[Hygienic maintenance of water mist-generating equipment that couples directly to the tap].

OBJECTIVES: Water mist is usually generated using equipment directly connected to the water tap, as its installation is relatively easy. However, there is no legal regulation regarding the maintenance of this equipment, and the quality of the mist has not been sufficiently well investigated. In this study, we sought to establish methods that allow the hygienic maintenance of this equipment. METHODS: We monitored the use of the mist generating equipment in five of the 61 institutions in the jurisdiction of Ichinomiya Health Center, examined the resulting water quality, and tested for Legionella bacteria in the mist. If equipment was found to contain bacteria, the contaminated part was identified by counting the number of bacteria in the water after sequentially washing and disinfecting parts of the equipment. We also identified the predominant bacterial species. RESULTS: In the water mists from three of 5 institutions, the number of bacteria greatly exceeded that permitted for drinking-water, even though the residual chlorine level was >0.1 mg/l. However, no Legionella bacteria were detected. Brevundimonas species were predominant in the water mists at each institute. The hose was found to be the contaminated component in each case. CONCLUSION: Our findings suggest that the number of bacteria in the water mist exceeded the drinking-water quality standard, even with a residual chlorine level of >0.1 mg/l. This study also revealed the importance of the continued drainage of water, following suitable cleaning and disinfection for maintenance of the mist-generating equipment.

Association between aggregative adherence fimbriae types including putative new variants and virulence-related genes and clump formation among aggR-positive Escherichia coli strains isolated in Thailand and Japan.

Enteroaggregative Escherichia coli (EAggEC) are an important cause of diarrhea. Four types of AAF have been identified; however, their prevalence and association with virulence properties remain unclear. E. coli strains carrying the aggR gene as EAggEC that were isolated in Japan and Thailand (n = 90) were examined for AAF subunit genes, two toxin genes (pet/astA), and clump formation. The most prevalent AAF gene was hdaA (28%), followed by aafA (20%), aggA (12%), and agg3A (4%), as well as a putative new AAF sequence (25.6%). Retention status of the toxin genes and intensities of clump formation appeared to vary according to the AAF type.

An enrichment medium for increasing a very small number of vibrio parahaemolyticus cells to the detection limit of the loop-mediated isothermal amplification (LAMP) assay.

We developed an enrichment medium for use with the loop-mediated isothermal amplification (LAMP) assay (enrichment media + LAMP assay) to quickly increase a very small number of Vibrio parahaemolyticus cells to the detection limit of the assay. Thirty-nine different enrichment media were prepared based on evaluating 12 potential ingredients. From our assessment of the 39 media, enrichment medium #36, which contained 2% sodium chloride, 1% proteose peptone no. 2, 0.1% trehalose, 0.5% α-ketoglutaric acid, 0.25% pyruvic acid, and 0.5% yeast extract (pH 8.6), was found to be most effective at enhancing the proliferation of V. parahaemolyticus during incubation for 3 h at 40ºC. We compared the detection limits of the LAMP assay, the enrichment medium #36 + LAMP assay, and the cultivation method using bacterial cell and spiked shrimp sample tests. The detection limits of the LAMP assay, the medium #36 + LAMP assay, and the cultivation method were 10(3), 10(0)-10(-1), and 10(-1) CFU ml(-1), respectively. Enrichment medium #36 promoted a 10(3)- to 10(4)-fold increase in the bacterial population, and the detection limit of the enrichment media + LAMP assay was the same as that of the cultivation method.

Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand.

Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae- and bfpA-positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n=27) and Thailand (n=26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp-2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.

Intimin types determined by heteroduplex mobility assay of intimin gene (eae)-positive Escherichia coli strains.

We developed a quick genetic approach to screen variants of the intimin gene (eae) by using a heteroduplex mobility assay (HMA) that targets the 5' conserved region of eae. The eae variants were categorized into 4 major HMA types and 10 minor subtypes.

[Mutations of gyrA gene and parC gene in fluoroquinolone-resistant Escherichia coli isolates from sporadic diarrheal cases].

