Search

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

UNLABELLED Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified in clades by single nucleotide polymorphisms (SNPs), but this analysis requires significant laboratory effort. As the distribution of insertion sequence (IS) 629 insertions has been reported to be biased among different clades, O157 isolates can be putatively classified in clades by comparison with an IS629 distribution database. A database of the IS629 distribution in O157 strains isolated in Chiba Prefecture and their classification in clades was determined by SNP analysis and IS-printing, an easy and quick analytical tool for IS629 in the O157 genome. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing. These strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. Concordance Ratios were calculated, which compared the number of strains putatively classified in a clade by Relative Likelihood to the number of strains classified in that clade by SNP analysis. For the Fukuoka and Yamagata strains, the Concordance Ratios for clades 3, 6 and 8 were 97-100%, for clade 7 about 88%, and for clades 2 and 12 over 90%. In conclusion, O157 clade 2, 3, 6, 7, 8 and 12 strains could be putatively classified by IS-printing. SIGNIFICANCE AND IMPACT OF THE STUDY This study demonstrated that enterohemorrhagic E. coli O157 (O157) strains could be putatively classified in clades using an IS-printing system. IS-printing was previously developed as a relatively quick and easy tool for analysis of insertion sequence 629 in the O157 genome. Since most local government public health institutes in Japan carry out IS-printing for early detection of O157 outbreaks, these data should be useful for putative classification of O157 strains in each area.

Recently, there has been a marked increase in the number of reports of fluoroquinolone-resistant Campylobacter jejuni and Campylobacter coli. The aim of this study was to evaluate the prevalence of antimicrobial resistance and its genetic determinants in Campylobacter species isolated from meat and human subjects in Fukuoka Prefecture, Japan. Between 2011 and 2013, 55 and 64 isolates were collected from meat (chicken meat and beef liver) and humans, respectively, in this prefecture. Antimicrobial susceptibility tests were conducted using the agar dilution method in accordance with the Clinical and Laboratory Standards Institute guidelines, using the following 11 antimicrobial agents : cephalexin, cefoxitin, nalidixic acid, ciprofloxacin, levofloxacin, tetracycline, minocycline, ampicillin, streptomycin, kanamycin and erythromycin. The susceptibility rates of the isolates to three quinolones (nalidixic acid, ciprofloxacin, levofloxacin) were 43.7%, 41.2%, 40.3%, respectively. All the isolates were multidrug resistant. Whereas 46.9%-51.6% of the human isolates were resistant to one or more of the quinolones, only 32.7%-34.5% of the meat isolates were resistant to one or more of the drugs. DNA sequencing showed that of the 50 quinolone resistant isolates 44 had position 86 isoleucine (Ile) substituted for threonine (Thr) in the GyrA protein (Thr86Ile). This amino acid substitution resulted from ACA to ATA and ACT to ATT mutations of codon 86 in C. jejuni and C. coli, respectively. Furthermore, two of the four C. jejuni isolates lacking the Thr86Ile mutation had combined Ser22Gly-Asn203Ser substitutions, while the remaining two isolates had combined Ser22Gly-Asn203Ser-Ala 206Val substitutions. These four isolates also had cmeABC sequences that differed from the quinolone sensitive C. jejuni ATCC33560(T) strain. In conclusion, C. jejuni and C. coli have relatively high quinolone resistance, and are resistant to other antibiotics. The new combination of amino acid substitutions in the GyrA protein could pose a potential threat to public health in Japan.

In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f-producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli.

We evaluated the sensitivity and specificity of an immunochromatography kit, Single-path Emetic Tox Mrk (Merck), which targets a marker protein for the detection of Bacillus cereus that produces emetic toxin. Strains were isolated after outbreaks of food poisoning, and from retail prepared foods and food products. The strains were examined for the presence of the emetic toxin-synthetase gene by PCR. All 58 emetic strains isolated from the food poisoning cases showed a positive reaction in the immunochromatography kit. No emetic strains gave false negative result. Among 47 non-emetic strains, only two strains isolated from the food poisonings and one strain isolated from food products showed a false positive reaction in the test. We concluded that this method has high sensitivity and specificity. The test can be used for detection of emetic toxin-producing B. cereus not only from food poisoning cases, but also in food products.

