Hidemasa Izumiya

Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor

A mismatch amplification mutation PCR assay was developed and validated for rapid detection of the biotype specific cholera toxin B subunit of V. cholerae O1. This assay will enable easy monitoring of the spread of a new emerging variant of the El Tor biotype of V. cholerae O1.

Life-Threatening Infantile Diarrhea from Fluoroquinolone-Resistant Salmonella enteric Typhimurium with Mutations in Both gyrA and parC

Salmonella Typhimurium DT12, isolated from a 35-day-old infant with diarrhea, was highly resistant to ampicillin, tetracycline, chloramphenicol, streptomycin, gentamycin, sulfamethoxazole/trimethoprim, nalidixic acid, and fluoroquinolones. The patient responded to antibiotic therapy with fosfomycin. Multidrug-resistance may become prevalent in Salmonella infections in Japan, as shown in this first case of a patient infected with fluoroquinolone-resistant Salmonella.

Identification of a Chitin-Induced Small RNA That Regulates Translation of the tfoX Gene, Encoding a Positive Regulator of Natural Competence in Vibrio cholerae

The tfoX (also called sxy) gene product is the central regulator of DNA uptake in the naturally competent bacteria Haemophilus influenzae and Vibrio cholerae. However, the mechanisms regulating tfoX gene expression in both organisms are poorly understood. Our previous studies revealed that in V. cholerae, chitin disaccharide (GlcNAc)₂ is needed to activate the transcription and translation of V. cholerae tfoX (tfoX(VC)) to induce natural competence. In this study, we screened a multicopy library of V. cholerae DNA fragments necessary for translational regulation of tfoX(VC). A clone carrying the VC2078-VC2079 intergenic region, designated tfoR, increased the expression of a tfoX(VC)::lacZ translational fusion constructed in Escherichia coli. Using a tfoX(VC)::lacZ reporter system in V. cholerae, we confirmed that tfoR positively regulated tfoX(VC) expression at the translational level. Deletion of tfoR abolished competence for exogenous DNA even when (GlcNAc)₂ was provided. The introduction of a plasmid clone carrying the tfoR(+) gene into the tfoR deletion mutant complemented the competence deficiency. We also found that the tfoR gene encodes a 102-nucleotide small RNA (sRNA), which was transcriptionally activated in the presence of (GlcNAc)₂. Finally, we showed that this sRNA activated translation from tfoX(VC) mRNA in a highly purified in vitro translation system. Taking these results together, we propose that in the presence of (GlcNAc)₂, TfoR sRNA is expressed to activate the translation of tfoX(VC), which leads to the induction of natural competence.

Application of λ Red recombination system to Vibrio cholerae genetics: Simple methods for inactivation and modification of chromosomal genes

The lambda Red-based recombination system is very useful for genetic manipulation of some Gram-negative bacteria. Here we report simple procedures for the inactivation and modification of genes of interest on Vibrio cholerae chromosome using this recombination technique. For this purpose, a polymerase chain reaction (PCR) fragment carrying an antibiotic resistance cassette flanked by regions homologous to the target locus was electroporated into recipient V. cholerae strains expressing a highly proficient lambda Red recombination system. Two PCR procedures were tested to generate an amplification product carrying an antibiotic resistance cassette flanked by short (50 or 100 nt) or long (1000 nt) homologous extensions, which allowed successful disruption of four chromosomal loci (ctxB, toxT, lacZ, and recA). Our results suggest that 100-nt homology between the PCR product and the target gene is sufficient to stimulate the lambda Red-dependent recombination. To increase recombination efficiency, however, the PCR procedure should be used to generate a product with 1000-nt homologous extensions. Furthermore, we applied this gene replacement method to create lacZ reporter fusion to the target gene. Transcriptional fusion to the V. cholerae ctxA gene was constructed using a PCR product that contains the 100-nt homologous extension to ctxA on each side of the lacZ::cat cassette, and was shown to respond appropriately to a null mutation in the regulatory gene, toxT. Use of the techniques presented here should prompt rapid and efficient mutagenesis/modification of V. cholerae chromosomal genes.

