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Previous Article | Next Article 
Applied and Environmental Microbiology, June 2001, p. 2421-2429, Vol. 67, No. 6
Center of Marine Biotechnology, University of Maryland
Biotechnology Institute, Baltimore, Maryland
212021; Department of Cell and
Molecular Biology, University of Maryland, College Park, Maryland
207425; Departamento de
Microbiologia, Imunologia e Parasitologia, Escola Paulista de
Medicina,2 and Departamento de
Microbiologia, Instituto de Ciências Biomédicas,
Universidade de São Paulo, CEP 05508-900,3
São Paulo, Brazil; Microbiology Department, Seoul
National University, Seoul, Korea 151-7424; and
School of Hygiene and Public Health, Johns Hopkins
University, Baltimore, Maryland 212056
Received 30 October 2000/Accepted 5 March 2001
Vibrio cholerae is an autochthonous inhabitant of
riverine and estuarine environments and also is a facultative pathogen
for humans. Genotyping can be useful in assessing the risk of
contracting cholera, intestinal, or extraintestinal infections via
drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA,
hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes,
using multiplex PCR. Based on tcpA and hlyA
gene comparisons, the strains could be grouped into Classical and El
Tor biotypes. The toxR, hlyA, and ompU genes
were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic
V. cholerae O1 and V. cholerae O139 strains
examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly,
among the isolates of V. cholerae non-O1/non-O139, two had
tcpA Classical, nine contained tcpA El Tor,
three showed homology with both biotype genes, and four carried the
ctxA gene. The stn/sto genes were present in
28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic
V. cholerae O1, and in 14.3% of the O139 serogroups.
Except for stn/sto genes, all of the other genes studied
occurred with high frequency in toxigenic V. cholerae O1
and O139 strains. Based on results of this study, surveillance of
non-O1/non-O139 V. cholerae in the aquatic environment,
combined with genotype monitoring using ctxA, stn/sto, and
tcpA ET genes, could be valuable in human health risk assessment.
Toxigenic Vibrio cholerae
O1 and V. cholerae O139 are etiological agents of epidemic
cholera. However, both V. cholerae O1 strains that do not
produce cholera toxin, i.e., that are nontoxigenic (NT), and
non-O1/non-O139 strains have also been associated with cholera,
gastroenteritis, septicemia, and/or extraintestinal infections (49, 51, 52, 63, 66, 71, 81). Outbreaks of cholera were
reported in Brazil during the third (1853 to 1854), fourth (1866 to
1868), and fifth (1893 to 1895) pandemics (3). In the
early 1970s, when cholera spread to Africa and Southern Europe, it was
forecast to arrive in countries across the Atlantic as well. This
prompted the establishment of a surveillance program in S. Paulo State,
Brazil, by the WHO (World Health Organization) and CETESB (Companhia de
Tecnologia de Saneamento Ambiental, S.P.-Brazil The seventh pandemic reached the Americas on 29 January 1991 in Lima,
Peru, and spread rapidly to the Peruvian Northern Andean and Amazon
regions (74). Brazil reported its first case of cholera on
8 April 1991 in Tocantins. Cholera cases then occurred in the Amapa,
Amazonas, Maranh The pathogenicity of V. cholerae O1 and O139 strains depends
on a combination of properties, including enterotoxin (cholera toxin
[CT], ctxA) and the ability to adhere to, and colonize, the small intestine (colonization factor, tcpA)
(27). The major virulence-associated factors are present
in clusters (23), with at least three regions in the
V. cholerae chromosome. The first is the CTX genetic element
(45), which has now been reported to comprise the genome
of a filamentous bacteriophage (CTX V. cholerae can be found in the environment both as a
free-living bacterium and in association with zooplankton
(30). Therefore, not surprisingly, non-O1/non-O139
V. cholerae is frequently isolated from the aquatic
environment and seafood (5, 8, 9, 29, 30, 31, 32, 41, 43, 44,
78). In fact, V. cholerae is a heterogeneous species,
with 206 serotypes identified to date (G. B. Nair, personal communication).
