Enterohaemorrhagic Escherichia coli O121:H19 acquired an extended-spectrum ß-lactamase gene during the development of an outbreak in two nurseries.

Enterohaemorrhagic Escherichia coli (EHEC) is an important human pathogen worldwide. Although serotype O157 is currently the most dominant and important EHEC strain, serotypes O26, O111, O91, O103 and O121 are also recognized as serious pathogens that affect public health. EHEC outbreaks often occur in nurseries and elderly care facilities. In 2012, a nursery outbreak of EHEC O121 occurred during which the bacterium acquired a plasmid-borne extended-spectrum ß-lactamase (ESBL) gene. ESBL-producing E. coli O86 was concurrently isolated from one of the EHEC patients. Therefore, we investigated the isolates by whole-genome sequence (WGS) analysis to elucidate the transmission dynamics of the EHEC strains and the ESBL plasmid. According to WGS-based phylogeny, all 17 EHEC O121 isolates were clonal, while E. coli O86 was genetically distant from the EHEC O121 isolates. The complete sequence of an ESBL plasmid encoding the CTX-M-55 ß-lactamase was determined using S1-PFGE bands, and subsequent mapping of the WGS reads confirmed that the plasmid sequences from EHEC O121 and E. coli O86 were identical. Furthermore, conjugation experiments showed that the plasmid was capable of conjugative transfer. These results support the hypothesis that EHEC O121 acquired an ESBL-producing plasmid from E. coli O86 during the outbreak. This report demonstrates the importance of implementing preventive measures during EHEC outbreaks to control both secondary infection and the spread of antimicrobial resistance factors.

ALDH2 Polymorphism rs671, but Not ADH1B Polymorphism rs1229984, Increases Risk for Hypo-HDL-Cholesterolemia in a/a Carriers Compared to the G/G Carriers.

It has been reported that polymorphisms within the gene-encoding enzymes related to alcohol metabolism are associated with levels of serum HDL-cholesterol (HDL-C) in East Asian populations. We evaluated the effects of genetic variants within the aldehyde dehydrogenase-2 (ALDH2) gene and the alcohol dehydrogenase-1B (ADH1B) gene on changes in the lipid profile in an 11-year longitudinal study. We genotyped rs1229984 within ADH1B and rs671 within ALDH2. We combined the genetic data with longitudinal clinical and biochemical data from 2002 to 2013 and designed a retrospective longitudinal study of 1436 Japanese males. There were significant negative relationships between rs671 within ALDH2 and HDL-C levels according to multiple linear regression analysis. Next, we assessed the association between the development of hypo-HDL cholesterolemia and rs1229984 within ADH1B or rs671 within ALDH2. In logistic regression analysis, rs671 A allele homozygote carriers have 2.65 times higher risk of developing hypo-HDL cholesterolemia than G allele homozygote carriers. Even after adjusting for possible confounding factors, a significant association was observed. However, no association between rs1229984 within ADH1B and the development of hypo-HDL cholesterolemia was observed. Rs671 within ALDH2 but not rs1229984 within ADH1B was associated with lower HDL-C levels in Japanese males.

No causal impact of serum vascular endothelial growth factor level on temporal changes in body mass index in Japanese male workers: a five-year longitudinal study.

PURPOSE: It has been reported that adipocytes secrete vascular endothelial growth factor. Therefore, we conducted a 5-year longitudinal epidemiological study to further elucidate the association between vascular endothelial growth factor levels and temporal changes in body mass index. METHODS: Our study subjects were Japanese male workers, who had regular health check-ups. Vascular endothelial growth factor levels were measured at baseline. To examine the association between vascular endothelial growth factor levels and overweight, we calculated the odds ratio using a multivariate logistic regression model. Moreover, linear mixed effect models were used to assess the association between vascular endothelial growth factor level and temporal changes in body mass index during the 5-year follow-up period. RESULTS: Vascular endothelial growth factor levels were marginally higher in subjects with a body mass index greater than 25 kg/m2 compared with in those with a body mass index less than 25 kg/m2 (505.4 vs. 465.5 pg/mL, P = 0.1) and were weakly correlated with leptin levels (ß: 0.05, P = 0.07). In multivariate logistic regression, subjects in the highest vascular endothelial growth factor quantile were significantly associated with an increased risk for overweight compared with those in the lowest quantile (odds ratio 1.65, 95 % confidential interval: 1.10-2.50). Moreover P for trend was significant (P for trend = 0.003). However, the linear mixed effect model revealed that vascular endothelial growth factor levels were not associated with changes in body mass index over a 5-year period (quantile 2, ß: 0.06, P = 0.46; quantile 3, ß: -0.06, P = 0.45; quantile 4, ß: -0.10, P = 0.22; quantile 1 as reference). CONCLUSIONS: Our results suggested that high vascular endothelial growth factor levels were significantly associated with overweight in Japanese males but high vascular endothelial growth factor levels did not necessarily cause obesity.

