Impact of skeletal muscle mass on functional prognosis in acute stroke: A cohort study.

INTRODUCTION: Changes in skeletal muscle mass affect physical performance in chronic stroke survivors. The skeletal muscle mass index is thus an important assessment factor in stroke; however, its value in the acute phase is unclear. OBJECTIVE: This study investigated the association between skeletal muscle mass and acute stroke outcome. DESIGN: This was a single-center cohort study design. PARTICIPANTS: A total of 189 consecutively hospitalized patients with acute stroke were included in the study. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The main outcome of the study was a good modified Rankin Scale (mRS) score at hospital discharge. We divided the participants into good (mRS score 0-2) and poor (mRS score 3-6) function groups. Logistic regression was performed to identify the factors associated with functional prognosis. RESULTS: Atrial fibrillation (odds ratio [OR], 14.95; 95% confidence interval [CI], 2.45-91.39; P = 0.003), pre-mRS (OR, 2.22; 95% CI, 1.05-4.68; P = 0.036), National Institutes of Health Stroke Scale (OR, 1.32; 95% CI, 01.12-1.56; P = 0.001), skeletal muscle mass index (OR, 0.31; 95% CI, 0.11-0.87; P = 0.027), and Lower Extremity Fugl-Meyer Assessment (OR, 0.68; 95% CI, 0.56-0.82; P = 0.000) were all independently associated with the functional prognosis of the patients included in the study. CONCLUSION: This study confirmed that skeletal muscle mass is a strong prognostic factor in acute stroke. Thus, prestroke skeletal muscle mass, along with stroke severity and lower limb paralysis, needs to be assessed to more accurately determine the prognosis of patients with stroke.

Escherichia cryptic clade I is an emerging source of human intestinal pathogens.

BACKGROUND: Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear. RESULTS: We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC. CONCLUSIONS: Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

A Salmonella enterica Serovar Oranienburg Clone Caused a Cluster of Bacteremia Cases in Persons With No Recognizable Underlying Diseases in Japan.

Background: Salmonella enterica subspecies enterica serovar Oranienburg (SO) is a foodborne pathogen but rarely causes systemic infections such as bacteremia. Between July and September 2018, bacteremia cases caused by SO were identified in 12 persons without any underlying medical conditions in the southern Kyushu area of Japan. Methods: Randomly amplified polymorphic DNA (RAPD) analysis was performed to investigate the genetic similarity of the 12 bacteremia-related strains and other Japanese isolates. Furthermore, a series of whole-genome sequence (WGS)-based phylogenetic analyses was performed with a global SO strain set (n = 1648). Results: The resolution power of RAPD was insufficient to investigate the genetic similarity between the bacteremia-related strains and other strains. WGS-based phylogenetic analyses revealed that the bacteremia-related strains formed a tight cluster along with 2 strains isolated from asymptomatic carriers in 2018 in the same area, with a maximum within-cluster single-nucleotide polymorphism (SNP) distance of 11. While several strains isolated in the United States and the United Kingdom were found to be closely related to the bacteremia-related strains, 2 strains isolated in 2016 in the southern Kyushu area were most closely related, with SNP distances of 4-11 and 5-10, and had the same plasmids as the bacteremia-related strains. Conclusions: The 12 bacteremia cases identified were caused by a single SO clone. As none of the bacteremia patients had any underlying diseases, this clone may be prone to cause bacteremia. Although further analyses are required to understand its virulence, particular attention should be given to this clone and its close relatives in the surveillance of nontyphoidal salmonellae.

Real-time PCR assays to detect 10 Shiga toxin subtype (Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) genes.

To develop subtyping methods for Shiga toxin (Stx)1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g genes for epidemiological analyses of Shiga toxin-producing Escherichia coli (STEC), we developed 10 simplex real-time polymerase chain reaction (PCR) assays with reference to 284 valid stx sequences and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates and recombinant plasmids, respectively. Three stx1 and 5 stx2 subtype genes, except for stx2c and stx2d, were detected with high specificity using STEC isolates. However, some stx2a sequences potentially being close to both Stx2a and Stx2d cluster in neighbor-joining cluster analysis were positive for stx2a and stx2d by real-time PCR. For the stx2c assay, the number of real-time PCR cycles was reduced to avoid unnecessary false-positive results. Based on these considerations, the real-time PCR assays developed here might aid epidemiological investigations of infections or outbreaks caused by STEC harboring any of the stx subtype genes.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

AIMS: Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. METHODS AND RESULTS: We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1 CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). CONCLUSIONS: Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Transmission dynamics of a linear vanA-plasmid during a nosocomial multiclonal outbreak of vancomycin-resistant enterococci in a non-endemic area, Japan.

