Environmental bacteriophages active on biofilms and planktonic forms of toxigenic Vibrio cholerae: Potential relevance in cholera epidemiology

Cholera epidemics have long been known to spread through water contaminated with human fecal material containing the toxigenic bacterium Vibrio cholerae. However, detection of V. cholerae in water is complicated by the existence of a dormant state in which the organism remains viable, but resists cultivation on routine bacteriological media. Growth in the mammalian intestine has been reported to trigger "resuscitation" of such dormant cells, and these studies have prompted the search for resuscitation factors. Although some positive reports have emerged from these investigations, the precise molecular signals that activate dormant V. cholerae have remained elusive. Quorum-sensing autoinducers are small molecules that ordinarily regulate bacterial gene expression in response to cell density or interspecies bacterial interactions. We have found that isolation of pathogenic clones of V. cholerae from surface waters in Bangladesh is dramatically improved by using enrichment media containing autoinducers either expressed from cloned synthase genes or prepared by chemical synthesis. These results may contribute to averting future disasters by providing a strategy for early detection of V. cholerae in surface waters that have been contaminated with the stools of cholera patients or asymptomatic infected human carriers.

<i>Vibrio cholerae</i>O1 El Tor and O139 Bengal Strains Carrying<i>ctxB</i>^ET, Bangladesh

Distribution of Vibrio cholerae in the Apalachicola (Florida) Bay Estuary

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.

Effect of iron and pH on the survival of Vibrio cholerae in water

The effects of alum [KAl(SO4)2] on free-living and copepod-associated Vibrio cholerae O1 and O139 were investigated by using plate counts and immunofluorescence direct viable counting (DVC). Growth of alum-treated cells in 0.5/1000 Instant Ocean seawater was inhibited, i.e., no growth was obtained on Luria-Bertani (LB) agar or thiosulfate-citrate-bile salt-sucrose (TCBS) agar. However, a significant number of the inhibited cells maintained viability, as measured by DVC. In comparison, a significant number of V. cholerae organisms associated with zooplankton, most of which were crustacean copepods, were viable but nonculturable, with only a small number of cells retaining culturability on LB and TCBS agar. Both DVC and viable plate counts (CFU) were significantly greater for V. cholerae O1 and O139 associated with zooplankton than for V. cholerae in water alone, i.e., without copepods. It is concluded that alum is an effective coagulant but not an effective killing agent for V. cholerae and that association with copepods offers protection for V. cholerae O1 and O139 against alum and chlorine treatments.

In the freshwater environment of north India, cholera appears seasonally in form of clusters as well as sporadically, accounting for a significant piece of the puzzle of cholera epidemiology. We describe a number of cholera outbreaks with an average attack rate of 96.5/1000 but an overall low case fatality (0.17). Clinical cholera cases coincided with high rainfall and elevated temperatures, whereas isolation of V. cholerae non-O1 non-O139 from water was dependent on temperature (p < 0.05) but was independent of rainfall and pH (p > 0.05). However, isolation from plankton samples correlated with increased temperature and pH (p < 0.05). A lag period of almost a month was observed between rising temperature and increased isolation of V. cholerae from the environment, which in succession was followed by an appearance of cholera cases in the community a month later. Our results suggested that the aquatic environment can harbor highly divergent V. cholerae strains and serve as a reservoir for multiple V. cholera virulence-associated genes that may be exchanged via mobile genetic elements. In agreement with PFGE, AFLP data also proved that the V. cholerae O1 population was not clonal but was closely related. Our investigation did not support the concept that seasonal cholera outbreaks occur by movement of a single clonal strain across the region, as the clinical isolates from the same years were clearly different, implying that continuous evolution of V. cholerae O1 strains occurs in the cholera endemic area. Interestingly, the viable but non-culturable (VBNC) V. cholerae O1 cells were demonstrated in 2.21% samples from natural water bodies in addition to 40.69% samples from cholera-affected areas respectively. This suggests that aquatic environs do harbor the pathogenic O1 strain, though the isolation of culturable V. cholerae O1 is a rare event in the presence of relatively abundant non-O1 non-O139 isolates.