[Influence of Stx2 Subtype on Bacteriological Identification of Outbreaks of EHEC O157: H- Infections in Saitama City].

In 2013, two outbreaks of enterohemorrhagic Escherichia coli (EHEC) occurred in Saitama city. According to reports from each of the medical institutions that detected the EHEC isolates, the isolates seemed to differ in their production of Vero Toxin (VT / Shiga Toxin: Stx) since one isolate produced only Stx1 and the other produced both Stx1 and Stx2. However, a patient survey conducted by a public health center revealed that common foodstuffs had been consumed in both outbreaks. Because, the two EHEC isolates were newly detected from two people in one patient's family, we analyzed the phenotypic and genetic relationships among four isolates in total. All the isolates were serotyped as O157: H-, and both stx1 and stx2 were detected. Subsequently, all four isolates were shown to have the same pulsed-field gel electrophoresis (PFGE) banding pattern. The findings suggested that these isolates belonged to the same strain group. Among these cases, the isolates had stx2c which is one of the stx2 subtypes. Reportedly, some cases with the Stx2 subtype can not be detected using conventional tests for toxin. In addition, Stx2 can be overlooked as a result of this limitation of Stx-production tests. Both epidemiological research by public health centers and genetic analysis by prefectural and municipal public health institutes (PHIs) are very important for clarifying possible relationships among outbreaks, as in the present cases. Moreover, collaborations and networks among medical institutions, PHIs and public health centers should be further strengthened to prevent the spread of infections.

The First Report of Vibrio parahaemolyticus Strain O10:K60 in Japan, a New Combination of O and K Serotypes Isolated from a Patient with Gastroenteritis.

Vibrio parahaemolyticus is an important pathogen that causes gastroenteritis in humans, generally associated with the consumption of contaminated seafood, particularly raw shellfish. There are many serotypes in V. parahaemolyticus resulting from a combination of O and K antigens. Among them, O3:K6 and their variants, which represent the pandemic clone, are the most widespread strains worldwide. In this study, we examined V. parahaemolyticus isolated from a gastroenteritis patient's stool at a hospital in Saitama City, Japan in 2013. Serotyping of the O and K antigens identified the strain as O10:K60. To our knowledge, this is the first reported case of a V. parahaemolyticus strain with this antigen combination in Japan. Subsequently, we used PCR to assay for pathogenicity-associated genes, and found that it was positive for tdh, T3SS1, and T3SS2α genes. Antibiotic susceptibility tests showed that the strain was susceptible to all selected antibiotics except ampicillin. Moreover, we detected specific marker genes for the pandemic clone with two kinds of PCR assay. Our results suggest that the isolate O10:K60 is a newly emerging serotype that belongs to the pandemic clone.

[Identification of resistance and susceptibility to cefotaxime in EHEC O121 strains isolated from an outbreak at two nurseries].

A Shiga toxin 2 producing enterohemorrhagic Escherichia coli (EHEC) O121: H19 was isolated from a 2-year-old child who attending a nursery. An EHEC O121 outbreak in two nurseries (A, B), involving a total of 17 infected persons including 12 children, was revealed through contact investigation. The symptoms of all infected persons were almost all mild, and no one developed the hemolytic uremic syndrome. The combination use of desoxycholate-hydrogen sulfide-lactose (DHL) and CHROMagar STEC as selective isolation media was employed for efficient fecal examination. Nursery A and nursery B were combined as one group after the outbreak in nursery A was confirmed. As a result, EHEC O121 infected persons were also detected in children from nursery B. The 17 strains of EHEC O121 obtained from the total population showed almost the same pulsed-gel electrophoresis (PFGE) pattern, suggesting that these strains were very closely related. However, 13 of these 17 strains obtained from nursery A were susceptible to cefotaxime, whereas the remaining 4 strains obtained from nursery B showed cefotaxime resistance. A cefotaxime resistant Escherichia coli (E. coli) O86 strain was isolated in the stool specimen from a child who had been infected with the cefotaxime resistant EHEC O121. Both the cefotaxime resistant EHEC O121 and E. coli O86 had the same drug resistant gene (bla(CTX-M-1) group). The child was the index case of these 4 later cases and had received no antibiotics therapy prior to the laboratory examination. These findings suggested the possibility that an EHEC O121 strain had acquired a drug resistant gene from E. coli O86 in the digestive tract of the child.

