Virulence and Multidrug Resistance Patterns of Vibrio cholerae O1 Isolates from Diarrheal Outbreaks of South India During 2006

Abstract

A total of 31 Vibrio cholerae O1 (4- Inaba and 27- Ogawa serotype) isolates collected during a three-year period (2006–2009) from acute diarrheal cases in Tamil Nadu, India were analyzed for antibiotic resistance profiling, virulence-associated factors, genetic profiling by enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC PCR), and biofilm-forming ability. Antibiotic resistance profile revealed that most of the strains have become multidrug-resistant strains. All the isolates are resistant to ampicillin and polymyxin B, 97% of the isolates are resistant to nalidixic acid, 90% to co-trimoxazole, 32.3% to norfloxacin and ciprofloxacin, 29% to doxycycline, 10% to gentamicin, whereas only 3% to chloramphenicol. Molecular characterization of virulence-associated genes by multiplex PCR revealed the presence of ace, ctxA, tcpA, toxR, and ompU as 93.5%, followed by ompW with 33.3%. The presence of zot was restricted to only one isolate and hlyA was not encountered in any of the strains. ERIC PCR produced more than 10 bands for each isolate and the dendrogram generated based on the cluster analysis showed the presence of 29 electrophoretic types among the 31 isolates. Isolates from different area or year of isolation are intermingled in all the clusters. With respect to biofilm formation, 24 isolates were found to be biofilm formers and eight of them produced strong biofilm. This study demonstrates the presence of critical virulence factors and antibiotic resistance in the diarrhea isolates, which signifies the importance of routine monitoring and proper treatment to prevent cholera outbreaks.

Introduction

The Gram-negative bacterium Vibrio cholerae has been identified as the main causative agent of the acute dehydrating diarrheal condition called cholera, which is still a major public health problem in many developing countries. India remains endemic for cholera²⁵ particularly, V. cholerae strains belonging to serogroups O1 and O139 are responsible for the vast majority of epidemic cholera. V. cholerae O1 is further divided into two biotypes, Classical and El Tor and two major serotypes, Inaba and Ogawa.

Cholera has been categorized as one of the emerging and remerging infections²³ threatening many developing countries. Several past events that mark epidemiological importance of the disease include the re-emergence of cholera in Latin America in 1991;^10,17 the explosive outbreak of cholera among Rwandan refugees in Goma, Zaire, which resulted in about 70,000 cases and 12,000 deaths in 1994;²⁴ and the emergence of V. cholerae O139 in the Indian subcontinent during 1992 to 1993, possibly marking the beginning of the eighth pandemic of cholera.^15,26

Clusters of acute diarrhea are common, but investigations are difficult to conduct and many clusters of diarrhea are not investigated using laboratory methods that could lead to the identification of the causative agent. Thus, cholera may be under-recognized, and many outbreaks were simply recorded as “diarrhea outbreaks”.³³ During 2004, in many parts of India, the re-emergence and progression of V. cholerae O1 serotype Inaba causing outbreaks and sporadic infections was documented by Taneja et al.²⁷

Cholera pathogenesis relies on the synergistic effect of a number of pathogenic factors produced by toxigenic V. cholerae. V. cholerae adopts several virulence factors, such as cholera toxin subunit A (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), outer membrane protein (omp), haemolysin (hylA), and toxin-coregulated pilus for intestinal colonization (tcpA), to establish itself in the host system. In addition, V. cholerae has the ability to forms biofilms, thereby protecting themselves from antibiotics, toxins, and other hostile environments.

Emergence of multiple drug resistance is a serious clinical problem in the treatment and containment of the disease. The occurrence of multiple antibiotic resistance in V. cholerae is being reported with increasing frequency.^5,15,20 In south India, Kerala is considered as endemic to the disease cholera and outbreaks involving multiple drug-resistant strains have been reported.^21,22 Arriving at the virulence and drug-resistant pattern is of paramount importance for the timely containment of cholera outbreak. Phenotypic methods, such as O typing with monoclonal antibodies, comprise frequently used rapid methods and will enable us to identify the serovar responsible for the outbreak in a particular region or locality, but do not cover the entire spectrum of existing or emerging pathogenic clones of V. cholerae. Whereas, multiplex polymerase chain reaction (PCR) can be employed to arrive at the virulence pattern. Enterobacterial repetitive intergenic consensus (ERIC) PCR (with a 22-mer primer) can be used for the comparison of genetic relationship among the V. cholerae isolates.

