Contrasting Results from Two Commercial Kits Testing for the Presence of Clostridium perfringens Enterotoxin in Feces from Norovirus-Infected Human Patients.

BACKGROUND: Detection of Clostridium perfringens enterotoxin (CPE) is critical for disease surveillance; however, commercial testing kits produce contrasting results. METHODS: We examined the cause of the differing results from a reversed passive latex agglutination (RPLA) assay (PET-RPLA Toxin Detection Kit) and an enzyme-linked immunosorbent assay (C. perfringens Enterotoxin ELISA Kit) using 73 human norovirus-positive fecal samples from gastroenteritis patients across 22 episodes in Japan. RESULTS: CPE was detected in 39/73 samples using the RPLA method; however, ELISA-based examination of 10 RPLA-positive samples produced negative results. Moreover, cpe was not detected in any of the RPLA-positive (n = 32) or -negative (n = 5) samples, and C. perfringens was only isolated from one RPLA-positive sample. CONCLUSIONS: An ELISA-based testing approach may be more reliable than RPLA assays for CPE detection from human fecal samples. These findings may also be applicable to the detection of other foodborne diseases.

Flagellum expression and swimming activity by the zoonotic pathogen Escherichia albertii.

Flagella are the well-known structural appendages used by bacteria for motility. Although generally reported to be non-motile, the enteropathogenic bacterial species Escherichia albertii produces flagella intermittently. We found that E. albertii expressed flagella under specific environmental conditions. After several generations (involving 4 to 12-h incubations), six of the twelve strains we investigated displayed swimming motility in various aquatic environments, including pond water containing nutrients from pigeon droppings (10% suspension) as well as in 20 × -diluted tryptic soy broth. The most significant motility determinant was a temperature between 15 and 30 °C. At 20 °C in the 10% pigeon-dropping suspension, microscopic observations revealed that some cells (1%-95% of six strains) showed swimming motility. Electron microscopy showed that the E. albertii cells expressed flagella. Lower concentrations of some substrates (including nutrients) may be of secondary importance for E. albertii flagella expression. Interestingly, the non-motile strains (n = 6/12) contained pseudogenes corresponding to essential flagella structural proteins. After being released from its host into surface water, E. albertii may express flagella to move toward nutrient sources or new hosts.

Epidemiological Aspects of Escherichia albertii Outbreaks in Japan and Genetic Characteristics of the Causative Pathogen.

Zoonotic pathogen Escherichia albertii has been identified as the cause of several human disease outbreaks; however, factors such as the general symptoms and incubation period of E. albertii infection have yet to be defined. Therefore, we aimed to determine the unique aspects of E. albertii outbreaks in Japan and to examine the genetic characteristics of the causative pathogen. We studied all known E. albertii outbreaks that occurred in Japan up until 2015, which consisted of five confirmed outbreaks and one putative outbreak (Outbreaks 1-6). Outbreaks were re-examined based on personal communications between researchers in prefectural and municipal public health institutes, and through examination of any published study conducted at the time. Draft genome sequences of outbreak-associated E. albertii isolates were also generated. The most common symptom displayed by patients across the six episodes was watery diarrhea (>80%), followed by abdominal pain (50-84%) and fever (37.0-39.5°C) (26-44%). The estimated average incubation period of E. albertii infection was 12-24 h. We assumed that most of the outbreaks were foodborne or waterborne, with restaurant foods, restaurant water, and boxed lunches being the suspected transmission vehicles. Three of the six outbreak-associated E. albertii isolates possessed intact ETT2 regions, while the remaining isolates contained disrupted ETT2-encoding genes. Virulence gene screening revealed that more than half (44/70) of the tested genes were present in all 5 strains examined, and that each of the strains contained more than 1 gene from 14 out of the 21 groups of virulence genes examined in this study. The five E. albertii strains were classified into four of the five known phylogroups. Therefore, we determined that multiple E. albertii genotypes in Japan have the potential to cause outbreaks of diarrhea, abdominal pain, and/or fever following infection of a human host.

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.

Decrease in the prevalence of extended-spectrum cephalosporin-resistant Salmonella following cessation of ceftiofur use by the Japanese poultry industry.