We studied 107 isolates of Escherichia coli O153 from sporadic diarrhea cases in Fukui, Toyama, Aichi, and Saga prefectures from 1991 to 2005 for antimicrobial susceptibility and mechanisms of fluoroquinolone resistance, based on standard disk diffusion. Of 12 drugs tested, ampicillin displayed resistance to 72.9% of isolates, streptomycin to 48.6%, tetracycline to 46.7%, sulfisoxazole to 46.7%, trimethoprim/sulfamethoxazole to 29.9%, nalidixic acid (NA) to 29.9%, and ciprofloxacin (CPFX) to 24.3%. Ten of 32 isolates resistant to 3-6 drugs and 16 of 18 isolates resistant to 7-10 drugs were resistant both to NA and CPFX. Mutations of amino acid in quinolone resistance-determining regions of gyrA and parC genes were detected in 24 isolates resistant both to NA and CPFX, and in 1 isolate resistant to NA. The former possessed a combination of double substitution (S83L and D87L) in GyrA and a single substitution (S80I) in ParC. Some 12 of 24 isolates possessed another single substitution (E84V or E84G or A108T) in ParC. The 25 isolates were classified into 4 types as follows. 1 isolate as type 1: GyrA (S83L) and ParC (S80I); 12 isolates as type 2: GyrA (S83L and D87N) and ParC (S80I); 8 isolates as type 3: GyrA (S83L and D87N) and ParC (S80I and E84G/S80R and E84V); and 4 isolate as type 4: GyrA (S83L and D87N) and ParC (S80I and A108T). In the relationship between amino acid mutations and minimal inhibitory concentrations (MIC) of fluoroquinolone, MICs of CPFX, ofloxacin, and norfloxacin showed 1microg/mL, 2microg/mL and 8microg/mL in type 1; 8 approximately 32microg/mL, 8 approximately 32microg/mL and 16 approximately 256microg/mL in type 2; and 32 approximately 256microg/mL' 32 approximately 128microg/mL and 128-->512microg/ mL in types 3 and 4. These results suggest that most of multiple-antimicrobial-resitant E. coli O153 isolates from sporadic diarrhea cases were resistant to fluoroquinolones and possessed mutations at gyrA and parC genes associated with fluoroquinolone resistance.

Typing of bfpA genes of enteropathogenic Escherichia coli isolated in Thailand and Japan by heteroduplex mobility assay.

We developed a rapid genetic approach for screening bfpA variants of enteropathogenic E. coli(EPEC) using a heteroduplex mobility assay (HMA). A total of 204 human EPEC strains were isolated in Thailand and Japan. Of 34 bfpA-positive EPEC strains, bfpA variants were classified into 5 HMA-types. Different HMA-types were found in EPEC of the same serotypes. The results suggest that HMA is a simple and easy method to analyze polymorphism of bfpA gene, and can be used in laboratories without large apparatus such as sequencers.

[Isolation of enteroaggregative escherichia coli (EAggEC) from patients with diarrheal disease in an outbreak case of overseas tour].

Twenty four patients out of 78 Cambodia tourists (31%) suffered from diarrhea and/or abdominal pain. Though the stools of 20 patients were examined in some local health centers and institutes, well-known pathogens were detected in only a low level and the cause of the outbreak remained unclear. We suspected the enteroaggregative Escherichia coli (EAggEC) as a cause of this outbreak. We examined E. coli strains isolated from stools of 8 patients (Okayama:7, Aichi:1) at first by the PCR method targeted both the aggR and the astA genes related to the virulence factors of EAggEC. As a result, the E. coli strains with positive aggR and/or astA genes were isolated from 8 patients. And the E. coli strains with positive both aggR gene and clump formation isolated from 3 patients adhered aggregatively to HEp-2 cells and accordingly identified as EAggEC. The plasmid profiles, PFGE patterns and drug resistance patterns of these EAggECs agreed completely. From these results, we concluded that at least 3 patients were infected with EAggEC of the same origin. Though we could not examine all samples from 20 patients, it is possible that the still uncommon EAggEC might be a cause of the outbreak. The E. coli strains with positive aggR gene did not always aggregatively adhered to HEp-2 cells. So we recommend to perform stepwise EAggEC screening tests by the PCR and the clump formation, and final confirmation test by the aggregative adhesion to HEp-2 cells.

[Epidemiological study of outbreaks and sporadic cases due to Vibrio parahaemolyticus--serotype O3:K6 in Aichi Prefecture, Japan, during 1988 and 2001].

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.

[Drug resistance of enterohemorrhagic Escherichia coli O157 and a possible relation of plasmids to the drug-resistance].

