Mechanism of Drug Resistance in Clonally Related Clinical Isolates of Vibrio fluvialis Isolated in Kolkata, India

The molecular mechanisms of drug resistance in 19 strains ofVibrio fluvialis isolated from 1998 to 2002 in Kolkata, India,were investigated. Class 1 integrons were detected in eightstrains, and four strains were found to carry SXT integrases.In the presence of carbonyl cyanide m-chlorophenylhydrazoneor reserpine, all nalidixic acid- and ciprofloxacin-resistantstrains became sensitive, suggesting that drug efflux playsa major role in quinolone resistance in V. fluvialis. It wasfurther seen that strains which had MICs of >25 µg/mlfor nalidixic acid had a sense mutation (Ser to Ile) at position83 of the quinolone resistance-determining region of gyrA. Allexcept one of the integron- and SXT integrase-bearing strainsbelonged to the same ribotype.

INTRODUCTION

Top

Introduction

Vibrio fluvialis, a halophilic Vibrio species, has been associatedwith sporadic outbreaks of diarrhea worldwide (10, 11, 13),which is clinically very similar to cholera. It is being isolatedwith an increased frequency from hospitalized patients in Kolkata,India, with cholera-like illnesses (our unpublished observations).The V. fluvialis strains isolated in the past were found tobe resistant to several antibiotics (13). However, there isa paucity of information on the genetic basis of drug resistancein these strains. Antibiotic resistance arises through multiplemeans and can be mediated by plasmids, integrons, and transposons,besides mutations in target genes and the overexpression ofefflux systems (4, 5, 6, 8). Only two reports (1, 2) have addressedthis issue. In those studies, the presence of the drug cassettesaac(3)-Id and aadA7, which confer resistance to gentamicin,streptomycin, and spectinomycin, were identified in V. fluvialis,as was an SXT constin (6). In this study, we performed antibioticsusceptibility tests with 19 strains of V. fluvialis isolatedfrom hospitalized patients in Kolkata, India, recovered from1998 to 2002 and also analyzed their drug resistance profiles.

MATERIALS AND METHODS

Top

Materials and Methods

V. fluvialis strains were isolated from patients with acutecholera-like diarrhea admitted to the Infectious Diseases Hospital,Kolkata, India, between 1998 and 2002.

Bacteriology. V. fluvialis isolates were identified by using the API 20E system(bioMerieux, Marcy l'Etoile, France) and also by 16S rRNA genesequencing.

Antimicrobial susceptibility testing and MIC determination. The isolates were examined for antibiotic resistance as describedpreviously (17). The MICs of ciprofloxacin and nalidixic acidwere determined as described in the CLSI standards (7).

Bacterial genomic DNA and plasmid isolation. Genomic and plasmid DNA was extracted from the isolates by amethod described earlier (17), except that for plasmid isolation,0.75 ml of culture and twice the suggested volumes of all threesolutions were used.

Bacterial transformation and plating on selective media. Escherichia coli JM109 electrocompetent cells, prepared accordingto the manufacturer's recommendations (Bio-Rad Laboratories,Richmond, Calif.), were transformed with 30 ng of plasmid preparationsand selected as described before (17).

Ribotyping. The rRNA gene restriction patterns (ribotypes) of the test strainswere determined exactly as described before (16).

PCR amplifications, sequence analyses, and GenBank accession numbers. PCRs for determination of the presence of class 1 integrons,SXT integrases, and mutations in the quinolone resistance-determiningregion (QRDRs) of the gyrA, gyrB, parC, and parE genes of quinolone-resistantstrains were carried out with the appropriate primers (Table1). Elution of the amplicons was done as described previously.Sequencing was carried out by following the manufacturer's instructions(BigDye Terminator kit; Applied Biosystems) with an ABI Prism310 instrument. The identities of the sequences were establishedthrough a database search by using the BLAST program (3), andmutations were identified by comparison of the sequences withthe V. cholerae genome sequence (9). Sequences with the followingaccession numbers were obtained from GenBank: AY605692, strainBD146 (dfrA15); AY605688, strain BD73 (dfrA1); AY605684, strainBD123 (aadA7); AY605683, strain BD51 [aac(3)-Id and aadA7];and AY103456, strain PL78/6 (dfrA1).

View this table:
[in this window]
[in a new window]
TABLE 1. Primers used in this study

Accumulation studies with quinolones. The accumulation of quinolones was examined by the method ofMortimer and Piddock (15), with minor modifications: phosphate-bufferedsaline (0.136 M NaCl, 0.0026 M KCl, 0.01 M Na₂HPO₄, 0.00176M KH₂PO₄; pH 7.0) was used instead of sodium phosphate buffer,and cell lysis was carried out for 6 h. Carbonyl cyanide m-chlorophenylhydrazone(CCCP) or reserpine was added to a final concentration of 20µg/ml. The fluorescence of the supernatant was measuredwith a spectrofluorimeter (Tegimenta AG type SFM 25).

