Chironomid Egg Masses as a Natural Reservoir of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Non-O1 and Non-O139 in Freshwater Habitats

Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year. The natural reservoir of the bacterium is environmental. A recent report indicated an association between V. cholerae and chironomid egg masses. Chironomids, the non-biting midges (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater. Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the waters edge where bacteria are abundant and may encounter the nutrient-rich substrate. Here we report the isolation of non-O1 and non-O139 V. cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa. In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region. The bacterial population rose as water temperature surpassed 25°C. Thirty-five different serogroups of V. cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity. Two strains of V. cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive. Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V. cholerae.     

Interaction of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1/non-O139 with Copepods,Cladocerans and Competing Bacteria in the Large Alkaline Lake Neusiedler See,Austria

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community.     

Dependent population dynamics between chironomids (nonbiting midges) and Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. Chironomid (nonbiting midges) egg masses were recently found to harbour V. cholerae non-O1 and non-O139, providing a natural reservoir for the cholera bacterium. Chironomid populations and the presence of V. cholerae in chironomid egg masses were monitored. All V. cholerae isolates were able to degrade chironomid egg masses. The following virulence associated genes were detected in the bacterial isolates: hapA (100%), toxR (100%), hlyA (72%) and ompU (28%). The chironomid populations and the V. cholerae in their egg masses followed the phenological succession and interaction of host-pathogen population dynamics. A peak in the chironomid population was followed by a peak in the V. cholerae population. If such a connection is further substantiated for the pathogenic serogroups of V. cholerae in endemic areas of the disease, it may lead to a better understanding of the role of chironomids as a host for the cholera bacterium.     

Occurrence of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1 in three wastewater treatment plants in Agadir (Morocco)

A total of 21 isolates of Vibrio cholerae non-O1 strains were isolated from three wastewater treatment plants in Agadir, Morocco. The isolates were analyzed by biochemical analysis, antibiogram, pulsed-field gel electrophoresis and the MALDI-TOF patterns of their protein masses were compared. Over 67% of isolates were susceptible to antimicrobial agents tested and 14% proved resistant to both trimethoprim-sulfamethoxazole and nalidixic acid. Typing by pulsed-field gel electrophoresis with NotI digestion revealed that the V. cholerae non-O1 strains from Agadir (Morocco) have a lower level of genetic homogeneity, the restriction patterns of whole-chromosomal DNA grouped the V. cholerae O1 and V. alginolyticus strains into a separate cluster from V. metschnikovii and V. cholerae non-O1 isolates. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used dendrogram based on MALDI-TOF spectral patterns generated by the BioTyper 1.1™ software. All m/z signatures of all strains tested indicate that the mass spectral data contained sufficient information to distinguish between strains of V. cholerae.     

Incidence of Non-01 Vibrio cholerae and Aeromonas spp. in Fresh Water in Araraquara, Brazil

The occurrence of Aeromonas spp., Vibrio cholerae, and Plesiomonas shigelloides in fresh water from various sources in Araraquara, State of S?o Paulo, Brazil was determined. Samples from ten distinct irrigation systems used in vegetable cultivation, from five distinct streams, from two reservoirs, from one artificial lake, and from three distinct springs were analyzed. All isolates were serotyped and tested for hemolysin, cytotoxin, heat-stable (ST) and heat-labile (LT) enterotoxins production; presence of plasmid; autoagglutination and drug resistance. V. cholerae isolates were also tested for cholera enterotoxin (CT) production, and Aeromonas isolates for suicide phenomenon. No P. shigelloides was found. V. cholerae non 01 was found in five irrigation water samples and in three stream samples. Aeromonas sp. were isolated in two samples of irrigation water, in three streams, and in one reservoir. All the V. cholerae and Aeromonas isolates were positive for β-hemolysin production, and all Aeromonas isolates were positive for suicide phenomenon; cytotoxic activities were observed in two Aeromonas strains. Cholera enterotoxin was not found in eight V. cholerae non-01 isolates tested by the Y-1 mouse adrenal cell. All isolates were also negative for the other virulence markers. V. cholerae isolates were found to be sensitive to the majority of drugs tested, while Aeromonas strains presented multiple drug resistance. Received: 4 November 1997 / Accepted: 23 January 1998     

Novel insights into Haemagglutinin Protease (HAP) gene regulation in Vibrio cholerae