The emergence of the new serogroup O139 as a second etiologic agent of
cholera epidemics (48), along with the discovery of
horizontal and vertical genetic transfer by phages (80)
and the elucidation of pathogenicity islands and other mobile genetic elements (36), has revived interest in the non-O1/non-O139
V. cholerae strains that had been previously implicated in
cholera-like epidemics (2, 13, 51, 71, 81). In addition,
the possible conversion of non-O1 to O1 serotype has provided added
interest (10).
Cholera surveillance is now under way in many countries, based
primarily on detection of V. cholerae O1 and O139 and
determining the presence of cholera toxin using biological and
molecular methods. However, virulence-associated factors in V. cholerae isolates from environmental sources are of concern.
The primary objective of this study was to evaluate the presence of
virulence-associated factors in V. cholerae
populations as potential pathogenic markers suitable for
environmental monitoring. Virulence-associated factors studied
here included cholera toxin (ctxA), hemolysin
(hlyA), non-O1 heat-stable enterotoxin (stn/sto), outer membrane protein (ompU), TCP (tcpA and
tcpI), ToxR regulatory protein (toxR), and zonula
occludens toxin (zot).
Bacterial strains.
A total of 69 V. cholerae
isolates were included in this study. V. cholerae O1 strains
comprised 19 toxigenic clinical and environmental isolates from Brazil
(14 isolates), Peru (3 isolates), Chile (1 isolate), and Mexico (1 isolate) and four nontoxigenic V. cholerae O1 isolates from
Brazil (1978 to 1980). Thirty-nine environmental isolates of
non-O1/non-O139 V. cholerae from Brazil, seven V. cholerae O139 clinical isolates from India, and five V. mimicus environmental isolates from Louisiana (United States) were
included in the study (Tables 1 and
2). The Brazilian environmental strains
were isolated in the CETESB Laboratory, S. Paulo, Brazil. All of the
isolates are part of a culture collection (RRC) at the Center of Marine
Biotechnology (Baltimore, Md.). Frozen stock cultures were subcultured
on Luria-Bertani (LB) broth (Difco Laboratories, Detroit, Mich.),
streaked onto LB agar, and then onto TCBS agar (Oxoid) to verify
purity.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2421-2429.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genotypes Associated with Virulence in
Environmental Isolates of Vibrio cholerae
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
State Agency for
Environmental Control). Sewage samples were monitored for V. cholerae in the community, and 12,867 samples were collected. From
these samples, four NT V. cholerae O1 strains were isolated, the first V. cholerae O1 NT strains to be isolated in
Brazil. The isolates were from sewage samples collected in 1978, 1980, and 1983 (42). Non-O1 V. cholerae strains were
subsequently isolated from sewage (77.3%), seawater (40.4%), and
freshwater (33.3%) samples collected in 1982 and 1983, at a time when
no cases of cholera or gastroenteritis had been reported in S. Paulo State (41). At the same time, in Rio de Janeiro State,
non-O1 V. cholerae was isolated from 12% of seawater and
oyster samples (64).
) (80). The second
region is a large pathogenicity island for V. cholerae (VPI)
(35) that encodes a toxin-coregulated pilus (TCP) gene cluster, a type IV pilus that functions as an essential colonization factor (75) and acts as CTX receptor (35).
The third gene cluster, the RTX toxin gene cluster, was described by
Lin et al. (40) in a V. cholerae O1 El Tor
strain and encodes cytotoxic activity for Hep-2 cells in vitro.
However, the implication of RTX in pathogenesis has yet to be confirmed
(16). Other factors have been associated with
enteropathogenicity and include an El Tor-like hemolysin
(hlyA) (82), heat-stable enterotoxin
(stn/sto) (1, 22, 55), hemagglutinins
(14), neuraminidase (nanH) (20), a
new CT (33), outer membrane protein (ompU)
(72), Shiga-like toxin (stx) (33),
a ToxR regulatory protein (46), and a zonula occludens
toxin (zot) (18).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Genotypic traits of toxigenic and NT V. cholerae O1, V. cholerae O139, and V. mimicus isolates examined in this study
TABLE 2.