RESULTS

Top

Results

Drug resistance in V. fluvialis. The V. fluvialis strains were found to be resistant to one ormore antibiotics, with 75% of the strains being resistant toampicillin, furazolidone, nalidixic acid, and streptomycin (Table2).

View this table:
[in this window]
[in a new window]
TABLE 2. Phenotypic and genotypic characterization of V. fluvialis strains

Class 1 integrons and mapping of resistance gene cassettes. Eight of the 19 strains produced a 0.8-kb amplicon with theprimers qacE ${Delta}$ 1-F and Sul1B (Table 1; Fig. 1). A 0.3-kb ampliconwas obtained with the L2 and L3 primer pair (Table 1; Fig. 1).Sequencing of the 0.8-kb and 0.3-kb amplicons followed by BLASTanalysis revealed 100% sequence identity of these ampliconswith the 3' conserved sequence (3' CS) and 5' CS, respectively,of class 1 integrons (8, 12). PCR carried out with primers in-F(5' CS) and in-B (3' CS) (Table 1; Fig. 1) produced ampliconsof four distinct size classes: 0.7, 1.0, 1.25, and 1.5 kb. Sequenceanalysis of the 0.7-kb amplicon revealed the presence of thedfrA15 gene cassette in strain BD146 (Table 2; Fig. 1). The1.25-kb amplicon, generated by strains PL78/6 and BD73, harboredthe dfrA1 gene cassette and the gene cassette of an unknowngene (orfC) (Table 2; Fig. 1). The aadA7 gene cassette was foundon the 1.0-kb PCR product obtained with BD123 (Table 2; Fig.1). The 1.5-kb amplicon from strain BD51 harbored two cassettes,aac(3)-Id and aadA7 (Table 2; Fig. 1). In strains PL78/7b, PL171b,and CRC233, no amplicon could be generated with primer pairin-F and in-B (Table 1; Fig. 1). However, a 1.2-kb ampliconwas obtained with primers L2 and Sul1B. Sequencing of this ampliconrevealed the presence of a "null" cassette (17) in this class1 integron.

View larger version (8K):
[in this window]
[in a new window]
FIG. 1. Diagrammatic representation of the different types of gene cassettes identified in the class 1 integrons in V. fluvialis strains. Solid lines, gene cassettes; solid circles, 59-base elements; hatched box, attI recombination site. The locations of the 5' CSs and the 3' CSs of class 1 integrons and those of the primer pair qacE ${Delta}$ I-F and Sul1B and primer pair in-F (5' CS) and in-B (3' CS) are shown at the bottom.

Occurrence of SXT integrase gene. To look for SXT, primers SXT-F and SXT-B, whose sequences areinternal to that of the SXT integrase gene (SXT int), were used(Table 1) for amplification of the putative SXT int. A 0.8-kbamplicon was obtained with only four strains, namely, strainsPL78/6, PL78/7b, PL171b, and BD51, which, upon sequencing, exhibited100% homology with the relevant portion of the SXT int gene(17), suggesting the presence of SXT constins or parts thereofin these strains.

Resistance to quinolones: analysis for QRDRs in target genes. Fourteen of the 19 strains were resistant to the nonfluorinatedquinolone nalidixic acid and two were additionally resistantto ciprofloxacin. The nalidixic acid-resistant strains couldbroadly be classified into two groups: one with MICs between10 and 20 µg/ml and the other with MICs $≥$ 25 µg/ml(Table 2). Quinolone resistance in bacteria is mainly linkedto the mutations in the QRDRs of the gyrAB and parCE genes.To check for the mutations in QRDRs, primers gyrA-F and gyrA-Rand primers parC-F and parC-R (Table 1), respectively, wereused to amplify these regions. Sequencing of the 0.25-kb ampliconobtained with all strains for the gyrA QRDR showed the presenceof a mutation at position 83, which led to the replacement ofserine with isoleucine in nine strains, which had MICs $≥$ 25 µg/mlfor nalidixic acid. No mutation could be detected in any ofthe amplicons, an amplicon of 0.25 kb for parC and an ampliconof 0.3 kb for both the gyrB and the parE QRDRs, with the appropriateprimers (Table 1).

Involvement of efflux pumps. Although strains BD36, BD56, BD73, CRC111, and CRC159 were resistantto 10 to 15 µg/ml of nalidixic acid, they did not haveany mutations in the QRDRs of target genes. This indicated thatother mechanisms could be responsible for this level of nalidixicacid resistance observed in these strains. To examine if multidrugresistance pumps are involved in conferring quinolone resistanceto V. fluvialis, the level of accumulation of quinolones inthe absence and the presence of the efflux pump inhibitors,namely, CCCP (an inhibitor of the proton motive force [PMF])and reserpine (a plant alkaloid known to inhibit the ATP bindingcassette [ABC transporters]), was measured. It was seen thatirrespective of the MICs, both CCCP and reserpine could preventthe accumulation of nalidixic acid in all nalidixic acid-resistantstrains (Fig. 2). These same two inhibitors could also preventthe accumulation of ciprofloxacin in two strains, strains PL171band BD146, which were additionally resistant to this drug. Theresults (Fig. 3) indicated that efflux pumps indeed had a rolehere also.

