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Vibrio cholerae is an inhabitant of aquatic environments worldwide. Here, we report the draft genome sequences of eight V. cholera non-O1, non-O139 isolates that were recovered from the corpses of two seabird chicks (common terns) following a mass mortality event in a German breeding colony in 2019.

An occurrence of Vibrio cholerae non-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. cholerae infection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae , suggesting that there might be additional virulence factors in non-O1/O139 V. cholerae yet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. cholerae isolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. cholerae in the interest of public health.

Non-O1/O139 Vibrio cholerae is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of V. cholerae non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for ctxB and harbored a rare genotype; 10 (3.4%) isolates carried several types of rstR sequences, eight of which carried rare types of toxin-coregulated pili ( tcpA ). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates ( n = 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The V. cholerae non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.

We report the detection of PER-1 extended-spectrum β-lactamase (ESBL) in a clinical non-O1, non-O139 Vibrio cholerae strain from China. IS CR1 -mediated bla PER-1 was embedded in a complex In 4 family class 1 integron belonging to the lineage of Tn 1696 on a conjugative IncA/C plasmid. A free 8.98-kb circular molecule present with the IS CR1-bla PER-1 –truncated 3′-conserved sequence (CS) structure was detected in this isolate. These findings may provide insight into the mobilization of bla PER-1 .

Mobile genetic elements play a pivotal role in the adaptation of bacterial populations, allowing them to rapidly cope with hostile conditions, including the presence of antimicrobial compounds. IncA/C conjugative plasmids (ACPs) are efficient vehicles for dissemination of multidrug resistance genes in a broad range of pathogenic species of Enterobacteriaceae . ACPs have sporadically been reported in Vibrio cholerae , the infectious agent of the diarrheal disease cholera. The regulatory network that controls ACP mobility ultimately depends on the transcriptional activation of multiple ACP-borne operons by the master activator AcaCD. Beyond ACP conjugation, AcaCD has also recently been shown to activate the expression of genes located in the Salmonella genomic island 1 (SGI1). Here, we describe MGI Vch Hai6, a novel and unrelated mobilizable genomic island (MGI) integrated into the 3′ end of trmE in chromosome I of V. cholerae HC-36A1, a non-O1/non-O139 multidrug-resistant clinical isolate recovered from Haiti in 2010. MGI Vch Hai6 contains a mercury resistance transposon and an integron In104-like multidrug resistance element similar to the one of SGI1. We show that MGI Vch Hai6 excises from the chromosome in an AcaCD-dependent manner and is mobilized by ACPs. Acquisition of MGI Vch Hai6 confers resistance to β-lactams, sulfamethoxazole, tetracycline, chloramphenicol, trimethoprim, and streptomycin/spectinomycin. In silico analyses revealed that MGI Vch Hai6-like elements are carried by several environmental and clinical V. cholerae strains recovered from the Indian subcontinent, as well as from North and South America, including all non-O1/non-O139 clinical isolates from Haiti. IMPORTANCE Vibrio cholerae , the causative agent of cholera, remains a global public health threat. Seventh-pandemic V. cholerae acquired multidrug resistance genes primarily through circulation of SXT/R391 integrative and conjugative elements. IncA/C conjugative plasmids have sporadically been reported to mediate antimicrobial resistance in environmental and clinical V. cholerae isolates. Our results showed that while IncA/C plasmids are rare in V. cholerae populations, they play an important yet insidious role by specifically propagating a new family of genomic islands conferring resistance to multiple antibiotics. These results suggest that nonepidemic V. cholerae non-O1/non-O139 strains bearing these genomic islands constitute a reservoir of transmissible resistance genes that can be propagated by IncA/C plasmids to V. cholerae populations in epidemic geographical areas as well to pathogenic species of Enterobacteriaceae . We recommend future epidemiological surveys take into account the circulation of these genomic islands.