The spread of vancomycin-resistant enterococci (VRE) is a major threat in nosocomial settings. A large-scale multiclonal VRE outbreak has rarely been reported in Japan due to low VRE prevalence. We evaluated the transmission of vancomycin resistance in a multiclonal VRE outbreak, conducted biological and genomic analyses of VRE isolates, and assessed the implemented infection control measures. In total, 149 patients harboring VanA-type VRE were identified from April 2017 to October 2019, with 153 vancomycin-resistant Enterococcus faecium isolated being grouped into 31 pulsotypes using pulsed-field gel electrophoresis, wherein six sequence types belonged to clonal complex 17. Epidemic clones varied throughout the outbreak; however, they all carried vanA-plasmids (pIHVA). pIHVA is a linear plasmid, carrying a unique structural Tn1546 containing vanA; it moves between different Enterococcus spp. by genetic rearrangements. VRE infection incidence among patients in the "hot spot" ward correlated with the local VRE colonization prevalence. Local prevalence also correlated with vancomycin usage in the ward. Transmission of a novel transferrable vanA-plasmid among Enterococcus spp. resulted in genomic diversity in VRE in a non-endemic setting. The prevalence of VRE colonization and vancomycin usage at the ward level may serve as VRE cross-transmission indicators in non-intensive care units for outbreak control.

Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic Escherichia coli Serogroups O157, O26, and O111 Based on a De Novo Look-Up Table Constructed by Regression Analysis.

Multilocus variable-number tandem-repeat analysis (MLVA) is a widely accepted molecular typing tool for enterohemorrhagic Escherichia coli (EHEC). However, ensuring the accuracy of MLVA data among multiple laboratories remains difficult. We developed a method of constructing adjusted look-up tables, which are necessary for MLVA profiling, at each laboratory using a regression analysis based on electrophoresis data from 24 in-house reference strains. On performing MLVA against 51 EHEC O157 isolates, the repeat numbers of 46 isolates were determined accurately using the look-up table with a 99% prediction interval, an outcome superior to that when using a 95% prediction interval. For the remaining five isolates, although the electrophoresis size fell outside the look-up table, we were able to predict the repeat number accurately by extrapolation or the nearest values of the look-up table. Our approach provides more accurate results than a nonadjusted conventional look-up table for calibrating MLVA profiles.

Prevalence and Characterization of Quinolone-Resistance Determinants in Escherichia coli Isolated from Food-Producing Animals and Animal-Derived Food in the Philippines.

Antimicrobial resistance to quinolones, which constitutes a threat to public health, has been increasing worldwide. In this study, we investigated the prevalence of quinolone-resistant determinants in Escherichia coli not susceptible to quinolones and isolated from food-producing animals and food derived from them, in the Philippines. A total of 791 E. coli strains were isolated in 56.4% of 601 beef, chicken, pork, egg, and milk samples, as well as environmental, cloacal, and rectal swab-collected samples from supermarkets, open markets, abattoirs, and poultry, swine, and buffalo farms. Using the disc diffusion method, it was determined that 78.6% and 55.4% of the isolates were resistant to at least one antimicrobial and multiple drugs, respectively. In 141 isolates not susceptible to quinolones, 115 (81.6%) harbored quinolone-resistant determinants and had mutations predominantly in the quinolone-resistance determining regions (QRDRs) of gyrA and parC. Plasmid-mediated, quinolone resistance (PMQR) and Qnr family (qnrA1, qnrB4, and qnrS1) genes were detected in all isolates. Forty-eight sequence types were identified in isolates harboring mutations in QRDR and/or PMQR genes by multilocus sequence typing analysis. Moreover, 26 isolates harboring mutations in QRDR and/or PMQR genes belonged mostly to phylogroup B1 and Enteroaggregative E. coli. In conclusion, a high prevalence of E. coli was found in food-producing animals and products derived from them, which could potentially spread high-risk clones harboring quinolone-resistance determinants.

Detection of Genetic Elements Carrying vanA in Vancomycin-Resistant Enterococcus saigonensis VE80T Isolated from Retail Chicken Meat.