Genotypic traits of non-O1/non-O139 V. cholerae Brazilian isolates examined in this study
Positive and negative controls. V. cholerae O1 Classical ATCC 11623, V. cholerae O1 El Tor ATCC 14033 (ctxAB negative), V. cholerae O1 Classical ATCC 14035, V. cholerae non-O1 ATCC 14547, V. cholerae non-O1 ATCC 25872 (ctxAB+), V. cholerae non-O1 ATCC 25874 (ctxAB+), V. mimicus ATCC 33653, V. cholerae O22, and V. cholerae O31 were used as positive controls, and Escherichia coli was used as a negative control.
Chromosomal DNA preparation. DNA was extracted by the CTAB (cetyltrimethylammonium bromide) method previously described (62). DNA extracts were resuspended in Tris-EDTA (10 mM Tris-HCl, 0.10 mM EDTA [pH 8.0]) buffer and stored at 4°C for further analysis. Dilutions of template DNA were made with sterile distilled water to obtain a concentration of ca. 100 ng/µl.
PCR primers and amplification conditions.
The
oligonucleotide primers for each of the selected virulence-associated
factors were designed based on available GenBank sequences for V. cholerae O1 Classical and V. cholerae O1 E1 Tor for all
genes, except the stn/sto genes, for which V. cholerae non-O1 and V. cholerae O1 sequences were used.
The sequence positions and accession numbers of the sequences or
sources are listed in Table 3.
Oligonucleotide primers were synthesized by Genosys Biotechnologies,
Inc.
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Multiplex PCR. The following reagents were added to each sample PCR mixture: 2.5 µl of 10× amplification buffer A (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2, 0.01% [wt/vol] gelatin) (Promega, Madison, Wis.); 0.5 µl each of 2.5 mM dATP, dCTP, dGTP, and dTTP (Promega); 1.0 µl each of forward and reverse primers (20 µM); 0.125 µl of Taq DNA polymerase at 5 U/µl (Promega); and Milli-Q water (to a final volume of 24 µl). PCR was carried out in 0.2-ml microcentrifuge tubes with 24 µl of the PCR mixture and 1 µl (ca. 0.10 µg) of template DNA. The solution was mixed, centrifuged briefly, and placed in an automated Peltier thermal cycler (PTC-200; M. J. Research).
PCR amplification conditions were as follows: denaturation at 94°C for 2 min, annealing for 1 min at the temperatures shown in Table 3, and extension at 72°C, as given in Table 3; with a final extension step at 72°C for 10 min at the end of 30 cycles, followed by maintenance at 4°C. PCR products were separated by agarose gel electrophoresis (1.4%) in 1× TAE buffer (0.04 Tris-acetate, 0.001 M EDTA [pH 8.0]), stained with ethidium bromide, and visualized by using UV light.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Multiplex PCR was carried out, using positive and negative
controls and the primers designed for the genes studied. The size of
each amplicon was verified. Optimal PCR conditions were determined for
ctxA/ompU-PCR, hlyA (Classical and El Tor),
zot/toxR-PCR, and tcpA (Classical and El Tor),
using multiplex PCR. For tcpI and stn/sto genes,
simple PCR was used. The multiplex and simple PCR products obtained for
each gene investigated are shown in Fig.
1, and the corresponding amplicon sizes
are given in Table 3.
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The virulence-associated factors for specific V. cholerae serogroups are summarized in Tables 1 and 2.
All of the V. cholerae O1, O139, and non-O1/non-O139 strains, regardless of whether they were toxigenic or NT, were found to possess the toxR regulatory sequence, a gene absent in the five V. mimicus strains tested.
CT and zonula occludens toxin (ZOT) were present in all (100%) of the toxigenic V. cholerae O1 and O139 strains tested and were absent in NT V. cholerae O1. In V. cholerae non-O1/non-O139 strains, we found four strains (RC60, RC61, RC233, and RC253) to be ctxA positive and zot negative. These isolates were positive for ctxA, using multiplex PCR assay, with a 564-bp amplicon identical to that of the O1 strains. The size of ctxA amplicons was confirmed by sequencing (data not shown).
