Yoshitsugu Nakaguchi

Pandemic Vibrio parahaemolyticus O3:K6, Europe

To the Editor: Vibrio parahaemolyticus is a halophilic member of the genus Vibrio that inhabits temperate and tropical marine environments worldwide. Strains that produce the thermostable direct hemolysin or the thermostable direct hemolysin-related hemolysin, which are encoded by tdh and trh genes, respectively, are considered pathogenic. While almost all clinical strains have these virulence factors, these strains represent <1% of all environmental strains.

Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel.

We developed a completely homogeneous and isothermal method of detecting RNA sequences and demonstrated ultrarapid and direct quantification of pathogenic gene expression with high sensitivity. The assay is based on performing isothermal RNA sequence amplification in the presence of our novel DNA probe, an intercalation activating fluorescence DNA probe, and measuring the fluorescence intensity of the reaction mixture. When detecting mecA gene expression of methicillin-resistant Staphylococcus aureus, we quantified starting copies ranging from 10 to 10(7) copies within 10min. The primer sequences were designed to bind to secondary structure-free sites of the target RNA, which enabled a totally isothermal protocol to quantify mRNA specifically in a sample of existing genomic DNA. When we applied this to quantifying the expression of marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG strain, the results correlated well with the viability of each bacterium. We also demonstrated monitoring Pab gene expression of M. bovis BCG during cultivation with antibiotics. The present method can potentially realize rapid antimicrobial susceptibility testing of slowly growing organisms, such as tuberculosis.

Genetic characterization of Escherichia coli O157:H7/- strains carrying the stx2 gene but not producing Shiga toxin 2

Nine Escherichia coli O157:H7/– strains isolated primarily from non‐clinical sources in Thailand and Japan carried the stx2 gene but did not produce Stx2 toxin in a reversed passive latex agglutination (RPLA) assay. A strain (EDL933) bearing a stx2 phage (933W) was compared to a strain (Thai‐12) that was Stx2‐negative but contained the stx2 gene. To study the lack of Stx2 production, the Thai‐12 stx2 gene and its upstream nucleotide sequence were analyzed. The Thai‐12 stx2 coding region was intact and Stx2 was expressed from a cloned stx2 gene using a plasmid vector and detected using RPLA. A lacZ fusion analysis found the Thai‐12 stx2 promoter non‐functional. Because the stx2 gene is downstream of the late promoter in the stx2 phage genome, the antitermination activity of Q protein is essential for strong stx2 transcription. Thai‐12 had the q gene highly homologous to that of Φ21 phage but not to the 933W phage. High‐level expression of exogenous q genes demonstrated Q antitermination activity was weak in Thai‐12. Replication of stx2 phage was not observed in Stx2‐negative strains. The q‐stx2 gene sequence of Thai‐12 was well conserved in all Stx2‐negative strains. A PCR assay to detect the Thai‐12 q‐stx2 sequence demonstrated that 30% of O157 strains from marketed Malaysian beef carried this sequence and they produced little or no Stx2. These results suggest that stx2‐positive O157 strains that produce little or no Stx2 may be widely distributed in the Asian environment.

Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

ABSTRACT Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

Variability of Properties of Vibrio parahaemolyticus Strains Isolated from Individual Patients

ABSTRACT Infections by strains belonging to the O3:K6 pandemic clone of Vibrio parahaemolyticus are prevalent in southern Thailand, and serovariants of these strains have also been detected. V. parahaemolyticus strains lacking important virulence genes (tdh and trh) were isolated from 6.5 to 10.9% of clinical specimens during the period from 2000 to 2003. In order to understand whether changes to the characteristics of V. parahaemolyticus occur during infection, 10 isolates collected from each of 63 patients who presented with diarrhea at the Hat Yai hospital from 2003 to 2004 were examined for the presence of the tdh and trh genes, the O:K serotype, and genetic markers for the pandemic clone. A total of 42 patients (66.7%) yielded identical isolates (homogeneous populations), and 21 of the patients (33.3%) yielded isolates that differed in at least one character from the other isolates (heterogeneous populations). The DNA fingerprints (examined by arbitrarily primed PCR and pulsed-field gel electrophoresis) of some, but not all, of the heterogeneous populations from single patients were indistinguishable. The results indicated that some patients were infected with a unique strain and that in vivo changes (tdh deletion or serotype conversion) might have occurred in certain individuals. It is therefore important to bear in mind that epidemiological studies based on the analysis of a single colony from a single patient might lead to misleading conclusions. Finally, the present study did not rule out the possibility that isolates lacking tdh and trh have unknown virulence mechanisms other than the tdh and trh genes.