The toxigenic bacterium Vibrio cholerae belonging to the O1 and O139 serogroups is commonly associated with epidemic diarrhea in tropical settings; other diseases caused by this environmental pathogen are seldom identified. Here we report two unassociated cases of nonfatal, nontoxigenic V. cholerae non-O1, non-O139 bacteremia in patients with comorbidities in Ho Chi Minh City, Vietnam, that occurred within a 4-week period.

Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttR A (locus tag A33_1664) and vttR B (locus tag A33_1675), are predicted to encode proteins that show similarity to the transcriptional regulator ToxR, which is found in all strains of V. cholerae . Strains with a deletion of vttR A or vttR B showed attenuated colonization in vivo , indicating that the T3SS-encoded regulatory proteins play a role in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the T3SS structural components identified growth in the presence of bile as a condition that modulates gene expression. Under this condition, VttR A and VttR B were necessary for maximal gene expression. In contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene, which encodes an effector protein. Increased vttR B reporter fusion activity was observed in a Δ vttR B strain background, suggesting that VttR B may regulate its own expression. The collective results are consistent with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the expression of selected T3SS genes.

The incidence of Vibrio cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% ( n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher colonization in an animal model compared to those lacking tcpA . PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA , rtxA , Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.

Vibrio pathogenicity island-2 (VPI-2) is a 57-kb region integrated at a transfer RNA (tRNA)-serine locus that encompasses VC1758 to VC1809 on the V. cholerae N16961 genome and is present in pandemic isolates. VPI-2 encodes a P4-like integrase, a restriction modification system, a Mu phage-like region, and a sialic acid metabolism region, as well as neuraminidase (VC1784), which is a glycosylhydrolase known to release sialic acid from sialoglycoconjugates to unmask GM1 gangliosides, the receptor for cholera toxin. We examined the tRNA-serine locus among the sequenced V. cholerae genomes and identified five variant VPI-2 regions, four of which retained the sialometabolism region. Three variant VPI-2 regions contained a type three secretion system. By using an inverse nested PCR approach, we found that the VPI-2 region can form an extrachromosomal circular intermediate (CI) molecule after precise excision from its tRNA-serine attachment site. We constructed a knockout mutant of VC1758 ( int ) with V. cholerae strain N16961 and found that no excision PCR product was produced, indicating that a functional cognate, VPI-2 integrase, is required for excision. The Vibrio seventh pandemic island-I (VSP-I) and VSP-II regions are present in V. cholerae O1 El Tor and O139 serogroup isolates. Novel regions are present at the VSP-I insertion site in strain MZO-3 and at the VSP-II insertion site in strain 623-39. VSP-II is a 27-kb region that integrates at a tRNA-methionine locus, is flanked by direct repeats, and encodes a P4-like integrase. We show that VSP-II can excise and form a CI and that the cognate VSP-II integrase is required for excision. Interestingly, VSP-I is not inserted at a tRNA locus and does encode a XerDC-like recombinase, but similar to VPI-2 and VSP-II, VSP-I does excise from the genome to form a CI. These results show that all three pathogenicity islands can excise from the chromosome, which is likely a first step in their horizontal transfer.

Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae . In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

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.

In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a β-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for bla TEM or primers for bla CARB gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the β-lactamase was determined by bidirectional sequencing of a Sau 3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this β-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the bla CARB-7 gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the bla CARB-7 gene shared 93% identity with a locus situated inside V. cholerae 's chromosome 2. These results strongly suggest the chromosomal location of the bla CARB-7 gene, making this the first communication of a β-lactamase gene located on the VCR island of the V. cholerae genome.

Vibrio cholerae NRT36S is a non-cholera toxin-producing, non-O1 strain that causes diarrhea in volunteers. The genome of NRT36S was sequenced to create a draft containing 174 contigs plus the superintegron region. Our analysis of the draft genome revealed several putative toxin genes and colonization factors. Besides confirming the existence of nonagglutinable heat-stable toxin, we also identified the genes for a type three secretion system, a putative exotoxin, two different RTX toxins, and four pilus systems.