Enterohemorrhagic Escherichia coli serovar O157 (O157) strains with highly similar pulsed-field gel electrophoresis (PFGE) patterns were isolated in Japan during 2007 and 2008. Several genetic features related to O157 evolution were investigated to indicate whether homoplasy might have contributed to the highly similar PFGE patterns in these strains. The O157 strains were classified in lineage I/II, as defined by a lineage-specific polymorphism assay-6 with an atypical allele in Z5935 (code: 231111). Analysis of the insertion sites of stx2 phage in these strains showed that the sites were “occupied” in yehV and “intact” in wrbA, indicating that the strains were derived from “Cluster 1” of “Subgroup C.” When a specific single-nucleotide polymorphism in ECs2357 in clade 8 strains was investigated, all of the strains in the present study were confirmed to be clade 8 strains. These results indicated that the O157 strains in this study had common genetic features, suggesting that the highly similar PFGE patterns of these strains were not due to homoplasy. Because no common source of these strains could be identified in 2007 to 2008 in Japan, these strains may have emerged from a unique O157 clade 8 clone and then spread by dissemination in Japan.

For infection control in pediatric hospitals, we investigated the risk of pertussis and diphtheria infections among pediatric healthcare workers. Forty-nine Japanese pediatric healthcare workers in 12 general hospitals were screened for antibodies of pertussis toxin (PT), filamentous hemagglutinin (FHA), and diphtheria toxin (DT). The seropositive rates of anti-PT IgG (protective level, > 10 U/mL), anti-FHA IgG (> 10 U/ mL), and anti-DT (> 0.11 U/mL) were 50, 82, and 59%, respectively. During this survey period (Oct. 2003-Feb. 2004), 16 (33%) of the healthcare workers were in contact with pertussis-infant (s). However, all culture and PCR tests for Bordetella pertussis were negative. One of the 16 exposed healthcare workers, a male pediatrician, had serological evidence of a pertussis infection, but no disease symptomatic of pertussis. Our observations indicate that i) 50 and 41% of Japanese pediatric healthcare workers were seronegative for pertussis (anti-PT IgG) and diphtheria antibodies, respectively, and ii) although the healthcare workers had a high rate of contact with pertussis-infant (s), the infection rate was low. For pertussis and diphtheria infection control in pediatric hospitals, it is important for healthcare workers to be aware of their own protection levels against these diseases.

Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) that causes more than 50 cases of foodborne illness in Japan each year. For quantitatively assessing the presence of K. septempunctata spores in the causative fish at food poisoning outbreaks, both a direct observation method using microscopy and a quantitative real-time PCR (qRT-PCR) method are officially accepted in Japan. However, lower correlations have been often noticed between the number of spores counted using the direct observation method and the DNA amount determined using the qRT-PCR method. To elucidate the cause of this discrepancy, we observed muscle tissues of infected olive flounders with K. septempunctata by transmission electron microscopy. The images demonstrated unsynchronized development of K. septempunctata spores in plasmodia found within myofibers; in other words, the plasmodium contained not only developed spores with completed shell valves but also developing spores (sporoblasts) composed of spore-forming cells without shell valves. Furthermore, the ratio between developed spores and sporoblasts varied at different parts of muscles. The direct microscopic observation method could count developed spores, whereas the qRT-PCR method could quantify the amount of not only spores but also sporoblastic cells regardless of the cellular development and differentiation. Considering that the food toxicity caused by K. septempunctata is induced by viable spores passing through the gastric environment, the direct observation method counting only developed spores is better than the qRT-PCR method for assessing the cause of foodborne illness at the outbreak as well as the risk of human illness in monitoring surveys of aquacultured or natural-water fish.

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.

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx 2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx 2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA O26, irp 2, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.

To estimate the prevalence and distribution of salmonellae, especially Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis), in Western Japan, an investigation was conducted of the chicken industry and environmental sources between 1995 and 1998. Salmonellae were isolated from 34 of 90 samples (37·8 %) of raw chicken parts, 34 of 98 faecal samples (34·7 %) at 35 broiler farms, 11 of 59 samples (18·6 %) of liquid eggs, and from 71 of 272 samples (26·1 %) of swab specimens from equipment and cracked or faecally soiled shell eggs at the processing facilities. Salmonellae, including S. Enteritidis, were also isolated from swab samples of henhouses associated with one of the shell-egg processing facilities (11 samples out of 55, 20 %). In the broiler meat production environment, S. Infantis was dominant. Twenty-two of 36 sewage samples (61·1 %) and 16 of 72 samples (22·2 %) taken from 5 rivers contained salmonellae including S. Enteritidis. S. Enteritidis isolates were analysed with pulsed-field gel electrophoresis using enzyme Bln I. Thirty-four isolates from shell-egg processing facilities and henhouses, obtained over several years, had the same pulsed-field profile as isolates obtained from four individual outbreaks that occurred in this location in 1997. One of the clonal lines of S. Enteritidis, among multiple serovars of salmonellae in the environment, was thought to have distributed in reservoirs, laying hens, for several years, and continued to cause outbreaks in this area.

