Masahiro Asakura

Cholera Toxin Production by the El Tor Variant of Vibrio cholerae O1 Compared to Prototype El Tor and Classical Biotypes

ABSTRACT Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.

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.

Unnoticed Spread of Class 1 Integrons in Gram‐Positive Clinical Strains Isolated in Guangzhou, China

A total of 46 gram‐positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1‐specific primers, and the intI1‐positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII‐orfF‐aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram‐positive clinical strains isolated in Guangzhou, China.

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.

CYTOLETHAL DISTENDING TOXIN (CDT): GENETIC DIVERSITY, STRUCTURE AND ROLE IN DIARRHEAL DISEASE

In 1987, cytolethal distending toxin (CDT) was discovered by Johnson and Lior as a new type of protein toxin produced by certain strains of Escherichia coli, which is different from heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli. Although LT causes only cell elongation, CDT causes cell elongation, cell distention, irreversible cell cycle arrest, and consequently, death of the cultured mammalian cells. Recently, CDT was recognized as a new family of bacterial toxin, as a genotoxin, produced by a diverse group of gram-negative bacteria, all of which are related to mucosal infection. Although tremendous efforts have been made to study the structure and mode of action of CDT, its role in bacterial pathogenesis still remains unclear. In this review, we focus mainly on CDT produced by enteric bacteria and describe the history of CDT, their gene and protein structure, structure-function relationship, and its mode of action particularly how CDT contributes to the gastrointestinal infections.

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.

Peruvian Vibrio cholerae O1 El Tor strains possess a distinct region in the Vibrio seventh pandemic island-II that differentiates them from the prototype seventh pandemic El Tor strains

A collection of environmental and clinical strains of Vibrio cholerae O1 isolated from the beginning of the Latin American epidemic of cholera in 1991 to 2003 from multiple locations in Peru were characterized and compared with V. cholerae O1 El Tor strains of the seventh pandemic from the rest of the world (Asia, Africa, Australia and Europe) using a multilocus virulence gene profiling strategy and DNA sequencing. Peruvian strains differed from El Tor strains from the rest of the world by the failure of PCR to amplify genes VC0512, VC0513, VC0514 and VC0515 in the Vibrio seventh pandemic island-II (VSP-II) gene cluster. Sequencing of the VSP-II gene cluster and its flanking regions in one Peruvian strain (PERU-130) confirmed the PCR results, indicating that the Peruvian strain had low DNA homology (46.6 %) compared to the reference strain N16961 within the VSP-II region encompassing genes VC0511 to VC0515. Based on these differences in VSP-II, and based on the overall similarity between the pulsotypes of the Peruvian strains and the El Tor reference strain N16961, we concluded that the Peruvian, Eurasian and African strains belonged to the same clonal complex, and that the Peruvian strains represented variants that had independently evolved for a relatively short time. Since these ORFs in VSP-II of Peruvian strains are unique and conserved, they could form the basis for tracking the origin of the Peruvian strains and therefore of the Latin American pandemic.