Vibrio spp. of clinical interest from the Arno River basin (Tuscany, Italy) were investigated in this study. Vibrios were isolated from 70% of water samples. Vibrio cholerae non-O1 was the most prevalent species (82% of isolates), followed by Vibrio mimicus (10%) and Vibrio metschnikovii (8%). Recovery of vibrios was correlated with temperature, pH, and various indicators of municipal pollution. None of the 150 Vibrio isolates carried ctx-related genomic sequences, whereas 18 (14.6%) of the 123 V. cholerae non-O1 isolates and 1 (6.7%) of the 15 V. mimicus isolates carried sto alleles. These findings indicate that considerable circulation of sto-positive vibrios may occur in temperate-climate freshwater environments.

Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage. This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin. Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY. Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5. NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold. The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin. Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop. Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region. Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V. cholerae non-O1 strains which produce this cytotoxin.

Cholera is a severe diarrheal disease typically caused by O1 serogroup strains of Vibrio cholerae. The pathogenicity of all pandemic V. cholerae O1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139 V. cholerae strains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139 V. cholerae has not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139 V. cholerae pathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected with V. cholerae O1. Unlike V. cholerae O1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain’s virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139 V. cholerae represents a new type of diarrheagenic mechanism., IMPORTANCE Cholera, which is caused by Vibrio cholerae, is an important cause of diarrheal disease in many developing countries. The mechanisms of virulence of nonpandemic strains that can cause a diarrheal illness are poorly understood. AM-19226, like several other pathogenic, nonpandemic V. cholerae strains, carries genes that encode a type III secretion system (TTSS), but not cholera toxin (CT) or toxin coregulated pilus (TCP). In this study, we used infant rabbits to study AM-19226 virulence. Infant rabbits orally inoculated with this strain rapidly developed a fatal diarrheal disease, which was accompanied by marked disruptions of the intestinal epithelium. This strain’s TTSS proved essential for its pathogenicity, and there was no diarrhea, intestinal pathology, or colonization in rabbits infected with a TTSS mutant. The effector proteins translocated by the TTSS all appear to contribute to AM-19226 virulence. Thus, our study provides insight into in vivo mechanisms by which a novel TTSS contributes to diarrheal disease caused by nonpandemic strains of V. cholerae.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.

A fish pathogen, Vibrio cholerae non-O1, was isolated from diseased ayu fish (Plecoglossus altivelis) collected from rivers in eight prefectural districts of Japan. This organism was found to have biochemical characteristics similar to those of V. cholerae non-O1, except that our isolates were negative for ornithine decarboxylase. Antiserum against an ayu isolate did not agglutinate with the majority of environmental V. cholerae non-O1 isolates, but a major O antigen was common among the ayu isolates. All strains were hemolytic to sheep erythrocytes, and oral administration of culture supernatants induced fluid accumulation in suckling mice. However, the crude toxin was not lethal to adult mice, and no cholera toxin-like enterotoxins were detected.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody (MAb) against synthetic heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was produced. The MAb, namely, 2F, belonged to the immunoglobulin G1 class. Ascitic fluid drawn from pristane-primed BALB/c mice injected with a 2F-producing clone demonstrated anti-NAG-ST activity which could be detected in enzyme-linked immunosorbent assay even at a dilution of 1:128,000. Fifty-fold-diluted ascitic fluid could completely neutralize the activity of NAG-ST (synthetic and native) and Vibrio mimicus ST (identical to NAG-ST) in suckling mice. In the same assay, 2F could also neutralize Yersinia enterocolitica ST (Y-ST) but did not neutralize Escherichia coli STh and STp. A similar pattern of reactivity occurred in a competitive enzyme-linked immunosorbent assay with homologous and heterologous toxins. Competitive inhibition curves with synthetic peptides representing NAG-ST and its shorter analogs revealed that aspartic acid located at position 2 from the N terminus of NAG-ST was the essential residue of the recognized epitope. Significantly, in Y-ST, to which 2F cross-reacted, aspartic acid is in the corresponding position as that of NAG-ST, thereby confirming our conclusions that the epitope defining this MAb is aspartic acid.

