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Campylobacter is a widespread pathogen in the food chain of chickens. Once chickens become infected, large numbers of Campylobacter cells are excreted in their feces.

Objectives Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have become a cause for great concern. Although some studies have reported the prevalence of ESBL-producing bacteria and ESBL-encoding genes in horses worldwide, the genetic structure surrounding the ESBL gene has not been analysed in detail. In the present study, we isolated two ESBL-producing Escherichia coli strains from diseased racehorses in Japan and demonstrated the mechanisms underlying the acquisition of their antimicrobial resistance (AMR) genes. Methods Two ESBL-producing E. coli strains (E148 and E189) were isolated from the heart and liver of horses with endocarditis and sepsis in 2014 and 2016, respectively, in Japan. Complete genomic sequences of the two strains were analysed using a PacBio RSII sequencer. Antimicrobial susceptibility testing was performed by the agar dilution method. Results The two isolates possessed a chromosomal AMR gene cluster containing blaCTX-M-1 that was similar to the pEQ1 plasmid found in E. coli isolated from a racehorse in the Czech Republic. In one of the two strains, tandem duplication of the 16-kb region containing blaCTX-M-1 and a class 1 integron, which occurred via IS26-mediated recombination, increased minimum inhibitory concentrations (MICs) associated with the duplicated AMR genes. Conclusion Chromosomal blaCTX-M-1 possibly derived from the pEQ1 or pEQ1-like plasmid was found in Japanese equine E. coli isolates. In Japanese strains, many AMR genes containing blaCTX-M-1 and the class 1 integron are highly accumulated in one region on the chromosome, and the AMR of E. coli was enhanced via the IS26-mediated duplication of the AMR gene cluster.

Chicken is a major source of human campylobacteriosis. Chicken meat originates not only from broilers but also from spent layers; however, few reports have documented the prevalence and antimicrobial resistance of Campylobacter spp. in layers in Japan. Therefore, we investigated the prevalence and antimicrobial susceptibility of Campylobacter spp. in 47 layer farms in Japan. Fecal samples were collected from the youngest and oldest flocks on the farm, and Campylobacter spp. was isolated from 46/47 (97.9%) farms. Among the C. jejuni isolates, the resistance rates to ampicillin, tetracycline, and ciprofloxacin were 29.6%, 22.2%, and 19.8%, respectively. The ciprofloxacin resistance rate (7.3%) in C. jejuni isolated from old flocks was significantly (P0.01) lower than that in young flocks (32.5%).

Campylobacter is one of the most important causes of food-borne infectious diseases. Antibiotics are rarely needed to treat campylobacteriosis, but occasionally used in severe or prolonged cases. Consumption of contaminated bovine liver is a source of campylobacteriosis. Bovine liver can be contaminated with Campylobacter on the surface and inside by the bile at slaughterhouses. Therefore, we investigated the current prevalence and characteristics of Campylobacter in bovine bile at a slaughterhouse. Campylobacter was isolated from 35.7% (55/154) of bile samples. C. jejuni and C. fetus were the two most frequent species. High antimicrobial resistant rates in C. jejuni were observed against tetracycline (63.0%) and ciprofloxacin (44.4%). Multi-locus sequence typing divided C. jejuni isolates (27 isolates) into 12 sequence types (STs) in which ST806 was the most frequent ST and accounted for 37.0%. All C. fetus were identified as C. fetus subsp. fetus which can cause systemic infections. High antimicrobial resistant rates in C. fetus were observed against ciprofloxacin (66.6%), streptomycin (58.3%) and tetracycline (33.3%). All the C. fetus isolates were divided into two STs, ST3 (16 isolates) and ST6 (8 isolates). Of the 16 ST3 isolates, 15 (93.8%) were resistant to both streptomycin and ciprofloxacin. Our data shows high prevalence of Campylobacter in bovine bile and their high rates of antimicrobial resistance. Preventing bile contamination of bovine liver at slaughterhouses is thus considered to be one of control measures to reduce the risk of Campylobacter infections.