In the present study, we have characterized V. cholerae O1 strains isolated from acute diarrheal cases of south India during 2006–2009. PCR analysis of the toxin genes and ERIC PCR were used for genotypic characterization of the isolates. The antibiotic susceptibility patterns of the isolates were also examined, because of increasing reports of antibiotic resistance in strains of V. cholerae O1.

Materials and Methods

Study Site and Bacterial Strains

V. cholerae strains (n=31) were collected from adult patients with severe/acute diarrhea in Tamil Nadu, India during 2006 to 2009. The strains were isolated from stool samples and rectal swabs of the patients after obtaining the informed consents. Preliminary laboratory, biochemical, and serological tests conducted on the strains confirmed them to be V. cholerae. Two V. cholerae reference strains, O1 and O139, obtained from the National Institute of Cholera and Enteric Diseases, Kolkata, were used as control strains. The stool samples were enriched in alkaline peptone water at 37°C for 6 hr (180 rpm) and 20 μl of the enriched samples were plated onto TCBS agar (HiMedia) plates and were incubated at 37°C for 18–24 hr. For serological typing, bacterial isolates were inoculated in a Brain Heart Infusion broth and incubated for 18–24 hr at 37°C. A serological slide agglutination test was done by mixing each V. cholerae O1 (serovar Ogawa and Inaba) and O139 antiserum with each bacterial culture suspension followed by slight rotation of the slide to mix the antiserum with the cells. Observation for agglutination was done within 1 to 2 min.

Antimicrobial Susceptibility Testing

Resistance profiles were determined by the NCCLS agar disk diffusion method.¹¹ V. cholerae strains were tested for antibiotic resistance by using antibiotic impregnated discs (Hi-Media) of ampicillin (10 μg), doxycycline (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), co-trimoxazole (25 μg), gentamicin (10 μg), norfloxacin (10 μg), nalidixic acid (30 μg), and polymyxin B (50 U). Escherichia coli ATCC10536 was used as the quality control strain.

Biofilm Assay

All the V. cholerae strains were screened for biofilm formation using the 24-well polystyrene plate assay method.²⁸ For quantitative analysis, 1:100 dilution of culture was made in the Luria-Bertani broth and allowed for forming biofilm by incubating at 37°C for 24 hr. After incubation, crystal violet staining was performed. The wells were destained with absolute alcohol and quantified for the biofilm formation spectrophotometrically at absorbance 570 nm. V. cholerae reference strains, O1 and O139, were used as positive controls and Streptococcus pyogenes (st2147),²⁸ a nonbiofilm former, was used as a negative control for biofilm assay. Staining of biofilms for light microscopy and Confocal Laser Scanning Microscopy (CLSM) were performed as described previously by Thenmozhi et al.²⁸ For CLSM, the biofilms formed on cover slips were stained with 0.02% acridine orange (HiMedia Laboratories) for 2 min and excess stains were gently removed with distilled water. The stained slides were subjected to visualization under CLSM (Zeiss LSM710 meta). Images were captured and processed by using Zeiss LSM Image Examiner Version 4.2.0.121.

Virulence-Associated Genes Profiling by PCR

In an attempt to characterize the virulence-associated genes of the 31 V. cholerae isolates, genomic DNA was extracted using the CTAB protocol as described by Ausubel et al.,¹ and the following genes were amplified by multiplex PCR analysis: ompU, zot, toxR, ompW, ctxA, ace, hylA, and tcpA.^6,16,19 Each multiplex PCR comprised of a 50 μl reaction mixture containing 2 μl (10 ng) of DNA as the template, each primer at a concentration of 0.5 μM, 1.5 mM MgCl₂, and each deoxynucleoside triphosphate at a concentration of 50 μM, as well as 2 U of Taq polymerase and buffer as recommended by the manufacturer (MBI Fermentas). After the initial denaturation for 5 min at 95°C, there were 30 cycles consisting of denaturation at 95°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 2 min, and then a final extension step consisting of 10 min at 72°C; GeneAmp PCR System (Applied Biosystems) was used. The multiplex PCR amplification was confirmed by running the amplicons in 2% agarose gel in 1×TAE. Visualization of the gel and analysis of the bands were done under the gel documentation system (Bio-Rad Laboratories, USA). The PCR products of the reference strains O1 and O139, and 50-bp ladders were included in the gel. Staining of the gel was done by using ethidium bromide for 10 min followed by destaining in distilled water for 10 min.

Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction

The variability of V. cholerae isolates were studied using ERIC PCR amplification as reported earlier.¹⁸ The PCR profile used was 1 cycle of 95°C for 7 min, 30 cycles of 94°C for 1 min, 52°C for 1 min, and 65°C for 2 min followed by a final extension of 65°C for 15 min. Reaction mixture (50 μl) contained 10 ng of template DNA, each primer at a concentration of 0.5 μM, 1.5 mM MgCl₂, each deoxynucleoside triphosphate at a concentration of 50 μM, as well as 1 U of Taq polymerase, buffer as recommended by the manufacturer (MBI Fermentas). The differential distribution of haplotypes between the isolates was assessed. Construction of dendrogram was done by using the pairwise distance matrix using the neighbor-joining procedure in the GelQuest version 3.1.1 and ClusterVis version 1.8.1 (Sequentix).

Results

Serological Test

In the slide agglutination test, all isolates were positive with the V. cholerae O1 antiserum and not with O139 antiserum. Out of the 31 strains, only 4 were positive with the V. cholerae serovar Inaba antiserum, while the remaining 27 were positive with the serovar Ogawa antiserum.

Antibiogram

Antibiogram assay showed that most of the V. cholerae strains isolated from diarrhea were multidrug resistant. All the isolates were resistant to ampicillin and polymyxin B. Most of the isolates (97%) were resistant to nalidixic acid, 90% to co-trimoxazole, 32.3% to norfloxacin and ciprofloxacin, 29% to doxycycline, 10% to gentamicin, whereas only 3% to chloramphenicol. The antibiotic resistance patterns are shown in Table 1.

Table 1.

Antibiotic Resistance Patterns and Virulence-Associated Gene Profiles of Vibrio cholerae Isolates

			Virulence factors
S. No.	Strain	Antibiotic resistance profile	Biofilm formation	ctxA	ace	ompU	ompW	toxR	tcpA
1	VC1	A, P, NA, Co, Nor	++	−	+	+	−	+	−
2	VC2	A, P, Na, Co, Nor, G	−	+	+	+	+	+	+
3	VC3	A, P, Na, C, D, Co	−	+	−	+	+	+	+
4	VC4	A, P, Na, Co	+	+	+	+	+	+	+
5	VC5	A, P, Na, Co	++	+	+	+	+	+	+
6	VC6	A, P, Na, Co	++	+	+	+	+	+	+
7	VC7	A, P, Na, D, C, Co, Nor	+	+	+	+	+	+	+
8	VC8	A, P, Co, G	−	+	+	+	+	+	+
9	VC9	A, P, Na, C, Co	+	+	+	+	+	+	+
10	VC10	A, P, Na, Co	−	+	+	−	−	−	+
11	VC11	A, P, Na, Co	−	+	+	+	−	+	+
12	VC12	A, P, Na, Co, N	+	+	+	+	−	+	+
13	VC13	A, P, Na, C, Co	+	+	+	+	−	+	+
14	VC14	A, P, Na, Co	++	+	+	+	−	+	+
15	VC15	A, P, D, Na, Cl, C, Co	+	+	+	+	−	+	+
16	VC16	A, P, Na, Co, Nor	−	+	+	+	+	+	+
17	VC17	A, P, Na, Co	++	+	+	+	−	+	+
18	VC18	A, P, Na, Co	+	+	+	+	+	+	+
19	VC19	A, P, Na, C, Co, Nor	+	+	−	−	−	+	+
20	VC20	A, P, Na, Co, G	++	+	+	+	−	+	+
21	VC21	A, P, Na, Co, Nor	+	+	+	+	−	+	+
22	VC22	A, P, Na, C, Co	++	+	+	+	−	+	+
23	VC23	A, P, Na, Co	+	+	+	+	−	+	+
24	VC24	A, P, Na, Co, Nor	+	+	+	+	−	+	+
25	VC25	A, P, D, Na, C, Co	−	+	+	+	−	+	+
26	VC26	A, P, Na, Co	++	+	+	+	−	+	+
27	VC27	A, P, Na, Co, Nor	+	+	+	+	−	+	+
28	VC28	A, P, D, Na, C, Co	+	+	+	+	−	+	+
29	VC29	A, P, Na, C, Co	+	+	+	+	−	+	+
30	VC30	A, P, D, Na, Co, Nor	+	+	+	+	−	+	+
31	VC31	A, P, Na, Co	+	−	+	+	−	−	−

The isolate VC2 alone showed the presence of zot and hlyA was not encountered in any of the isolates.