Extended-spectrum cephalosporin (ESC)-resistant Salmonella in chicken meat is a significant food safety concern. We previously reported that the prevalence of ESC-resistant Salmonella in chicken meat, giblets, and processed chicken (chicken meat products) increased in Japan between 2005 and 2010, with 27.9% (17/61) of Salmonella isolated from chicken meat products in 2010 showing resistance to ESC. The aims of the present study were to clarify trends in the prevalence of ESC-resistant Salmonella in chicken meat products in Japan between 2011 and 2015, and to determine the genetic profiles of bla-harboring plasmids, including replicon types, using next-generation sequencing. Our results showed that the prevalence of ESC-resistant Salmonella, mainly consisting of AmpC ß-lactamase CMY-2-producing isolates, in chicken meat products had increased to 45.5% (10/22) by 2011. However, following the voluntary cessation of ceftiofur use by the Japanese poultry industry in 2012, the prevalence of ESC-resistant Salmonella steadily decreased each year, to 29.2% (7/24), 18.2% (4/22), 10.5% (2/19), and 10.5% (2/19) in 2012, 2013, 2014, and 2015, respectively. Furthermore, no AmpC ß-lactamase CMY-2-producing isolates were identified in 2014 and 2015. However, the prevalence of Salmonella enterica subspecies enterica serovar Manhattan isolates harboring a blaTEM-52-carrying IncX1 plasmid remained steady even after the cessation of ceftiofur use. Therefore, continuous monitoring of ESC resistance amongst Salmonella isolates from chicken meat products is required for food safety.

Pulsed-field profile diversities of Salmonella Enteritidis, S. Infantis, and S. Corvallis in Japan.

The diversity of pulsed-field profiles (PFPs) within non-typhoidal Salmonella subtypes influences epidemiological analyses of Salmonella outbreaks. Therefore, determining the PFP diversity of each Salmonella serovar is important when evaluating current circulating strains. This study examined the PFP diversity of three important public health Salmonella enterica subspecies enterica serovars, S. Enteritidis (n=177), S. Infantis (n=205), and S. Corvallis (n=90), using pulsed-field gel electrophoresis. Isolates were collected from several sources, primarily from chicken-derived samples, in the Kyushu-Okinawa region of Japan between 1989 and 2005. S. Enteritidis isolates displayed 51 distinct PFPs (E-PFPs), with 92 (52.0%) and 32 (18.1%) isolates displaying types E-PFP1 and E-PFP10, respectively. The 205 S. Infantis isolates showed 54 distinct PFPs (I-PFPs), with 87 (42.4%) and 36 (17.6%) isolates being I-PFP4 and I-PFP2, respectively. I-PFP18 was the dominant I-PFP of layer chicken isolates across a 5-year period. Fourteen distinct S. Corvallis PFPs were detected. Simpson's index results for the genetic diversities of S. Enteritidis, S. Infantis, and S. Corvallis isolates were 0.70, 0.79, and 0.78, respectively. None of the E-PFPs or I-PFPs of layer chicken isolates overlapped with those of broiler chicken isolates, and the dominant clonal lines existed for >10 years. In conclusion, limited PFP diversities were detected amongst S. Enteritidis, S. Infantis, and S. Corvallis isolates of primarily chicken-derived origins in the Kyushu-Okinawa region of Japan. Therefore, it is important to take into account these limitations in PFP diversities in epidemiological analyses of Salmonella outbreaks.

Simultaneous oral administration of Salmonella Infantis and S. Typhimurium in chicks.

BACKGROUND: To confirm the hypothesis that Salmonella enterica subspecies enterica serovar (S.) Infantis has higher basic reproductive rates in chicks compared with other Salmonella serovars, 1-day-old specific-pathogen-free chicks (n = 8) were challenged simultaneously with S. Infantis and S. Typhimurium per os. Challenged chicks (Group A) were then housed with non-infected chicks (Group B, n = 4) for 6 days (from 2 to 8 days of age). Group B birds were then housed with other non-infected birds (Group C, n = 4), which were then transferred to cages containing a further group of untreated chicks (Group D, n = 2). A control group consisting of four non-infected chicks was used for comparison. All chickens were humanely sacrificed at 18 days of age, and Salmonella from bowel and liver samples were enumerated. RESULTS: Both serovars were isolated from all groups except the control group. S. Typhimurium was isolated at a greater frequency than S. Infantis from the bowel samples of chicks from Groups B, C and D, while no differences in colonisation rates were observed between the two serovars in liver samples from Groups B, C and D. S. Typhimurium, but not S. Infantis, was immunohistochemically detected in the lamina propria of the cecum and rectum in five birds of Group A. Despite the competitive administration, neither of the two serovars completely excluded the other, and no differences were observed in basic reproductive rates between the two serovars. CONCLUSIONS: These findings, together with data from previous studies, suggest that the initial quantitative domination of S. Infantis in chicken flocks may explain why this serovar is predominant in broiler chickens.