We have evaluated the ability of a new genotyping method for Campylobacter jejuni based on restriction fragment length polymorphisms using random chromosomal gene probes. DNAs from C. jejuni strains digested with each of three restriction enzymes, Hha I, Hae III, and Hpa II, were analyzed by Southern hybridization using each of two unrelated cosmid clones, P14 and P15 (respectively containing 30- and 35-kb genomic DNA fragments of C. jejuni strain OH4384). The method reported provides a stable and discriminating means for identifying C. jejuni strains and should be useful for epidemiological analyses.

We investigated the conditions of enrichment cultures preceding the immunomagnetic separation (IMS) procedure to detect Escherichia coli O157 (E. coli O157) from human stool specimens in routine laboratory examinations. Samples were made by adding either of the three selected strains of E. coli O157 to stools from three healthy human subjects in three different doses. The enrichment cultures were done for 18 hours at 37 degrees C or 42 degrees C, using five different media such as trypticase soy broth (TSB). TSB containing cefixime, tellurite and vancomycin, modified EC broth (mEC), mEC containing novobiocin (N-mEC) and BGLB. The IMS procedure following enrichment culture increased the detection rate of E. coli O157, irrespective of the kinds of the media and the temperatures. It recovered E. coli O157 in 42 samples out of 90, while only 31 samples were positive when the IMS was not applied. The N-mEC showed the best recovery rate of the five enrichment media, and it was the only media that recovered the E. coli O157 Gunmma 298 strains at a level of 2-3 cells per ml. In 73 stool samples collected from probable patients with E. coli O157 infection and subjects who made close contact with the patients, positive results were obtained in six samples with the N-mEC enrichment followed by the IMS procedure, while only three samples were positive by the direct isolation culture. It was concluded, therefore, that, in routine laboratory examinations of E. coli O157 from human stools, the N-mEC enrichment culture for 18 hours followed by the IMS procedure is a sensitive method even when the dose of E. coli O157 in the stool is minimal.

A total of 11 strains of Escherichia coli (E. coli) belonging to serogroup O157 were examined for the expression of long-chain lipopolysaccharide (LPS) and major outer-membrane proteins (OMPs) by means of SDS-PAGE. The strains belonged to either one of four different flagellar (H) types or did not express flagella. Four of the eleven strains carried genes encoding Shiga-like toxins (SLTs). All the strains exhibited one of four LPS profiles, designated A, B, C or D. Electron microscopic analysis with the freeze-substitution technique demonstrated the differences in the cell surface structures of strains with each LPS profile. Strains with LPS profile A, B or C had layers of thin fibers 10, 20 and 20 nm long, respectively, on the outer membrane but strains with LPS profile D had no such structure. An analysis of the OMPs showed that all the strains had one of four OMP profiles, designated I, II, III or IV. Both LPS and OMP profiles were dependent on H-serotypes, and the combination pattern of LPS and OMP profiles of the strains was unique for each H-serotype. These data support the existence of heterogeneous groups of O157 strains.

We have studied 167 epidemiologically unlinked strains of enterohemorrhagic Escherichia coli O157 (O157) isolated from patients with hemorrhagic colitis (HC), 87 strains from patients not with HC and 35 asymptomatic carriers (the not HC group), and differentiated these strains into clades using high resolution melting analysis. In addition, lineage analysis was carried out using lineage-specific polymorphism assay-6 and analysis of the distribution of IS629 insertion sites was carried out using IS-printing. Most strains were correctly clustered by minimum spanning tree analysis, and strains in the major clades showed linkage disequilibrium, confirming the clade differentiation in this study. The number of O157 strains in the different clades isolated from HC patients and the not HC group was significantly different (Chi square test, P < 0.05), indicating that strains in different clades had different pathogenicities for hemorrhagic colitis. Pairwise comparison of the number of strains in different clades isolated from HC patients indicated that clade 12 strains were weakly pathogenic for HC. Stx2 titers and the number of strains carrying an stx2 gene were significantly different for different clades (Kruscal–Wallis test and Chi square test, P < 0.05). Pairwise comparison of the Stx2 titer and the number of strains with an stx2 gene in different clades showed that the weak HC pathogenicity of clade 12 strains would be related to the low number of clade 12 strains with an stx2 gene and the low Stx2 production in those strains. Interestingly, the Stx2 titer and the prevalence of strains with an stx2 gene were significantly higher among clade 6 and 8 strains compared to clade 7 strains, although clades 6, 7, and 8 were all in lineage I/II. These results were discordant with the O157 evolutionary model, suggesting that insertion of an stx2 gene in lineage I/II strains after divergence of each clade.