Diagnostic Limitations to Accurate Diagnosis of Cholera

ABSTRACT The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.

Whole-Genome Analysis of Salmonella enterica Serovar Typhimurium T000240 Reveals the Acquisition of a Genomic Island Involved in Multidrug Resistance via IS1 Derivatives on the Chromosome

ABSTRACT Salmonella enterica serovar Typhimurium is frequently associated with life-threatening systemic infections, and the recent global emergence of multidrug resistance in S. enterica isolates from agricultural and clinical settings has raised concerns. In this study, we determined the whole-genome sequence of fluoroquinolone-resistant S. enterica serovar Typhimurium T000240 strain (DT12) isolated from human gastroenteritis in 2000. Comparative genome analysis revealed that T000240 displays high sequence similarity to strain LT2, which was originally isolated in 1940, indicating that progeny of LT2 might be reemerging. T000240 possesses a unique 82-kb genomic island, designated as GI-DT12, which is composed of multidrug resistance determinants, including a Tn2670-like composite transposon (class 1 integron [intI1, blaoxa-30 , aadA1, qacEΔ1, and sul1], mercury resistance proteins, and chloramphenicol acetyltransferase), a Tn10-like tetracycline resistance protein (tetA), the aerobactin iron-acquisition siderophore system (lutA and lucABC), and an iron transporter (sitABCD). Since GI-DT12 is flanked by IS1 derivatives, IS1-mediated recombination likely played a role in the acquisition of this genomic island through horizontal gene transfer. The aminoglycoside-(3)-N-acetyltransferase (aac(3)) gene and a class 1 integron harboring the dfrA1 gene cassette responsible for gentamicin and trimethoprim resistance, respectively, were identified on plasmid pSTMDT12_L and appeared to have been acquired through homologous recombination with IS26. This study represents the first characterization of the unique genomic island GI-DT12 that appears to be associated with possible IS1-mediated recombination in S. enterica serovar Typhimurium. It is expected that future whole-genome studies will aid in the characterization of the horizontal gene transfer events for the emerging S. enterica serovar Typhimurium strains.

Characterization of Isolates of Salmonella enterica Serovar Typhimurium Displaying High-Level Fluoroquinolone Resistance in Japan

ABSTRACT Strains of the multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium isolated in Japan were examined for high-level fluoroquinolone resistance. Since the first isolation in 2000 (described in reference 13), we have identified 12 human and 5 nonhuman isolates with high-level fluoroquinolone-resistance (ciprofloxacin MIC of 24 μg/ml or more). Most of these isolates shared some features including definitive phage type (DT12/193), resistance type (ACSSuTNCp; resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, nalidixic acid, and ciprofloxacin), and genotype on pulsed-field gel electrophoresis that were different from those of the MDR S. enterica Typhimurium DT104. Mutations in quinolone resistance-determining regions of gyrA and parC were also conserved in almost all of the isolates despite the absence of any apparent epidemiological relationships among cases. This suggests that a specific clonal group of the serovar Typhimurium with high levels of fluoroquinolone resistance is disseminating among animals and humans in Japan.

Characterization of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated in Japan

ABSTRACT A total of 221 isolates of multidrug-resistant Salmonella enterica serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.

Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

A multiplex PCR assay was developed based on atpA-sequence diversification for molecular identification of 3 major pathogenic Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. It specifically identified them from among 133 strains of various Vibrio species and other genera, and was applicable for testing seawater, suggesting its usefulness.

Antimicrobial Susceptibility of Shigella sonnei Isolates in Japan and Molecular Analysis of S. sonnei Isolates with Reduced Susceptibility to Fluoroquinolones

ABSTRACT We performed susceptibility testing with Shigella sonnei isolates from imported and domestic cases of infection in Japan during 2001 and 2002. Some S. sonnei isolates were resistant to nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole. Most of the nalidixic acid-resistant strains showed reduced susceptibility to fluoroquinolones but did not show fluoroquinolone resistance.