Non-biogroup 1 or 2 Strains of the Emerging Zoonotic Pathogen Escherichia albertii, Their Proposed Assignment to Biogroup 3, and Their Commonly Detected Characteristics.

Escherichia albertii, a zoonotic enteropathogen, is responsible for outbreaks of disease in humans. Identifying strains of E. albertii by phenotypic characterization tests is difficult because of its poorly defined properties. Screening its phenotypic characteristics is, nevertheless, a necessary prerequisite for further genetic analysis of its properties, and species-specific polymerase chain reaction (PCR) analysis can be used to type the pathogen. While two E. albertii biogroups (1 and 2) have been described, strains with characteristics divergent from both biogroups have been reported worldwide. The aim of the present study was to evaluate the characteristics of non-biogroup 1 or 2 strains, and discern the characteristics common to all of the E. albertii strains from this study. Altogether, 107/414 field isolates were selected for examination based on pulsed-field gel electrophoresis analysis. The 107 strains were isolated from 92 sources, including humans and pigeon feces, other wild birds, and retail chicken livers. All strains were then examined using various culture-based, biochemical (API 50CHE tests, API Zym test, and others) and molecular (virulence gene screening, multi-locus sequence analysis) testing methods. Our results revealed that all field strains (n = 107) showed non-biogroup 1 or 2 characteristics, with multiple sequence differences. Variations in indole production and the lysine decarboxylase activity profiles among the isolates made identification of E. albertii very difficult. Therefore, we propose that non-biogroup 1 or 2 of E. albertii should be assigned to biogroup 3 to make screening of them easier in public health and clinical laboratory settings. Clearly, having group criteria for indole-negative/lysine-positive, indole-positive/lysine-negative, and indole-positive/lysine-positive E. albertii biogroups 1, 2, and 3 strains, respectively, should provide for more accurate identification of E. albertii isolates. Based on our findings, we recommend that isolates displaying phenotype mobility-negativity (sulfide-indole-motility medium, 37°C), hydrogen sulfide production-negativity (triple sugar iron medium), acid production-negativity from xylose, negative ß-glucuronidase activity properties, and showing indole production and lysine decarboxylase activity profiles in accordance with one of the three biogroups, should be further assessed using an E. albertii-specific PCR assay.

Meningococcal disease outbreak related to the World Scout Jamboree in Japan, 2015.

PROBLEM: Six invasive meningococcal disease cases occurred among Scottish and Swedish nationals associated with the World Scout Jamboree (WSJ), an international mass gathering, held in Japan. The index case developed symptoms while returning home. The strains from all six cases were identical and seldom seen in Japan. CONTEXT: Over 33 000 participants from 155 countries attended WSJ. At the Jamboree site, participants of the North of Scotland's and Sweden's units camped within the same subcamp and kept the same schedule of events. No information was available about the Swedish and Scottish cases' close personal contact history. ACTION: Health Protection Scotland investigated Scottish cases, conducted active case finding, provided chemoprophylaxis, vaccinated close contacts and advised Scottish WSJ participants and contacts to seek medical care if they developed symptoms. The Public Health Agency of Sweden recommended chemoprophylaxis to all participants in Sweden. In Japan, the Ministry of Health, Labour and Welfare (MHLW) requested the Scout Association of Japan advise all participants to seek medical attention if they developed symptoms. MHLW shared information about the event with local authorities, medical associations, and the Ministry of Education, Culture, Sports, Science and Technology. OUTCOME: No additional case related to WSJ has been reported. This outbreak highlighted the risk for international spread of invasive meningococcal disease at international mass gatherings. DISCUSSION: Assessing risk, educating participants, enhancing surveillance and sharing timely information among related countries are significant for prevention and response against invasive meningococcal disease outbreaks at mass gatherings.

National surveillance for meningococcal disease in Japan, 1999-2014.

We summarize the epidemiology of Japanese meningococcal disease with serogroup distribution. One hundred seventy-eight meningococcal meningitis cases were reported from April 1999 to March 2013 to the national surveillance system. From April 2013, bacteremia was added to the condition of reporting invasive meningococcal disease (IMD). Since then, 59 IMD cases were reported by the end of 2014. Approximately two thirds of the cases were male and the median age was 56years (range: 0-93years). Only 3% of the cases were <5years old. One third of reported cases were meningitis and the others were bacteremia. The annual incidence (2014) for IMD was 0.028 per 100,000 and case fatality rate (CFR) was 19%. Serogroup Y (42%) was the most dominant serogroup, followed by C (12%), B (7%) and W (3%). Even though the number of reported cases has increased after the amendment of reporting requirements, the incidence of IMD is still low in Japan. Underreporting may play a role in this low incidence. Improving on the limitations of the surveillance system is necessary to capture the true epidemiology and accurate serogroup distribution of IMD cases in Japan, which is essential for making effective recommendations on newly licensed vaccine.