Occurrence of preservatives and antimicrobials in Japanese rivers.

We established a method for the simultaneous analysis of seven preservatives and five antimicrobials in water. These chemicals are widely used in cosmetics, and their presence in river water is of concern. We used the method to test 13 sites in Japanese rivers and streams contaminated by domestic wastewater, effluent, or industrial wastewater. 2-Phenoxyethanol (2-PE), isopropylmethylphenol, resorcinol, and triclosan were detected at most sites; the maximum concentration of 2-PE detected was 14000 ng L(-1). The results suggest that the major sources of 2-PE were cosmetics and household detergents. The ratio of methylparaben to n-propylparaben in river waters with direct wastewater drainage was similar to that in common cosmetics. This is the first research on levels of 2-PE, isopropylmethylphenol, and chlorphenesin in river water.

Culture temperature affects gene expression and metabolic pathways in the 2-methylisoborneol-producing cyanobacterium Pseudanabaena galeata.

A volatile metabolite, 2-methylisoborneol (2-MIB), causes an unpleasant taste and odor in tap water. Some filamentous cyanobacteria produce 2-MIB via a two-step biosynthetic pathway: methylation of geranyl diphosphate (GPP) by methyl transferase (GPPMT), followed by the cyclization of methyl-GPP by monoterpene cyclase (MIBS). We isolated the genes encoding GPPMT and MIBS from Pseudanabaena galeata, a filamentous cyanobacterium known to be a major causal organism of 2-MIB production in Japanese lakes. The predicted amino acid sequence showed high similarity with that of Pseudanabaena limnetica (96% identity in GPPMT and 97% identity in MIBS). P. galeata was cultured at different temperatures to examine the effect of growth conditions on the production of 2-MIB and major metabolites. Gas chromatograph-mass spectrometry (GC-MS) measurements showed higher accumulation of 2-MIB at 30 °C than at 4 °C or 20 °C after 24 h of culture. Real-time-RT PCR analysis showed that the expression levels of the genes encoding GPPMT and MIBS decreased at 4 °C and increased at 30 °C, compared with at 20 °C. Furthermore, metabolite analysis showed dramatic changes in primary metabolite concentrations in cyanobacteria grown at different temperatures. The data indicate that changes in carbon flow in the TCA cycle affect 2-MIB biosynthesis at higher temperatures.

[Residual pesticide concentrations after processing various types of tea and tea infusions].

The effects of processing to produce various types of tea or infusion on the levels of pesticide residues in tea were investigated for three insecticides (chlorfenapyr, pyrimiphos-methyl, and clothianidin). Tea plants were sprayed with one of the three pesticides and cultivated under cover. The levels of pesticide residues in tea decreased after processing according to the time and temperature of heating, as well as fermentation. Although significant differences were not observed among the three pesticides in the ratio of decreased of pesticide concentration after processing to green tea, clothianidin, which is a neonicotinoid insecticide and has a lower log Pow value, tended to be transferred more than the other two insecticides into infusions. However, no significant difference in the ratios of clothianidin transferred to infusions was observed among green tea with three different leaf sizes.

Characterization of group B streptococcus isolated from women in Saitama city, Japan.

Streptococcus agalactiae (group B streptococcus; GBS) is a common cause of neonatal sepsis and meningitis. Intrapartum antibiotic prophylaxis is effective in reducing neonatal GBS disease. Penicillin is recommended for intrapartum antibiotic prophylaxis; however, other antibiotics are administered to pregnant women with penicillin allergy. Serotyping and antibiotic susceptibility testing were performed on 376 GBS isolates collected from vaginal swabs in Saitama City. Of the 376 isolates, 328 (87.2%) were obtained from obstetrics and gynecology clinics. Although approximately 80% of the isolates (299/376) were from women of reproductive age (age, 15-49 years), no definite information on their pregnancy status was obtained. The most frequent serotype was V (19.1%) followed by Ib (18.6%), III (16.2%), VI (14.9%), and Ia (14.6%). None of the isolates were resistant to penicillins and cephalosporins. Isolates that were resistant to erythromycin (12.8%), clindamycin (9.0%), ofloxacin (19.4%), levofloxacin (18.4%), and tetracycline (46.5%) were detected. There was a high prevalence of resistance to erythromycin (39.3%) and clindamycin (27.9%) in serotype III. In addition, almost all serotype Ib isolates were resistant to ofloxacin and levofloxacin (both, 91.4%). Pulsed-field gel electrophoresis analysis on certain GBS isolates (serotype Ib, III, and V) indicated that there was genetic diversity among the resistant isolates obtained from a limited area of Japan. In conclusion, present intrapartum antibiotic prophylaxis with penicillins would be effective in Japan; however, performing susceptibility testing before administering other antibiotics is important in order to ensure activity against the relevant isolate.

