a critical threat to the clinical management of diarrheal cases

  • We report the relative abundance of different ARGs in the diarrheal gut microbiota in Kolkata and the suburban areas

  • Twenty-four of these samples were from patients who were admitted to Infectious Diseases Hospital (IDH) for 1–4 days for treatment of acute diarrhea and in these patients diarrhea lasted for 1–5 days

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Summary

Introduction

Antimicrobial resistance (AMR) among diarrheal pathogens has emerged as a critical threat to the clinical management of diarrheal cases. In this study we have attempted to report about the profile of selected resistance determinants obtained from fecal samples using simple polymerase chain reaction (PCR) with specific primers to detect a spectrum of antimicrobial resistance determinants that are involved in diverse antimicrobial resistance mechanisms in bacteria and which are associated with the most common classes of antibiotics advocated for diarrheal treatment. These include tetracyclines, macrolides, amphenicol, aminoglycosides, carbapenem, trimethoprim, sulfamethoxazole, and quinolones.

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