A mixture of five nitroarenes, 3‐nitrofluoranthene, 2‐nitrofluorene, 2,7‐dinitrofluorene, 2,7‐dinitro‐9‐fluorenone and 9‐nitroanthracene, was administered to ddY male mice by intraperitoneal injection. At 48 h after treatment with the mixture, the frequency of micronucleated reticulocytes increased significantly (p < 0.01), and was 0.55, 0.60, 0.85 and 0.77%, at 50, 100, 200 and 400 mg/kg, respectively. However, the frequency of micronucleated reticulocytes did not increase over control values after intraperitoneal administration of each nitroarene alone at 80 mg/kg.

飲用井戸水におけるX-GAL-MUG法と上水試験方法に採用されているLB-BGLB法およびMMO-MUG法との大腸菌群検出についての比較検討を行い以下の結果がえられた.1. X-GAL-MUG法と, LB-BGLB法の大腸菌群検出率の一致率は92.6%であった.2. X-GAL-MUG法とMMO-MUG法の大腸菌群検出率の一致率は91%であった.また推定大腸菌の検出率の一致率は100%であった.以上の結果から, X-GAL-MUG法はLB-BGLB法およびMMO-MUG法と比較してもなんら遜色なく使用できるものと考えられる.

Canine leptospirosis, which is caused by infection with pathogenic Leptospira species, occurs worldwide, but information regarding the causative Leptospira serotypes and genotypes and their effects on virulence in dogs remains limited. Monitoring acute leptospirosis in dogs as sentinels can also aid in estimating the risk of human leptospirosis, particularly when the disease is rare, as it currently is in Japan. Among 283 clinically suspected cases of leptospirosis diagnosed from August 2007 to March 2011 in Japan, 83 cases were laboratory diagnosed as leptospirosis by blood culture, a rise in antibody titres in paired sera using a microscopic agglutination test (MAT) and/or DNA detection using flaB-nested PCR. The infected dogs comprised hunting dogs (31 dogs) and companion animals (50 dogs) and two unknown; 63.4 % of the infected dogs were males. The mortality rate was 53.2 %. A rise of at least fourfold in MAT titre was detected in 30 dogs whose paired serum samples were obtained, and the predominant reactive serogroup was Hebdomadis (53.3 %), followed by Australis (16.7 %) and Autumnalis (16.7 %). Leptospira interrogans was isolated from 45 dogs of the following serogroups: Australis (16), Autumnalis (six), Canicola (one), Hebdomadis (21) and Icterohaemorrhagiae (one). All of these serogroups caused lethal infections (57.1-100 %). Genetic heterogeneity was demonstrated in serogroups Australis, Autumnalis and Hebdomadis by multilocus sequence typing (MLST) and/or RFLP analysis based on PFGE. In serogroup Hebdomadis, each genotype determined by MLST had a unique mortality rate in the infected dogs. Although classic canine leptospirosis is associated with serovars Canicola and Icterohaemorrhagiae, serogroup Hebdomadis has become the predominant serogroup causing high mortality in Japan. This study suggests that the virulence of members of serogroup Hebdomadis in dogs may be associated with the genotypes in this serogroup.

The distribution of insertion sequence (IS) 629 among strains of enterohemorrhagic Escherichia coli serovar O157 (O157) was investigated and compared with the strain lineages defined by lineage specific polymorphism assay-6 (LSPA-6) to demonstrate the effectiveness of IS629 analysis for population genetics analysis. Using pulsed-field gel electrophoresis and variable-number tandem repeat typing, 140 strains producing both VT1 and VT2 and 98 strains producing only VT2 were selected from a total of 592 strains isolated from patients and asymptomatic carriers in Chiba Prefecture, Japan, during 2003-2008. By LSPA-6 analysis, six strains had atypical amplicon sizes in their Z5935 loci and five strains had atypical amplicon sizes in their arp-iclR intergenic regions. Sequence analyses of PCR amplified DNAs showed that five of the six loci used for LSPA-6 analysis had tandem repeats and the allele changes were due to changes in the number of tandem repeats. Subculturing and long-term incubation was found to have no detectable effect on the lineages defined by LSPA-6 analysis, demonstrating the robustness of LSPA-6 analysis. Minimum spanning tree analysis reconstruction revealed that strains in lineage I, I/II, and II clustered on separate branches, indicating that the distribution of IS629 was biased among O157 strains in different lineages. Strains with LSPA-6 codes 231111, 211113, and 211114 had atypical amplicon sizes and were clustered in lineage I/II branch, and strains with LSPA-6 codes 212114, 221123, 221223, 222123, 222224, 242123, 252123, and 242222 had atypical amplicon sizes and clustered in lineage II branches. Linkage disequilibrium was observed in strains in every lineage when the standardized index of association was calculated using IS629 distribution data. Therefore, the distribution analysis of IS629 may be effective for population genetics analysis of O157 due to the biased IS629 distribution among strains in the three O157 lineages.

