Atsushi Hinenoya

Development of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube(-1) by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus.

Aims:  To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human.

An inducible lambdoid prophage encoding cytolethal distending toxin (Cdt-I) and a type III effector protein in enteropathogenic Escherichia coli

Cytolethal distending toxins (CDTs) are inhibitory cyclomodulins, which block eukaryotic cell proliferation and are produced by a diverse group of Gram-negative bacteria, including Escherichia coli strains associated with intestinal and extraintestinal infections. However, the mode of transmission of the toxin gene clusters among diverse bacterial pathogens is unclear. We found that Cdt-I produced by enteropathogenic E. coli strains associated with diarrhea is encoded by a lambdoid prophage, which is inducible and infectious. The genome of Cdt-I converting phage (CDT-1Φ) comprises 47,021 nucleotides with 60 predicted ORFs organized into six genomic regions encoding the head and tail, virulence, integrase, unknown functions, regulation, and lysis. The genomic organization of CDT-1Φ is similar to those of SfV, a serotype-converting phage of Shigella flexneri, and UTI89, a prophage identified in uropathogenic E. coli. Besides the cdtI gene cluster, the virulence region of CDT-1Φ genome contains sequences homologous to a truncated cycle inhibiting factor and a type 3 effector protein. Mutation analysis of susceptible E. coli strain C600 suggested that the outer membrane protein OmpC is a putative receptor for CDT-1Φ. CDT-1Φ genome was also found to integrate into the host bacterial chromosome forming lysogens, which produced biologically active Cdt-I. Furthermore, phage induction appeared to cause enhanced toxigenicity of the E. coli strains carrying lysogenic CDT-1Φ. Our results suggest that CDT-1Φ is the latest member of a growing family of lambdoid phages encoding bacterial cyclomodulins and that the phage may have a role in horizontal transfer of these virulence genes.

Capsaicin, a potential inhibitor of cholera toxin production in Vibrio cholerae.

The use of natural compounds as inhibitory agents for virulence factor production is a new approach to overcome increased antimicrobial resistance in pathogenic bacteria. In this study, we examined whether red chilli (Capsicum annuum) contains any such compound(s) that can repress the cholera toxin (CT) production in Vibrio cholerae. We found that the methanol extract of red chilli could inhibit CT production in recently emerged V. cholerae O1 El Tor variant strains without affecting their viability. Interestingly, capsaicin, a well-studied active component of red chilli, also drastically inhibited CT production in V. cholerae strains belonging to various serogroups including variants. Real-time quantitative reverse transcription-PCR assay revealed that capsaicin effectively repressed the transcription of ctxA, tcpA and toxT genes, but not of toxR and toxS genes. On the contrary, capsaicin significantly enhanced the transcription of the hns gene, the product of which is known to regulate negatively the transcription of ctxAB, tcpA and toxT genes. These results suggest that capsaicin might act as a potent repressor for CT production possibly by enhancing the transcription of hns.

Identification of Vibrio campbellii isolated from diseased farm-shrimps from south India and establishment of its pathogenic potential in an Artemia model

Shrimp diseases are frequently reported to be caused by closely related vibrios, and in many cases they are tentatively but inaccurately identified as Vibrio harveyi and related vibrios. In the present study, 28 biochemically identified V. harveyi-related strains isolated from diseased shrimps were randomly selected for further characterization by molecular tools. Twenty-six strains were identified as Vibrio campbellii and two as V. harveyi by sequence analysis of 16S rRNA and uridylate kinase genes. Haemolysin-gene-based species-specific multiplex PCR also confirmed these results. Experimental challenge studies using Artemia as a model showed that eight isolates were highly pathogenic, three were moderately pathogenic and the remaining 17 were non-pathogenic. Ribotyping with BglI clearly distinguished V. campbellii from V. harveyi, but it failed to separate pathogenic and non-pathogenic clusters. Artemia nauplii challenged with a fluorescently labelled highly pathogenic strain (IPEY54) showed patches in the digestive tract. However, no patches were observed for a non-pathogenic strain (IPEY41). Direct bacterial counts also supported colonization potential for the highly pathogenic strain. To our knowledge, this is the first report on the isolation and accurate identification of large numbers of V. campbellii associated with shrimp disease in aquacultural farms. V. campbellii has long been considered to be non-pathogenic and classified with V. harveyi-related bacteria. However, we show that this species may be an emerging aquaculture pathogen. This study will help to formulate suitable strategies to combat this newly identified pathogen.

Identification of Vibrio harveyi as a causative bacterium for a tail rot disease of sea bream Sparus aurata from research hatchery in Malta.

A bacterial disease was reported from gilthead sea bream (Sparus aurata) within a hatchery environment in Malta. Symptoms included complete erosion of tail, infection in the eye, mucous secretion and frequent mortality. A total of 540 strains were initially isolated in marine agar from different infected body parts and culture water sources. Subsequently 100 isolates were randomly selected, identified biochemically and all were found to be Vibrio harveyi-related organisms; finally from 100 isolates a total of 13 numbers were randomly selected and accurately identified as V. harveyi by 16S rRNA gene sequencing and species-specific PCR. Ribotyping of these strains with HindIII revealed total of six clusters. In vivo challenge study with representative isolates from each cluster proved two clusters each were highly pathogenic, moderately pathogenic and non-pathogenic. All 13 isolates were positive for hemolysin gene, a potential virulence factor. Further analysis revealed probably a single copy of this gene was encoded in all isolates, although not in the same locus in the genome. Although V. harveyi was reported to be an important pathogen for many aquatic organisms, to our knowledge this might be the first report of disease caused by V. harveyi and their systematic study in the sea bream hatchery from Malta.

Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus.

Aim:  To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus.

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year‐long surveillance study. A PCR‐RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt‐I, three for cdt‐II, four for cdt‐III, three for cdt‐IV and four samples were positive for cdt‐V, as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt‐I, one cdt‐II, three cdt‐III, three cdt‐IV and four with cdt‐V. Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup O2. The other virulence genes such as astA, cnf1, eaeA, cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence‐associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR‐RFLP in Providencia alcalifaciens. Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.

Molecular Characterizations of Cytolethal Distending Toxin Produced by Providencia alcalifaciens Strains Isolated from Patients with Diarrhea

ABSTRACT Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides—CdtA, CdtB, and CdtC—encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206–215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G2/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.

Novel Cholix Toxin Variants, ADP-Ribosylating Toxins in Vibrio cholerae Non-O1/Non-O139 Strains, and Their Pathogenicity

ABSTRACT Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.