Pili of Vibrio cholerae non-O1 strain S7 were purified and characterized. The pili of S7 were morphologically, electrophoretically, and immunologically (as far as polyclonal antibody was used) indistinguishable from the 16-kilodalton pili of V. cholerae O1 strain 82P7. The purified pili and organisms had D-mannose- and L-fucose-resistant hemagglutinin. The hemagglutinating activity of the purified pili was inhibited by the Fab fraction of antipilus antibody, but the hemagglutinating activity of live organisms was not inhibited completely. The purified pili or Fab fraction of antipilus antibody did not inhibit the adhesion of V. cholerae non-O1 to rabbit intestines. Therefore, the pili were not regarded as a colonization factor of V. cholerae non-O1. A total of 148 V. cholerae non-O1 and O1 clinical isolates were screened for the presence of S7 pili by using an agglutination test with anti-S7 pilus serum; 12 of 49 V. cholerae non-O1 strains and 25 of 99 V. cholerae O1 strains were positive for agglutination. These agglutination reactions were not correlated with adhesion of the organisms to intestines.

Pili of the Vibrio cholerae non-O1 strain V10 were purified and characterized. The V10 pili were physicochemically and immunologically different from those of the previously reported V. cholerae non-O1 strain S7, although the pili of the two strains had homologous N-terminal amino acid sequences. V10 plus antigen was detected only in V. cholerae non-O1 strains.

We present 2 cases of primary pulmonary non-O1 Vibrio cholerae infection. We believe that these are the first documented cases of primary pulmonary infection due to this organism from a freshwater source.

We report a case of a cholera-like gastroenteritis subsequent with bacteremia in a healthy man following consumption of raw clams. Although we failed to recover the organism from the patient's stool culture, his blood culture was positive for a non-cholera toxin-producing yet cytotoxin-producing non-O1 and non-O139Vibrio cholerae.

A strain of Vibrio cholerae non-O1 was isolated from a 48-year-old male in California who presented with a chief complaint of watery diarrhea. Laboratory investigations of this strain revealed the bacterium to be resistant to trimethoprim-sulfamethoxazole and to the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine).

Vibrio cholerae non-O1 strain E8498, isolated in 1978 from fresh water in Louisiana, produced a vascular permeability factor when cultured in shallow resting cultures of Casamino Acids-yeast extract-glucose medium for 24 h at 30 degrees C. Undiluted resting culture filtrates contained heat-labile permeability factor activity which was only partially neutralized by cholera antitoxin and GM1 ganglioside. Supernatants concentrated with PM-10 membranes caused hemorrhage and necrosis in rabbits within 1 h after intracutaneous injection, whereas appropriate dilutions of both filtrates and concentrates demonstrated delayed permeability factor activity, without hemorrhage or necrosis, which was indistinguishable in appearance from that caused by purified cholera enterotoxin produced by V. cholerae O1 Inaba strain 569B. Crude E8498 filtrates contained the biological equivalent of about 5 ng/ml of purified enterotoxin. Permeability factor activity in the fraction obtained by 20 to 50% saturation of filtrate concentrate with ammonium sulfate could be completely neutralized by reference standard cholera antitoxin prepared against purified 569 B enterotoxin. Hemorrhagic activity was unaffected by cholera antitoxin. A 5,000-fold concentrate of the culture supernatant yielded a line of identity with purified cholera enterotoxin in an agar gel double-diffusion test against cholera antitoxin purified by affinity column chromatography with BrCN-activated Sepharose 4B-linked purified cholera enterotoxin as the adsorbent. These findings indicate that V. cholerae non-O1 E8498 produces a permeability factor which is immunologically and biologically indistinguishable from that produced by a strain of V. cholerae O1 classical biotype.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.