Meningococcal carriage rates in healthy individuals in Japan determined using Loop-Mediated Isothermal Amplification and oral throat wash specimens.

The detailed epidemiology of meningococcal diseases in Japan has yet to be determined and, moreover, the healthy carriage rate is also unknown. In this study, to obtain insight into the carriage rate of Neisseria meningitidis in healthy individuals in Japan, we developed a new method to detect the N. meningitidis-specific ctrB gene, one of the genes encoding enzymes for capsule synthesis, by Loop-Mediated Isothermal Amplification (LAMP) and examined the meningococcal carriage rate by using self-collected oral throat wash specimens from 836 students at a university. Examination by LAMP showed that 7 out of 836 samples were positive for N. meningitidis DNA, and the results were also verified by the nested PCR method for the meningococcus specific ggt gene. The N. meningitidis carriage rate in healthy individuals was estimated to be 0.84%. Moreover, we further confirmed by the nested-PCR-based serogroup typing method that 5 of the positive samples belonged to serogroup Y, 1 belonged to group B and 1 was unidentifiable. Considering the epidemiology for meningococcal diseases in Japan, the carriage rate and the serogroup profile seem to be consistent with low incidence of meningococcal diseases and serogroup distribution of clinical meningococcal isolates in Japan, respectively.

Enterohemorrhagic Escherichia coli outbreaks related to childcare facilities in Japan, 2010-2013.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) is an important cause of gastroenteritis in Japan. Although non-O157 EHEC infections have been increasingly reported worldwide, their impact on children has not been well described. METHODS: We collected national surveillance data of EHEC infections reported between 2010 and 2013 in Japan and characterized outbreaks that occurred in childcare facilities. Per Japanese outbreak investigation protocol, faecal samples from contacts of EHEC cases were collected regardless of symptomatic status. Cases and outbreaks were described by demographics, dates of diagnosis and onset, clinical manifestations, laboratory data, and relation to specific outbreaks in childcare facilities. RESULTS: During 2010-2013, a total of 68 EHEC outbreaks comprised of 1035 cases were related to childcare facilities. Among the 66 outbreaks caused by a single serogroup, 29 were serogroup O26 and 22 were O157; 35 outbreaks were caused by stx1-producing strains. Since 2010, the number of reported outbreaks steadily increased, with a rise in cases and outbreaks caused by stx1-producing O26. Of 7069 EHEC cases reported nationally in 2010-2011, the majority were caused by O157 (n = 4938), relative to O26 (n = 1353) and O111 (n = 195). However, relative to 69 cases of O157 (2%) associated with childcare facility EHEC outbreaks, there were 131 (10%) such cases of O26, and this trend intensified in 2012-2013 (O157, 3%; O26, 24%; O111, 48%). Among family members of childcare facility cases, the proportion of cases that were symptomatic declined with age; 10/16 cases (63%) aged 6 years or younger, 16/53 cases (30%) 6-19 years old, 23/120 cases (19%) 20-49 years old and 2/28 cases (7%) 50 years or older were symptomatic. Thirty one of the 68 outbreaks (46%) were classified as foodborne-related. CONCLUSIONS: Childcare facility EHEC outbreaks due to non-O157 serogroups, particularly O26 and O111, increased during 2010-2013. These facilities should pay extra attention to health conditions in children. As older family members of childcare facility cases appear to be less symptomatic, they should be vigilant about hand-washing to prevent further transmission.

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.

Coordinate control of the locus of enterocyte effacement and enterohemolysin genes by multiple common virulence regulators in enterohemorrhagic Escherichia coli.