Antimicrobial susceptibility was examined using 89 enterohemorrhagic Escherichia coli O157 isolates obtained from diarrhea patients in Aichi Prefecture, Japan between June 1996 and June 1997. Among the 89 isolates, 15 (16.9%) were found to be resistant to 6 of 9 antibiotics examined. These 6 antibiotics were ampicillin (ABPC), cefaloridine (CER), chloramphenicol (CP), kanamycin (KM), streptomycin (SM), and tetracycline (TC). Among the 15 drug-resistant isolates, 7 were resistant to 4 drugs (ABPC, CER, SM, TC), 3 were resistant to 3 (ABPC and 2 of CER, SM, TC), 2 were resistant to 2 (SM, TC), one each to KM or SM. Another isolate showed resistance to 5 drugs (ABPC, CP, KM, SM, TC). Selected 13 drug-sensitive and selected 12 multi-drug resistant isolates were tested for the presence of plasmids. All of the drug-sensitive isolates had 54 MDa plasmid and the majority (8/13) had 2.0 MDa plasmids, whereas; all of the drug-resistant isolates except one (1/12) had 54 MDa plasmid and the majority had 8.0 MDa (9/12) and 4.2 MDa (11/12) plasmids. The first transformation test revealed that plasmids of 8.0 MDa (3/4) and 46 MDa (1/4) were transferred to a donor cell with ABPC resistance. 54 MDa plasmid was transferred to a donor cell with both of ABPC and TC resistance. In the second transformation test, only the 8.0 MDa plasmid was confirmed to be transferred to a donor cell with ABPC resistance. Accordingly, it was indicated that the ABPC resistant gene was carried on 8.0 MDa plasmid, and it was suggested that resistant genes for ABPC and TC, and ABPC were carried on 54 MDa, and on 46 MDa plasmids, respectively.

[Presence of the genes regarding adherence factors of Escherichia coli isolates and a consideration of the procedure for detection of diarrheagenic strain].

Diarrheagenic Escherichia coli are differentiated from non-pathogenic members with enterotoxin production, enteroinvasiveness and serotyping. However, the serotypic members are rarely sufficient to reliably identify a strain as diarrheagenic on E. coli. Recently, there are many definite articles which the adhesive E. coli strain against intestinal epithelial cells is enterovirulent. In this study, 1,748 E. coli isolates of diarrheagenic and non-diarrheagenic categories which belonged to EHEC, ETEC, EIEC EPEC and non-EPEC were examinated by PCR method for the presence of eaeA, aggR and bfpA regarding adherence factor genes, and astA of EAST1. The strains examined were recognized to variable carrying geno-patterns, and a large number of EHEC, EPEC and non-EPEC had carried either eaeA or aggR genes. In EHEC isolates, a carrying pattern with the most high frequency was only eaeA, and this type was recognized in the isolates of serotype O157, O26 and O111. EPEC and non-EPEC isolates were recognized eaeA or aggR which harboring with astA or not. Of 508 EPEC isolates from human, a total of 137 isolates (27.0%) carried aggR, and a total of 74 isolates (14.6%) had eaeA, while of the 91 isolates from non-human were recognized aggR and eaeA with 2.2% (2 isolates) and 12.1% (11 isolates), respectively. Also, of 266 non-EPEC isolates from human, a total of 16 isolates (6.0%) carried aggR, and a total of 58 isolates (21.8%) had eaeA. On the other hand, 22 (7.0%) of 316 isolates examined from non-human had eaeA, however no isolate had aggR. Thirteen isolates of EIEC and 218 ETEC isolates were screened, and only 6 ETEC isolates had either eaeA or aggR. The astA gene was recognized in the isolates of all categories, and ETEC strains had more frequently. The bfpA gene was recognized with more frequently in a serotype O157: H45, which is obtained from human with diarrhea, however, this strain was not recognized a member of the EPEC serotype. There is no diagnostic system for the strain of E. coli that cause diarrheal diseases, therefore more laboratories are unable to identify them. The authors had confirmed which PCR technique is a useful simple and rapid method for the detection of adherence factor genes on E. coli strains. From the these results, we showed a differentiation method using PCR technique which have relation with adherence factor, enterotoxin-production and invasiveness, and we firmly believe that application of the procedure is a reasonable and useful method for the identification of diarrheagenic E. coli.