Differential dynamics and impacts of prophages and plasmids on the pangenome and virulence factor repertoires of Shiga toxin-producing Escherichia coli O145:H28.

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

Association of Cell-adhesion Activities with Virulence in Shiga toxin-producing Escherichia coli O103：H2.

The characteristics of 11 strains of Stx1-producing and Stx2-non-producing STEC O103：H2 were analyzed to investigate the differences in virulence in a single serotype of Shiga toxin (Stx) -producing Escherichia coli (STEC). Differences in the cell-adhesion activity to Caco-2 cells were observed among the strains. The activity of the one strain, isolated from a patient with hemolytic uremic syndrome was 4-20-fold higher than those of the other strains. Although the strains with high cell-adhesion activity showed high expressions of eae, espB, espD, and tir in the locus of enterocyte effacement related with cell-adhesion, those were not specific for this strain. In addition, the Stx1 production level of the strain was not particularly high. It was indicated that the high adhesion activity might be a potential factor to associate serious symptom.

[Molecular Characterization of Salmonella enterica subsp. enterica Serovar 4: b: -].

Salmonella is a major causative agent of food borne diseases. Recently, monophasic strains of Salmonella, such as S. enterica 4: i: -, have been frequently reported. Here, we investigated the genetic background of S. enterica 4: b: - using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. A total of 10 strains of S. enterica (I) 4: b: - were examined and compared with 34 strains including serovar Paratyphi B and Paratyphi B var Java, Schleissheim, and II b: -. All I 4: b: - strains were negative for hin which encodes an invertase that converts the H phases, and six were also negative for fljB, which encodes the second phase of the H antigen. An MLST analysis identified 12 sequence types (ST) and 6 ST complexes (STC) from the 44 strains. A clustering analysis of PFGE patterns almost corresponded to the STC. The monophasic I 4: b: - strains were assigned to 3 STCs (19, 32 and 155), corresponding to those of Paratyphi B var. Java or a monophasic strain according to the data of this and previous studies. These findings suggest that the monophasic strains examined in this study might have been derived from multiple clones of Paratyphi B var Java. This study shows the usefulness of molecular typing as complementation tools of the conventional serotyping system.

Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Evaluation of the culture method NIHSJ-02 alternative to ISO 10272-1:2006 for the detection of campylobacter jejuni and campylobacter coli in chicken: collaborative study.

For the surveillance of the prevalence of Campylobacter jejuni and Campylobacter coli in raw chicken products in Japan, a qualitative method, National Institute of Health Sciences Japan (NIHSJ)-02, was developed as an alternative to International Organization for Standardization (ISO) 10272-1:2006. In the NIHSJ-02 culture method, the enrichment step is carried out in a reduced volume of Preston broth at 42 +/- 1 degrees C to reduce cost and space, and to prevent the overgrowth of background bacteria. To evaluate the performance of NIHSJ-02, a collaborative study was conducted, and the results obtained by NIHSJ-02 were compared with those obtained using the reference method, ISO 10272-1:2006. Fifteen laboratories participated; each examined 48 minced chicken samples consisting of test samples uninoculated, inoculated with C. jejuni at a low or high level, and inoculated with C. coli at a low level. The average probabilities of detection by NIHSJ-02 across laboratories were 0.033, 0.222, 0.678, and 0.267 in samples uninoculated, inoculated with C. jejuni at a low and high level, and with C. coli at a low level, respectively. Those by ISO 10272-1:2006 were 0.051, 0.128, 0.551, and 0.090. Significantly higher probabilities of detection were determined by NIHSJ-02 compared to ISO 10272-1:2006, except for uninoculated samples. On the other hand, significantly lower frequency of occurrence of background bacteria was observed by NIHSJ-02 (43.1%) compared with ISO 10272-1:2006 (92.6%). NIHSJ-02 showed better performance than ISO 10272-1:2006 with regard to the selective detection of C. jejuni and C. coli in chicken.

Virulence gene profiles and population genetic analysis for exploration of pathogenic serogroups of Shiga toxin-producing Escherichia coli.