The oligonucleotide primers targeting tcpA exploited sequence differences between the tcpA of the El Tor (ET) and Classical V. cholerae biotypes. All toxigenic V. cholerae O1 and O139 and three of four nontoxigenic V. cholerae O1 isolates examined showed amplicons of the same size as that obtained for the tcpA El Tor gene. Of 39 non-O1/non-O139 V. cholerae strains, 14 yielded amplicons, using tcpA primers. The amplicon size (451 bp) of nine strains (RC66, RC71, RC233, RC239, RC240, RC246, RC253, GM32, and TM50022) was identical to that obtained for V. cholerae El Tor ATCC 14033, while the amplicon size (620 bp) of two strains (RC61 and RC62) was identical to that obtained for V. cholerae O1 Classical ATCC 14035. Interestingly, three strains carried both genes (RC60, RC69, and TMA52) (Table 2).
The tcpI gene was frequently found in toxigenic V. cholerae O1 (84.2%) and O139 (71.4%) serogroups. However, this gene was also present in 50% of the NT V. cholerae O1 and in 56.4% of the non-O1/non-O139 serogroups.
The hlyA El Tor gene was found in all toxigenic and NT V. cholerae O1 and in V. cholerae O139. Among non-O1/non-O139 serogroup strains, 94.9% showed homology to El Tor hemolysin, 2.6% were associated with Classical hemolysin, and 2.5% were negative for both genes. The amplified fragment sizes were 727 bp, specifically for the Classical biotype (ATCC 11623 and ATCC 14035) and both 481 bp and 738 bp for the El Tor biotype. Occasionally, a larger fragment (~1.4 kb) was observed in the El Tor biotype strains.
The ompU gene was found in all strains of toxigenic and NT V. cholerae O1, V. cholerae O139, and in 76.9% of the environmental non-O1/non-O139 V. cholerae isolates.
Genes homologous to stn/sto were observed in toxigenic V. cholerae O1 (2 of 19), V. cholerae O139 (1 of 7), and non-O1/non-O139 V. cholerae (11 of 39) strains, yielding an amplicon size of 172 bp. However, we observed an amplicon of 800 bp in one isolate from sediment (RC68).
The genotypes found in each V. cholerae serogroup are listed
in Table 4. The genotype most frequently
observed in clinical toxigenic V. cholerae O1 (68.4%) and
O139 (71.4%) isolates was ctxA hlyAET ompU
tcpAET tcpI toxR zot. Other genotypes found in toxigenic V. cholerae O1 strains were similar, but the
tcpI and/or stn/sto genes were absent. In
non-O1/non-O139 V. cholerae serogroups, the genotypes were
diverse; however, the most frequent genotypes were hlyAET
ompU tcpI toxR (7 of 39) and hlyA toxR (6 of 39).
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The presence of virulence-associated genes in non-O1/non-O139 V. cholerae isolates was analyzed by origin, i.e., water and sediment
for marine ecosystems, mussels, and wastewater, and the results are
summarized in Table 5.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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The majority of V. cholerae strains in the environment are considered to be harmless estuarine microorganisms. However, specific strains appear to have evolved that cause disease in humans by effectively colonizing the small intestine and releasing a potent enterotoxin. Eight chromosomal regions coding for virulence-associated factors in V. cholerae (ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot) were included in this study.
Distribution of virulence-associated genes in V. cholerae.
The major CTX genetic element has the structure of
a compound transposon, with a 4.5-kb central core region (ctxAB,
zot, ace, orfU, and cep) that encodes for both CT and
for functions required for virion morphogenesis and is flanked by one
or more copies of a 2.7-kb repetitive sequence that encodes functions
required for regulation, replication, and integration of CTX
(36, 80). In this region, we tested for the presence of
two genes: CT subunit A (ctxA) and ZOT (zot).
