Isolation of Vibrio cholerae and Vibrio vulnificus from Estuarine Waters, and Genotyping of V. vulnificus Isolates Using Loop-Mediated Isothermal Amplification.

Bacteria in the genus Vibrio are ubiquitous in estuarine and coastal waters. Some species (including Vibrio cholerae and Vibrio vulnificus) are known human pathogens causing ailments like cholera, diarrhea, or septicemia. Notably, V. vulnificus can also cause a severe systemic infection (known as vibriosis) in eels raised in aquaculture facilities. Water samples were periodically collected from the estuary of the Asahi River, located in the southern part of Okayama City, Japan. These samples were directly plated onto CHROMagar Vibrio plates, and colonies displaying turquoise-blue coloration were selected. Thereafter, polymerase chain reaction was used to identify V. cholerae and V. vulnificus. A total of 30 V. cholerae strains and 194 V. vulnificus strains were isolated during the warm season when the water temperature (WT) was higher than 20 °C. Concurrently, an increase in coliforms was observed during this period. Notably, V. vulnificus has two genotypes, designated as genotype 1 and genotype 2. Genotype 1 is pathogenic to humans, while genotype 2 is pathogenic to both humans and eels. The loop-mediated isothermal amplification method was developed to rapidly determine genotypes at a low cost. Of the 194 strains isolated, 80 (41.2%) were identified as genotype 1 strains. Among the 41 strains isolated when the WTs were higher than 28 °C, 25 strains (61.0%) belonged to genotype 1. In contrast, of the 32 strains isolated when the WTs were lower than 24 °C, 27 strains (84.4%) belonged to genotype 2. These results suggest that the distribution of the two genotypes was influenced by WT.

Investigation of the Expression of Serine Protease in Vibrio vulnificus.

Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a metalloprotease-negative strain isolated from a diseased eel, produces a 45-kDa chymotrypsin-like alkaline serine protease known as VvsA. The gene encoding vvsA also includes another gene, vvsB with an unknown function; however, it is assumed to be an essential molecular chaperone for the maturation of VvsA. In the present study, we used an in vitro cell-free translation system to examine the maturation pathway of VvsA. We individually expressed the vvsA and vvsB genes and detected their mRNAs. However, the sample produced from vvsA did not exhibit protease activity. A sodium dodecyl sulfate (SDS) analysis detected the VvsB protein, but not the VvsA protein. A Western blotting analysis using a histidine (His)-tag at the amino terminus of proteins also showed no protein production by vvsA. These results suggested the translation, but not the transcription of vvsA. Factors derived from Escherichia coli were used in the in vitro cell-free translation system employed in the present study. The operon of the serine protease gene containing vvsA and vvsB was expressed in E. coli. Although serine proteases were produced, they were cleaved at different sites and no active mature forms were detected. These results indicate that the operon encoding vvsA and vvsB is a gene constructed to be specifically expressed in V. vulnificus.

Second extracellular protease mediating maturation of Vibrio mimicus hemolysin.

Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg151 and Ser152. The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile33-Ser403). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH.

Inhibitory Effects of Polymyxin B and Human LL-37 on the Flagellin Expression in Vibrio vulnificus.

Vibrio vulnificus, an opportunistic human pathogen responsible for primary septicemia, initiates pathogenesis by attachment to the intestinal epithelial cells for which the motility by the polar flagellum plays an essential role. The proteomic analysis of outer membrane proteins showed that the treatment with the 1/2 minimum inhibitory concentration (MIC) of polymyxin B (a bacterial antimicrobial peptide) led to the reduced production of flagellin (a major component of the polar flagellum). Furthermore, the bacterial motility was inhibited in the presence of 1/2 MIC of polymyxin B. V. vulnificus has six flagellin genes organized into the flaFBA and flaCDE loci. The flaA was found to be expressed higher than flaC, and its expression was significantly decreased by polymyxin B. As well as polymyxin B, the 1/2 MIC of LL-37 (a human intestinal antimicrobial peptide) reduced the expression of flaA. In addition, among four fragments of LL-37, KI-18 and FK-13 containing F17KRIVQRIKDELR29 could lead to the decreased expression of flaA. Because the motility closely relates to virulence of V. vulnificus, the findings obtained herein indicate that LL-37 may reduce the bacterial virulence through inhibition of the motility via the polar flagellum.

Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India.

Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25°C and 37°C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25°C, but that was low when cultured at 37°C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future.

