Genomic plasticity associated with antimicrobial resistance in Vibrio cholerae

Abstract

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae, the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens.

Keywords: antimicrobial resistance; cholera; genome; mobile genetic elements; proteome.

Fig. 1.

(A) Antimicrobial resistance diversity in V. cholerae isolates. (B) Number of antibiotics against which resistance was detected. The V. cholerae strains were isolated during 2008 to 2015. (C) The resistance diversities showed significant variations between isolates belonging to different serotypes. Lines extending vertically from the boxes in B and C showing variability outside the upper and lower quartiles.

Fig. 2.

Resistance profile of V. cholerae against different antibiotics. Bar graph showing the number of isolates in which resistance was detected against different antibiotics. The highest number of isolates showed resistance to sulfamethoxazole. Minimum resistance was detected against neomycin.

Fig. 3.

Differential resistance pattern between V. cholerae O1 and V. cholerae nonO1-nonO139 clinical isolates. Resistance is high in O1 isolates against tetracycline, chloramphenicol, streptomycin, and trimethoprim. NonO1-nonO139 isolates showed higher resistance to polymixin B compared with O1 isolates.

Fig. 4.

Yearwise resistance pattern of V. cholerae strains isolated during 2008 to 2015. Resistance against polymixin B is reduced over the year (except 2014). Tetracycline resistance was also low until 2014. 1′, 2008; 2, 2009; 3, 2010; 4, 2011; 5, 2012; 6, 2013; 7, 2014; 8, 2015.

Fig. 5.

Abundance of different antimicrobial resistance genes in the whole genome-sequenced V. cholerae isolates. Size of the bubbles corresponds to the abundance of resistance genes in the genomes of the four isolates. Subclasses of the resistance genes are also mentioned within the bubble. The picture was drawn to scale. Details of the resistance genes are provided in SI Appendix.

Fig. 6.

Mobile genetic elements linked with the antibiotic resistance genes in the genome of V. cholerae. Mobile element proteins like transposase/integrase/site-specific recombinase are used as signatures of MGEs. For β-lactamase, genes A and D denote serine-β-lactamase, whereas B denotes metallo-β-lactamase. Details of the resistance genes are provided in SI Appendix.

Fig. 7.

Whole-cell proteome analysis revealed expression of different antimicrobial resistance genes in the presence and absence of antibiotic. The V. cholerae IDH06781 strain was cultivated in the presence and absence of imipenem. Whole-cell proteins were labeled using iTRAQ and detected by TripleTOF 5600 mass spectrometer.