Quorum sensing is the phenomenon, whereby bacteria use signal molecules to communicate with each other. For example, to establish a successful infection, pathogenic bacteria become virulent only when they reach a certain local concentration in their host. Bassler and others have highlighted the surprising observation that quorum sensing seems to repress Vibrio cholerae virulence factor expression (e.g. cholera toxin), in contrast to what has been observed for virulence gene expression in other bacteria. Here, I present a novel insight that may clarify the way V. cholerae quorum‐sensing signals regulate its genes. Chironomids (Diptera; Chironomidae), which occur worldwide and are frequently the insect found most abundantly in fresh water bodies, are natural reservoirs of V. cholerae. Quorum‐sensing signals in V. cholerae up‐regulate the production of an extracellular enzyme, haemagglutinin protease (HAP), which degrades chironomid egg masses and prevents the eggs from hatching. HAP, therefore, is a virulence factor against chironomids. Indeed, in a survey carried out over the course of a year, V. cholerae and chironomids showed a pattern that mirrored the dynamics of predator‐prey populations. Globally, the numbers of chironomids are much larger than those of humans, so quorum‐sensing signals of V. cholerae and HAP gene regulation should be understood with regard to their role in chironomids rather than humans. Further research is needed to understand the role of cholera toxin in the environmental existence of V. cholerae.     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.     

Vibrio cholerae Hemagglutinin/Protease Degrades Chironomid Egg Masses

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.     

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 °C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4–98.4 %) in the biofilm production was observed at 37 °C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 °C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103–143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139–244 %, at pH 7.5 136–164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151–319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113–177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204–329 % at pH 8.5, and 123–316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).     

The association between non‐biting midges and Vibrio cholerae

Vibrio cholerae is a natural inhabitant of aquatic ecosystems, yet its interactions within this habitat are poorly understood. Here we describe the current knowledge on the interaction of V. cholerae with one group of co‐inhabitants, the chironomids. Chironomids, non‐biting midges (Chironomidae, Diptera), are an abundant macroinvertebrate group encountered in freshwater aquatic habitats. As holometabolous insects, chironomids start life when their larvae hatch from eggs laid at the water/air interface; through various feeding strategies, the larvae grow and pupate to become short‐lived, non‐feeding, adult flying insects. The discovery of the connection between V. cholerae and chironomids was accidental. While working with Chironomus transavaalensis, we observed the disintegration of its egg masses and searched for a possible microbial agent. We identified V. cholerae as the primary cause of this phenomenon. Haemagglutinin/protease, a secreted extracellular enzyme, degraded the gelatinous matrix surrounding the eggs, enabling bacterial growth. Observation of chironomids in relation to V. cholerae continuously for 7 years in various types of water bodies in Israel, India, and Africa revealed that environmental V. cholerae adhere to egg‐mass surfaces of various Chironomini (‘bloodworms’). The flying adults' potential to serve as mechanical vectors of V. cholerae from one water body to another was established. This, in turn, suggested that these insects play a role in the ecology of V. cholerae and possibly take part in the dissemination of the pathogenic serogroups during, and especially between, epidemics.     

Presence of genes for type III secretion system 2 in <Emphasis Type="Italic">Vibrio</Emphasis><Emphasis Type="Italic">mimicus</Emphasis> strains

Background  

Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.     

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.     

Virulence profile and clonal relationship among the <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from ground and surface water in a cholera endemic area during rainy season

All the V. cholerae non-O1, non-O139 isolates from ground and surface water samples collected during the rainy season (rainfall contributes significantly in the spread of cholera) contained ompW and a regulatory toxR gene, while many others possessed accessory cholera toxin (ace), hemolysin (hlyA) and outer membrane protein (ompU) genes. All the isolates lacked ctxAB, tcp, zot, rfbO1 and rfbO139 genes. The strains could be grouped into two main clusters colligating the isolates from ground water and surface water samples. The results suggest that surface water harbors various virulent V. cholerae strains that contaminate the ground water due to rain or poor hygienic practices, and result in the emergence of new toxigenic strains for cholera.     

Identification of chironomid species as natural reservoirs of toxigenic Vibrio cholerae strains with pandemic potential

Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cholerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chironomids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cholerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns. A similar pattern is found for cholera epidemics in the Bengal Delta region. Thus, we hypothesize that monitoring chironomids in endemic areas of the disease may provide a novel tool for predicting and preventing cholera epidemics. Moreover, serogroup O139 was detected only in two chironomid species that have a restricted distribution in the Indian subcontinent, possibly explaining why the distribution of the O139 serogroup is limited.     