Inhibitory Effects of Escherichia coli on the Formation and Development of Staphylococcus epidermidis Biofilm.

In the present study, we examined whether a commensal gut bacterium Escherichia coli might prevent the formation and development of the biofilm of Staphylococcus epidermidis, a nosocomial extraintestinal pathogen but not a gut microorganism. When co-cultured with S. epidermidis, E. coli strain ATCC 35218, a non-pathogenic strain, was found to be dominant in the biofilm formed on the surface of wells of a microtiter plate. In addition, E. coli significantly incorporated and grew in a niche preoccupied by S. epidermidis biofilm. Two other E. coli strains (strain K-12 and B) also showed to interfere the biofilm formation by S. epidermidis. In contrast, S. epidermidis could not grow in a niche preoccupied by E. coli biofilm. These results suggest that, through inhibition of the formation and development of the biofilm, E. coli may eliminate S. epidermidis from the gastrointestinal tract.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

Cholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred. IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

Regulation of Chitin-Dependent Growth and Natural Competence in Vibrio parahaemolyticus.

Vibrios can degrade chitin surfaces to soluble N-acetyl glucosamine oligosaccharides (GlcNAcn) that can be utilized as a carbon source and also induce a state of natural genetic competence. In this study, we characterized chitin-dependent growth and natural competence in Vibrio parahaemolyticus and its regulation. We found that growth on chitin was regulated through chitin sensors ChiS (sensor histidine kinase) and TfoS (transmembrane transcriptional regulator) by predominantly controlling the expression of chitinase VPA0055 (ChiA2) in a TfoX-dependent manner. The reduced growth of ΔchiA2, ΔchiS and ΔtfoS mutants highlighted the critical role played by ChiA2 in chitin breakdown. This growth defect of ΔchiA2 mutant could be recovered when chitin oligosaccharides GlcNAc2 or GlcNAc6 were supplied instead of chitin. The ΔtfoS mutant was also able to grow on GlcNAc2 but the ΔchiS mutant could not, which indicates that GlcNAc2 catabolic operon is dependent on ChiS and independent of TfoS. However, the ΔtfoS mutant was unable to utilize GlcNAc6 because the periplasmic enzymes required for the breakdown of GlcNAc6 were found to be downregulated at the mRNA level. We also showed that natural competence can be induced only by GlcNAc6, not GlcNAc2, because the expression of competence genes was significantly higher in the presence of GlcNAc6 compared to GlcNAc2. Moreover, this might be an indication that GlcNAc2 and GlcNAc6 were detected by different receptors. Therefore, we speculate that GlcNAc2-dependent activation of ChiS and GlcNAc6-dependent activation of TfoS might be crucial for the induction of natural competence in V. parahaemolyticus through the upregulation of the master competence regulator TfoX.

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation.

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.

Low Viability of Cholera Toxin-Producing Vibrio cholerae O1 in the Artificial Low Ionic Strength Aquatic Solution.

It has been well known that Vibrio cholerae inhabit in environmental water. As many patients infected with cholera toxin-producing V. cholerae O1 (toxigenic V. cholerae O1) emerge in Kolkata, India, it has been thought that toxigenic V. cholerae O1 is easily detected in environmental water in Kolkata. However, we could not isolate toxigenic V. cholerae O1 from environmental water in Kolkata, though NAG Vibrio (generic name of V. cholerae non-O1/non-O139) is constantly detected. To clear the reason for the non-isolation of toxigenic V. cholerae O1, we examined the viability of V. cholera O1 and NAG Vibrios in the artificial low ionic strength aquatic solution. We found that the viability of toxigenic V. cholerae O1 in the solution is low, but that of NAG Vibrios is high. Subsequently, we examined the viability of NAG Vibrios possessing cholera toxin gene (ctx) in the same condition and found that the viability of these NAG Vibrios is low. These results indicate that the existence of ctx in V. cholerae affects the viability of V. cholerae in the aquatic solution used in this experiment. We thought that there was closely relation between the low viability of toxigenic V. cholerae O1 in the artificial low ionic strength aquatic solution and the low frequency of isolation of the strain from environmental water.

Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata, India: generation of novel types of genomic islands containing plural antibiotic resistance genes.

Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance-determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non-O1/non-O139 strains through the action of newly generated genomic islands.

Comparative proteomic analysis to characterize temperature-induced viable but non-culturable and resuscitation states in Vibrio cholerae.

