Epidemiology and genotypic characterisation of dissemination patterns of uropathogenic Escherichia coli in a community.

To characterise the dissemination patterns of uropathogenic Escherichia coli (UPEC) in a community, we conducted a study utilising molecular and fundamental descriptive epidemiology. The subjects, consisted of women having community-acquired acute urinary tract infection (UTI), were enrolled in the study from 2011 to 2012. UPEC isolates were subjected to antibacterial-susceptibility testing, O serogrouping, phylotyping, multilocus-sequence typing with phylogenetic-tree analysis and pulsed-field-gel electrophoresis (PFGE). From the 209 unique positive urinary samples 166 UPEC were isolated, of which 129 were fully susceptible to the tested antibiotics. Of the 53 sequence types (STs), the four most prevalent STs (ST95, ST131, ST73 and ST357) accounted for 60% of all UPEC strains. Antimicrobial resistance was less frequently observed for ST95 and ST73 than for the others. A majority of rare STs and a few common STs constituted the diversity pattern within the population structure, which was composed of the two phylogenetically distinct clades. Eleven genetically closely related groups were determined by PFGE, which accounted for 42 of the 166 UPEC isolates, without overt geo-temporal clustering. Our results indicate that a few major lineages of UPEC, selected by unidentified factors, are disseminated in this community and contribute to a large fraction of acute UTIs.

Web survey-based selection of controls for epidemiological analyses of a multi-prefectural outbreak of enterohaemorrhagic Escherichia coli O157 in Japan associated with consumption of self-grilled beef hanging tender.

An outbreak of enterohaemorrhagic Escherichia coli O157 occurred in multiple prefectures of Japan in November 2009. We conducted two case-control studies with trace-back and trace-forward investigations to determine the source. The case definition was met by 21 individuals; 14 (66.7%) were hospitalised, but no haemolytic uraemic syndrome, acute encephalopathy or deaths occurred. Median age was 23 (range 12-48) years and 14 cases were male (66.7%). No significant associations with food were found in a case-control study by local public health centres, but our matched case-control study using Internet surveys found that beef hanging tender (or hanger steak), derived from the diaphragm of the cattle, was significantly associated with illness (odds ratio = 15.77; 95% confidence interval, 2.00-124.11). Pulsed-field gel electrophoresis analysis of isolates from patients and the suspected food showed five different patterns: two in faecal and food samples, and another three in patient faecal samples only, although there were epidemiological links to the meat consumed at the restaurants. Trace-back investigation implicated a common food processing company from outside Japan. Examination of the logistics of the meat processing company suggested that contamination did not occur in Japan. We concluded that the source of the outbreak was imported hanging tender. This investigation revealed that Internet surveys could be useful for outbreak investigations.

Epidemiological analysis of a large enterohaemorrhagic Escherichia coli O111 outbreak in Japan associated with haemolytic uraemic syndrome and acute encephalopathy.

A large outbreak of enterohaemorrhagic Escherichia coli (EHEC) O111 and O157 occurred in Japan in April 2011. We conducted an unmatched case-control study and trace-back investigation to determine the source of EHEC O111 infection and risk factors for severe complications. Pulsed-field gel electrophoresis was performed to help define cases. A total of 86 individuals met the case definition. Of these, 40% experienced haemolytic uraemic syndrome (HUS), 24% acute encephalopathy, and 6% died. Illness was significantly associated with eating the raw beef dish yukhoe (odds ratio 19·64, 95% confidence interval 7·03-54·83), the likely food vehicle. EHEC O111 and its closely related stx-negative variants were found in the beef. HUS occurred most frequently in individuals aged 5-9 years, and this age group was significantly associated with acute encephalopathy. The prevalence of HUS and acute encephalopathy was higher than in previous non-O157-related outbreaks, indicating a high risk of severe complications.

Salmonella enterica serovar Typhi in Japan, 2001-2006: emergence of high-level fluoroquinolone-resistant strains.

The phage types and antimicrobial susceptibilities of 226 isolates of Salmonella enterica serovar Typhi from imported cases in Japan between 2001 and 2006 were investigated. Most (93.8%) had travelled to Asian countries, particularly South East Asia. Twenty-one phage types were identified with E1 (30.5%), UVS (15.9%) and B1 (9.3%) being the most common. The frequency of multidrug-resistant strains reached 37.0% in 2006 with phage types E1 and E9 predominating. Almost half (48.2%) of the isolates were resistant to nalidixic acid and two isolates displayed high-level fluoroquinolone resistance. Three mutations, two in gyrA and one in parC, were identified in both isolates.