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) is a serious public health concern, causing severe diarrhea and hemolytic-uremic syndrome. Patient symptoms are varied among STEC strains, possibly implying the presence of markers for STEC virulence other than Stx. To reveal the genotypic traits responsible for STEC virulence, we investigated 282 strains of various serogroups for the presence of 17 major virulence genes, i.e., stx1, stx2a, stx2c, stx2d, stx2e, stx2f, eae, tir, espB, espD, iha, saa, subA, ehxA, espP, katP, and stcE. Next, we examined the prevalence of virulence genes according to the seropathotypes in which serotypes were classified (seropathotypes A through E) based on the reported frequencies in human illness, as well as known associations with outbreaks and with severe disease. Our results demonstrate that the presence of both katP and stcE in STEC, in addition to the genes located in the locus of enterocyte effacement (LEE), including eae, tir, espB, and espD, may indicate the most pathogenic genotype of STEC. A population structure analysis of the profiles of virulence genes statistically supported the pathogenic genotype and, furthermore, revealed that there are serogroups with potentially higher pathogenicity than previously thought. Some strains in serogroups O26, O145, and O165 may have high virulence equivalent to that of serogroup O157. Several serogroups, including O14, O16, O45, O63, O74, 119, O128, and O untypeable, also may be potentially pathogenic, although rarely in humans.

Determination of enterohemorrhagic Escherichia coli serotype O165:HNM infection in a hemolytic uremic syndrome patient with adenovirus seroype 41.

A 4-year-old girl who was positive for adenovirus according to a rapid immunochromatographic test conducted at a hospital, progressed to hemorrhagic diarrhea and hemolytic uremic syndrome (HUS). The presence of adenovirus serotype 41 (AdV-41) was confirmed by TaqMan real-time PCR and sequence analysis. However, most enteric viral infections cause mild to moderate diarrhea. In the present case, enterohemorrhagic Escherichia coli (EHEC) O165:HNM was isolated concomitantly with AdV-41. In addition, O165 antibody was specifically detected in patient sera. The EHEC isolate was positive for the virulence genes stx1, stx2a, eae type ε, ehxA, and norV. Therefore, we concluded that EHEC O165:HNM was the precise pathogen leading to HUS in this patient.

Differences in the stress tolerances of Vibrio parahaemolyticus strains due to their source and harboring of virulence genes.

To investigate the diversity of stress tolerance levels in Vibrio parahaemolyticus, 200 V. parahaemolyticus strains isolated from various coastal environments, seafood, and human clinical cases were exposed to acid, low-osmolality, freezing-thawing, and heat stresses. Tolerance against acid stress was higher in the virulent (tdh- and/or trh-positive) strains than in the avirulent (tdh- and trh-negative) strains. Tolerance against low-osmolality, freezing-thawing, and heat stresses was higher in the clinical strains of tdh- and/or trh-positive V. parahaemolyticus than in the coastal environment- and seafood-originated strains of tdh and/or trh-positive V. parahaemolyticus. Tolerance against acid stress was higher in the strains isolated from coastal seawater at ≤15°C than in the strains isolated at ≥20°C. Tolerance against heat stress was higher in the avirulent strains than the virulent strains, and in the strains isolated from coastal seawater at ≥20°C than the strains isolated from coastal seawater at ≤15°C. Therefore, this study demonstrated that the diversity of stress tolerance levels in V. parahaemolyticus strains depended on their source and whether they harbored virulence genes. In particular, there was significantly greater tolerance against acid in the virulence gene-harboring strains and strains isolated from low-temperature seawater. Because the stress tolerances of V. parahaemolyticus have direct influences for the survival in environment and food, it is important for the prevention of foodborne infection to control the stress-tolerant strains.

Isolation and identification of Escherichia albertii from a patient in an outbreak of gastroenteritis.

A microbial strain harboring the eae gene, which is known as the virulence gene of enteropathogenic Escherichia coli (EPEC) and most enterohemorrhagic E. coli, was isolated from a patient in a gastroenteritis outbreak that occurred in 22 patients in Akita Prefecture, Japan, in November 2011. The biochemical characteristics of the isolate were more similar to those of a novel Escherichia sp., E. albertii than E. coli. Partial 16S rRNA gene sequences of the isolate were identical to those of a certain E. albertii strain, but also showed a high degree of similarity to those of E. coli strains. Finally, we identified this isolate as E. albertii by performing PCR analysis that targeted the uidA, lysP, mdh, and cdtB genes in addition to stx and eae genes to differentiate between the EPEC and E. albertii strains.

Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients.

Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.

Application of a multilocus variable number of tandem repeats analysis to regional outbreak surveillance of Enterohemorrhagic Escherichia coli O157:H7 infections.

A total of 18 strains of EHEC O157:H7 were isolated from distinct cases in Akita Prefecture, Japan from July to September 2007. The genetic relatedness of these isolates was investigated by performing a multilocus variable number of tandem repeats analysis (MLVA) and a pulsed-field gel electrophoresis (PFGE) analysis using XbaI. The PFGE analyses allowed us to group these 18 isolates into three major clusters. The MLVA results correlated closely with those obtained by PFGE, although some variants were found within the clusters obtained by PFGE, thus highlighting the utility of this technique for determining a precise classification when it is difficult to differentiate between isolates with indistinguishable or very similar PFGE patterns. In addition, MLVA is a much easier and more rapid method than PFGE for analysis of the genetic relatedness of strains. Thus, as a second molecular epidemiological subtyping method, MLVA is useful for the regional outbreak surveillance of EHEC O157:H7 infections.

[A case of classical tsutsugamushi disease confirmed after an interval of 15 years in Akita Prefecture, Japan].

We report the confirmation of classical tsutsugamushi disease in August 2008. A 17-year-old woman seen for fever and eschar on the back reported having been bitten by an insect nine days earlier while fishing on the Omonogawa river. The suspected culprit was Leptotrombidium akamushi. During convalescence serum IgM and IgG antibody titers rose significantly against the Kato serotype antigen in indirect immunoperoxidase staining. Epidemiology, clinical symptoms and the antibodies detected suggested classical tsutsugamushi disease infection. Such disease transmitted by L. akamushi have not been reported since 1993 in Akita Prefecture. The public should thus be informed about Orientia tsutsugamushi prevention, in case such disease re-care in this area in the future.

[Legionella contamination risk factors in non-circulating hot spring water].

We examined water from 182 non-circulating hot spring bathing facilities in Japan for possible Legionella occurrence from June 2005 to December 2006, finding Legionella-positive cultures in 119 (29.5%) of 403 samples. Legionellae occurrence was most prevalent in bathtub water (39.4%), followed by storage tank water (23.8%), water from faucets at the bathtub edge (22.3%), and source-spring water (8.3%), indicating no statistically significant difference, in the number of legionellae, having an overall mean of 66 CFU/100mL. The maximum number of legionellae in water increased as water was sampled downstream:180 CFU/100 mL from source spring, 670 from storage tanks, 4,000 from inlet faucets, and 6,800 from bathtubs. The majority--85.7%--of isolated species were identified as L. pneumophila : L. pneumophila serogroup (SG) 1 in 22%, SG 5 in 21%, and SG 6 in 22% of positive samples. Multivariate logistic regression models used to determine the characteristics of facilities and sanitary management associated with Legionella contamination indicated that legionellae was prevalent in bathtub water under conditions where it was isolated from inlet faucet/pouring gate water (odds ratio [OR] = 6.98, 95% confidence interval [CI] = 2.14 to 22.8). Risk of occurrence was also high when the bathtub volume exceeded 5 m3 (OR = 2.74, 95% CI = 1.28 to 5.89). Legionellae occurrence was significantly reduced when the bathing water pH was lower than 6.0 (OR = 0.12, 95% CI = 0.02 to 0.63). Similarly, occurrence was rare in inlet faucet water or the upper part of the plumbing system for which pH was lower than 6.0 (OR = 0.06, 95% CI = 0.01 to 0.48), and when the water temperature was maintained at 55 degrees C or more (OR = 0.10, 95% CI = 0.01 to 0.77). We also examined the occurrence of amoeba, Mycobacterium spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus in water samples.

[The risk of pertussis and diphtheria infections among pediatric healthcare workers in Japan].