Vibrio cholerae can survive environmental adversities by entering into a viable but non-culturable (VBNC) state and is able to resuscitate under favourable conditions. In this study, an environmental strain of V. cholerae (AN59) showed a decrease in culturability from 4×107 to ≤ 3 c.f.u. ml -1 in artificial seawater media at 4 °C within 35 days. During the course of VBNC progression, viability was confirmed by real-time RT-PCR which showed reduced but stable expression of molecular chaperones groEL and dnaK. Resuscitation was induced in VBNC microcosm by a temperature increase from 4 to 37 °C for 24 h. The results obtained from resuscitation and growth experiments suggest that 103-104 c.f.u. ml -1 of VBNC cells should recover upon temperature increase and grow to attain 107 c.f.u. ml -1. We used comparative proteomics to differentiate recovery from the VBNC state and selected 19 proteins whose expression was significantly variable between these two states. These proteins were mainly related to carbohydrate metabolism, phosphate utilization, stress response, transport and translation. The main difference in the proteome profile was higher protein expression in the recovery state compared to VBNC state. However, during recovery Pi-starvation led to expression of PhoX, PstB and Xds, which might help in utilization of extracellular DNA to promote growth after resuscitation. In addition, the expression of EctC suggests that osmotic adaptation is necessary to grow at high salinity. Detection of AhpC in the VBNC and recovery state indicates the significance of the oxidative stress response. A temperature-induced VBNC and recovery state is a combination of adaptive and survival responses under nutrient limitation.

Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, India.

Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years.

International collaborative research on infectious diseases by Japanese universities and institutes in Asia and Africa, with a special emphasis on J-GRID.

In developed countries including Japan, malignant tumor (cancer), heart disease and cerebral apoplexy are major causes of death, but infectious diseases are still responsible for a high number of deaths in developing countries, especially among children aged less than 5 years. World Health Statistics published by WHO reports a high percentage of mortality from infectious diseases in children, and many of these diseases may be subject to transmission across borders and could possibly invade Japan. 　Given this situation, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan initiated Phase I of the Program of Founding Research Centers for Emerging and Reemerging Infectious Disease, which ran from FY 2005 to 2009, and involved 8 Japanese universities and 2 research centers. The program was established for the following purposes: 1) creation of a domestic research structure to promote the accumulation of fundamental knowledge about infectious diseases, 2) establishment of 13 overseas research collaboration centers in 8 countries at high risk of emerging and reemerging infections and at which Japanese researchers are stationed and conduct research in partnership with overseas instructors, 3) development of a network among domestic and overseas research centers, and 4) development of human resources. 　The program was controlled under MEXT and managed by the RIKEN Center of Research Network for Infectious Diseases (Riken CRNID). Phase II of the program was set up as the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), and has been running in FY 2010-2014. 　Phase III will start in April 2015, and will be organized by the newly established Japanese governmental organization "Japan Agency for Medical Research and Development (AMED)", the so-called Japanese style NIH. 　The Collaborative Research Center of Okayama University for Infectious Diseases in India (CRCOUI) was started up in 2007 at the National Institute of Cholera and Enteric Disease, Kolkata, India. Major projects of CRCOUI are concerned with diarrheal diseases such as, 1) active surveillance of diarrheal patients, 2) development of dysentery vaccines, 3) viable but nonculturable (VBNC) Vibrio cholerae, and 4) pathogenic mechanisms of various diarrhogenic microorganisms. 　This review article outlines project of J-GRID and CRCOUI which the authors carried out collaboratively with NICED staff members.

Stepwise changes in viable but nonculturable Vibrio cholerae cells.

Many bacterial species are known to become viable but nonculturable (VBNC) under conditions that are unsuitable for growth. In this study, the requirements for resuscitation of VBNC-state Vibrio cholerae cells were found to change over time. Although VBNC cells could initially be converted to culturable by treatment with catalase or HT-29 cell extract, they subsequently entered a state that was not convertible to culturable by these factors. However, fluorescence microscopy revealed the presence of live cells in this state, from which VBNC cells were resuscitated by co-cultivation with HT-29 human colon adenocarcinoma cells. Ultimately, all cells entered a state from which they could not be resuscitated, even by co-cultivation with HT-29. These characteristic changes in VBNC-state cells were a common feature of strains in both V. cholerae O1 and O139 serogroups. Thus, the VBNC state of V. cholerae is not a single property but continues to change over time.