Two types of intoxication, caused by intake of carp gallbladder or mainly its muscle (Sasimi), have been reported. In order to identify the substances responsible for these carp intoxications, we initially examined the distribution of toxin in normal carp viscera and muscle by means of an acute toxicity test in ddY male mice (19-20g). It was found that the toxic substances were in gallbladder and liver-pancreas, especially bile, but not muscle. Thus, intoxication by carp gallbladder could be attributable to components normally present in carp bile. Based on the LD50 values, it was concluded that essentially all of the bile toxicity can be explained by the toxicity of cyprinol sulfate, a major component of carp bile. Furthermore, the acute toxic symptoms induced by raw bile and cyprinol sulfate were similar. On the other hand, from the result of a 50% hemolysis test, it was found that some bile acids contained in raw bile were more hemolytic than cyprinol sulfate. Taking these results into consideration, it was suggested that carp intoxication resulting from ingestion of the gallbladder could be caused by not only cyprinol sulfate but also other components, especially bile acid.

The mutagenicity of SRM 1649 and 1650 was tested in the presence of rat liver S9 mix which was induced by polychlorinated biphenyl (PCB) or by the combination of phenobarbital and 5,6-benzoflavone. The S9 mix induced by PCB activated benzo[ a ]pyrene strongly. The S9 mix induced by phenobarbital-5,6-benzoflavone activated the complex mixtures to approximately the same extent as that induced by PCB. This finding indicates that phenobarbital-5,6-benzoflavone instead of PCB may be suitable as an inducer under some conditions. The preincubation procedure for the mutagenicity test was performed by preincubating the test compound, S9 mix and bacteria for 20 min in a water bath. This procedure was as effective as the plate incorporation test.

3,6-Dinitrobenzo[a]pyrene, a new mutagen, was detected in airborne particulates collected in Santiago (Chile). The quantity of the compound in the airborne particulates was very small, accounting for 0.01 μg/g of total particulates (0.002 ng/m3 of air) at the lowest concentration. It was found that 3,6-dinitrobenzo[a]pyrene is readily decomposed by UV irradiation at 312 nm. The decomposed product was identified as 3-nitrobenzo[a]pyrene-6-quinone by means of mass spectrometry and proton nuclear magnetic resonance analysis. The mutagenicity of 3,6-dinitrobenzo[a]pyrene was 137,000 revertants/nmole for Salmonella typhimurium strain TA98, less than that for strain TA98/1,8-DNP6, an acetyltransferase-deficient mutant, and more than that for strain YG1024, an acetyltransferase-rich mutant

The isolation and characterization of methicillin-resistant Staphylococcus aures (MRSA) strains from the bilateral nares of nurses and their gowns are described. MRSA strains could be isolated from eigth of fifty bilateral nares of nurses and two of their gowns. Ten MRSA strains were typed using coagulase typing, and divided into two types, coagulase II and III. In this study, we found a new group (producing toxic shock syndrome toxin -1, coagulase III and staphylococcal enterotoxin C) in Japanese MRSA. Furthermore, we confirmed that MRSA strains originating from bilateral nares of three nurses were identical and two strains isolated from the left naris of one nurse and her gown were also identical by pulsed-field gel electrophoresis.