A, Ampicillin; P, Polymyxin B; NA, Nalidixic acid; Co, co-trimoxazole; D, Doxycycline; Nor, Norfloxacin; Cl, Chloramphenicol; C, Ciprofloxacin; G, Gentamicin.

(++) Strong biofilm former; (+) Positive & weak biofilm former and (−) Negative.

Molecular Characterization of Isolates

Distribution of the virulence-associated genes in the V. cholerae diarrhea isolates are summarized in Table 1. The prevalence of the virulence genes ace, ctxA, tcpA, toxR, and ompU were 93.5% followed by ompW with 33.3%. The presence of zot was restricted to one isolate and hlyA was not detected in any of the isolates. Nine of the 31 isolates (29%) had the combination of virulence genes ace, ctxA, tcpA, toxR, ompW, and ompU, and 55% (n=17) of the isolates had the combination of ace, ctxA, tcpA, toxR, and ompU.

ERIC PCR Patterns

ERIC PCR produced more than 10 bands for each isolate (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/mdr). The dendrogram generated based on the cluster analysis reflects the clonal nature of the isolates (Fig. 1). Clonal clusters of haplotypes were found from 29 electrophoretic types. Isolates from different area or year of isolation are intermingled in all the clusters. Dendrogram showing the grouping of different isolates in a single clade indicates the genetic similarity between the V. cholerae isolates.

FIG. 1.

Dendrogram (generated based on enterobacterial repetitive intergenic consensus polymerase chain reaction pattern) illustrating the genetic similarity among 31 Vibrio cholerae O1 isolates from diarrhea patients. Bar represents the distance scale.

Screening of V. cholerae Biofilm Formation

The biofilm formation of the isolates were analyzed spectrophotometrically at 570 nm (Fig. 2) and qualitatively by a microscopic examination. Among the 31 V. cholerae isolates screened, 24 were turned out to be biofilm formers. Eight of the biofilm formers (VC1, VC5, VC6, VC14, VC17, VC20, VC22, and VC26) formed strong biofilms.

FIG. 2.

Biofilm forming ability of the 31 V. cholerae O1 isolates from diarrhea patients. V.cholerae O1- & O139-Positive controls and Streptococcus pyogenes st2147-Negative control.

Discussion

The aims of this study were to document the emergence of multiple drug resistance in O1 Ogawa/Inaba stains, genetic and virulence profiles of V. cholerae isolates from diarrhea patients in three regions of south India. The serological test showed that all the 31 diarrheal isolates belonged to either O1 Inaba (12.9%) or Ogawa (87%). In congruence to this, majority of the frequent diarrheal outbreaks in India were mainly caused by V. cholerae O1 Ogawa and O139 serogroup.^3,12,27 It is also interesting to note that there was no representation for the serotype O139, which is in total contrast to the results obtained from Orissa (66% Inaba vs 33.9% Ogawa)¹³ and Iran and Senegal (only Ogawa was reported with 100 and 92.1%, respectively).¹⁴ However, reports showed that diarrhea outbreaks were caused by both V. cholerae O1 serotypes, Inaba and Ogawa, showing dominance over O139.^2,20,27,32

The use of antimicrobial agents is generally an effective method against diarrhea outbreaks. Due to the extensive and improper usage of antimicrobials, multidrug resistance in diarrheal pathogens has become a common phenomenon in developing countries and has reduced the effectiveness of the drugs in disease containment. Most of the diarrhea isolates from the present study showed multidrug resistance. Particularly, all the isolates showed resistance to ampicillin and polymyxin B and more than 90% of the isolates were resistant to nalidixic acid and co-trimoxazole. These results are in agreement with the reporting of the multidrug-resistant V. cholerae O1 serotypes, Ogawa and Inaba, from two diarrheal outbreaks in West Bengal²⁵ and V. cholerae non-O1/non-O139 from waters in south India.⁶

