Summary: Vibrio cholerae is an aetiological agent of cholera that inhabits marine and estuarine environments. It can survive harsh environments by entering the viable but non‐culturable (VBNC) state, but the related changes in gene expression have not been described. Here, we experimentally induced the VBNC state in V. cholerae O1, by incubation in artificial seawater at 4°C. Bacterial cells that were incubated for 70 days retained their membrane integrity and were pathogenic, colonizing the gut of iron‐dextran‐treated mice, even though they formed no colonies on tryptic soy agar (TSA) or TSA amended with pyruvate. We therefore used this stage of cells as the VBNC bacteria. We compared the global transcription pattern of the VBNC cells with that of stationary‐phase cells grown in rich medium. A total of 100 genes were induced by more than fivefold in the VBNC state, and the modulated genes were mostly those responsible for cellular processes. Furthermore, real‐time RT‐PCR analysis verified the changes in the expression levels, showing that the VC0230 [iron(III) ABC transporter], VC1212 ( polB ), VC2132 ( fliG ) and VC2187 ( flaC ) mRNAs were increased in the non‐culturable state. Thus, these genes may be suitable markers for the detection of VBNC V. cholerae . To our knowledge, this is the first report of a comprehensive transcriptome analysis of V. cholerae in the VBNC state. The significance of this gene expression profile compared with those of in vivo isolates and non‐stressed bacteria (culturable in vitro ) is its potential to provide information about the public health risk from dormant bacteria.

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human β-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a ∼2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-κB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-κB activation and thus plays an important role in up-regulation of the innate immune response.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997; 5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assays, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx₁ and stx₂, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.

Campylobacter jejuni is a microaerophilic bacterium that causes diarrhea in humans. The first step in establishing an infection is adherence to a hos

An outbreak caused by salted salmon roe contaminated with enterohemorrhagic Escherichia coli O157 occurred in Japan in 1998. Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated. Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water. Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state. Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity. O157 that originated in roe, but not patients, killed mice and was isolated from the intestine. However, these isolates had become sensitive to NaCl. O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive. We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased. If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection. Finally, the establishment of a simple recovery system for VNC cells should be established.