We studied 101 strains of Enterohemorrhagic Escherichia coli (EHEC) O26 isolated from diarrhea patients in six prefectural institutes of public health in Japan during June 1996 and December 1997 and tried to establish an isolation medium for EHEC O26. None of the 101 EHEC O26 strains fermented rhamnose; Whereas all of the other EHEC including O157 and non-EHEC (166 strains) fermented rhamnose except 1 strain of non-EHEC. All of the randomly selected EHEC O26 (14 strains of O26:H11.2 strains of O26:H-) showed a very high resistance to potassium tellurite (Minimal Inhibitory Concentration (MIC) > or = 50 micrograms/ml), whereas all of the randomly selected non-EHEC (26 strains) but 1 showed a high sensitivity (MIC < or = 6.25 micrograms/ml) to this compound. On the basis of these results, we developed a Rhamnose MacConkey (RMAC) medium in which lactose in the MacConkey medium was replaced by rhamnose, and Cefixime-Potassium Tellurite-RMAC (CT-RMAC) medium in which Cefixime (0.05mg/l) and Potassium Tellurite (25mg/l) was added to RMAC for the isolation of EHEC O26 strains. We then evaluated the specifcity of these selective media by growing a selected number of O26 (24 strains) and 9 selected strains of bacteria. All of the EHEC O26 strains generated rhamnose non-fermented colonies (white color) on both media. In contrast to the EHEC O26, the vast majority of E. coli strains (166/167 = 99.4%) other than EHEC O26 were theoretically assumed to generate red colonies on the RMAC medium because of their rhamnose fermenting character and most of them were assumed not to grow on CT-RMAC medium because of their sensitivity to potassium tellurite. These findings and results indicate that EHEC O26 can be easily distinguished from other strains of E. coli including O157. Although EHEC O26 strains showed somewhat poor growth on CT-RMAC medium compared with that on RMAC medium, these O26 showed almost the same degree of growth on CT-RMAC as they showed on DHL media. The results of the present study demonstrated that the use of RMAC and CT-MRAC media for the isolation of EHEC O26 is very reliable and efficient with RMAC having good sensitivity and CT-RMAC having a better specificity for the isolation of this strain of EHEC.

To determine the extent of genetic diversity of Salmonella choleraesuis subspecies choleraesuis serovar Infantis and whether environmental isolates were similar or identical to human isolates, a total of 110 isolates from humans, broiler samples, egg production facilities, riverwater, sewage, and chicken meat were analyzed epidemiologically by pulsed-field gel electrophoresis. While the isolates showed 35 distinct pulsed-field profiles, none had the genotype of the human isolates. One pulsed-field profile was shared by 43 (39%) of the 110 isolates. These results indicate that relatively fewer clonal lines of S. serovar Infantis had spread widely while multiple clonal lines, including the strain involved in the outbreak, exist in Western Japan.

We evaluated the usefulness of bacterial agglutination antibodies for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 infections. We examined 50 serum samples from 50 control children (whiout diarrhea 31, with diarrhea 19), 24 samples from 8 diarrhea cases due to O157:H7, 37 samples from 14 cases of hemolytic uremic syndrome (HUS) for antibodies to heat-killed E. coli E32511 (O157:H.-) strain using the bacterial agglutination technique. Of the control sera all but one (x80) showed 20 > or = in the antibody. All the diarrhea patients due to O157:H7 showed a significant rise (x160-x5120) of the titers in the sera at 5-7 days on illness, after that the titers fell rapidly. Significant antibody rise (x160-x5120) was detected in twelve out of 14 HUS patients at the early stage of the illness which fell in the convalescent phase. The assay appeared to be a useful serodiagnostic technique because of its easiness and simplicity as well as because of its high sensitivity and specificity.

To elucidate a relationship between lung cancer and tumor induction of environmental chemicals, the presence of 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P) in 137 Japanese (97 male and 40 female), and 21 Chinese lung specimens was examined by HPLC, and GC-MS and environmental exposure discussed. Mortality due to lung cancer in Fuyuan County, China, is much higher than that of other cancers. We investigated 21 patients who were residents of Fuyuan County. All were female aged 28-64 years and were non-smoking farmers and cooks. The histological features of the tumors were adenocarcinoma, squamous cell carcinoma and small cell carcinoma. The incidence of cancer was due to inhaling soot from the combustion of coal used for cooking and indoor heating. The polycyclic aromatic hydrocarbons, chrysene, benzo[k]fluoranthene, B[a]P, benzo[g,h,i]perylene and pyrene, but not their nitrated derivatives, were detected in substantial amounts in the resected lungs. These mutagens and carcinogens normally originate in combustion products of coal, and are discussed as the possible initiators of the tumors in the lungs of these patients. All the Japanese lung specimens were also obtained from non-smokers. The concentrations of 1-NP averaged 21.3 +/- 12.4 and 5.9 +/- 2.4 pg/g of Japanese and Chinese samples respectively. The concentrations of B[a]P averaged 180.2 +/- 103.7 and 608.7 +/- 477.1 pg/g of Japanese and Chinese samples respectively. Thus, Japanese and Chinese lung specimens were mainly contaminated by 1-NP and B[a]P, respectively. Typical tissues from a carcinomatous human lung were examined. The patient was a 64-year-old Japanese male non-smoker and farmer who had raised chickens over a period of 40 years. The histological features of the tumor were those of keratinizing squamous cell carcinoma. 1-Nitropyrene, 1-nitro-3-hydroxypyrene, 1,3-dinitropyrene and chrysene were detected at concentrations of 0.11, 0.036, 0.095 and 0.16 ng respectively per gram of lung tissue. This cancer was due to long-term exposure to the combustion products of heavy oil used in a chicken house.