Subtyping of Type 1 Diabetes as Classified by Anti-GAD Antibody, IgE Levels, and Tyrosine kinase 2 (TYK2) Promoter Variant in the Japanese.

OBJECTIVE: Type 1 diabetes (T1D) is known to be caused by Th1 cell-dependent autoimmunity. Recently, we reported that TYK2 promoter variant serves as a putative virus-induced diabetes susceptibility gene associated with deteriorated interferon-dependent antiviral response. TYK2 is also related to HIES, that is, Th2 cell-dependent. Therefore, TYK2 promoter variant may be also associated with the pathogenesis of T1D, modulating Th1/Th2 balance. RESEARCH DESIGN AND METHODS: We assessed the association between anti- GAD Ab, IgE levels, and TYK2 promoter variant among 313 T1D patients, 184 T2D patients, and 264 YH controls in the Japanese. RESULTS: T1D patients had elevated IgE (median, 56.7U/ml; p<0.0001) compared with T2D patients (22.5U/ml) and controls (43.3U/ml). Contrary to our expectations, there was no correlation between TYK2 promoter variant and IgE levels. We found that T1D could be subtyped as four groups based on anti-GAD Ab and IgE profile: Subtype 1, anti-GAD Ab positive and non-elevated IgE (47.0%); Subtype 2, anti-GAD Ab negative and non-elevated IgE (35.1%); Subtype 3, anti-GAD Ab positive and elevated IgE (10.9%); and Subtype 4, anti-GAD Ab negative and elevated IgE (7.0%). In Subtype 2, a significantly higher incidence was observed in T1D cases carrying the TYK2 promoter variant (OR, 2.60; 95%CI, 1.03-6.97; p=0.032), and also showing a flu-like syndrome at diabetes onset (OR, 2.34; 95%CI, 1.27-4.35; p=0.003). INTERPRETATION: Anti-GAD Ab and IgE profiling helps classifying T1D into four groups that recognize variable pathogenic bases of T1D.

New Identification of Three or More Campylobacter Species on the Basis of a Degenerate PCR-RFLP Method Targeting gyrB Gene.

This method was aimed targeting more Campylobacter species than conventional PCR-based identifications. They generally use species-specific primers focusing on clinically common species like C. jejuni, resulting in failure to recognize other species. We made the PCR-based identification more flexible using degenerate primers and DdeI- and MboI-separately used RFLP assay, which were designed on the basis of gyrB nucleotide sequence data of 14 Campylobacter species including C. jejuni, C. coli, and C. fetus. Ninety-four clinical isolates from patients with Campylobacter gastroenteritis and 13 biochemically identified C. fetus were used for its evaluation. In consequence, this method succeeded in identifying C. jejuni, C. coli, and C. fetus with tentative sensitivity (93.4-98.0%) and specificity (89.0-99.0%). According to our data-based analysis, the primers can possibly target other related species including Helicobacter and Arcobacter. This method may be a universal identification for Campylobacter and related organisms and would provide an alternative identification in clinical microbiology.

Typing Method for the QUB11a Locus of Mycobacterium tuberculosis: IS6110 Insertions and Tandem Repeat Analysis.

QUB11a is used as a locus for variable number of tandem repeats (VNTR) analysis of Mycobacterium tuberculosis Beijing lineage. However, amplification of QUB11a occasionally produces large fragments (>1,400 bp) that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110 insertion may complicate VNTR analysis of large QUB11a fragments in M. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110 insertion in the QUB11a locus of M. tuberculosis using capillary electrophoresis analysis and BsmBI digestion. All 29 large QUB11a fragments (>1,200 bp) investigated contained IS6110 insertions and varied in the number of repeats (18 patterns) and location of IS6110 insertions. This method allows VNTR analysis with high discrimination.

