Effects of chitin and its soluble derivatives on survival of Vibrio cholerae O1 at low temperature

Chitin concentrations greater than 0.04% (wt/wt) protected cholera vibrios against killing at low temperature. This protective effect was detected with both the soluble form of chitin, glycol chitin, and the insoluble particulate form of chitin. Some amino acids or peptides also showed the same protective effect.     

Effects of temperature and salinity on survival of toxigenicVibrio cholerae O1 in seawater

In 1991 and 1992, the Latin American epidemic strain of Vibrio cholerae O1 was isolated from ballast water, bilge water, and sewage taken from cargo ships docked in Mobile Bay, Alabama. The findings raised questions regarding the organism's ability to survive long-term aboard ships and to withstand the exchange of ballast at sea. The effects of temperature (6, 18, and 30°C) and salinity (8, 16, and 32 ppt) on survival of V. cholerae O1 strains C6706 and C6707 and a ballast water isolate in sterile seawater were determined. The ballast water isolate, which had a D-value (number of days required to produce a 1 log₁₀ reduction in colony-forming units per milliliter) of 240 days at 18°C, 32 ppt salinity, had the longest survival time. The range of D-values was 36–240 days at 18°C, 60–120 days at 30°C, and 5–20 days at 6°C. In sterile seawater short-term survival was temperature dependent, whereas long-term survival was salinity dependent. In raw seawater, survival time of the ballast water isolate was reduced to 12–27 days, implying the existence of biological influences. As also shown in our previous work, the organism appeared to be able to survive for several months under relatively stable conditions in ballast water aboard ships; however, viability may be reduced to only a few weeks after the organism is introduced into estuarine or marine environments. Correspondence to: Susan A. McCarthy.     

Effects of temperature and salinity on Vibrio cholerae growth.

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.     

Effects of temperature and salinity on Vibrio cholerae growth

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.     

Effect of alum on free-living and copepod-associated Vibrio cholerae O1 and O139.

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.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae, tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae , tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

Viability of the nonculturable Vibrio cholerae O1 and O139

Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Characterization of enterotoxin and soluble hemagglutinin from Vibrio mimicus: identity with V. cholerae O1 toxin and hemagglutinin

Abstract Eight strains of Vibrio mimicus isolated from patients with diarrhoea in Bangladesh were all found to produce an extracellular toxin identical to cholera toxin produced by Vibrio cholerae O1 bacteria, with regard to subunit structure and immunological properties. Like cholera toxin, but in contrast to heat-labile enterotoxin from Escherichia coli most of the toxin from V. mimicus was found extracellularly and was proteolytically 'nicked' in its A subunit. This may relate to the finding that V. mimicus also produced an extracellular hemagglutinin which was immunologically indistinguishable from the soluble hemagglutinin/nicking protease of V. cholerae O1.     

Development of hyperfimbriated strains of Vibrio cholerae O1

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

Filamentous Phage fs1 of Vibrio cholerae O139

Filamentous phage, fs1, was obtained from Vibrio cholerae O139. The lysogenized strains produced a large amount of fs1 phage in the culture supernatant. This phage was previously reported as novel fimbriae of that organism. The genome of the phage was a 6.5 kb single-stranded DNA. The capsid of fs1 consists of a small molecule peptide (about 2.5 kDa).     

Effects of nutrient deprivation on Vibrio cholerae.

Environmental and clinical strains of Vibrio cholerae were exposed to nutrient-free artificial seawater and filtered natural seawater microcosms for selected time intervals and examined for changes in cell morphology and number. Cells observed by transmission electron and epifluorescence microscopy were found to undergo gross alterations in cell morphology with time of exposure. The vibroid cells decreased in volume by 85% and developed into small coccoid forms surrounded by remnant cell walls. The initial number of cells inoculated into nutrient-free microcosms (culturable count and direct viable count) increased 2.5 log10 within 3 days, and even after 75 days the number of viable cells was still 1 to 2 log10 higher than the initial inoculum size. Nutrient-depleted coccoid-shaped cells were restored to normal size and assumed a bacillary shape within 3 h and began to divide within 5 h after nutrient supplementation. The increase in cell number and decrease in cell volume under nutrient-depleted conditions, as well as the rapid growth response after nutrient supplementation, may describe some of the survival mechanisms of V. cholerae in the aquatic environment.     

Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae

Vibrio cholerae is a highly infectious bacterium responsible for large outbreaks of cholera among humans at regular intervals. A seasonal distribution of epidemics is known but the role of naturally occurring habitats are virtually unknown. Plankton has been suggested to play a role, because bacteria can attach to such organisms forming a biofilm. Acanthamoebea castellanii is an environmental amoeba that has been shown to be able to ingest and promote growth of several bacteria of different origin. The aim of the present study was to determine whether or not an intra-amoebic behaviour of V. cholerae O139 exists. Interaction between these microorganisms in co-culture was studied by culturable counts, gentamicin assay, electron microscopy, and polymerase chain reaction. The interaction resulted in intra-amoebic growth and survival of V. cholerae in the cytoplasm of trophozoites as well as in the cysts of A. castellanii. These data show symbiosis between these microorganisms, a facultative intracellular behaviour of V. cholerae contradicting the generally held view, and a role of free-living amoebae as hosts for V. cholerae O139. Taken together, this opens new doors to study the ecology, immunity, epidemiology, and treatment of cholera.     

Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae O1 and non O1 in Romania

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.     

Occurrence of Vibrio cholerae serotype O1 in Maryland and Louisiana estuaries.

Vibrio cholerae serotype O1 has been isolated from Chesapeake Bay in Maryland and estuaries and sewers in Louisiana. The occurrence of V. cholerae O1 in the aquatic environment in the absence of human disease suggests that this organism survives and multiples in the natural environment.