Production of a novel monoclonal antibody applicable for an immunochromatographic assay for Kudoa septempunctata spores contaminating the raw olive flounder (Paralichthys olivaceus).

Kudoa septempunctata, a myxosporean parasite of the olive flounder (Paralichthys olivaceus), causes foodborne gastroenteritis after ingestion of contaminated raw flounder. Available methods to detect K. septempunctata require expensive equipment, well-trained personnel, and lengthy procedures. Here we generated a novel monoclonal antibody (MAb 15G11) against K. septempunctata and used it to produce a prototype immunochromatographic assay (prototype Kudoa-ICA). Within 15min, the prototype Kudoa-ICA detected ≥1.0×105spores/mL in a spore suspension and ≥2.0×104spores/g of P. olivaceus muscle. The prototype Kudoa-ICA weakly cross-reacted with spores of K. lateolabracis and K. iwatai. cDNA sequence, expression, and western blot analyses revealed that MAb 15G11 detected an approximately 24-kDa protein encoded by a 573bp mRNA. The cDNA nucleotide and predicted amino acid sequences were not significantly similar to any sequence in the GeneBank database. Immunoelectron microscopy revealed that MAb 15G11 reacted with the sporoplasmic cells and mainly with the capsulogenic cells of the K. septempunctata spore. Although the Kudoa-ICA was weakly cross-reactive with two other Kudoa species, it detected >1.0×106spores/g of K. septempunctata in P. olivaceus muscle, which is the criterion used to indicate a violation of the Food Hygiene Law of Japan. We conclude that MAb 15G11 may be suitable for use in an immunochromatographic assay for screening P. olivaceus muscle contaminated with K. septempunctata at food distribution sites such as food wholesalers, grocery stores, and restaurants.

A double-quadratic model for predicting Vibrio species in water environments of Japan.

Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.

The emerging ST8 methicillin-resistant Staphylococcus aureus clone in the community in Japan: associated infections, genetic diversity, and comparative genomics.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major concern worldwide. In the United States, ST8 CA-MRSA with SCCmecIVa (USA300) has been predominant, affecting the entire United States. In this study, we investigated Japanese ST8 CA-MRSA with new SCCmecIV1 (designated ST8 CA-MRSA/J), which has emerged in Japan since 2003. Regarding community spread and infections, ST8 CA-MRSA/J spread in 16.2-34.4% as a major genotype in the community in Japan, and was associated with skin and soft tissue infections (SSTIs), colitis, and invasive infections (sepsis, epidural abscesses, and necrotizing pneumonia), including influenza prodrome cases and athlete infections, similar to USA300. It spread to even public transport and Hong Kong through a Japanese family. Regarding genetic diversity, ST8 CA-MRSA/J included ST and spa variants and was classified into at least three pulsed-field gel electrophoresis types, ST8 Jα to γ. Of those, ST8 Jß was associated with severe invasive infections. As for genomics, ST8 CA-MRSA/J showed high similarities to USA300, but with marked diversity in accessory genes; e.g., ST8 CA-MRSA/J possessed enhanced cytolytic peptide genes of CA-MRSA, but lacked the Panton-Valentine leukocidin phage and arginine catabolic mobile element, unlike USA300. The unique features of ST8 CA-MRSA/J included a novel mosaic SaPI (designated SaPIj50) carrying the toxic shock syndrome toxin-1 gene with high expression; the evolution included salvage (through recombination) of hospital-acquired MRSA virulence. The data suggest that ST8 CA-MRSA/J has become a successful native clone in Japan, in association with not only SSTIs but also severe invasive infections (posing a threat), requiring attention.

Evaluation of streptococcal toxic shock-like syndrome caused by group B streptococcus in adults in Japan between 2009 and 2013.

Infection with Streptococcus agalactiae has long been recognized in infants. In recent years, S. agalactiae is an important cause of morbidity and mortality among adults and among those with underlying medical condition. Several cases of GBS infection and more fulminant disease similar to streptococcal toxic shock syndrome have recently been reported. We report here that 19 S. agalactiae strains were isolated from streptococcal toxic shock-like syndrome cases involving adult patients in Japan between 2009 and 2013. The average age of the patients was 66.3 years. At least one underlying disease was present in 47.4% (9/19) of the patients. The most prevalent serotype among these strains was Ib. All serotype Ib strains belonged to clonal complex 10 and were ciprofloxacin resistant. In contrast, all strains were susceptible to penicillin G, ampicillin, cefazolin, cefotaxime, imipenem, panipenem, and linezolid. The characteristic type distributions of streptococcal toxic shock-like syndrome isolates differed between isolates obtained from vaginal swabs of women and infants with invasive infections.