In this study, we aimed to detect genetic elements carrying vanA in Enterococcus saigonensis VE80T isolated from retail chicken in Vietnam. The structures of vancomycin-resistance determinants and the location of vancomycin-resistance genes were detected by sequencing the vanA gene cluster, Southern hybridization analyses, and whole-genome sequence analyses. The Tn1546-related elements harboring vanA clusters, which exhibited a characteristic structure with five point mutations compared with the prototype Tn1546, were located on the 76-kb plasmid pVE80-1 of VE80T. The vanS sequence of VE80T harboring three point mutations was 100% identical to those of vancomycin-resistant enterococci isolated from poultry in Taiwan and Japan, indicating that the element may be prevalent in poultry production farms in Asia.

First isolation and characterization of vancomycin-resistant Enterococcus faecium harboring vanD5 gene cluster recovered from a 79-year-old female inpatient in Japan.

This study reports the first isolation and characterization of a vanD5 genotype vancomycin-resistant Enterococcus faecium strain (E. faecium IPHb306) recovered from a 79-year-old Japanese female inpatient. Species identification was determined by biochemical testing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and species-specific PCR. Susceptibility tests indicated that E. faecium IPHb306 was resistant to vancomycin but susceptible to teicoplanin. Southern hybridization analyses indicated that E. faecium IPHb306 harbored a vanD5 gene cluster on chromosomal DNA. Growth curve analyses showed that a vancomycin resistance phenotype could be inducible. Sequencing analyses of the vanD5 gene cluster and the ddlE. faecium gene demonstrated several point mutations were present. Because this strain belongs to ST203, a major hospital-adapted lineage, spread of the vanD5 genotype E. faecium ST203 is considered a clinical threat in Japan.

The Virtual Kitchen Challenge: preliminary data from a novel virtual reality test of mild difficulties in everyday functioning.

Background: Efficient, objective measures of mild functional difficulties are lacking. Preliminary data from a novel, non-immersive virtual reality, performance-based task (Virtual Kitchen Challenge; VKC) were obtained to address this gap. Methods: 14 older and 21 younger adults completed cognitive tests and two everyday tasks (breakfast, lunch) in the VKC with virtual objects and a touch-screen and in the Real Kitchen with real objects (order counterbalanced). Automated performance measures were obtained from the VKC program and human coders scored VKC and Real Kitchen videos for errors. Results: Older adults made more errors than younger adults on the VKC and Real Kitchen, with similar error patterns across measures. VKC automated measures were significantly related to measures from human coders, performance on the Real Kitchen, and cognitive test scores. Conclusion: The VKC is a valid and highly efficient performance-based measure of subtle functional difficulties with great potential for future clinical and research applications.

Prevalence and Antimicrobial Susceptibility of Bacterial Pathogens in Ready-to-Eat Foods Retailed in Osaka Prefecture, Japan.

The potential human health risk of Japanese ready-to-eat (RTE) foods was investigated by determining the contamination by foodborne bacterial pathogens, the prevalence of opportunistic and nosocomial pathogens, and the antibiotic susceptibility of the isolates recovered from 96 samples of lightly pickled vegetables, 88 samples of Western-style desserts, and 98 samples of RTE fish and seafood products sold at retail in Osaka, Japan. Staphylococcus aureus, including isolates producing staphylococcal enterotoxin (SE), were isolated from six lightly pickled vegetable products, seven Western-style dessert products, and three RTE fish and seafood products. Of these isolates, one SEC-producing isolate from a cake was identified as community-acquired methicillin-resistant S. aureus, which was multilocus sequence type 8 and staphylococcal cassette chromosome mec type IV. Enterobacteriaceae species, including Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens, Citrobacter freundii-Citrobacter braakii, and/or the Enterobacter cloacae complex, were isolated from 92 (95.8%) of the lightly pickled vegetable products, 39 (44.3%) of the Western-style dessert products, and 74 (75.5%) of the RTE fish and seafood products. On the basis of the antimicrobial susceptibility profiles of the opportunistic and nosocomial Enterobacteriaceae pathogens, the third-generation cephalosporin, fosfomycin, and quinolone resistance determinants were investigated. We detected AmpC products of the CIT group and a qnrB9 product in 5 and 1 C. freundii-C. braakii isolates, respectively, and fosA gene products in 15 E. cloacae complex isolates. Because RTE foods are consumed without a heating process, the spread of bacterial pathogens from contaminated food to human consumers is possible. RTE foods must be handled using hygienic procedures from the processing steps to the table to reduce the prevalence of potentially pathogenic or pathogenic bacteria and to prevent bacterial growth.