Evaluation and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping Vibrio parahaemolyticus: an international multicenter collaborative study.

The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Development and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio cholerae.

PulseNet is a network that utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols with the purpose of conducting laboratory-based surveillance of foodborne pathogens. PulseNet standardized PFGE protocols are subject to rigorous testing during the developmental phase and careful evaluation during a validation process assessing its robustness and reproducibility in different laboratories. Here we describe the development and validation of a rapid PFGE protocol for subtyping Vibrio cholerae for use in PulseNet International activities. While the protocol was derived from the existing PulseNet protocol for Escherichia coli O157, various aspects of this protocol were optimized for use with V. cholerae, most notably a change of the primary and secondary restriction enzyme to SfiI and NotI, respectively, and the use of a two-block electrophoresis program. External validation of this protocol was undertaken through a collaboration between three PulseNet Asia Pacific laboratories (Public Health Laboratory Centre, Hong Kong, National Institute of Infectious Diseases, Japan, and International Center for Diarrhoeal Diseases Research-Bangladesh) and PulseNet USA. Comparison of PFGE patterns generated by each of the participating laboratories demonstrated that the protocol is robust and reproducible.

Characterization of multidrug-resistant Salmonella enterica serovar Typhimurium isolated in Japan.

A total of 221 isolates of multidrug-resistant Salmonella enterica serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients.

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.

Molecular epidemiological investigation of enterohaemorrhagic Escherichia coli isolates in Japan.

We have established several measures for control and prevention of EHEC infection including designation of the disease as notifiable since there was the sudden increase in the incidence of infection with EHEC O157:H7 in Japan in 1996, involving multiple outbreaks. Improvements in methodologies for isolation of these organisms and enhanced laboratory screening have revealed a variety of sources in food and animals. Although there seems to be a bovine reservoir for O157 EHEC in Japan as well as North America and UK, different foods have been linked to EHEC infection including salads, radish sprouts and salmon roe. There is clear evidence that divergent clones of EHEC O157:H7 are prevalent throughout Japan based on laboratory surveillance, however, we still need to better define the role of EHEC serogroups other than Escherichia coli O157 as important causes of human infection.

Salmonella typhimurium DT104 from livestock in Japan.

We examined the distribution of multidrug-resistant Salmonella enterica serotype Typhimurium definitive phage type 104 (DT104) among Japanese livestock from 1973 to 1998. The 144 S. Typhimurium field isolates were tested for susceptibility to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, kanamycin, trimethoprim, nalidixic acid, and norfloxacin. Thirty-six of 68 strains which exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104. Results of plasmid profiling showed that all DT104 strains retain a 90-kb virulence plasmid, while 20 of 36 strains possessed a few additional small plasmids ranging from 2 to 4 kb. These results showed that DT104 strains have existed in Japanese livestock since 1990, and that this phage type may be an important pathogen for cattle in Japan.

Preincubation of recombinant Ipa proteins of Shigella sonnei promotes entry of non-invasive Escherichia coli into HeLa cells.

Invasion plasmid antigens of Shigella sonnei, IpaB, C, D, were expressed as fusion proteins either with maltose-binding protein (MBP) or Strept-tag sequence. Affinity-purified IpaB and IpaD were separated from MBP by digestion with Factor Xa. Recombinant IpaC having Strept-tag sequence at its C-terminal was also purified by avidin affinity column chromatography. These recombinant proteins showed the ability to cause non-invasive Escherichia coli K-12 to internalize HeLa cell only when all of the proteins were preincubated with the bacterial prior to the inoculation of the mixture into HeLa cell culture. Electron microscopy also showed internalized bacteria within HeLa cells suggesting that functional complex of invasins (IpaB, C and D) were formed in vitro.

Prolonged incubation period of salmonellosis in an outbreak of Salmonella enteritidis infection.