[Molecular epidemiological study of community-acquired methicillin-resistant staphylococcus aureus (CA-MRSA) - an examination of commercially distributed meat as a possible vehicle for CA-MRSA].

Staphylococcus aureus has occupied an important position in public health as a cause of food poisoning and hospital-acquired MRSA (HA-MRSA) infections. The spread of community-acquired MRSA (CA-MRSA) infections has also recently become a concern. However, the sources of this infection remain unclear, and there are few reports of epidemiology information. In order to understand MRSA spread in the community, we investigated the distribution of MRSA strains in commercially distributed raw meat samples (n=305) and stool samples from outpatients with diarrhea (n=1,543) from the same meat distribution region in Oita Prefecture, Japan. 301 Staphylococcus aureus strains were isolated and 18 of them were MRSA (2 from chicken meat, 1 from duck meat, 1 from pork meat, and 14 from patients with diarrhea). All 18 MRSA strains were negative for Panton-Valentine leucocidin gene. In this study conducting a comparison of properties and a molecular epidemiological analysis of MRSA isolated from commercially distributed meat and diarrhea patient stools, the results suggest that commercially distributed meat could play a role in the prevalence of CA-MRSA in the community.

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.

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.

The emerging ST8 methicillin-resistant Staphylococcus aureus clone in the community in Japan: associated infections, genetic diversity, and comparative genomics.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major concern worldwide. In the United States, ST8 CA-MRSA with SCCmecIVa (USA300) has been predominant, affecting the entire United States. In this study, we investigated Japanese ST8 CA-MRSA with new SCCmecIVl (designated ST8 CA-MRSA/J), which has emerged in Japan since 2003. Regarding community spread and infections, ST8 CA-MRSA/J spread in 16.2-34.4% as a major genotype in the community in Japan, and was associated with skin and soft tissue infections (SSTIs), colitis, and invasive infections (sepsis, epidural abscesses, and necrotizing pneumonia), including influenza prodrome cases and athlete infections, similar to USA300. It spread to even public transport and Hong Kong through a Japanese family. Regarding genetic diversity, ST8 CA-MRSA/J included ST and spa variants and was classified into at least three pulsed-field gel electrophoresis types, ST8 Jα to γ. Of those, ST8 Jß was associated with severe invasive infections. As for genomics, ST8 CA-MRSA/J showed high similarities to USA300, but with marked diversity in accessory genes; e.g., ST8 CA-MRSA/J possessed enhanced cytolytic peptide genes of CA-MRSA, but lacked the Panton-Valentine leukocidin phage and arginine catabolic mobile element, unlike USA300. The unique features of ST8 CA-MRSA/J included a novel mosaic SaPI (designated SaPIj50) carrying the toxic shock syndrome toxin-1 gene with high expression; the evolution included salvage (through recombination) of hospital-acquired MRSA virulence. The data suggest that ST8 CA-MRSA/J has become a successful native clone in Japan, in association with not only SSTIs but also severe invasive infections (posing a threat), requiring attention.

Commercially distributed meat as a potential vehicle for community-acquired methicillin-resistant Staphylococcus aureus.

The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has been increasing; however, the sources of infection remain unclear. Therefore, we investigated the involvement of meat as a possible mediator of CA-MRSA infection. We examined the distribution of MRSA strains in commercially distributed raw meat samples (n = 197) and diarrheal stool samples of outpatients (n = 1,287) that were collected in Oita Prefecture, Japan, between 2003 and 2009 for routine legal inspections. Fourteen MRSA strains were isolated from three meat and 11 stool samples. Among these, seven isolates from three meat and four stool samples exhibited the same epidemiological marker profiles [coagulase type III, staphylococcal enterotoxin C, staphylococcal chromosomal cassette mec (SCCmec) type IV, ST8, spa type 606 (t1767), and toxic shock syndrome toxin-1 (TSST-1) producing type]. Furthermore, of the seven strains, three isolates from two meat samples and one stool sample collected in 2007 exhibited completely identical characteristics with respect to phage open reading frame (ORF) typing, pulsed-field gel electrophoresis, and drug susceptibility profiles. The results suggest that commercially distributed meat could play a role in the prevalence of CA-MRSA in the community.