Interestingly, we found four of the non-O1/non-O139 V. cholerae isolates contained ctxA gene but not the
zot gene. The ZOT was described by Fasano et al.
(18) as a toxin that increases permeability of the small
intestinal mucosa by affecting the structure of the intercellular tight
junction, or zonula occludens. However, Waldor and Mekalanos
(80) found that zot and orfU
correspond to genes involved in CTX morphogenesis and that the
biological activity previously designated "zonula occludens toxin"
is probably not directly associated with the zot gene
product, unless its product possesses a dual function. Additionally,
the presence of the zot gene and the absence of the
ctx gene in V. cholerae and V. mimicus
strains has been reported (7, 21, 38). To explore the
CTX genome integrated in the V. cholerae chromosome, sequencing of the CTX genetic element is under way.
, produced by vibrios containing VPI
(37, 77).
In this study, the presence of strains containing tcpA
genes, either identical to those of biotype El Tor or of Classical, is
in agreement with results obtained in Australia, where V. cholerae serogroup O6 was shown to contain the tcpAET
gene and another strain of serogroup O23 presented as tcpA
Classical, both strains having been isolated from water
(67).
TcpI, an integral inner membrane protein, involved in environmental
sensing and signal transduction (chemosensors), negatively regulates
the synthesis of the major pilin subunit of TCP (TcpA) (24,
76). Harkey et al. (24) suggested that regulators
such as TcpI, that act downstream of ToxR and ToxT may function to fine-tune the expression of the TCP virulence determinant throughout the pathogenic cycle of V. cholerae. However, our results
showing the presence of tcpI gene in 65.5% of V. cholerae strains suggests that the importance of this gene may be
physiological and not pathogenesis alone.
Outer membrane protein, OmpU, was reported to be a potential adherence
factor for V. cholerae (72). However, later
studies suggested that OmpU is not involved in the adhesion of V. cholerae to the intestinal epithelium (53). In this
study, the gene was present in 87% of the V. cholerae
strains tested, except in nine non-O1/non-O139 V. cholerae
strains isolated from wastewater. These findings suggest that this gene
may be mainly physiological in its activity.
The hemolysin traditionally has been employed to differentiate between
the two biotypes of V. cholerae O1. The sequence of the
Classical biotype has an 11-bp deletion within the hlyA
structural gene, compared to the El Tor biotype (57).
Using information on these genes, we designed primers to differentiate
both biotypes and verified the presence of this gene in 98.6% of the
V. cholerae strains, in agreement with previous reports
(4, 68, 71, 82, 83). The majority belonged to the El Tor
biotype, regardless of the hemolytic phenotype (67 of 69). The
multiplex PCR for hemolysin effectively distinguished the two biotypes
in non-O1/non-O139 V. cholerae strains compared with
V. cholerae O1 Classical ATCC 11623 and ATCC 14035 and
V. cholerae O1 El Tor ATCC 14033. Interestingly, the
hlyA gene is located on chromosome 2 (26).
V. cholerae non-O1/non-O139 strains may also produce a
17-amino-acid heat-stable enterotoxin (NAG-ST) (stn) that is
closely related to the heat-stable toxins produced by enterotoxigenic E. coli and other enteric pathogens (1, 55).
Also, a heat-stable enterotoxin in V. cholerae O1 strains
(sto) has been described (22). We designed
primers based on the stn/sto genes associated with
heat-stable enterotoxin production and found the genes to be homologous
to those of toxigenic V. cholerae O1 (2 of 19), V. cholerae O139 (1 of 7), and non-O1/non-O139 V. cholerae
(11 of 39) strains. This is the first report showing higher values than
those reported in other studies, using hybridization with a NAG-ST
probe, in V. cholerae non-O1/non-O139 population (12, 28, 56, 58). Interestingly, the stn/sto gene occurred
more frequently in isolates from seawater (7 of 9) and sediment (4 of
7) and was absent in isolates from sewage (0 of 14) or oysters and
mussels (0 of 9). It should be mentioned, however, that Caldini et al.