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have become a cause for great concern. Although some studies have reported the prevalence of ESBL-producing bacteria and ESBL-encoding genes in horses worldwide, the genetic structure surrounding the ESBL gene has not been analysed in detail. In the present study, we isolated two ESBL-producing Escherichia coli strains from diseased racehorses in Japan and demonstrated the mechanisms underlying the acquisition of their antimicrobial resistance (AMR) genes.Two ESBL-producing E. coli strains (E148 and E189) were isolated from the heart and liver of horses with endocarditis and sepsis in 2014 and 2016, respectively, in Japan. Complete genomic sequences of the two strains were analysed using a PacBio RSII sequencer. Antimicrobial susceptibility testing was performed by the agar dilution method.The two isolates possessed a chromosomal AMR gene cluster containing blaChromosomal bla

Campylobacter jejuni is one of the most important causes of food-borne diseases in industrialized countries. Amino acids are an important nutrient source for this pathogen because it lacks enzymes related to glycolysis. However, the metabolic characteristics of C. jejuni grown in a nutrient-restricted medium with specific amino acids have not been fully elucidated. This study shows that C. jejuni NCTC 11168 grows well in a nutrient-restricted medium containing serine, aspartate, glutamate, and proline. Subtracting serine significantly reduced growth, but the removal of the three other amino acids did not, suggesting that serine is a priority among the four amino acids. A transcriptomic analysis of C. jejuni NCTC 11168 grown in a medium with serine as the main energy source was then performed. Serine seemed to be sensed by some chemoreceptors, and C. jejuni reached an adaptation stage with active growth in which the expression of flagellar assembly components was downregulated and the biosyntheses of multiple amino acids and nucleotide sugars were upregulated. These data suggest that C. jejuni NCTC 11168 requires serine as a nutrient.

Salmonella Typhimurium and its monophasic variant (Salmonella 4,[5],12:i:-) are classified into nine clades. Clade 9 is composed of ST34 strains, including the Salmonella 4,[5],12:i:-European clone, which has rapidly disseminated in humans and animals worldwide since the 2000s. To reveal the microevolution of ST34/clade 9, we analyzed the whole-genome-based phylogenetic relationships of 230 Salmonella Typhimurium and Salmonella 4,[5],12:i:-strains isolated between 1998 and 2017 in Japan and other countries. We identified clade 9-2, a novel sublineage derived from the conventional Salmonella 4,[5],12:i:-clade 9 that exclusively consists of Japanese isolates. Clade 9-2 was generated by mobile genetic element-mediated stepwise genome reductions caused by IS26 and prophages. Furthermore, the recently identified strains developed further resistance to antimicrobials via the acquisition of resistance genes. During this microevolution, Salmonella 4,[5],12:i:-clade 9-2 has dominated among recent isolates from food animals in Japan via the emergence of clonally expanding and/or multiple host-adapted subclades.

SUMMARYA nationwide study of Shiga toxin-producingEscherichia coli(STEC) was performed to determine the prevalence, characteristics and risk factors for fecal shedding of STEC among cattle in Japan. Information on rearing practices was also collected to identify risk factors for fecal shedding of STEC. STEC was isolated from 24·1% of samples (133/551) collected from 59·1% of farms (65/110). Bayesian clustering using the virulence marker profiles of the isolates subdivided the isolates into four genetically distinct groups, two of which corresponded toeae- orsaa-positive STEC, which can cause severe disease in human. Both STEC groups exhibited characteristic phylogeny and virulence marker profiles. It is noteworthy that the tellurite resistance gene was not detected in allsaa-positive STEC isolates, suggesting that the standard isolation method using tellurite might lead to an underestimation of the prevalence ofsaa-positive STEC. A multivariate logistic regression model using epidemiological information revealed a significantly (P< 0·01) high odds ratio on STEC fecal shedding in tie-stall housing and a low odds ratio in flat feed box and mechanical ventilation. Information on isolate characteristics of the two major pathotypes and risk factors in rearing practices will facilitate the development of preventative measures for STEC fecal shedding from cattle.