Distribution of non-locus of enterocyte effacement pathogenic island-related genes in Escherichia coli carrying eae from patients with diarrhea and healthy individuals in Japan.

The relationship to diarrhea of genes located on the pathogenicity islands (PAI) other than the locus of enterocyte effacement (LEE) was investigated. Enteropathogenic Escherichia coli (EPEC), the retention of espC on the EspC PAI, the OI-122 genes (efa1/lifA, nleB), the phylogenetic marker gene yjaA, and the bundle-forming pilus gene bfpA on the EPEC adherence factor (EAF) plasmid were studied. E. coli strains carrying the intimin gene (eae) without the Shiga toxin gene, isolated from patients with diarrhea (n = 83) and healthy individuals (n = 38) in Japan, were evaluated using PCR. The genotypes of eae and espC were identified by heteroduplex mobility assay (HMA). The proportions of strains isolated from individuals with and without diarrhea that carried these genes were as follows: bfpA, 13.3 and 7.9%, respectively; espC, 25.3 and 36.8%; efa1/lifA, 32.5 and 13.2%; nleB, 63.9 and 60.5%; yjaA, 42.2 and 55.3%. Statistical significance (P < 0.05) was achieved only for efa1/lifA. The proportion of strains lacking espC and carrying efa1/lifA was higher for patient-derived strains (30.1%) than for strains from healthy individuals (13.2%), but the difference was not significant. Strains carrying both espC and efa1/lifA were rare (2 strains from patients). Statistical analyses revealed significant relationships between espC and yjaA and between efa1/lifA and nleB, as well as significant inverse relationships between espC and efa1/lifA and between efa1/lifA and yjaA. espC was found in eae HMA types a1, a2, and c2, whereas efa1/lifA was found in types b1, b2, and c1. In addition, 6 polymorphisms of espC were found. The espC, yjaA, efa1/lifA, and nleB genes were mutually dependent, and their distributions were related to eae type, findings that should be considered in future epidemiological studies.

Surveillance of severe invasive group G streptococcal infections in Japan during 2002-2008.

Group G Streptococcus strains isolated from patients with severe invasive infections in the period 2002-2008 were surveyed and their prevalence compared with that observed in the period 1995-2001 in Japan. Strains with genotypes stg485, stg6792, stc36, stg6, and stg652 were isolated in both periods, whereas various new genotypes appeared in 2002-2008 and some genotypes found in 1995-2001 were not found subsequently, thus indicating a change in the prevalent genotyped strains causing severe invasive streptococcal infections.

Surveillance of group B streptococcal toxic shock-like syndrome in nonpregnant adults and characterization of the strains in Japan.

Nine group B streptococci (GBS) strains were isolated from five toxic shock-like syndrome cases of nonpregnant adults in Japan from 2001 to 2005. All of them were identified as Streptococcus agalactiae. The serotypes of these strains were Ib, III, V, and VII. Pulsed-field gel electrophoresis revealed that the patterns of the strains isolated from the different patients were variable. Antimicrobial susceptibility tests showed that all of the strains were susceptible to penicillin G, ampicillin, cefotaxime, clindamycin, and telithromycin. One strain showed intermediate resistance to erythromycin.

[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.

Close correlation of streptococcal DNase B (sdaB) alleles with emm genotypes in Streptococcus pyogenes.

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emm genotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non-synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emm genotype-specific. Only sdaB7 was found in both emm2 and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.

Antimicrobial susceptibility survey of Streptococcus pyogenes isolated in Japan from patients with severe invasive group A streptococcal infections.

We assessed antimicrobial susceptibility against 211 Streptococcus pyogenes strains isolated from patients with severe invasive group A streptococcal infections. Overall, 3.8, 1.4, 1.4, and 0.5% of the isolates were resistant to erythromycin, clindamycin, telithromycin, and ciprofloxacin, respectively, and 10.4% had intermediate resistance to ciprofloxacin. All isolates were susceptible to ampicillin and cefotaxime.