View larger version (13K):
[in this window]
[in a new window]
FIG. 2. Accumulation of nalidixic acid (NAL) by V. fluvialis after the addition of CCCP (A) or resperine (B). The fluorescence of the supernatant was measured with a spectrofluorimeter at an excitation wavelength of 330 nm and an emission wavelength of 417 nm for nalidixic acid. The results for three representative strains, namely, strains BD56, CRC233, and PL78/6, are shown here. The arrows indicate the time of addition of CCCP and reserpine. The graphs reflect the differences in fluorescence shown by the bacterial cell in the presence and the absence of the inhibitors. All experiments were carried out at least three times.

View larger version (11K):
[in this window]
[in a new window]
FIG. 3. Studies with ciprofloxacin (CIP) performed as described in the legend to Fig. 2, except that the fluorescence of the supernatant was measured with a spectrofluorimeter at an excitation wavelength of 275 nm and an emission wavelength of 440 nm. The accumulation of ciprofloxacin by V. fluvialis after the addition of CCCP (A) or reserpine (B) is shown. The arrows indicate the time of addition of CCCP and reserpine.

Plasmid carriage and resistance conferred by plasmids. Eleven of the 19 strains had plasmids, of which 10 had plasmidsranging in size from 2 to 8 kb. Strain PL78/6 had a 240-kb plasmid(Table 2). All plasmids in the 2- to 8-kb range, except theones from strain CRC111, could transform E. coli JM109 to ampicillinresistance (data not shown). No transformant could be obtainedwith the 6-kb plasmid from CRC111. The 240-kb plasmid from PL78/6could transfer resistance to streptomycin, sulfamethoxazole,and ampicillin (data not shown).

DISCUSSION

Top

Discussion

The study described in this paper was undertaken to unravelthe molecular basis of drug resistance in V. fluvialis strainsisolated in Kolkata, India. Compared to only 37% of the isolatesrecovered in 1998, 100% of the isolates recovered in 2002 wereresistant to quinolones. Eight strains were found to carry class1 integrons. Although an integron in a V. fluvialis strain wasdetected for the first time in 2002 (1), our work revealed thatintegrons have actually been present in V. fluvialis at leastsince 1998. Although the "linked" gene cassettes aadA7 and aac(3)-Idwere detected in V. fluvialis earlier (1), the aadA7 gene cassettedetected in BD123 (Table 2 and Fig. 1), which occurs singly,has not been found in any Vibrio strain so far. Our studiesfurther showed that although all the strains were drug resistant,only five harbored integrons with drug cassettes which accountedfor resistance to only a few antibiotics (Table 2). Althoughalmost all of the strains were resistant to trimethoprim, onlythree strains, strains PL78/6, BD73, and BD146, had dfrA1 anddfrA15 genes, which confer resistance to this drug. Quite surprisingly,with the exception of four strains, SXT integrases could notbe detected in any other strain (Table 2). We found that 11strains harbored plasmids, but except in 2 strains, they carriedgenes only for ampicillin resistance. It was of interest thatexcept for strain CRC233, all integron- and SXT int-bearingstrains belonged to a single ribotype (data not shown). Quinoloneresistance in these strains could be traced either to mutationsin the QRDRs of gyrA or to the presence of efflux pumps, orboth. Efflux pumps alone were responsible for the nalidixicacid resistance phenotype in the strains with MICs between 10and 15 µg/ml, whereas both mutations in the gyrA QRDRand efflux pumps were responsible for nalidixic acid resistancein strains with MICs $≥$ 25 µg/ml.

ACKNOWLEDGMENTS

Financial support was received from the Department of Biotechnologyand the Council of Scientific and Industrial Research, Governmentof India. Vijaya Srinivasan gratefully acknowledges the Councilof Scientific and Industrial Research, Government of India,for a senior research fellowship. Rupinder Kaur Virk was supportedby a research associateship from the Department of Biotechnology,Government of India.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Microbial Technology, Molecular Biology Division, Sector 39A, Chandigarh 160036, India. Phone: 91-1722-690751. Fax: 91-1722-690585. E-mail: vijaya{at}imtech.res.in.

V. B. Srinivasan and R. K. Virk contributed equally to thiswork.

Present address: School of Biological Sciences, Indian Instituteof Advanced Research (The Puri Foundation), Block 2, 1st Floor,Udyog Bhavan, Sector 11, Gandhinagar, Gujarat, India.

REFERENCES

Top