[New clean-up method with dispersive solid-phase extraction for simultaneous determination of pesticide residues in livestock products by GC-MS/MS].

A simple clean-up method was developed for the simultaneous determination of pesticide residues in livestock products by GC-MS/MS. The pesticide residues were extracted with acetonitrile-ethanol (1 : 1), and matrix components such as adipose were effectively eliminated by a combination of refrigerated centrifugation, dispersive solid-phase extraction, and multifunctional column chromatography. In this method, samples are treated quickly and easily without the need for gel-permeation chromatography. Among 131 pesticides tested, 115 showed recovery within the range from 70 to 120%, with relative standard deviations of less than 15%. The quantification limits for the 115 pesticides in livestock products were 0.001 to 0.01 µg/g.

Retrospective longitudinal study on the relationship between 8-year weight change and current eating speed.

Most of the studies that have examined the relationship between the speed of eating and obesity have been cross-sectional. We investigated 529 male workers who received health check-ups provided by the employer in 2000 and 2008. We obtained information on the subjects' alcohol consumption, smoking status, self-reported speed of eating, and exercise in 2008, and height and weight in both 2000 and 2008. We compared weight change from 2000 to 2008 between 2 groups classified according to the speed of eating: a group of fast eaters, and a combined group of medium and slow eaters. The fast-eating group had a higher average weight gain (1.9 kg) than the medium and slow eating group (0.7 kg). Although statistically significant only for the 20-29-year age group, weight gain was greater in the fast-eating group for all age groups and was statistically significant when the age groups were combined. The relationship between eating fast and weight change was statistically significant even after adjusting for age and body mass index in 2000, drinking, smoking, and exercise. Our results suggested that the speed of eating is related to the rate of weight gain.

[Quick simultaneous determination of residual veterinary drugs in processed food].

Development of rapid and high accuracy analysis, and tightening of regulations for veterinary drugs are required because many examples of detection of veterinary drugs in many kinds of processed food have been reported. In this study, we constructed an improved method for simultaneous determination of veterinary drugs, based on the glass bead homogenization method with EDTA-2Na and batch purification for QuEChERS analysis. Our extraction procedure is suitable for handling multiple samples quickly and easily. Furthermore, our improved extraction solvent allowed simultaneous determination of tetracyclines in processed food. In a test of 69 veterinary drugs, recovery of over 60 ranged from 70 to 120%, with a coefficient of variation of less than 25% and with quantification limits of 0.01 µg/g (S/N≥10 ). This improved method is expected to be useful for quick simultaneous determination of multiple residues.

Occurrence and profiles of organic sun-blocking agents in surface waters and sediments in Japanese rivers and lakes.

Sun-blocking agents including eight UV filters (UVF) and 10 UV light stabilizers (UVLS) were measured in water and sediment collected from 22 rivers, four sewage treatment plant effluents (STPE) and three lakes in Japan. Total sun blocking agents levels ranged from N.D. to 4928 ng/L and from 2.0 to 3422 µg/kg dry wt in surface water and in sediment, respectively. Benzyl salicylate, benzophenone-3, 2-ethyl hexyl-4-methoxycinnamte (EHMC) and octyl salicylate were dominant in surface water receiving wastewater effluents and STPE, although UV-328, benzophenone and EHMC were dominant in other surface water except background sites. Three UVF and nine UVLS were observed from all sediment and their compositions showed similar patterns with UV-328 and UV-234 as the most prevalent compounds. Homosalate, octocrylene, UV-326, UV-327, UV-328 and UV-234 were significantly correlated with Galaxolide® in sediments. Concentrations of UV-327 and UV-328 also had strong correlation between those of UV-326 in sediment.

Measurement of high-molecular-weight adiponectin is not useful in assessing coronary stenosis.