An outbreak of Salmonella enteritidis infections occurred in Otaru, Japan, in September 1997. A total of 143 cases of salmonellosis were reported to the local Public Health Center. In this outbreak, one case had a 214-hr incubation period. We investigated 5 isolates including this case by phage typing and pulsed-field gel electrophoresis (PFGE) to determine the genetic heterogeneity of S. enteritidis. Five isolates were phage typed as reacted, but did not conform (RDNC) with identical reaction patterns and had quite similar PFGE patterns. Thus, the prolonged incubation period may not be attributed to genetic heterogeneity of the organism but rather to other factors.

Molecular analysis of enterohemorrhagic Escherichia coli isolates in Japan and its application to epidemiological investigation.

Many outbreaks of enterohemorrhagic Escherichia coli (EHEC) O157 infections occurred in Japan in 1996. Molecular epidemiological analyses of the isolates by methods such as XbaI-digested, pulsed-field gel electrophoresis (PFGE), revealed that a variety of PFGE type strains have spread all over Japan. They also showed that such molecular methods are very useful for confirming the epidemiologically related isolates and in assisting the epidemiological investigation. Recent characteristic features of the infection are that the incidence of patients experiencing symptoms such as abdominal cramps, diarrhea and hemorrhagic colitis is higher in children younger than 9 years old; however, that of asymptomatic carriers seems to be higher in adults; and that EHEC O157:H7-serotyped strains are still the main isolates, while the isolation frequency of other serotype strains, such as O26 and O111, is increasing.

[Isolation of Shiga toxin-producing Escherichia coli O157:H7 from processed salmon roe associated with the outbreaks in Japan, 1998, and a molecular typing of the isolates by pulsed-field gel electrophoresis].

Shiga toxin-producing Escherichia coil (STEC) O157 were isolated from processed salmon roe which had been a suspected food item in sporadic infections which occurred in Japan in 1998. A total of 45 samples of the processed salmon roe were pre-enriched in trypticase soy broth (TSB) at 36 degrees C for 6 h and novobiocin-supplemented modified EC broth (mEC-NB) at 42 degrees C for 18 h. After the pre-enrichments, the cultures were examined for possible occurrence of STEC O157, using an immunomagnetic separation (IMS) method. From the examination, a total of 84 strains of STEC O157:H7 that were positive for both stx 1 and stx 2 genes were isolated. By applying the most-probable-number technique, it was estimated that the number of STEC O157 was in the range of 0.73-1.5 per 10 g of the processed salmon roe. Subsequent analysis of the isolates by a pulsed-field gel electrophoresis (PFGE) revealed a pattern commonly seen in 82 isolates and another pattern in two isolates. Clinical isolates from 7 patients also showed an identical pattern to those of the 82 isolates and one isolate from a patient showed the other pattern identical to those of the two isolates. The isolates were found to belong to the phage type 14.

[Drug-resistance and definitive type 104 of Salmonella serovar typhimurium isolated from sporadic cases in Tokyo, 1980-1998].

A total of 674 Salmonella serovar Typhimurium (S. Typhimurium) strains consisting of 522 domestic strains and 152 imported strains isolated in Tokyo, 1980-1998, were examined regarding their drug-resistance and phage-type. Domestic strains accounted for 6.2% of all Salmonella (8,359 strains) isolated from domestic cases, and imported strains accounted for 3.7% of all Salmonella (4,083 strains) isolated from imported cases. A drug-resistance test using 9 drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM, and NFLX) showed that 245 strains (46.9%) of the domestic strains and 109 strains (71.7%) of the imported strains were resistant to some of the drugs, excluding FOM and NFLX. Drugs with a high resistance rate were TC, SM, ABPC, and CP for both groups. Drug-resistance patterns of the resistant strains varied among the 40 types. Among those, prevalent patterns recognized were CP.TC.SM.ABPC, CP.TC.SM.KM.ABPC, TC.SM, SM, and TC.KM in the domestic strains, and TC, CP.TC.SM.ABPC, CP.TC.SM.KM.ABPC, CP.TC.SM.KM.ABPC.ST and TC.KM in the imported strains. The results of the phage-typing test revealed that 31 strains of 52 domestic strains tested, and 13 strains of 46 imported strains tested were definitive type 104 (DT104). Those resistance patterns were CP.TC.SM.ABPC.SU (43 strains) and CP.TC.SM.KM.ABPC.SU (1 strain).