The locus of enterocyte effacement (LEE) pathogenicity island is required for the intimate adhesion of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelial cells. GrlR and GrlA are LEE-encoded negative and positive regulators, respectively. The interaction of these two regulators is important for controlling the transcription of LEE genes through Ler, a LEE-encoded central activator for the LEE. The GrlR-GrlA regulatory system controls not only LEE but also the expression of the flagellar and enterohemolysin (Ehx) genes in EHEC. Since Ehx levels were markedly induced in a grlR mutant but not in a grlR grlA double mutant and significantly increased by overexpression of GrlA in a ler mutant, GrlA is responsible for this regulation (T. Saitoh et al., J. Bacteriol. 190:4822-4830, 2008). In this study, additional investigations of the regulation of ehx gene expression determined that Ler also acts as an activator for Ehx expression without requiring GrlA function. We recently reported that the LysR-type regulator LrhA positively controls LEE expression (N. Honda et al., Mol. Microbiol. 74:1393-1411, 2009). The hemolytic activity of the lrhA mutant strain of EHEC was lower than that of the wild-type strain, and LrhA markedly induced ehx transcription in an E. coli K-12 strain, suggesting that LrhA also activates the transcription of ehx without GrlA and Ler. Gel mobility shift assays demonstrated that Ler and LrhA directly bind to the regulatory region of ehxC. Together, these results indicate that transcription of ehx is positively regulated by Ler, GrlA, and LrhA, which all act as positive regulators for LEE expression.

Transcription of the ehx enterohemolysin gene is positively regulated by GrlA, a global regulator encoded within the locus of enterocyte effacement in enterohemorrhagic Escherichia coli.

The pathogenicity island termed locus of enterocyte effacement (LEE) encodes a type 3 protein secretion system, whose function is required for full virulence of enterohemorrhagic Escherichia coli (EHEC). GrlR and GrlA are LEE-encoded negative and positive regulators, respectively, for controlling transcription of the ler gene, which encodes a central activator of LEE gene expression. We previously reported that the GrlR-GrlA regulatory system controls not only the LEE genes but also flagellar gene expression in EHEC (S. Iyoda et al., J. Bacteriol. 188:5682-5692, 2006). In order to further explore virulence-related genes under the control of the GrlR-GrlA regulatory system, we characterized a grlR-deleted EHEC O157 strain, which was found to have high and low levels of expression of LEE and flagellar genes, respectively. We report here that the grlR deletion significantly induced enterohemolysin (Ehx) activity of EHEC O157 on plates containing defibrinated sheep erythrocytes. Ehx levels were not induced in the grlR grlA double mutant strain but increased markedly by overexpression of GrlA even in the ler mutant, indicating that GrlA is responsible for this regulation. Ehx of the EHEC O157 Sakai strain is encoded by the ehxCABD genes, which are carried on the large plasmid pO157. The expression of ehxC fused with FLAG tag or a promoterless lacZ gene on pO157 was significantly induced under conditions in which GrlA was overproduced. These results together suggest that GrlA acts as a positive regulator for the ehx transcription in EHEC.

A new immunoglobulin-binding protein, EibG, is responsible for the chain-like adhesion phenotype of locus of enterocyte effacement-negative, shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) are important enteropathogens causing severe diseases such as hemorrhagic colitis and hemolytic-uremic syndrome in humans. The majority of STEC strains of serogroups O157, O26, or O111 associated with severe cases of these diseases possess a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE, which is responsible for the formation of attaching-and-effacing lesions on intestinal epithelial cells, is important for the full virulence of STEC. Nonetheless, LEE-negative STEC strains have repeatedly been reported to be associated with severe diseases in humans. In this study, we characterized adhesion to cultured epithelial cells of certain LEE-negative STEC isolated from humans with or without bloody diarrhea. Several LEE-negative STEC belonging to serogroup O91 showed an unusual, chain-like adhesion pattern to HEp-2 cells. Using Tn5-based transposon mutagenesis, we identified the gene essential for the chain-like adhesion phenotype of this O91 STEC strain. Sequence analysis of the Tn5-inserted allele identified a novel chromosomal open reading frame (ORF) encoding a polypeptide with a high degree of similarity to the E. coli immunoglobulin-binding (Eib) proteins EibA, -C, -D, -E, and -F. Therefore, the ORF was designated EibG. Laboratory E. coli strain MC4100 transformed with a multicopy plasmid carrying eibG showed chain-like adhesion to HEp-2 cells, and whole-cell lysates of the strain bound to human-derived immunoglobulin G (IgG) Fc and IgA. These results indicate that EibG acts as an IgG Fc- and IgA-binding protein, as well as an adhesin of LEE-negative STEC.

The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli.

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.