Physical compatibility of remimazolam with opioid analgesics, sedatives, and muscle relaxants during simulated Y-site administration.

PURPOSE: There is a lack of information on the compatibility of remimazolam with opioid analgesics, muscle relaxants, and other sedatives. This study aimed to evaluate the physical compatibility of remimazolam with these drug classes. METHODS: Remimazolam was combined with 1 or 2 target drugs (remifentanil, fentanyl, rocuronium, vecuronium, dexmedetomidine, and midazolam). Ten physical compatibility tests were conducted, including four 3-drug compatibility tests. Remimazolam was dissolved in 0.9% sodium chloride injection to a final concentration of 5 mg/mL. Other medications were diluted in 0.9% sodium chloride injection to obtain clinically relevant concentrations. Compatibility tests were conducted with 3 test solutions, wherein remimazolam and the target drugs were compounded at equal volume ratios (1:1 or 1:1:1). Visual appearance was assessed and testing of Tyndall effect, turbidity, and pH was performed immediately after mixing and then again 1 hour and 4 hours after mixing. Appearance and turbidity were evaluated by comparison with the control solution of each target drug diluted with 0.9% sodium chloride injection to the same concentration as the test solution. RESULTS: All drugs tested were determined to be compatible with remimazolam. The drug combination with the highest change of turbidity was remimazolam and vecuronium (a mean increase of 0.16 NTU relative to the remimazolam control solution), 4 hours after mixing. The combination with the highest pH was remimazolam, fentanyl, and vecuronium (mean [SD], 3.76 [0.01]), 4 hours after mixing. The combination of remimazolam and fentanyl showed a larger change in pH at 4 hours after mixing (a mean increase of 2.6%) than immediately after mixing. CONCLUSION: Remifentanil, fentanyl, rocuronium, vecuronium, dexmedetomidine, and midazolam are physically compatible with remimazolam during simulated Y-site administration.

Microflora of Mycotoxigenic Fungi in Rice Grains in Kyushu Region of Japan and Their Changes during Storage under non-Controlled Conditions.

Contamination of agricultural crops by mycotoxins has increased because of the expansion of mycotoxin-producing fungi along with global warming. In this study, the fungal microflora of brown rice grains cultivated in Kyushu region in the southern part of Japan was investigated. A total of 75% of rice samples examined in this study showed less than 30% of fungal contamination rates with a median rate of 12.5%. Some isolates of Aspergillus flavus showed the ability to produce aflatoxins (AFs) (AFB1 production was 62.5-70.4 ng/mL) . Furthermore, AF-producing Aspergillus flavus survived during storage and Aspergillus creber, which produced sterigmatocystin, was detected in a stored rice sample. Although AFs or sterigmatocystin-contamination was not detected in any rice samples, these mycotoxin-producing fungi are distributed and can survive during storage under the natural conditions in Japan. Employing suitable storage conditions is important for preventing mycotoxin contamination of brown rice grains.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

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.

Inter-Laboratory Study to Validate New Rapid Screening Methods for Kudoa septempunctata.

Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is time-consuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification (LAMP) method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method (10(5) spores/g olive flounder), have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 10(4) spores/g; however, these methods were able to detect more than 10(5) spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

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.

[Antimicrobial Susceptibility and Resistance Mutations in Campylobacter jejuni and C. coli Isolates from Human and Meat Sources].

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.

Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

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.

Shiga toxin 2f-producing Escherichia albertii from a symptomatic human.

The previously identified Shiga toxin (Stx) 2f-producing Escherichia coli O115:HNM strain F08/101-31, isolated from a symptomatic human, was confirmed to be E. albertii in the present study by whole genome DNA-DNA hybridizations, by sequencing (cpn60, dnaJ, and 16S rRNA genes), and by multi-locus sequence typing. The F08/101-31 strain was originally identified as E. coli rather than the relatively new bacterial species E. albertii, which was first described in 2003, because it did not display any of the biochemical characteristics of E. albertii. This new classification will impact public health management strategies in Japan because the present study showed that some E. albertii strains, which are often misidentified as E. coli, produce Stx and likely cause diarrhea in humans. Therefore, further guidelines for the management and identification of Stx-producing E. albertii are required in Japan.

Evolutionary model of the divergence of enterohemorrhagic Escherichia coli O157 lineage I/II clades reconstructed from high resolution melting and Shiga-like toxin 2 analyses.

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.

Isolation and characteristics of Shiga toxin 2f-producing Escherichia coli among pigeons in Kyushu, Japan.

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.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. METHODS: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. RESULTS: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. CONCLUSIONS: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.

Electron microscopic study of Kudoa septempunctata infecting Paralichthys olivaceus (olive flounder).

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.

Molecular and serological investigation of Leptospira and leptospirosis in dogs in Japan.

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.

Clinical significance of Escherichia albertii.

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.

[Evaluation of the immunochromatography kit for detection of emetic-toxin producing Bacillus cereus].

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.

Emergence of enterohemorrhagic Escherichia coli serovar O157 strains in clade 8 with highly similar pulsed-field gel electrophoresis patterns.

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 stx(2) 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.

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.

Biased distribution of IS629 among strains in different lineages of enterohemorrhagic Escherichia coli serovar O157.

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.

Isolation of Shiga toxin 2f-producing Escherichia coli (O115:HNM) from an adult symptomatic patient in Fukuoka Prefecture, Japan.

Shiga toxin 2f-producing Escherichia coli (O115:HNM) with eae was isolated from a symptomatic patient in Fukuoka Prefecture, Japan. The patient was a 23-year-old male and his symptoms were diarrhea, abdominal pain, headaches and a fever (37.7 degrees C). He had eaten raw chicken meat, raw chicken eggs, cooked chicken meat and raw vegetables about 13 h prior to the onset of the symptoms. The patient's specimen was examined, and no diarrheagenic agents were detected except for Shiga toxin 2f-producing E. coli (STEC(2f)) with eae. This is the first report of the serotype O115:HNM possessing stx(2f). We discuss the necessity of routinely using stx(2f)-detecting PCR primers for detection of this enteric pathogen.

Features of Salmonella serovars among food handlers in Kyushu, Japan.

Salmonella were isolated from 106 (0.032%) of 331,644 fecal samples from food handlers, and from 144 of 11,478 fecal samples from symptomatic patients in Japan to determine the incidence and features of Salmonella serovars among food handlers. S. enterica subspecies enterica serovar Infantis (S. serovar Infantis) was the dominant serovar (accounting for 48.1%), followed by S. serovar Corvallis, which showed poor genetic diversity, and S. serovar Enteritidis among food handlers. The former two serovars were not dominant among symptomatic patients. The present study demonstrates the need for education on the sanitary handling of chicken eggs and chicken meat, which are possible infectious sources of these Salmonella serovars.