Cholera is a major public health problem in the African Great Lakes basin. Two hypotheses might explain this observation, namely the lakes are reservoirs of toxigenic Vibrio cholerae O1 and O139 bacteria, or cholera outbreaks are a result of repeated pathogen introduction from the neighboring communities/countries but the lakes facilitate the introductions. A prospective study was conducted in Uganda between February 2015 and January 2016 in which 28 selected surface water sources were tested for the presence of V. cholerae species using cholera rapid test and multiplex polymerase chain reaction. Of 322 water samples tested, 35 (10.8%) were positive for V. cholerae non O1/non O139 and two samples tested positive for non-toxigenic atypical V. cholerae O139. None of the samples tested had toxigenic V. cholerae O1 or O139 that are responsible for cholera epidemics. The Lake Albert region registered the highest number of positive tests for V. cholerae non O1/non O139 at 47% (9/19). The peak period for V. cholerae non O1/non O139 positive tests was in March–July 2015 which coincided with the first rainy season in Uganda. This study showed that the surface water sources, including the African Great Lakes in Uganda, are less likely to be reservoirs for the observed V. cholerae O1 or O139 epidemics, though they are natural habitats for V. cholerae non O1/non O139 and atypical non-toxigenic V. cholerae O139. Further studies by WGS tests of non-toxigenic atypical V. cholerae O139 and physicochemical tests of surface water sources that supports V. cholerae should be done to provide more information. Since V. cholerae non O1/non O139 may cause other human infections, their continued surveillance is needed to understand their potential pathogenicity.

Cholera has been reported in the state of Orissa, India during the past decades. An outbreak of diarrheal disease occurred during November 1 to November 9, 2000 in Rusipada village near Puri, which was inhabited by a population of approximately 560. During the outbreak, Vibrio cholerae O139 strains were isolated from clinical specimens collected from patients with acute diarrhea admitted to the infectious diseases (ID) hospital in Puri and from environmental samples collected from multiple bodies of water in the village. The index case with acute diarrhea was a 60-year-old female resident of Rusipada who had not visited any known outbreak-related areas, including an outbreak 1 week prior to her symptom onset in a nearby village. All the isolated strains were positive for ctxA, tcpA, ace, and zot genes, produced cholera toxin, and exhibited a similar antibiogram pattern. Comparison of DNA fingerprinting analysis by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) method and dendrogram constructed from RAPD revealed that genetic homogeneity exist between the clinical and environmental O139 strains. Epidemic and endemic cholera is a major public health problem in many developing countries and continues to be an important cause of morbidity in many areas of Asia, Africa, and Latin America. Among more than 200 serogroups of V. cholerae so far identified, 1 only O1 and recently identified O139 serogroup 2–5 are capable of causing epidemic cholera. It is now widely accepted that O139 Bengal like O1 and non-1 and non-O139 Vibrio may survive better in aquatic environments 6–8 and environmental water is the reservoir for infectious V. cholerae. 7,9 In this report we investigated the clonal relationship between strains of V. cholerae collected both from stool specimens from persons with acute diarrhea and from environmental sources of water. During the outbreak of diarrhea, 23 non-randomly selected rectal swabs were collected from the hospitalized diarrhea patients in ID hospital, Puri in Cary-Blair Transport (CBT, DIFCO,USA) medium and transported to the Microbiology Department of Regional Medical Research Centre (RMRC), Bhubaneswar and bacteriologically analyzed following standard technique. 10 Subsequently after the isolation of V. cholerae O139 as the etiological agent of the outbreak, 20 water samples non-randomly selected were collected from water bodies situated at various distances in that village to determine the source of contamination following a previous method. 11 Presumptive identification of 10 and 6 V. cholerae were isolated from rectal swabs and water samples respectively and the strains were agglutinated with monoclonal O139 antiserum supplied by NICED, Kolkata, India and were confirmed to belong to V. cholerae serogroup O139. To investigate the similarities of clinical and environmental strains of V cholerae O139, drug susceptibility test, cholera toxin assay, detection of virulent genes by polymerase chain reaction (PCR) assay, RAPD finger printing assay, and PFGE were performed. A monosialoganglioside (GM1) enzyme-linked immunosorbent assay (ELISA) was used to examine cholera toxin production in V. cholerae O139 strains by the method Svennerholm and Holmgren. 12 Drug susceptibility test was performed following the method described elsewhere, 13 with the antibiotics (Hi-media Laboratories, Bombay, India) ampicillin (A, 10 g), chloramphenicol (C, 30 g), co-trimoxazole (Co, 25 g), ciprofloxacin (Cf, 5 g), furazolidone (fz, 100 g), gentamicin (G, 10 g), neomycin (N, 30 g), nalidixic acid (Na, 30 g), norfloxacin (Nx, 10 g), streptomycin (S, 10 g) and tetracycline (T, 30 g). Characterization of strains as susceptible or resistant was based on size of the inhibition zone around each disc according to manufacturer’s instructions, which matched interpretive criteria recommended by the WHO. 14 Strains showing an intermediate zone of inhibition were interpreted as resistant to that drug on the basis of previous MIC studies conducted with V. cholerae. 15

Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification

Vibrio cholerae O1 in 2 Coastal Villages, Papua New Guinea

Vibrio cholerae organisms are known to exist in more than 200 different serogroups based on their “O” antigenic characters. Out of these, strains belonging to O1 or O139 serogroups have been implicated as the causative agent of cholera in the epidemic form. On the other hand, V. cholerae non-O1, non-O139 organisms, ubiquitously present in the aquatic environment, are usually nonpathogenic in nature. However, these strains can be isolated from sporadic cases or occasional outbreaks of gastroenteritis in man. Recently, there has been an upsurge in their isolation rate from diarrheal patients in the cholera endemic areas. Detailed characterization of these strains has demonstrated considerable diversity in their phenotypic as well as genotypic properties that may not be related to their pathogenic potential. While non-O1, non-O139 strains can cause gastroenteritis by mechanisms unrelated to those involved in epidemic cholera, some of these are shown to harbor gene clusters for cholera toxin (CTX) and toxin coregulated pilus (TCP) that are linked to the virulence potential of V. cholerae O1 or O139 strains. Data available so far suggest that the evolution of pathogenic strains (CTX+ TCP+) of non-O1, non-O139 V. cholerae may follow different mechanisms involving horizontal transfer of Vibrio pathogenicity island (VPI) and CTX gene clusters, exchange of “O” antigen biosynthesis genes, etc. However, mere acquisition of these gene clusters does not necessarily endow a non-O1, non-O139 strain with the ability to cause epidemic form of the disease which probably requires additional genetic traits that are unique to V. cholerae O1 or O139 strains.

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.

To evaluate SMART, Medicos Dip Stick and an Institut Pasteur (IP) cholera dipstick tests for accuracy and ease of use. Every 50th patient presenting with diarrhoea at ICDDR,B between 1 April 2003 and 30 November 2003 was enrolled. The rapid diagnostic tests were performed by field and laboratory technicians, and sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values calculated. We isolated Vibrio cholerae O1 from 116 (38%) of 304 patients. The Se, Sp, PPV and NPV of the SMART test were 58%, 95%, 84% and 84% for field technicians, and 83%, 88%, 83% and 88% for laboratory technicians. The Se, Sp, PPV and NPV of the IP dipstick test were 93%, 67%, 63% and 94% for field technicians, and 94%, 76%, 70% and 95% for laboratory technicians. The Se, Sp, PPV and NPV of the Medicos test were 84%, 79%, 71% and 90% for field technicians, and 88%, 80%, 72% and 92% for laboratory technicians. A high proportion of indeterminates (30%) hampered the performance of the SMART test. The IP dipstick had the highest Se, irrespective of technician skill level. The IP dipstick is the most appropriate rapid diagnostic assay for the detection of V. cholerae O1 in locations where the skill level of personnel may be low, such as remote areas or refugee camp settings. High cost may limit the utility of any diagnostic test in the developing world.

Development of a multiplex single-tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection