Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic<i>Vibrio cholerae</i>O1 and O139

Hoshino, Katsuaki; Yamasaki, Shinji; Mukhopadhyay, Asish K.; Chakraborty, Soumen; Basu, Arnab; Bhattacharya, Sujit; Nair, G. Balakrish; Shimada, Toshio; Takeda, Yoshifumi

doi:10.1111/j.1574-695x.1998.tb01128.x

Cited by 254 publications

(50 citation statements)

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“…Samples preincubated overnight with 0.025% yeast extract and 0.002% nalidixic acid were subjected to M-PCR, using the procedure described in ref. 41. Primer sequences for genes, wbe, ctxA, and rstR2 are shown in SI Table 3.…”

Section: M-pcrmentioning

confidence: 99%

Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission

Alam

Sultana

Nair

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Vibrio cholerae persists in aquatic environments predominantly in a nonculturable state. In this study coccoid, nonculturable V. cholerae O1 in biofilms maintained for 495 days in Mathbaria, Bangladesh, pond water became culturable upon animal passage. Culturability, biofilm formation, and the wbe, ctxA, and rstR2 genes were monitored by culture, direct fluorescent antibody (DFA), and multiplex PCR. DFA counts were not possible after formation of biofilm. Furthermore, wbe, but not ctxA, were amplifiable, even after incubation for 54 and 68 days at room temperature (Ϸ25°C) and 4°C, respectively, when no growth was detectable. Slower biofilm formation and extended culturability were observed for cultures incubated at 4°C, compared with Ϸ25°C, suggesting biofilm production to be temperature dependent and linked to loss of culturability. Small colonies appearing after incubation in microcosms for 54 and 68 days at 25°C and 4°C, respectively, were wbe positive and ctxA and rstR2 negative, indicating loss of bacteriophage CTX⌽. The coccoid V. cholerae O1 observed as free cells in microcosms incubated for 495 days could not be cultured, but biofilms in the same microcosms yielded culturable cells. It is concluded that biofilms can act as a reservoir for V. cholerae O1 between epidemics because of its long-term viability in biofilms. In contrast to biofilms produced in Mathbaria pond water, V. cholerae O1 in biofilms present in cholera stools and incubated under identical conditions as the Mathbaria pond water biofilms could not be cultured after 2 months, indicating that those V. cholerae cells freshly discharged into the environment are significantly less robust than cells adapted to environmental conditions. Bangladesh ͉ bacteriophage CTX⌽ ͉ DFA ͉ multiplex-PCR ͉ ctxA C holera continues to pose a serious health threat globally, notably in those countries where clean drinking water is not available to local populations. Vibrio cholerae serogroups O1 and O139 are associated with epidemic and pandemic cholera. Cholera is endemic in the Ganges delta, occurring twice yearly in epidemic form (1). It is also a major health problem for countries of Africa, Latin America, and Asia (2). V. cholerae O1 is native to both marine and fresh water environments and exists in association with plankton (3). In general, it can be isolated from only 1% of water samples collected during epidemic periods and rarely, if ever, between epidemics (4). However, fluorescent antibody-based studies show that V. cholerae O1 is, nevertheless, present in aquatic environments throughout the year (5). Furthermore, the presence of nonculturable V. cholerae O1 is confirmed by molecular methods (6). The question of whether such nonculturable cells in aquatic environments are capable of returning to an actively growing state to initiate cholera epidemics has been debated.Extensive studies have shown that V. cholerae O1 becomes coccoid and enters into a nonculturable state in the environment when conditions are not conducive to active growth (5, 7). Some of...