For infection control in pediatric hospitals, we investigated the risk of pertussis and diphtheria infections among pediatric healthcare workers. Forty-nine Japanese pediatric healthcare workers in 12 general hospitals were screened for antibodies of pertussis toxin (PT), filamentous hemagglutinin (FHA), and diphtheria toxin (DT). The seropositive rates of anti-PT IgG (protective level, > 10 U/mL), anti-FHA IgG (> 10 U/ mL), and anti-DT (> 0.11 U/mL) were 50, 82, and 59%, respectively. During this survey period (Oct. 2003-Feb. 2004), 16 (33%) of the healthcare workers were in contact with pertussis-infant (s). However, all culture and PCR tests for Bordetella pertussis were negative. One of the 16 exposed healthcare workers, a male pediatrician, had serological evidence of a pertussis infection, but no disease symptomatic of pertussis. Our observations indicate that i) 50 and 41% of Japanese pediatric healthcare workers were seronegative for pertussis (anti-PT IgG) and diphtheria antibodies, respectively, and ii) although the healthcare workers had a high rate of contact with pertussis-infant (s), the infection rate was low. For pertussis and diphtheria infection control in pediatric hospitals, it is important for healthcare workers to be aware of their own protection levels against these diseases.

[Development of O-serogroup serodiagnosis for patients with hemolytic uremic syndrome by Ec-LPS array].

We extracted lipopolysaccharides from 58 O-serogroup strains of Escherichia coli with phenol-water for use as antigens for an Ec-LPS array. The Ec-LPS array was made by dot-blotting of E. coli LPS on PVDF membrane. Commercial anti-E. coli O-serogroup antisera reacted with homologous O-serogroup LPS in Ec-LPS arrays. Convalescent sera of 6 patients with hemolytic uremic syndrome reacted strongly with O157 LPS when IgM and IgA antibodies in patient sera analyzed by Ec-LPS arrays. When IgG antibody was analyzed in this array, it was difficult to diagnose the O-serogroup because of the reactivity of patient sera against many O-serogroup LPS. These results match those by ELISA and western blotting. Compared to these serological techniques, Ec-LPS array appears superior to ELISA and western blotting in cost performance, time performance, and technical complexity.

Nosocomial outbreak of ceftazidime-resistant Serratia marcescens strains that produce a chromosomal AmpC variant with N235K substitution.

Serratia marcescens is a Gram-negative bacterium that is often associated with nosocomial infections. Here we analyzed the resistance mechanism of the ceftazidime-resistant S. marcescens nosocomial strains. The five S. marcescens urinary tract infection-associated isolates were positive for chromosomal ampC and bla(TEM-1). Four of the five strains, ES11, ES31, ES42, and ES46, were single clone and ceftazidime resistant. The fifth strain, ES71, was susceptible to ceftazidime. Analysis of the deduced amino acid sequence revealed a Glu-235-Lys substitution in the third amino acid of the third motif of AmpC from both ES46 and ES71, and a site-directed mutagenesis experiment confirmed that this substitution is involved in the ceftazidime resistance phenotype. However, the resistance phenotypes of strains ES46 and ES71 to ceftazidime were quite different from one another, indicating that another mechanism, in addition to the AmpC mutation, is also involved in the determination of the resistance phenotype of these strains. Basal AmpC activity was more than two times higher in strain ES46 than in ES71, which could result in the differing resistance phenotypes of these two strains. The clinical significance and prevalence of extended-spectrum cephalosporin-resistant S. marcescens strains harboring the mutated chromosomal ampC gene are unclear in Japan and remain to be elucidated.

Campylobacter jejuni isolated from retail poultry meat, bovine feces and bile, and human diarrheal samples in Japan: comparison of serotypes and genotypes.

To determine the significance of poultry and bovine as infectious sources of Campylobacter jejuni in Japan, the serotype distribution and pulsed-field gel electrophoresis (PFGE) patterns of poultry and bovine isolates were compared with those of isolates from patients with diarrhea in Akita (Japan). Serotypes O:2 and O:4-complex were common in human, poultry, and bovine isolates, and serotype O:23,36,53 was common in human and bovine isolates. SmaI PFGE patterns of isolates belonging to these serotypes were generated. Eight PFGE patterns were shared by poultry and human isolates and three patterns were shared by human and bovine isolates. Further analysis of the isolates having the same SmaI PFGE pattern by KpnI PFGE confirmed that four patterns and two patterns were still shared by poultry and human isolates, and bovine and human isolates, respectively. Thus, serotypic and genotypic data indicated a possible link between sporadic human campylobacteriosis and C. jejuni from retail poultry and bovine bile and feces, suggesting that bovine serves as an infectious source of C. jejuni in Japan, as is observed in other countries.