A toxin which causes rapid fluid accumulation in a suckling mouse assay and which was produced by Vibrio cholerae non-O1 was investigated. The toxin was purified from the culture supernatant of V. cholerae non-O1 (strain A-5) by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatographies on SP-Sephadex C-50 and DEAE-Sephadex A-25, and high-pressure liquid chromatography on a Lichrosorb RP-8 column. About 1.4 X 10(5)-fold purification was achieved, with a recovery of about 12%. Although the crude preparation was heat labile, the purified toxin was heat stable. The minimum effective dose of purified toxin was about 5 ng in the suckling mouse assay. The amino acid composition of the purified toxin was determined to be Asp(3), Glu(1), Ala(1), half-Cys(6), Ile(2), Leu(1), Phe(1), and Pro(1). These data show the production of a new type of heat-stable enterotoxin (NAG-ST) by V. cholerae non-O1.

Hemolysins purified from non-O1 Vibrio cholerae (non-O1 hemolysin) and a Vibrio cholerae O1, biotype El Tor (El Tor hemolysin) were investigated for their homology. The hemolysins were isolated from the culture supernatant fluids by ammonium sulfate precipitation and gel filtration on Sephadex G-100 columns. The purified hemolysins gave single bands with an identical mobility on conventional polyacrylamide gel disc electrophoresis. The molecular weights of the non-O1 and El Tor hemolysins were estimated to be about 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the amino acid compositions of the hemolysins were very similar. The specific activities of the hemolysins were identical, and both hemolysins were neutralized to the same extent with antisera against the homologous and heterologous hemolysins. Ouchterlony double immunodiffusion tests with both hemolysins and antihemolysin serum gave a common (fused) precipitin line. These data indicate that the non-O1 hemolysin is biologically, physicochemically, and immunologically indistinguishable from the El Tor hemolysin.

Clinically isolated Vibrio cholerae non-O1 strains produced more cell-associated hemagglutinins (HAs) on colonization factor antigen agar after ca. 3 h than after ca. 20 h of incubation at 37 degrees C. A high cell-associated HA producer variant of strain TVN-318, grown for 3 h at 37 degrees C, was entrapped in a native mucus coat covering the human ileal mucosa and displayed a striking ability to adhere to the surface of a Formalin-treated mucus coat, in contrast to a poor cell-associated HA producer variant of TVN-318, grown for 20 h at 37 degrees C. Adherence to the Formalin-treated human mucus coat was confirmed with all of the strains tested. V. cholerae non-O1 strains also possessed the ability to adhere to the epithelial surfaces of Formalin-treated human and rabbit ileal or jejunal villi, as well as human lymphoid follicles, in proportion to cell-associated HA levels. The epithelial surface of the lymphoid follicles provided most of the adherence sites for V. cholerae non-O1 strains under the test conditions. We conclude that a mucus coat covering the human small intestinal mucosa is a primary adherence target for V. cholerae non-O1 strains in human intestinal infections and that cell-associated HAs have at least a partial role in the adherence of V. cholerae non-O1 strains to the human small intestine, suggesting a potential role for V. cholerae non-O1 strains in an oral live vaccine.

Two large, independently obtained collections of vibrio cholerae non O1, containing 59 and 67 reference strains, respectively, were compared serologically in four laboratories. Twenty strains in each collection were considered identical, and an additional 16 strains in each collection were probably identical. Twenty-eight unique strains were identified, and inconclusive results were obtained with 25 strains. Nine strains were not considered V. cholerae in at least one testing laboratory. Of these, five showed insufficient correspondence to V. cholerae O1 by DNA hybridization to be considered V. cholerae.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.

This report compares recovery of non-O1 Vibrio cholerae strains from seven California coastal sites during the winter and summer of 1983. A total of 41 identified and 27 presumptive nn-O1 V. cholerae strains were recovered from six of seven coastal sites in the summer. A 5-to 56-fold increase in the numbers of organisms isolated from different sites occurred in the summer months, when water temperatures were 1.9 to 5.1 degrees C higher. At the three sites where the highest levels of non-O1 V. cholerae were found, pollution, as measured by the total number of coliforms, exceeded the legal limit (less than 1,000 coliforms per 100 ml.).

Nineteen strains of Vibrio cholerae non-O1 were isolated from five separate marine sites along the Santa Cruz County coast. This environmental study was initiated after a human case of non-O1 cholera-like diarrhea was acquired endemically.