Development of a Real-Time PCR Assay for Detection of Kudoa iwatai (Myxosporea: Multivalvulida) in Japanese Seabass ( Lateolabrax japonicus).

Kudoa iwatai, a myxosporean parasite, has low host fish specificity, and consumers encounter commercial marine fish or marketed marine fish infected with this parasite in Japan. Although the presence of this parasite infection in fish samples is traditionally determined by the microscopic morphological examination of extracted spores, this method lacks sensitivity and specificity. In this study, we developed a real-time PCR assay for the detection of K. iwatai 18S rDNA to achieve the rapid and specific identification of K. iwatai in foreign substance inspection. We also evaluated the usefulness of real-time PCR for Japanese seabass ( Lateolabrax japonicus) with or without K. iwatai cysts. Our real-time PCR assay was able to reliably detect the target plasmid DNA over a 7-log range (from 4.0 × 101 to 4.0 × 107 copies per reaction) and displayed a linear relationship, with a correlation of determination value of 0.9993 and slope of -3.3651. Moreover, the mean value of the intra-assay coefficient of variation was 0.89% in triplicate assays, and the detection limit of this method was 2.5 copies of K. iwatai 18S rDNA per reaction. The sensitivity of the real-time PCR was the same or higher than that of an established conventional PCR when DNA extracts from eight Japanese seabass with or without K. iwatai were used as templates. The specificity of the real-time PCR was comparable with that of conventional PCR by using DNA extracts from fish samples infected with nine Kudoa species. Together, these results indicate that our real-time PCR assay is highly sensitive, reproducible, and specific for detecting K. iwatai 18S rDNA in foreign substance inspection. We believe that this highly sensitive real-time PCR may also be useful for understanding the gastrointestinal diseases associated with K. iwatai and for studying the yet unknown life cycle of K. iwatai.

Changes in Borg scale for resistance training and test of exercise tolerance in patients undergoing allogeneic hematopoietic stem cell transplantation.

PURPOSE: We aimed to investigate the relationship between Borg scale and intensity of resistance training in patients who had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). Furthermore, the relationship between Borg scale, heart rate (HR), and intensity of exercise tolerance test was also studied. METHODS: The study included 28 patients (19 men and 9 women) who had undergone allo-HSCT between June 2015 and February 2017. Their knee extension strengths and exercise tolerances were evaluated. Patients were asked to grade between 0 and 10 on Borg scale based on the level of difficulty experienced during exercising, after 10 repetitions in randomized 20, 40, and 60% resistance training for knee extension. Additionally, we evaluated Borg scale, HR, and load intensity during exercise tolerance test, every minute of the exercise for 2 weeks before and 3 weeks after HSCT. RESULTS: Knee extension strength and exercise tolerance were significantly decreased 3 weeks after HSCT from those before HSCT (p < 0.01). Additionally, rise in Borg scale with increase in load intensity during knee extension resistance training, both before and after HSCT (p < 0.01), was noted. Furthermore, Borg scale was found to be associated with HR and load intensity during exercise tolerance test in patients both before and after HSCT (p < 0.01). CONCLUSIONS: A correlation was found between Borg scale with intensity of resistance training and exercise tolerance in patients who had undergone allo-HSCT. Therefore, Borg scale could be useful to determine the intensity of physical exercise in patients who have undergone allo-HSCT.

Foodborne Outbreak of Group G Streptococcal Pharyngitis in a School Dormitory in Osaka, Japan.

In September 2016, 140 patients with primary symptoms of sore throat and fever were identified in a school dormitory in Osaka, Japan. Epidemiological and laboratory investigations determined that these symptomatic conditions were from a foodborne outbreak of group G streptococcus (GGS), with GGS being isolated from samples from patients, cooks, and foods. The strain of GGS was identified as Streptococcus dysgalactiae subsp. equisimilis of two emm types (stG652.0 and stC36.0). The causative food, a broccoli salad, was contaminated with the two types of S. dysgalactiae subsp. equisimilis, totaling 1.3 × 104 CFU/g. Pulsed-field gel electrophoresis (PFGE) of samples from patients, cooks, and foods produced similar band patterns among samples with the same emm type. This result suggested the possibility of exposure from the contaminated food. The average onset time was 44.9 h and the prevalence rate was 62%. This is the first report to identify the causative food of a foodborne outbreak by Streptococcus dysgalactiae subsp. equisimilis.

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

In Escherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli.

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.