Reduced Tyk2 gene expression in ß-cells due to natural mutation determines susceptibility to virus-induced diabetes.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic ß-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by ß-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic ß-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Detection of Escherichia albertii from chicken meat and giblets.

Escherichia albertii occasionally causes food-borne outbreaks of gastroenteritis in humans; however, little is known about the vehicle of transmission. To screen retail chicken products for the presence of E. albertii, 104 retail chicken products were investigated. Portions of enrichment cultures that were PCR-positive for E. albertii (n=3) were sub-cultured on agar medium. Only 2 strains obtained from 2 chicken giblet samples were identified as E. albertii by multi locus sequence typing. Antimicrobial susceptibility testing showed that 1 strain was resistant to streptomycin and sulfisoxazole. Both strains harbored the virulence genes cdt and eae. This study is the first description of E. albertii isolation from retail food, suggesting that chicken products are a potential vehicle of E. albertii transmission.

Increase in resistance to extended-spectrum cephalosporins in Salmonella isolated from retail chicken products in Japan.

Extended-spectrum ß-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996-2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the bla(CMY), bla(CTX-M), bla(TEM), and bla(SHV) resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (bla(CMY-2)), and nine ESBL-producing isolates harboring bla(CTX-M) (n = 4, consisting of two bla(CTX-M-2) and two bla(CTX-M-15 genes)), bla(TEM) (n = 4, consisting of one bla(TEM-20) and three bla(TEM-52) genes), and bla(SHV) (n = 1, bla(SHV-12)). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing bla(CMY-2) in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated bla(CMY-2) in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health.

Toll-like receptors recognize distinct proteinase-resistant glycoconjugates in Campylobacter jejuni and Escherichia coli.

Campylobacter jejuni causes gastroenteritis and autoimmune neuropathy Guillain-Barré syndrome. The mechanism by which C. jejuni infection results in such the hyperimmunity is not completely understood. Host immunity plays an important role in the disease pathogenesis; however, little is known how immune system recognizes this human pathogen. In this study, we report that Toll-like receptors recognize distinct proteinase K-resistant glycoconjugates in C. jejuni and Escherichia coli. Lipopolysaccharide is solely proteinase-resistant glycoconjugate in E. coli. In contrast, C. jejuni possesses at least five different components that are resistant to proteinase digestion and are capable of inducing NF-κB activation through TLR2 and TLR4. Possession of multiple activators of Toll-like receptors may be the unique strategy of C. jejuni to trigger hyperimmunity.

Easy washing of lysed cell plugs for bacterial typing by pulsed-field gel electrophoresis using simple equipment.

We designed and tested equipment to wash plugs following cell lysis in pulsed-field gel electrophoresis (PFGE). Our system can wash 30 plugs simultaneously in 1h using 15L of Tris-EDTA buffer, which makes plug washing for PFGE less labor-intensive.

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.

Penicillin susceptibility of non-serotypeable Streptococcus pneumoniae from ophthalmic specimens.

Nontypeable (NT) Streptococcus pneumoniae strains isolated from eyes were examined for both penicillin susceptibility by E-test and penicillin-binding protein (PBP) gene alterations using PCR. Of the 25 ophthalmic isolates, 15 proved to be sensitive (PSSP, MIC < or = 0.06 microg/ml) and 10 were shown as intermediately resistant to penicillin (PISP, MIC = 0.1-1 microg/ml). No penicillin-resistant S. pneumoniae (PRSP, MIC > or = 2 microg/ml) were found. PBP gene (pbp1a and pbp2b) alteration PCR indicated that 12 (80.0%) of the 15 ophthalmic PSSPs had unaltered pbp genes and that 3 (20.0%) had alterations in either pbp1a or pbp2b, whereas 8 (80.0%) of the 10 PISPs had unaltered pbp genes and 2 (20.0%) had alterations in both pbp1a and pbp2b. These data suggest that penicillin resistance is spread among NT pneumococci typically associated with ophthalmic infections.