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin.

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N-terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin-like substrate specificity was purified from the bacterial culture supernatant. The N-terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg(151)-Ser(152) bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

Isolation of viable but nonculturable Vibrio cholerae O1 from environmental water samples in Kolkata, India, in a culturable state.

Previously, we reported that viable but nonculturable (VBNC) Vibrio cholerae was converted into a culturable state by coculture with several eukaryotic cell lines including HT-29 cells. In this study, we found that a factor converting VBNC V. cholerae into a culturable state (FCVC) existed in cell extracts of eukaryotic cells. FCVC was nondialyzable, proteinase K-sensitive, and stable to heating at <60°C for 5 min. We prepared thiosulfate citrate bile salts sucrose (TCBS) plates with FCVC (F-TCBS plates). After confirming that VBNC V. cholerae O1 and O139 formed typical yellow colonies on F-TCBS plates, we tried to isolate cholera toxin gene-positive VBNC V. cholerae from environmental water samples collected in urban slum areas of Kolkata, India and succeeded in isolating V. cholerae O1 El Tor variant strains harboring a gene for the cholera toxin. The possible importance of VBNC V. cholerae O1 as a source of cholera outbreaks is discussed.

Defensive effects of human intestinal antimicrobial peptides against infectious diseases caused by Vibrio mimicus and V. vulnificus.

Of human pathogenic Vibrio species, V. mimicus causes gastroenteritis whereas V. vulnificus causes fatal septicemia after consumption of contaminated seafood. These two pathogens produce hemolytic toxins termed V. mimicus hemolysin (VMH) and V. vulnificus hemolysin (VVH), respectively. These toxins elicit the cytolysis of various eukaryotic cells, as well as erythrocytes. The human intestine secretes cationic antimicrobial peptides (AMPs) to prevent infectious diseases. Paneth cells in the small intestine secrete α-defensin 5 (HD-5) and epithelial cells in the large intestine produce LL-37. In the present study, we examined the bactericidal activities of AMPs against V. mimicus and V. vulnificus. Although HD-5 showed no bactericidal activity, LL-37 revealed significant activity against both Vibrio species, suggesting that neither V. mimicus nor V. vulnificus can multiply in the large intestine. We also tested whether AMPs had the ability to inactivate the hemolytic toxins. Only HD-5 was found to inactivate VMH, but not VVH, in a dose-dependent manner through the direct binding to VMH. Therefore, it is considered that V. mimicus cannot penetrate the small intestinal epithelium because the cytolytic action of VMH is inactivated by HD-5.

Ecological study of pathogenic vibrios in aquatic environments.

An ecological study of pathogenic vibrios in aquatic environments of Okayama was carried out. The number of Vibrio parahaemolyticus detected in the sea area was comparatively smaler than that found in the survey of about two decades ago. Various reasons for the decrease in the case of food poisoning by V. parahaemolyticus have been suggested but the lower number of the vibrio in aquatic environments may be one explanation. Although the number of V. vulnificus was also not as large, most of the isolates possessed the pathogenic genes, vvp and vvh, suggesting the potential for fatal pathogenicity to patients having underlying diseases. As for V. cholerae, some non-O1/non-O139 serovar isolates were detected in a fresh water area, and many of them had hlyA, the gene for hemolysin which acts as a pathogenic factor in sporadic cases of diarrhea. Thus, the total number of pathogenic vibrios detected was not of concern. However, the marine products of these areas are shipped in wide area and are for general consumption. Therefore, it is necessary to continue to survey pathogenic vibrios in aquatic environments in order to ensure food hygiene.

An extracellular serine protease produced by Vibrio vulnificus NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel.

Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in immunocompromised humans, cultured eels or shrimps. An extracellular metalloprotease VVP/VvpE has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA, but not VVP/VvpE. In the present study, we found that these strains had lost the 80 kb genomic region including the gene encoding VVP/VvpE. We also purified VvsA from the culture supernatant through ammonium sulfate fractionation, gel filtration and ion-exchange column chromatography, and the enzyme was demonstrated to be a chymotrypsin-like protease, as well as those from some vibrios. The gene vvsA was shown to constitute an operon with a downstream gene vvsB, and several Vibrio species were found to have orthologues of vvsAB. These findings indicate that the genes vvp/vvpE and vvsAB might be mobile genetic elements.