3,9- and 3,7-Dinitrofluoranthene (3,9- and 3,7-DNF), 3-nitrofluoranthene (3-NF) and benzo[a]pyrene (B[a]P) were tested for pulmonary carcinogenicity by intrapulmonary implantation of the compounds into rat lung. These chemicals were given in various doses as suspensions in beeswax-trycaprylin and the animals were observed for 100 weeks. The control group received no drugs. The incidences of lung tumors were 19/21 (90.5%), 7/10 (70%) and 1/10 (10%) in rats treated with 200, 100 and 50 micrograms of 3,9-DNF, 4/9 (44.4%), 3/10 (30%) and 0/10 (0%) in rats treated with 200, 100 and 50 micrograms of B[a]P, 12/22 (54.5%) in rats treated with 200 micrograms of 3,7-DNF and 1/20 (5%) in rats treated with 1000 micrograms of 3-NF respectively. No lung tumors were found in control rats. The incidence of lung tumors induced by 3,9-DNF was twice as high as that induced by B[a]P, when the equivalent dose levels of the two compounds were compared. Histologically, most of the tumors induced by 3,9- and 3,7-DNF and B[a]P were squamous cell carcinomas.

Of the dinitroarenes, dinitropyrene and dinitrofluoranthene are classified as extraordinary mutagens, and small amounts of them are emitted by incomplete combustion of fossile fuel; these compounds are mostly coexistent with mononitroarenes in particles from diesel emission, kerosene heater and city gas emission, and airborne pollutants (see review by Tokiwa and Ohnishi, 1986).

Nitroarenes are nitrated by-products of the reaction of various environmental agents with polycyclic aromatic hydrocarbons (PAHs); the presence of nitroarenes in the environment appears to result mainly from manTs activities. Most nitroarenes are strongly associated with mutagenicity, genotoxicity, and carcinogenicity. Some nitroarenes, which are potent mutagens in bacteria, have been reported to be carcinogenic at various sites in animals [see review by Tokiwa and Ohnishi, (7)], suggesting a correlation between mutagenicity and carcinogenicity, at least in nitroarenes. However, there is no clear evidence that indicates an epidemiological relation with human carcinogenesis. On the other hand, it has been found that most nitroarenes are induced from diesel exhaust emissions, wood burning, kerosene heaters, city gas, and liquified petroleum gas, and are widespread in the environment (8). In relation to cigarette smoking, some nitroarenes were also found to be induced when cigarette smoke condensates were treated with nitric acid or Chinese cabbage pickles (4).

Two dinitrofluoranthenes (DNFs) derived from 3-nitrofluoranthene were purified to over 99%, and tested for tumorigenicity in F344/DuCrj male rats. Rats were inoculated s.c. with 0.05 mg 3,7- or 3,9-DNF twice a week for 10 weeks. All 21 rats given 3,7-DNF and 10 of 11 rats (91%) given 3,9-DNF developed tumors at the injection site by 48 weeks after the first injection. Twenty of the 21 tumors induced by 3,7-DNF and seven of the 10 tumors induced by 3,9-DNF were classified as malignant fibrous histiocytoma, but one tumor in the 3,7-DNF-treated group and three tumors induced by 3,9-DNF showed typical features of rhabdomyosarcoma. The first tumors in 3,9-DNF-treated rats appeared on day 88 (on day average 117), 10 weeks earlier than in 3,7-DNF-treated ones (on day average 186).

Mutagenic activity in particulate airborne pollutants in several samples collected in a wide variety of industrial, residential, and small-scale factory districts over the past seven years was detected by the Ames test. The particulate air samples that were not contaminated with several chemicals such as NO (less than 0.001 ppm), NO2 (less than 0.001 ppm), and SO2 (less than 0.001 ppm) showed only low mutagenic activity (1 revertant/m3) when they were tested with Salmonella typhimurium strain TA98 in the presence of S9 mix. However, most of the samples polluted by particulate matters showed high mutagenicity, with responses varying from 2.4 to 445 revertants per m3: 67 samples from an industrial area induced an average of 44 revertants per m3; 60 from a residential area, 16.2; and 10 from a small-scale factory area, 72.1. For assessing the mutagenic potential of the pollution in the atmosphere, the frequency of mutation determined with strain TA98 in the presence of S9 mix was used to divide the samples tentatively into five groups (A-E) on the basis of the normal logarithmic distribution curve of 137 samples. Air samples belonging to group A gave less than 2.3 revertants per m3 of air (1.12 +/- 0.12, no pollution); those of group B gave a range of 2.4 to 8.6 (5.93 +/- 1.91, slight pollution); those of group C gave a range of 8.7 to 30.2 (16.0 +/- 5.36, moderate pollution); those of group D gave a range of 30.3 to 115 (56.7 +/- 20.1, considerable pollution); and those of group E gave more than 116 (234 +/- 119, heavy pollution). Of the 137 samples tested, 6 samples (4.4%) were assigned to group A, 38 (27.7%) to group B, 52 (38.0%) to group C, 34 (24.8%) to group D, and 7 (5.1%) to group E. Furthermore, the samples in an industrial area were classified in the order of group C (35.8%), group B (26.9%), group D (22.4%), group E (8.96%), and group A (5.97%), and those in a residential area in the order of group C (46.7%), group B (33.3%), group D (18.3%), and group A (1.67%).