Peptidoglycan (PG) acetylation of Gram-positive bacteria confers lysozyme resistance and contributes to survival in the host. However, the importance of PG acetylation in Gram-negative bacteria has not been fully elucidated. The genes encoding putative PG acetyltransferase A (PatA) and B (PatB) are highly conserved in Campylobacter jejuni , the predominant cause of bacterial diarrhea worldwide. To evaluate the importance of PatA and PatB of C. jejuni , we constructed patA and patB isogenic mutants and compared their phenotypes with those of the parental strains. Although transmission electron microscopy did not reveal morphological changes, both mutants exhibited decreased motility and biofilm formation in vitro . The extent of acetylation of the PG purified from the patA and patB mutants was significantly lower than the PG acetylation in the parental strains. Both mutants exhibited decreased lysozyme resistance and intracellular survival in macrophage cells. In a chick colonization experiment, significant colonization deficiency was observed for both mutants. These results suggest that PatA and PatB of C. jejuni play important roles in maintaining cell wall integrity by catalyzing PG O -acetylation and that the loss of these enzymes causes decreased motility and biofilm formation, thus leading to colonization deficiency in chicken infection. IMPORTANCE The importance of peptidoglycan (PG) acetylation in Gram-negative bacteria has not been fully elucidated. The genes encoding putative PG acetyltransferase A (PatA) and B (PatB) are highly conserved in Campylobacter jejuni , the predominant cause of bacterial diarrhea worldwide. We evaluated the importance of these enzymes using isogenic mutants. The results of this study suggest that PatA and PatB of C. jejuni play important roles in maintaining cell wall integrity. The loss of these factors caused multiple phenotypic changes, leading to colonization deficiency in chicken infection. These data should be useful in developing novel control measures to prevent chicken colonization by C. jejuni . Inhibitors of the PG acetylation enzymes PatA and PatB might serve as potent anti- C. jejuni agents.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:–, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:– isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR. The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Salmonellagenomic island 3 (SGI3) was first described as a chromosomal island inSalmonella4,[5],12:i:-, a monophasic variant ofSalmonella entericasubsp.entericaserovar Typhimurium. The SGI3 DNA sequence detected fromSalmonella4,[5],12:i:-isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic resistance operon in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter mating experiments using theS. entericaserovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genespheVorpheR. The length of the target site was 52 or 55 bp, and a 55-bpattIsequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4compared with the recipient strains under anaerobic conditions. Resistance was defined by thecusgene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4compared with recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic resistance ofS. enterica.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The bla NDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: bla NDM-1 in 2 isolates, bla NDM-5 in 7 isolates, and bla NDM-7 in 5 isolates. The bla CTX-M gene was detected in 112 isolates (66.3%): bla CTX-M-15 in 108 isolates and bla CTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the bla NDM -positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

Salmonella enterica serovar Typhimurium ( Salmonella Typhimurium) and its monophasic variant ( Salmonella 4,[5],12:i:−) are the major causes of gastroenteritis in both humans and animals. Pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis have been used widely as subtyping methods for these pathogens in molecular epidemiological analyses, but the results do not precisely reflect phylogenetic information. In this study, we performed a phylogenetic analysis of these serovars using whole-genome sequencing data and identified nine distinct genotypic clades. Then, we established an allele-specific PCR-based genotyping method detecting a clade-specific single nucleotide polymorphism to rapidly identify the clade of each isolate. Among a total of 815 isolates obtained from cattle in Japan between 1977 and 2017, clades 1, 7, and 9 contained 77% of isolates. Obvious replacement of the dominant clone was observed five times in this period, and clade 9, which mostly contains Salmonella 4,[5],12:i:−, is currently dominant. Among 140 isolates obtained from swine in Japan between 1976 and 2017, clades 3 and 9 contained 64% of isolates. Clade 9 is the latest clone as is the case in cattle isolates. Clade 9 is similar to an epidemic clone from Europe, which is characterized by sequence type 34 (ST34), chromosomal Salmonella genomic island 3, and a composite transposon containing antimicrobial resistance genes. The increased prevalence of clade 9 among food animals in Japan might be a part of the pandemic of the European Salmonella 4,[5],12:i:− clone.

The sewage treatment plant (STP) is one of the most important interfaces between the human population and the aquatic environment, leading to contamination of the latter by antimicrobial-resistant bacteria. To identify factors affecting the prevalence of antimicrobial-resistant bacteria, water samples were collected from three different STPs in South India. STP1 exclusively treats sewage generated by a domestic population. STP2 predominantly treats sewage generated by a domestic population with a mix of hospital effluent. STP3 treats effluents generated exclusively by a hospital. The water samples were collected between three intermediate treatment steps including equalization, aeration, and clarification, in addition to the outlet to assess the removal rates of bacteria as the effluent passed through the treatment plant. The samples were collected in three different seasons to study the effect of seasonal variation. Escherichia coli isolated from the water samples were tested for susceptibility to 12 antimicrobials. The results of logistic regression analysis suggest that the hospital wastewater inflow significantly increased the prevalence of antimicrobial-resistant E. coli, whereas the treatment processes and sampling seasons did not affect the prevalence of these isolates. A bias in the genotype distribution of E. coli was observed among the isolates obtained from STP3. In conclusion, hospital wastewaters should be carefully treated to prevent the contamination of Indian environment with antimicrobial-resistant bacteria.

