Role of integrons, plasmids and SXT elements in multidrug resistance of Vibrio cholerae and Providencia vermicola obtained from a clinical isolate of diarrhea

Abstract

The isolates of Vibrio cholerae and Providencia vermicola obtained from a diarrheal patient were investigated for genetic elements governing their drug resistance phenotypes. Out of 14 antibiotics tested, V. cholerae Vc IDH02365 isolate showed resistance to nine antibiotics, while P. vermicola Pv NBA2365 was found to be resistant to all the antibiotics except polymyxin B. Though SXT integrase was depicted in both the bacteria, class 1 integron was found to be associated only with Pv NBA2365. Integrons in Pv NBA2365 conferred resistance to β-lactams, aminoglycosides, and trimethoprim. Pv NBA2365 carried two transformable plasmids imparting distinct antibiotic resistance traits to their Escherichia coli transformants. In rabbit ileal loop assays, Pv NBA2365 did not show any fluid accumulation (FA) in contrast with Vc IDH02365 that showed high FA. To the best of our knowledge, this is the first report of a highly drug resistant P. vermicola and additionally co-existence of multidrug resistant V. cholerae and P. vermicola. Both the microbes appeared to possess a wide array of mobile genetic elements for a large spectrum of antimicrobial agents, some of which are being used in the treatment of acute diarrhea.

Keywords: Providencia vermicola; SXT element; Vibrio cholerae; integron; multidrug resistance; plasmid.

FIGURE 1

Identification and differentiation of constituent bacteria of a clinical isolate of diarrhea. Agarose gel (1%) analysis of (A) PCR products for confirming V. cholerae species with primer pair OmpW- F/OmpW-R; (B) PFGE of DNA from Vc IDH02365 and P. vermicola Pv NBA2365 digested with SfiI restriction enzyme; (C) RAPD profile of clinical isolates with 1281 and 1283 primers; and (D) PCR with species-specific primers for P. rettgeri/P. vermicola and only P. vermicola. All the DNA samples used as templates are indicated on top of each lane. The clinical isolates of V. cholerae IDH01526, IDH01572, IDH01581, N16961, MO10, P. vermicola BAB 812, P. rettgeri MTCC8099 and mixture of Vc IDH02365 and Pv NBA2365 as obtained from the original stab culture (mixed) were used as controls for various assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.

FIGURE 2

Agarose gel (1%) of PCR products for analysis of SXT element and class 1 integron showing amplification with (A) primer pair SXT-F/SXT-R for detection of SXT integrase; (B) primer pair L2/L3 for detection of 5′ CS of class 1 integron; (C) primer pair qacEΔ1-F/Sul1-B for amplification of 3′ CS of class 1 integron; (D) primer pair In-F/In-B for amplification of variable regions associated with class 1 integrons. All the DNA samples used as templates are indicated on top of each lane. V. cholerae MO10 and V. fluvialis BD146 were used as controls for various PCR assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.

FIGURE 3

Schematic representation of class 1 integron containing 2.8 and 1.2 kb variable regions harboring different kinds of gene cassettes. Integrons consist of a gene intI1 encoding a site-specific recombinase called “integrase” belonging to tyrosine-recombinase family and a recombination site attI1 into which exogenous gene cassettes (here blaVIM-1, aadB, dfrA1, and orfC) harboring the recombination site attC are inserted through site-specific recombination. These exogenous cassettes that vary from one integron to the other, together constitute variable region of the integron. In the 5′ conserved sequences (5′ CS), a promoter located within intI1 drives transcription of the captured genes. qacEΔ1 and sul1 are conserved regions in 3′ conserved sequences (3′ CS) which contribute resistance to ethidium bromide and sulfonamides. Purple arrows indicate the direction of transcription for each gene. Black arrows indicate the position of primers used for detection and analysis of class 1 integrons.

FIGURE 4

Agarose gel analysis for genomic DNA and plasmid DNA of Vc IDH02365 and Pv NBA2365. All the DNA samples used as templates are indicated on top of each lane. Marker positions in kb have been indicated. g. denotes total genomic DNA preparation and p. denotes plasmid DNA preparation.

FIGURE 5

Rabbit ileal loop assay to assess entrotoxigenic activity. Pictorial view of rabbit ileal loop of different clinical strains (A). Analysis of fluid accumulation of different clinical strains (B). Rabbit ileal loops were inoculated with 10⁸ CFU of each strain V. cholerae N16961 (O1 El Tor, clinical), Vc IDH02365 (O1 El Tor, clinical), Pv NBA2365 in PBS and incubated for 18 h. Results are expressed as fluid accumulation (FA; in milliliters) per loop length (in centimeters). PBS and the mixture of Pv NBA2365 and Vc IDH02365 were taken as controls in this assay.