O-antigen biosynthesis gene clusters of Escherichia albertii: their diversity and similarity to Escherichia coli gene clusters and the development of an O-genotyping method.

Escherichia albertii is a recently recognized human enteropathogen that is closely related to Escherichia coli. In many Gram-negative bacteria, including E. coli, O-antigen variation has long been used for the serotyping of strains. In E. albertii, while eight O-serotypes unique to this species have been identified, some strains have been shown to exhibit genetic or serological similarity to known E. coli/Shigella O-serotypes. However, the diversity of O-serotypes and O-antigen biosynthesis gene clusters (O-AGCs) of E. albertii remains to be systematically investigated. Here, we analysed the O-AGCs of 65 E. albertii strains and identified 40 E. albertii O-genotypes (EAOgs) (named EAOg1-EAOg40). Analyses of the 40 EAOgs revealed that as many as 20 EAOgs exhibited significant genetic and serological similarity to the O-AGCs of known E. coli/Shigella O-serotypes, and provided evidence for the inter-species horizontal gene transfer of O-AGCs between E. albertii and E. coli. Based on the sequence variation in the wzx gene among the 40 EAOgs, we developed a multiplex PCR-based O-genotyping system for E. albertii (EAO-genotyping PCR) and verified its usefulness by genotyping 278 E. albertii strains from various sources. Although 225 (80.9 %) of the 278 strains could be genotyped, 51 were not assigned to any of the 40 EAOgs, indicating that further analyses are required to better understand the diversity of O-AGCs in E. albertii and improve the EAO-genotyping PCR method. A phylogenetic view of E. albertii strains sequenced so far is also presented with the distribution of the 40 EAOgs, which provided multiple examples for the intra-species horizontal transfer of O-AGCs in E. albertii.

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.

Postprandial Glucose Surges after Extremely Low Carbohydrate Diet in Healthy Adults.

Carbohydrate-restricted diets are prevalent not only in obese people but also in the general population to maintain appropriate body weight. Here, we report that extreme carbohydrate restriction for one day affects the subsequent blood glucose levels in healthy adults. Ten subjects (median age 30.5 years, BMI 21.1 kg/m2, and HbA1c 5.5%), wearing with a continuous glucose monitoring device, were given isoenergetic test meals for 4 consecutive days. On day 1, day 2 (D2), and day 4 (D4), they consumed normal-carbohydrate (63-66% carbohydrate) diet, while on day 3, they took low-carbohydrate/high-fat (5% carbohydrate) diet. The daily energy intake was 2,200 kcal for males and 1,700 kcal for females. On D2 and D4, we calculated the mean 24-hr blood glucose level (MEAN/24h) and its standard deviation (SD/24h), the area under the curve (AUC) for glucose over 140 mg/dL within 4 hours after each meal (AUC/4h/140), the mean amplitude of the glycemic excursions (MAGE), the incremental AUC of 24-hr blood glucose level above the mean plus one standard deviation (iAUC/MEAN+SD). Indexes for glucose fluctuation on D4 were significantly greater than those on D2 (SD/24h; p = 0.009, MAGE; p = 0.013, AUC/4h/140 after breakfast and dinner; p = 0.006 and 0.005, and iAUC/MEAN+SD; p = 0.007). The value of MEAN/24h and AUC/4h/140 after lunch on D4 were greater than those on D2, but those differences were not statistically significant. In conclusion, consumption of low-carbohydrate/high-fat diet appears to cause higher postprandial blood glucose on subsequent normal-carbohydrate diet particularly after breakfast and dinner in healthy adults.

Defining the Genome Features of Escherichia albertii, an Emerging Enteropathogen Closely Related to Escherichia coli.

Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen.

Identification of O serotypes, genotypes, and virulotypes of Shiga toxin-producing Escherichia coli isolates, including non-O157 from beef cattle in Japan.

Bovines are recognized as an important reservoir of Shiga toxin-producing Escherichia coli (STEC). Although STEC strains are significant foodborne pathogens, not all of the STEC held by cattle are pathogenic, and which type of STEC that will become epidemic in humans is unpredictable. Information about the prevalence of serotype and virulence gene distribution in beef cattle is insufficient to develop monitoring and controlling activities for a food safety and security program. Thus, this study investigated the prevalence of O157 and non-O157 STEC in Japanese beef cattle and characterized the isolates by the type of O antigen and several virulence markers to help predict the pathogenicity. In this study, 64.2% (176 of 274) of enrichment cultures of fecal samples collected from an abattoir and farms were stx1 and/or stx2 positive by PCR. STEC strains were isolated from 22.1% (39 of 176) of the positive fecal samples, and these isolates represented 17 types of O antigen (O1, O2 or O50, O5, O8, O55, O84, O91, O109, O113, O136, O150, O156, O157, O163, O168, O174, and O177). Two selective media targeting major STEC groups, cefixime-tellurite sorbitol MacConkey agar and CHROMagar O26/O157, allowed isolation of a variety of STEC strains. The most frequently isolated STEC was O113 (8 of 39), which has previously been reported as a cause of foodborne infections. Although most of the O113 STEC isolated from infected patients possessed the enterohemolysin (hlyA) gene, none of the O113 STEC cattle isolates possessed the hlyA gene. The second most common isolate was O157 (6 of 39), and all these isolates contained common virulence factors, including eae, tir, lpf1, lpf2, and hlyA. This study shows the prevalence of O157 and non-O157 STEC in Japanese beef cattle and the relationship of O antigen and virulotypes of the isolates. This information may improve identification of the source of infection, developing surveillance programs or the current understanding of virulence factors of STEC infections.