The aim of this study was to investigate the effects of lipooligosaccharide (LOS) core truncations on the ability of Campylobacter jejuni to attach to glass and form biofilms under aerobic conditions. We compared the wild-type and previously constructed glycosyltransferase mutants of two C. jejuni strains, NCTC 11168 and 81-176, in terms of the numbers of attached cells, biofilm formation, autoagglutination (AAG) activity, and extracellular DNA (eDNA) release. All LOS mutants of NCTC 11168, except cj1138 mutant, and 81-176 waaC mutant with most severe LOS truncation, exhibited increased attachment and biofilm formation. Conversely, waaC and lgtF mutant of both NCTC 11168 and 81-176 showed significantly reduced AAG activity. There was no significant difference between all LOS mutants and the wild-type with respect to eDNA production. The biofilm formation levels correlated significantly with the attachment numbers (p < 0.05, R2 = 0.95) but not with the AAG activity and eDNA levels (p ≥ 0.48, R2 ≤ 0.72). These results suggest that various LOS core truncations have different impacts on C. jejuni attachment, biofilm formation under aerobic conditions, and the AAG activity levels, whereas they may not affect eDNA production. Bacterial attachment, but not the AAG activity and eDNA release levels, may cause changes in C. jejuni biofilm formation because of LOS core truncations. Discipline: Food Additional key words: Autoagglutination activity, extracellular DNA release * Corresponding author: akiba@affrc.go.jp † Taketoshi Iwata and Vu Tuan Nguyen contributed equally to this study Received 15 August 2014; accepted 24 December 2014. Introduction Campylobacter jejuni is an important foodborne bacterial pathogen, which is frequently associated with gastrointestinal diseases in humans worldwide (Kirkpatrick & Tribble 2011). Attachment to abiotic surfaces plays an important role in the survival of C. jejuni in food systems and the environment, thereby leading to infections (Nguyen et al. 2012). Because of the importance of bacterial attachment, we previously investigated factors that potentially influence the ability of C. jejuni to attach to abiotic surfaces, thereby elucidating the mechanisms of attachment. We found that cell surface hydrophobicity affected the ability of many C. jejuni strains to attach to abiotic surfaces such as stainless steel and glass (Nguyen et al. 2011). Although it has been suggested that capsular polysaccharides (CPS) and lipooligosaccharides (LOS) contribute to the degree of hydrophobicity of various bacteria (Guerry & Szymanski 2008), CPS removal and LOS core truncation caused by kpsE and waaF mutation, respectively, did not affect cell surface hydrophobicity and attachment in C. jejuni (Nguyen et al. 2013). Various LOS core truncations caused by the mutation of various genes encoding heptose biosynthesis enzymes and glycosyltransferases were found to increase the cell surface hydrophobicity of both C. jejuni NCTC 11168 and 81-176 strains (Iwata et al. 2013). However, it is currently unknown whether LOS core truncations in these sugars affect the attachment ability of C. jejuni. In addition to surface attachment, biofilm formation by C. jejuni has a role in enhanced survival (Joshua et al. 2006

Insertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria; however, they also play important roles in genome evolution. We recently identified a protein called IS excision enhancer (IEE) in enterohemorrhagic Escherichia coli (EHEC) O157. IEE promotes the excision of IS elements belonging to the IS 3 family, such as IS 629 , as well as several other families. IEE-mediated IS excision generates various genomic deletions that lead to the diversification of the bacterial genome. IEE has been found in a broad range of bacterial species; however, among sequenced E. coli strains, IEE is primarily found in EHEC isolates. In this study, we investigated non-EHEC pathogenic E. coli strains isolated from domestic animals and found that IEE is distributed in specific lineages of enterotoxigenic E. coli (ETEC) strains of serotypes O139 or O149 isolated from swine. The iee gene is located within integrative elements that are similar to SpLE1 of EHEC O157. All iee -positive ETEC lineages also contained multiple copies of IS 629 , a preferred substrate of IEE, and their genomic locations varied significantly between strains, as observed in O157. These data suggest that IEE may have been transferred among EHEC and ETEC in swine via SpLE1 or SpLE1-like integrative elements. In addition, IS 629 is actively moving in the ETEC O139 and O149 genomes and, as in EHEC O157, is promoting the diversification of these genomes in combination with IEE.