V. cholerae strains with the ability to cause cholera possess a set of virulence genes required for pathogenesis in humans. The pathogenesis of cholera is a complex process, involving a number of factors that help the pathogen to colonize the epithelium of the small intestine and produce enterotoxins that disrupt ion transport. Although production of cholera toxin by V. cholerae, encoded by the ctxAB genes, is directly responsible for the manifestation of diarrhea, cholera pathogenesis relies on the synergistic action of a number of other genes and part of the cholera toxin genetic elements comprising zot, which increases the permeability of the small intestinal mucosa by affecting the structure of the tight junction,⁴ and ace, which is capable of causing fluid accumulation in ileal loops.²⁹ In addition, the tcp gene cluster is also required for pathogenesis of cholera. In the present study, the virulence genes ace, ctxA, tcpA, toxR, and ompU were found in 29 isolates out of 31 strains, which emphasises that these diarrheal isolates are toxic enough to cause cholera. Throughout the world, the prevalence of toxin genes was more than 90% and the results of our study is in total agreement with previous reports.^8,14 Similar to our results, V. cholerae strains not carrying and carrying the ctx, zot, ace, and tcpA genes either alone or in combination have been reported.^7,9 A recent study by Valeru et al.,³⁰ experimentally proved that toxR plays some regulatory role in biofilm formation and ompU expression. However, in the study, it was found that the distribution of toxR was even among strong biofilm formers (23 out of 24 isolates) and weak biofilm formers (6 out of 7). This shows that apart from toxR, some other factors appear to negatively regulate biofilm or involve in dispersal of biofilm. The V. cholerae isolates characterized in the present study possessed all the virulence-associated genes, which are required for pathogenesis. The presence of the ompW gene was very crucial as the sequence is highly conserved among the V. cholerae belonging to different serogroups and is targeted for the species-specific identification of V. cholera.⁸ The presence of nearly all virulence-associated genes (with the exception of zot and hlyA) in 93.5% of the diarrheal isolates clearly implies the pathogenic potential.

Biofilm formation is recognized as an important virulence factor for a disease-causing bacteria and provides a protection against antibiotics.²⁸ Biofilm formation in V. cholerae is dependent on exopolysaccharide production.³¹ Biofilm formation by diarrhea isolates implies that they would be resistant to antibiotic treatment upon biofilm formation. The relatedness of the toxigenic V. cholerae isolates have been studied extensively by ERIC PCR. Both O1 Inaba and Owaga isolates are intermingled, which once again affirms that V. cholerae strains are genetically related and can interconvert between the Ogawa and Inaba serotypes.²⁰ In addition, a study by Zo et al.,³² showed that even O1 and O139 strains formed tight clusters in ERIC PCR analysis. Isolates from a different area or year of isolation are intermingled in all the clusters.

In conclusion, the present study demonstrates the presence of critical virulence factors and antibiotic resistance in the diarrheal isolates, which signifies the importance of routine monitoring and proper treatment to prevent cholera outbreaks. In addition, the ability to identify and differentiate clones makes it possible to detect the source and track the spread of pathogenic strains. The findings indicate that simple diarrhea cases could be a potential reservoir for multidrug-resistant V. cholerae strains and a routine surveillance by health authorities is required for early control of the outbreak.

Footnotes

Acknowledgments

This work was supported, in part, by the Grants from Indian Council of Medical Research (ICMR), University Grants Commission (UGC), and Department of Biotechnology (DBT), India to Prof. S. Karutha Pandian. Financial support provided to Mr. K. Balaji by Council of Scientific and Industrial Research (CSIR) in the form of Senior Research Fellowship (SRF) is thankfully acknowledged. Authors also acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by DBT, GOI; Grant No. BT/BI/25/001/2006). Authors express their sincere thanks to Dr. S. Dhanapal, Department of Microbiology, K. A. P. Viswanatham, Government Medical College in Tiruchirappalli for providing the clinical isolates.

Author Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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Virulence and Multidrug Resistance Patterns of Vibrio cholerae O1 Isolates from Diarrheal Outbreaks of South India During 2006–2009

Abstract

Introduction

Materials and Methods

Study Site and Bacterial Strains

Antimicrobial Susceptibility Testing

Biofilm Assay

Virulence-Associated Genes Profiling by PCR

Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction

Results

Serological Test

Antibiogram

Molecular Characterization of Isolates

ERIC PCR Patterns

Screening of V. cholerae Biofilm Formation

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Supplementary Material