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.

Imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus.

A Japanese man suffered from acute respiratory tract infection after returning to Japan from Bali, Indonesia in 2007. Miyazaki-Bali/2007, a strain of the species of Nelson Bay orthoreovirus, was isolated from the patient's throat swab using Vero cells, in which syncytium formation was observed. This is the sixth report describing a patient with respiratory tract infection caused by an orthoreovirus classified to the species of Nelson Bay orthoreovirus. Given the possibility that all of the patients were infected in Malaysia and Indonesia, prospective surveillance on orthoreovirus infections should be carried out in Southeast Asia. Furthermore, contact surveillance study suggests that the risk of human-to-human infection of the species of Nelson Bay orthoreovirus would seem to be low.

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.

Relationship between stx genotype and Stx2 expression level in Shiga toxin-producing Escherichia coli O157 strains.

To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.

Transmission of Bordetella holmesii during pertussis outbreak, Japan.

We describe the epidemiology of a pertussis outbreak in Japan in 2010-2011 and Bordetella holmesii transmission. Six patients were infected; 4 patients were students and a teacher at the same junior high school. Epidemiologic links were found between 5 patients. B. holmesii may have been transmitted from person to person.

Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay.

A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.

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.

Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand.

Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae- and bfpA-positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n=27) and Thailand (n=26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp-2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.

Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types.

We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980-2008. Most (80.2%) belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95% CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9% of all isolates). Moreover, 79.7% of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulence-associated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n=6), ST120 (n=5) and ST138 (n=5). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies.

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.

[Largest outbreak of legionellosis associated with spa baths: comparison of diagnostic tests].

In July 2002, a large outbreak of legionellosis occurred in a bathhouse with spa facilities in Miyazaki Prefecture. Among the visitors, 295, including suspected cases had pneumonia and/or symptoms of fever, coughing, etc. Of these, 37% were hospitalized and 7 died. Clinical samples from 95 mainly inpatients were collected and microbiologically tested at our laboratory. Legionella pneumophila serogroup (SG) 1 was isolated from 3 of 24 in sputum culture, and none of the 3 had been treated effectively with antibiotics at sputa collection. L. pneumophila antigen in urine was detected by using enzyme immunoassay and/or immunochromatographic kits in 23 of 75 patients. Serum antibodies to L. pneumophila SG1 and Legionella dumoffii were detected in 5 each of 66 patients--9 cases including a case at mixed infection-by microplate agglutination test and/or indirect immunofluorescence assay. At our laboratory, 32 were diagnosed with legionellosis. In this outbreak, 14 were diagnosed at other laboratories, resulting in 46 confirmed cases. Urine antigen was detected more frequently by Binax NOW immunochromatographic assay than by Biotest EIA-31% versus 16% of cases tested. Both assays detected urine antigen only in samples collected within 4 weeks after onset. Antigen concentration in urine enhanced sensitivity-58% and 51%-and extended the period of antigen detection beyond 5 weeks. Both antibody titers to L. pneumophila SG1 and L. dumoffii in more than 90% of sera collected within 3 weeks after onset were < 1:16. The rate of serum antibody titer to > or = 1:128 within 3 weeks was 1.6%, during 4 to 6 weeks less than 10%, and after 7 weeks or more 8 to 25%. After an administrative report was published, L. pneumophila DNA in sputa was detected in 5 of 17 patients by nested PCR, resulting in extra 3 cases. Altogether, urinary antigen detection and PCR were more effective in laboratory diagnostic tests than culture and serology. Culture combined with molecular epidemiology is critical, however, for confirming the source of infection.

[Symptoms of food-borne diseases and gastroenteritis in Kyushu, Japan].

In this study we analyzed the symptoms of gastroenteritis or food-borne disease caused by the 10 most prevalent pathogens: Norovirus, Salmonella, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, Shiga toxin-producing Escherichia coli (STEC), enterotoxigenic E. coli (ETEC), Shigella sonnei/flexneri (Shigella), Staphylococcus aureus, and emetic-type Bacillus cereus. The symptoms diarrhea, vomiting, fever, abdominal pain, and headache, and the incubation period in 646 cases in 10 districts of Kyushu between January 2000 and December 2004 were recorded. The pathogen with the shortest mean incubation period was B. cereus (0.8 h), and was followed by S. aureus (3.3 h), C. perfringens (10.7 h) and V. parahaemolyticus (16.4 h). All the patients infected with B. cereus and S. aureus developed symptoms within 6 hours, and those infected with V. parahaemolyticus and C. perfringens developed symptoms within 24 hours. Bloody diarrhea was associated with STEC and Shigella, but rare with other pathogens. Vomiting was associated with almost all cases of S. aureus and B. cereus infection, and occurred in 71.5% of the Norovirus cases and 56.1% of the V. parahaemolyticus cases. Vomiting was less common in the C. perfringens (22.0%) and the ETEC and STEC (both about 5%). Bloody diarrhea, abdominal pain, and vomiting were statistically significantly more common with STEC 0157 infection than with STEC non-0157 infection. Since the cases analyzed in this study included all degrees of illness, mild to severe, and a wide range of ages, the information obtained will serve as a good reference material for administrative and laboratory work when an outbreak takes place.