We report epidemic occurrences of fatal salmonellosis caused by Salmonella Enteritidis in captive maras (Dolichotis patagonum) in a zoological garden in Japan. The deaths were sudden or followed a peracute course within a few hours of the first observations of clinical abnormalities. Gross lesions included haemorrhages in the subcutis and skeletal muscles, liver, spleen, lung, heart and gastrointestinal tract. Microscopically, there were haemorrhagic and necrotizing lesions with gram-negative bacilli in the liver, spleen, small intestine and Peyer's patches. These bacilli showed strong immunolabelling for Salmonella O9 antigen and S. Enteritidis was isolated from the lesions. To the best of our knowledge, this is the first report of salmonellosis in maras.

Campylobacter jejuni is a major cause of bacterial foodborne illness in humans worldwide. Bacterial entry into a host eukaryotic cell involves the initial steps of adherence and invasion, which generally activate several cell-signaling pathways that induce the activation of innate defense systems, which leads to the release of proinflammatory cytokines and induction of apoptosis. Recent studies have reported that the unfolded protein response (UPR), a system to clear unfolded proteins from the endoplasmic reticulum (ER), also participates in the activation of cellular defense mechanisms in response to bacterial infection. However, no study has yet investigated the role of UPR in C. jejuni infection. Hence, the aim of this study was to deduce the role of UPR signaling via induction of ER stress in the process of C. jejuni infection. The results suggest that C. jejuni infection suppresses global protein translation. Also, 12 h of C. jejuni infection induced activation of the eIF2α pathway and expression of the transcription factor CHOP. Interestingly, bacterial invasion was facilitated by knockdown of UPR-associated signaling factors and treatment with the ER stress inducers, thapsigargin and tunicamycin, decreased the invasive ability of C. jejuni. An investigation into the mechanism of UPR-mediated inhibition of C. jejuni invasion showed that UPR signaling did not affect bacterial adhesion to or survival in the host cells. Further, Salmonella Enteritidis or FITC-dextran intake were not regulated by UPR signaling. These results indicated that the effect of UPR on intracellular intake was specifically found in C. jejuni infection. These findings are the first to describe the role of UPR in C. jejuni infection and revealed the participation of a new signaling pathway in C. jejuni invasion. UPR signaling is involved in defense against the early step of C. jejuni invasion and thus presents a potential therapeutic target for the treatment of C. jejuni infection.

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenic E. coli strains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenic E. coli . In the present study, we determined the O serogroups of 967 E. coli isolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup of Shigella boydii type 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.

Yersiniosis, which is caused by pathogenic Yersinia enterocolitica or Yersinia pseudotuberculosis, poses a serious problem for zoological gardens engaged in breeding nonhuman primates. In Japan, Y. pseudotuberculosis in particular frequently causes fatal infection, and affected nonhuman primates may die unexpectedly or after a very short illness. Our epidemiological study in 17 zoological gardens in Japan suggested that Yersinia pseudotuberculosis-derived mitogen (YPM), which is a kind of super-antigenic toxin, might be the cause of, or at least the most important factor in, the high mortality of breeding nonhuman primates infected by Y. pseudotuberculosis in Japan. Furthermore, seroepidemiological study proved that pathogenic Yersinia is highly prevalent among breeding squirrel monkeys in Japan. It is likely that the monkeys that are pathogenic Yersinia positive have been inapparently or mildly infected by low pathogenic strains of Yersinia, not highly pathogenic strains of Yersinia, such as YPM-producing Y. pseudotuberculosis. In this review, we will describe the epidemiology of yersiniosis in breeding nonhuman primates in Japan.