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Section: M-pcrmentioning

confidence: 99%

Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission

Alam

Sultana

Nair

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

209

175

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“…The amplification of V. cholerae species-specific conserved intergenic spacer region between 16S and 23S rRNA genes as carried out by the method of Chun et al [2]. The rfb genes specific for V. cholerae O1, O139 serogroups, and the ctxA-encoding subunit A of CT were amplified by using multiplex PCR as carried out by the method of Hoshino et al [3]. The hexaplex PCR for rapid detection of virulence and regulatory genes viz.…”

Section: Pcr Analysismentioning

confidence: 99%

Detection of Virulence Genes in Vibrio cholerae Isolated from Aquatic Environment in Kerala, Southern India

Kumar

Peter

Thomas

2008

Appl Biochem Biotechnol

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Vibrio cholerae is the etiologic agent of cholera. It is an autochthonous inhabitant of all aquatic environments. The virulence of V. cholerae is maintained by the CTX genetic element and tcpA gene. In the present investigation, environmental strains of V. cholerae isolated from different aquatic biotopes in Kerala were identified and serotyped. The antibiotic resistance pattern and presence of virulence and regulatory genes were examined. We found the presence of toxigenic non-O1/non-O139 strains harboring the CTX genetic element, heat-stable enterotoxin, rtxA gene, El Tor hemolysin, and Vibrio pathogenicity island (VPI). The strains also produced the cholera toxin (CT) as determined by monosialoganglioside enzyme-linked immunosorbent assay. A few strains belonging to the O1 serogroup but lacking the CTX genetic element were also observed. The majority of the environmental strains belonged to non-O1/non-O139 serogroup with many possessing toxR, ompU, heat-stable enterotoxin, and rtxA gene. The toxigenic non-O1/non-O139 strains exhibited resistance to trimethoprim, ampicillin, and polymixin B and intermediate resistance to co-trimoxazole. However, all other environmental strains were found resistant to ampicillin and polymixin B. Our findings demonstrate that the virulence genes are dispersed among the environmental strains of V. cholerae and a complex aquatic environment can give rise to pathogenic V. cholerae.

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“…At each site, 12 liters of epipelagic water (whole water, plankton-free water [PFW], and plankton fraction), 20 to 25 oysters, and 80 to 100 g of sediment were collected, and V. cholerae was isolated by using alkaline peptone water enrichment according to standard protocols (26). Briefly, three volumes of water (1 liter, 100 ml, and 10 ml), homogenized oysters (10 g, 1 g, and 0.1 g), and sediment samples, each added to 10ϫ alkaline peptone water, were incubated statically at 35°C for 16 to 18 h. One milliliter of each enrichment culture grown overnight (O/N) was used for DNA extraction by boiling (27) and tested by multiplex PCR for ctxA and toxigenic V. cholerae O1 and O139 (28). A loopful of pellicle from each enrichment culture grown overnight was streaked onto the selective media CHROMagar Vibrio (CHROMagar, USA) and thiosulfate citrate bile salts sucrose (TCBS) agar (Difco, USA) and incubated overnight at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay, Maryland

Ceccarelli

Chen

Hasan

et al. 2015

Appl Environ Microbiol

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f Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyA ET , the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health. Vibrio cholerae, a waterborne bacterial pathogen, is an autochthonous inhabitant of riverine and estuarine aquatic environments. There are Ͼ200 serogroups, based on O antigenic characters, but only serogroups O1 and O139 have been associated with epidemic cholera, and both are considered a major public health threat for developing countries (1).Developed countries today rarely witness cholera cases caused by epidemic strains of V. cholerae, and outbreaks are typically travel associated (2). However, infections other than cholera can be caused by nonepidemic V. cholerae serogroups that are collectively referred to as non-O1/non-O139 V. cholerae and are generally acquired through the consumption of raw or undercooked seafood. Non-O1/non-O139 V. cholerae infections are continuously reported worldwide (3, 4), emphasizing their clinical significance. Although non-O1/non-O139 V. cholerae strains generally do not produce cholera toxin, other virulence factors contribute to their pathogenicity, including the hemolysin gene hlyA (5), the protease gene hapA (6), the cytotoxic actin cross-linking repeats in toxin gene rtxA (7), the sialidase gene nanH (8), the heat-stable toxin (NAG-ST) (9), a type 6...

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Development and evaluation of a multiplex PCR assay for rapid detection of toxigenicVibrio choleraeO1 and O139

Cited by 254 publications

References 23 publications

Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission

Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission

Detection of Virulence Genes in Vibrio cholerae Isolated from Aquatic Environment in Kerala, Southern India

Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay, Maryland

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