Transfer of Campylobacter jejuni from raw to cooked chicken via wood and plastic cutting boards

Aims:  We quantified Campylobacter jejuni transferred from naturally contaminated raw chicken fillets and skins to similar cooked chicken parts via standard rubberwood (RW) and polyethylene cutting boards (PE).

Distribution of Virulent and Pandemic Strains of Vibrio parahaemolyticus in Three Molluscan Shellfish Species (Meretrix meretrix, Perna viridis, and Anadara granosa) and Their Association with Foodborne Disease in Southern Thailand

Distribution of pandemic strains of Vibrio parahaemolyticus in seafood, particularly in molluscan shellfish, and their serological and molecular relationships to clinical strains were examined from Hat Yai City in southern Thailand. During 2000 to 2002, virulent strains (tdh+ or trh+) were isolated from 13 of 230 molluscan shellfish samples using alkaline peptone water enrichment followed by immunomagnetic separation. The isolates included 12 pandemic strains (tdh+, trh-, group-specific PCR positive) from five Oriental hard clam samples, five green mussel samples, and one bloody clam sample. Among the pandemic strains, eight belonged to serogroup O3:K6, three belonged to O1:K25, and one was O1:K untypeable. One hundred eighty-seven strains of V. parahaemolyticus were isolated from clinical specimens obtained from a hospital in this city during 2000 to 2001. The pandemic strains comprised 64 and 68% of the isolates in 2000 and 2001, respectively. Among the serotypes of the pandemic strains, O3:K6 was dominant at 73% in 2000 and 76% in 2001 followed by O1:K25 at 20% in 2000 and 13% in 2001. Pulsed-field gel electrophoresis profiles of the pandemic strains from molluscan shellfish were indistinguishable or very similar to those of patient isolates. Similarity of the serotype distribution and DNA fingerprints occurring between the molluscan shellfish strains and clinical strains suggests that molluscan shellfish may be an important source of pandemic V. parahaemolyticus infection in southern Thailand. For public health, proper cooking of molluscan shellfish in this area is strongly recommended.

Simulation of cross-contamination and decontamination of Campylobacter jejuni during handling of contaminated raw vegetables in a domestic kitchen.

Campylobacter jejuni was found to occur at high prevalence in the raw salad vegetables examined. Previous reports describe cross-contamination involving meat; here we investigated the occurrence of cross-contamination and decontamination events in the domestic kitchen via C. jejuni-contaminated vegetables during salad preparation. This is the first report concerning quantitative cross-contamination and decontamination involving naturally contaminated produce. The study was designed to simulate the real preparation of salad in a household kitchen, starting with washing the vegetables in tap water, then cutting the vegetables on a cutting board, followed by slicing cucumber and blanching (heating in hot water) the vegetables in 85 degrees C water. Vegetables naturally contaminated with C. jejuni were used throughout the simulation to attain realistic quantitative data. The mean of the percent transfer rates for C. jejuni from vegetable to wash water was 30.1 to 38.2%; from wash water to cucumber, it was 26.3 to 47.2%; from vegetables to cutting board, it was 1.6 to 10.3%; and from cutting board to cucumber, it was 22.6 to 73.3%. The data suggest the wash water and plastic cutting board as potential risk factors in C. jejuni transmission to consumers. Washing of the vegetables with tap water caused a 0.4-log reduction of C. jejuni attached to the vegetables (most probable number/gram), while rapid blanching reduced the number of C. jejuni organisms to an undetectable level.

Rapid and Specific Detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by Transcription-Reverse Transcription Concerted Reaction with an Automated System

ABSTRACT Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 103 to 107 copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups.

Multiplex PCR for the concurrent detection and differentiation of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium

Salmonellosis outbreaks involving typhoid fever and human gastroenteritis are important diseases in tropical countries where hygienic conditions are often not maintained. A rapid and sensitive method to detect Salmonella spp., Salmonella Typhi and Salmonella Typhimurium is needed to improve control and surveillance of typhoid fever and Salmonella gastroenteritis. Our objective was the concurrent detection and differentiation of these food-borne pathogens using a multiplex PCR. We therefore designed and optimized a multiplex PCR using three specific PCR primer pairs for the simultaneous detection of these pathogens. The concentration of each of the primer pairs, magnesium chloride concentration, and primer annealing temperature were optimized before verification of the specificity of the primer pairs. The target genes produced amplicons at 429 bp, 300 bp and 620 bp which were shown to be 100% specific to each target bacterium, Salmonella spp., Salmonella Typhi and Salmonella Typhimurium, respectively.