(5) reported the presence of the sto gene in
12.7% (19 of 150) of the V. cholerae non-O1 isolates from
freshwater in the river basin of central Italy, but the incidence of
NAG-ST in V. cholerae non-O1 was not clearly established.
Results of a study involving human volunteers demonstrated that,
besides the production of NAG-ST, the virulence of non-O1/non-O139
V. cholerae depends on its ability to colonize the intestine
(50).
The regulation and expression of genes for growth and survival depend
on the regulon ToxR, coordinately regulated by a cascade mechanism
involving three known components: ToxR, ToxS, and ToxT (15). ToxR, a 32-kDa transmembrane protein, is the master
regulator, and its expression is dependent upon environmental growth
conditions (incubation temperature, pH, osmolarity, bile salts, oxygen
tension, hydrostatic pressure, and amino acid composition of the
medium) (15, 46). The toxR gene encodes a
transcriptional activator controlling CT gene expression
(ctxA), TCP biogenesis (tcpA), outer membrane
protein expression (ompU), and at least 17 distinct genes in
O139 and O1 strains (15, 27, 46). In this study, the
presence of the toxR gene was verified in all V. cholerae studied, regardless of serogroup or source of isolation,
a finding in agreement with previous reports (21, 71).
The presence of toxR (100%), hlyA (98.6%),
ompU (87%), and tcpI (65.5%) genes in the
V. cholerae isolates suggest that they are required for
functioning of the organism in the environment and are not solely
related to pathogenesis.
Five V. mimicus environmental strains were included in our
study because of their close relationship to V. cholerae.
None carried the toxR gene. The presence of CT in four of
the five V. mimicus strains was confirmed, while the
zot gene was found in only three of the strains, results
similar to those reported earlier by Chowdhury et al. (7).
The stn/sto gene was found in three of five environmental
strains, as reported elsewhere, using PCR (79). In
contrast, Pal et al. (56) reported the presence of NAG-ST
gene in 13.7 and 22.6% of V. mimicus isolates from
environmental and clinical sources, respectively, and
Ramamurthy et al. (59) suggested that V. mimicus may act as a genetic reservoir for these genes.
Multiplex PCR was found to be sensitive and specific for assessing the
pathogenicity of clinical and environmental V. cholerae isolates. We tested multiplex PCR for ctxA/tcpA El Tor genes
using four primers94F, 614R, 72F, and 477Ras a primary screening
for V. cholerae during epidemiological surveillance with
good success (data not shown).
V. cholerae genotypes. A single factor cannot explain enteropathogenicity. In this study eight virulence-associated genes were detected in V. cholerae strains isolated from both clinical and environment sources. Previous findings, in an earlier study of 172 V. cholerae non-O1/non-O139 environmental isolates from seawater and sediment samples collected in São Paulo, Brazil, showed that 60.4% of the strains were hlyAC+, nanH+, and toxR+ and ctxAB, elt (33), and zot negative as determined by using probes and radioactive hybridization. The frequency of occurrence of the genes among the strains tested was 98.8% for toxR, 97.1% for hlyAC, 66.9% for nanH, 5.8% for slt, 1.2% for zot, and 0.6% for ctxAB (D. E. Alvarado, V. H. Pellizari, T. A. T. Gomes, and I. N. G. Rivera, Abstr. 8th Int. Symp. Microb. Ecol. p. 89, 1998). In this study, we included V. cholerae O1 toxigenic and NT strains and V. cholerae O139 strains, using multiplex PCR, finding that 100% of the V. cholerae O1 and O139 strains carried ctxA, hlyAET, ompU, tcpAET, toxR, zot, and tcpI and sometimes the stn/sto gene.
Non-O1/non-O139 V. cholerae strains tested revealed the genotype hlyAET+ ompU+ tcpI+ toxR+ and negative for ctxA, stn/sto, tcpAET, and zot for isolates from mussels (4 of 9), wastewater (2 of 14), and seawater (1 of 9). Interestingly, we observed the genotype hlyAET+ toxR+ and negative for ctxA, ompU, stn/sto, tcpAET, and zot in 6 of 14 non-O1/non-O139 V. cholerae isolates from wastewater, a finding similar to the virulence pattern obtained for 15 clinical strains associated with an unusual upsurge of cholera-like disease in India (71) and in non-O1/non-O139 V. cholerae strains isolated from volunteers in a vaccine trial in Peru (13). A low frequency of the combination ctxA+ tcpAClass+ (1 of 39), and ctxA+ tcpAET+ (3 of 39) was observed in environmental non-O1/non-O139 V. cholerae strains, as reported earlier (21). However, we found 23.1% (9 of 39) of CT-negative, but TcpAET-positive strains, potentially convertible to toxigenic strains by CTX, either inside
the host intestine or in the environment.
V. cholerae is autochthonous to estuarine and coastal
environments (nutrient poor) and also colonize the human intestine
(nutrient rich). In the study reported here, we observed a varied
incidence of virulence-associated factors in V. cholerae
isolates, depending on the source or ecosystem (seawater, seafood,
wastewater, and clinical specimens). The response to changing
conditions occurs by expressing appropriate sets of genes that promote
growth in each niche.
Aquatic and marine ecosystems are subjected to large spatial and
temporal nutrient fluxes arising from seasonal and geographic variations in temperature, salinity, nutrient input, pH, oxygen tension, etc. (65). The mechanisms by which environmental
conditions affect the coordinated expression of virulence factors by
V. cholerae remain poorly understood. Hase and Mekalanos
(25) proposed a model where both ToxR and -S and TcpP and
-H are involved in sensing various environmental and internal stimuli
and are required for the production of TCP in V. cholerae.
Furthermore, the V. cholerae genome encodes 43 methyl-accepting chemotaxis proteins that regulate swimming behavior in
response to aminoacids, sugars, and oxygen (16).
Non-O1/non-O139 V. cholerae and risk assessment. Non-O1/non-O139 V. cholerae strains can no longer be ignored. The rationale for continuous monitoring is based first on the emergence of serogroup O139 (Bengal) in Bangladesh (CT positive) and Argentina (CT negative), each of which clearly evolved independently (73). Second, the sixth pandemic, the seventh pandemic, and U.S. Gulf Coast isolates represent three different clones, each independently evolved from environmental non-O1 V. cholerae isolates (34). Third, other epidemic serogroups have emerged, including V. cholerae O31 (50), O37 (11), O53 (52), O10, and O12 (13, 71). Finally, the emergence of a new clone of the V. cholerae O1 El Tor in Calcutta, India (70), and in Bangladesh (17) has been reported. The possibility exists that additional new strains of toxigenic V. cholerae with epidemic potential may emerge in the future.
We reported earlier an ERIC-PCR method for detecting emergent pathogenic V. cholerae strains, whereby specific fingerprints for pathogenic strains (CT and ZOT toxin positive) were different from nonpathogenic strains (61). In this study, we report probable parental toxigenic genotypes present in small numbers in the Brazilian environment. While these strains did not cause epidemics, there are environmental factors that may change, enhance multiplication or dominance, or select for genotypes of V. cholerae, and the strains themselves need to be tested for potential selective advantage under selected environmental conditions.| |
ACKNOWLEDGMENTS |
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Irma N. G. Rivera was supported by a Fellowship from UNESCO-ASM during the research period and from CNPQ (National Council for Research Support [Brazil]) during the postdoctoral period.This research was supported by NIH (grant 1R01A139129-01) and EPA (grant R824995-01).
We thank P. S. Sanchez and M. I. Sato from the State Agency for Environmental Control-CETESB, S. Paulo, Brazil, for providing the strains in this study.
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FOOTNOTES |
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*
Corresponding author. Mailing address: Departamento de
Microbiologia, ICB-USP, 1374, Lineu Prestes Av.Edif. ICB II,
USP-S.P., São Paulo, Brazil CEP 05508-900. Phone: 55-11-38187205. Fax: 55-11-38187354. E-mail: igrivera{at}usp.br.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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