Isolation,identification, and characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from the Danube River in Slovakia

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.     

Arctic Actinomycetes as Potential Inhibitors of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Biofilm

The aim of this study was to identify novel biofilm inhibitors from actinomycetes isolated from the Arctic against Vibrio cholerae, the causative agent of cholera. The biofilm inhibitory activity of actinomycetes was assessed using biofilm assay and was confirmed using air–liquid interphase coverslip assay. The potential isolates were identified using 16S rRNA gene sequencing. Of all, three isolates showed significant biofilm inhibition against V. cholerae. The results showed that 20% of the actinomycetes culture supernatant could inhibit up to 80% of the biofilm formation. When different extracted fractions were assessed, significant biofilm inhibition activity was only seen in the diethyl ether fraction of A745. At 200 μg ml⁻¹ of diethyl ether fraction, 60% inhibition of V. cholerae biofilm was observed. The two potential isolates were found to be Streptomyces sp. and one isolate belonged to Nocardiopsis sp. This is the first report showing a Streptomyces sp. and Nocardiopsis sp. isolated from the Arctic having a biofilm inhibitory activity against V. cholerae. The spread of drug resistant V. cholerae strains is a major clinical problem and the ineffectiveness in antibiotic treatment necessitates finding new modes of prevention and containment of the disease, cholera. The formation of biofilms during the proliferation of V. cholerae is linked to its pathogenesis. Hence, the bioactive compound from the culture supernatant of the isolates identified in this study may be a promising source for the development of a potential quorum sensing inhibitors against V. cholerae.     

Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139

Vibrio cholerae is a waterborne bacterium native to the aquatic environment. There are over 200 known serogroups yet only two cause cholera pandemics in humans. Direct contact of human sewage with drinking water, sea-born currents and marine transportation, represent modes of dissemination of the bacteria and thus the disease. The simultaneous cholera outbreaks that occur sometimes in distant localities within continental landmasses are puzzling. Here we present evidence that flying, non-biting midges (Diptera; Chironomidae), collected in the air, carry viable non-O1 non-O139 serogroups of V. cholerae. The association of V. cholerae with chironomid egg masses, which serve as a V. cholerae reservoir, was further confirmed. In simulated field experiments, we recorded the transfer of environmental V. cholerae by adult midges from the aquatic environment into bacteria-free water-pools. In laboratory experiments, flying adult midges that emerged from V. cholerae (O1 or O139) contaminated water transferred the green fluorescent protein (GFP)-tagged pathogenic bacteria from one laboratory flasks to another. Our findings show that aerial transfer by flying chironomids may play a role in the dissemination of V. cholerae in nature.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Fish as Reservoirs and Vectors of Vibrio cholerae

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments, but despite intensive efforts its ecology remains an enigma. Recently, it was suggested that copepods and chironomids, both considered as natural reservoirs of V. cholerae, are dispersed by migratory waterbirds, thus possibly distributing the bacteria between water bodies within and between continents. Although fish have been implicated in the scientific literature with cholera cases, as far as we know, no study actually surveyed the presence of the bacteria in the fish. Here we show for the first time that fish of various species and habitats contain V. cholerae in their digestive tract. Fish (n = 110) were randomly sampled from freshwater and marine habitats in Israel. Ten different fish species sampled from freshwater habitats (lake, rivers and fish ponds), and one marine species, were found to carry V. cholerae. The fish intestine of Sarotherodon galilaeus harboured ca. 5×10³ V. cholerae cfu per 1 gr intestine content—high rates compared with known V. cholerae cfu numbers in the bacteria''s natural reservoirs. Our results, combined with evidence from the literature, suggest that fish are reservoirs of V. cholerae. As fish carrying the bacteria swim from one location to another (some fish species move from rivers to lakes or sea and vice versa), they serve as vectors on a small scale. Nevertheless, fish are consumed by waterbirds, which disseminate the bacteria on a global scale. Moreover, V. cholerae isolates had the ability to degrade chitin, indicating a commensal relationship between V. cholerae and fish. Better understanding of V. cholerae ecology can help reduce the times that human beings come into contact with this pathogen and thus minimize the health risk this poses.