Gaseous substances such as nitrogen dioxide (NO2) and sulfur dioxide (SO2) stimulate the process of nitration of polycyclic aromatic hydrocarbons, and the transformation products display a broad spectrum of mutagenicity, genotoxicity, and carcinogenicity. Bacterial mutation by nitroarenes is specific. Tetracyclic nitroarenes are thought to be the most mutagenic compounds in the Salmonella test system, and some are carcinogenic in rats and mice. Furthermore, it was found that the mutational nitroarenes produced mostly DNA damage, which is subject to recombination repair in the rec assay system using Bacillus subtilis. Nitroarenes in the environment seem to be ubiquitous; the majority of the compounds are emitted directly from diesel emissions, kerosene heaters, and gas and liquefied-gas burners or heaters. In nitroarenes induced during incomplete combustion, nitropyrene and nitrofluoranthene derivatives are the most important mutagens/carcinogens for determining the chronic toxicity of nitroarenes overall.

Of the many nitroarenes, dinitropyrenes (DNPs) have the potential to revert Salmonella typhimurium his− mutants. This study was conducted to investigate the potential mutagens present in airborne particulate matter collected in Santiago, Chile. 5 organic substances extracted with dichloromethane showed mutagenic rates of from 38.9 to 287 revertants per m3 of air for S. typhimurium his− strain TA98 without S9 mix. 4 of the samples had greatly reduced mutagenicity for strain TA98/1, 8DNP6 but not for strain TA98NR. The 1-nitropyrene (1-NP) content accounted for 0.06-0.15 μg per g of particulate, as determined by high-performance liquid chromatography (HPLC), but the contribution of the compound to mutagenicity was less than 1% of the total activity. On the other hand, by using two columns in the HPLC, DNPs of 1,6- and 1,8-isomers were detected in the samples pooled after the determination of 1-NP, and the amount of the derivatives was about 0.2 μg per g of particulate matter.

By renitrating 3-nitrofluoranthene in the presence of fuming nitric acid, some additional nitro-derivatives were induced; they were identified as 3,7-, 3,9- and 3,4-dinitrofluoranthene (DNF), and two trinitrofluoranthenes (TNF, 3,4,7- and 3,4,8- or 3,4,9-isomers) on the basis of the results of mass spectrometry and 1H-nuclear magnetic resonance. The yield of 3,7- and 3,9-DNF was about 61.3% in all of the derivatives induced. All of the DNFs yielded positive results in the rec-assay system, inducing DNA-damaging activity in Bacillus subtilis. Both 3,7- and 3,9-DNF converted Salmonella typhimurium His- strains TA98, TA97 and TA1538 from autotrophy to prototrophy, indicating a frameshift-type mutation for both; for strain TA98, 3,7-, 3,9- and 3,4-DNF gave mutagenicity of 422, 355 and 15.5 His+ revertants, respectively, per nanogram, corresponding to the specific activity of 1,6-dinitropyrene (DNP), a powerful mutagen. These DNFs are known to be potential mutagens which are eluted at adjacent retention times with 1,3-, 1,6- and 1,8-DNP on a column for high-performance liquid chromatography.

In tests on the carcinogenicity of 1,6-dinitropyrene [(1,6-DNP) CAS: 42397-64-8] and 1-nitropyrene [(1-NP) CAS: 5522-43-0], 0.1 mg of each compound was inoculated sc into BALB/c mice once a week for 20 weeks. In the group given injections of 1,6-DNP the first tumor appeared on day 112, and 10 of the 20 mice developed tumors at the injection site by 45 weeks after the first injection. However, no tumors were induced in any of the mice that received injections of 1-NP. All of the induced tumors were transplantable for more than five generations in male BALB/c mice. Most of the tumors showed the characteristic histologic features of malignant fibrous histiocytoma.