BACKGROUND: In many studies, high-molecular-weight (HMW) adiponectin has been considered the active form of adiponectin. However, whether HMW adiponectin is a good surrogate marker for coronary artery disease still needs to be elucidated. METHODS: We conducted a hospital-based cross-sectional study to examine the relationship between total, HMW or non-HMW adiponectin concentrations and coronary stenosis in 83 male patients and 138 male controls. RESULTS: Patients with coronary stenosis had significantly lower total adiponectin concentrations compared with controls. Non-HMW adiponectin concentrations in cases were significantly lower than the controls. However, there were no significant differences between cases and controls in HMW adiponectin concentrations. From the receiver operating characteristic (ROC) analysis, the area under the curve (AUC) for total and non-HMW adiponectin was significantly larger than that for HMW adiponectin concentrations. Of the three models, that for non-HMW adiponectin showed the largest AUC (total adiponectin 0.74, HMW adiponectin 0.54, and non-HMW adiponectin 0.79). CONCLUSIONS: Despite associations between total adiponectin levels and coronary stenosis, our data go against any apparent association between HMW adiponectin concentrations and coronary stenosis.

Inverse correlation between adiponectin and the risk of metabolic syndrome in middle-aged Japanese male workers.

Despite a close association between adiponectin and both hypertension and type 2 diabetes, the relationship between adiponectin and metabolic syndrome has not yet been well-investigated. To examine and evaluate the association between serum adiponectin levels and metabolic syndrome based on Japanese diagnostic criteria, we analyzed adiponectin and anthropometric parameters in 869 male employees aged 40-59 who belonged to a health insurance society in Fukuoka Prefecture and who underwent annual health check-ups from August 2006 to July 2007. Two hundred and thirty-two of the 869 subjects (26.7%) were diagnosed with metabolic syndrome. The serum adiponectin levels were significantly higher in the non-metabolic syndrome group. In a multiple logistic regression analysis, the subjects in the top quartile of serum adiponectin (adjusted odds ratio:0.36;95% confidence interval:0.21-0.63) and the second (adjusted odds ratio:0.51;95% confidence interval:0.31-0.84) quartile had a significantly decreased risk for metabolic syndrome in comparison to the bottom quartile. The dose-response relationship between serum adiponectin levels and metabolic syndrome was significant (p for trend 0.0001) after adjusting for age, body mass index, smoking status, and drinking status. The current findings suggest that hypoadiponectinemia is inversely correlated with the risk of metabolic syndrome in middle-aged Japanese male workers.

[Study of simultaneous determination of residual veterinary drugs including tetracycline antibiotics in milk and dairy products].

It is considered to be difficult to detect tetracycline antibiotics in all-at-once simultaneous analysis with other drugs by liquid chromatography with tandem mass spectrometry, because tetracycline antibiotics chelate with bivalent metal ions such as calcium in samples. Therefore, we studied simultaneous determination of tetracycline antibiotics after removal of calcium with disodium ethylenediaminotetraacetate (EDTA-2Na). Tetracycline antibiotics could be assayed in all-at-once analysis by adding EDTA-2Na during the extraction procedure. It was possible to determine 65 veterinary drugs in milk, 70 in yogurt, 59 in whipped cream, 67 in cheese and 60 in ice cream. Recovery ranged from 70 to 120%, with a coefficient of variation of less than 25% and with a quantification limit of 0.01 microg/g (S/N>or=10).

[Genomic analysis of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains isolated in Saitama City].

To elucidate the origin of infection, we conducted epidemiological and bacteriological studies to clarify the origin of five sporadic outbreaks of enterohemorrhagic Escherichia coli (EHEC) O157:H7 between May and July 2007 in Saitama City and its outskirts. Of the 20 subjects were reported; including 6 patients and 5 infected persons, none of the 9 symptomatic subjects developed hemolytic-uremic syndrome. No association was confirmed between infection and food materials, but 11 organisms showed almost the same chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis. Our results suggested that these 5 sporadic outbreaks were part of a diffuse outbreak induced by an EHEC O157:H7 strain having a single origin.

An outbreak of measles in Saitama City in 2007. What is the vaccination strategy to eliminate measles in Japan?

In the present study, to elucidate an outbreak of measles in Saitama City, Japan, we analyzed the data for all notified subjects with measles. According to an active surveillance program, a total of 464 subjects were notified in 2007. The clinical criteria for the diagnosis of measles were defined as at least 3 days of a generalized maculopapular rash; a fever of 38.0 degrees C or more; and cough, mucus, or pharyngitis. Two peaks according to age group were recognized: namely, children less than 2 years of age and adolescents from 15 to 19 years of age. The latter peak was associated with the period of time when the measles-mumps-rubella vaccine had become a social problem (40.9% of vaccinees and 41.6% of non-vaccinees in this group). Japan is said to be a developing country regarding its measles vaccination strategy. In addition, no national program against measles has yet been established. Continuous efforts to increase immunization coverage are needed to interrupt indigenous measles transmission. The Japanese Ministry of Health, Labor and Welfare should therefore plan and implement a nationwide program to eliminate measles in Japan.