From July 2004 to April 2005, total of 494 flies, including 128 houseflies (Musca domestica) and 366 blowflies (Genus Calliphora), were captured at 6 pig farms, at a slaughterhouse and at 2 wet markets in the Mekong Delta, Vietnam to know the role of flies as a transmitter of Salmonella. Salmonella were isolated from 38 (7.7%) of the 494 flies samples. The isolation rates of Salmonella from flies captured at a slaughterhouse (15.5%) were significantly higher than that in pig farms (4.5%) and in wet markets (6.5%). The isolation rates of Salmonella from blowflies (9.8%) were higher than those houseflies (1.6%). From those 38 Salmonella-positive fly samples, 41 Salmonella strains were isolated and 14 serovars were identified. The predominant serovars were S. Typhimurium, S. Panama, S. Newport, S. Derby, S. Bareilly, S. Lexington and S. Anatum. Of the 41 Salmonella isolates examined, 38 (92.7%) showed resistance to at least one or following antibiotics ; SM, OTC, KM, NA, ABPC, SMX, CP, and GM. No isolates showed resistance to CEZ, CTRX and CPFX. Among the resistant isolates, 22 isolates were multi-drug resistance. Flies seem to play an important role, as an epidemiological link between humans and domestic animals in the Mekong Delta, Vietnam.

From August 2001 to July 2002 and between June and December 2004, fecal samples from 922 children with acute diarrhea admitted to hospitals at Can Tho City, Dong Thap and Soc Trang provinces were examined for the presence of Salmonella and Escherichia coli O157. Salmonella was isolated from 45 (4.9%) of 922 diarrheic children. From the 45 patients, 47 Salmonella strains were isolated and 9 serovars were identified. The predominant serovars were S. London, S. Bareilly, S. Agona and S. Typhimurium. Of the 47 Salmonella isolates examined, 12 (25.5%) of isolates showed resistance to one or more antibiotics. Of 12 resistance isolates, 11 (23.4%) showed multi-drug resistance. Salmonella seems to be an important diarrhea-causing pathogen in children in the Mekong Delta, Vietnam. E. coli O157 was not isolated from any children's fecal samples with or without bloody stools.

Between April 2001 and 2007, 18 Yersinia pseudotuberculosis outbreaks occurred in breeding monkeys at 12 zoological gardens in Japan, and 28 monkeys of 8 species died. A total of 18 Y. pseudotuberculosis strains from the dead monkeys, comprising one strain per outbreak, were examined for serotype and the presence of the virulence genes virF, inv, ypm (ypmA, ypmB and ypmC) and irp2. Of the 18 Y. pseudotuberculosis strains, 7 (38.9%) were serotype 4b, 7 (38.9%) were serotype 1b, and there was one each of serotypes 2b, 3, 6 and 7. All the 18 strains examined harbored virF and inv. Sixteen (88.9%) strains, including the strain of serotype 7, harbored ypmA. However, no strain harbored ypmB, ypmC and irp2. This study demonstrated that among other pathogenic factors, almost all the Y. pseudotuberculosis isolated from the outbreaks had the ypm gene encoding the superantigenic toxin, YPM. As most of the monkeys who died in those outbreaks originated from South America and other regions, where the presence of the ypm gene have not been reported, YPM might be the cause, or at least the most important factor for, the high mortality of the breeding monkeys infected by Y. pseudotuberculosis in Japan. This is also the first report of a fatal case due to Y. pseudotuberculosis serotype 7 infection in the world.

A total of 230 Salmonella isolates representing 33 serotypes originated from food (pork, beef, chicken meat, duck meat, and shrimp), domestic animals (pig, chicken, and duck), and human (children with diarrhea) in the Mekong Delta, Vietnam were examined for the antimicrobial resistance to 10 antibiotics. Of the 230 Salmonella isolates examined, 49 (21.3%) showed antimicrobial resistance. Thirty-eight isolates (16.5%) were resistant to oxytetracycline, 26 (11.3%) to chloramphenicol, 17 (7.4%) to nalidixic acid, 16 (7.0%) to streptomycin, 5 (2.2%) to kanamycin, and 4 (1.7%) to ampicillin. No isolate showed resistance to gentamicin, cefazolin, ceftriaxone, and ciprofloxacin. Among the resistant isolates, nineteen isolates were resistant to one antimicrobial agent, 10 to two, 15 to three, 3 to four, and 2 to five antimicrobial agents. The resistance rate of Salmonella isolates from the Mekong Delta, Vietnam to these antimicrobial agents seems to be relatively lower than the results of developed countries and even those of the neighboring countries.

To develop an effective method to isolate an injured pathogenic Yersinia enterocolitica O:8 organism from environmental samples, we compared the isolation of freeze-injured and non-injured Y. enterocolitica O:8 and found that the isolation was more successful when immuno-magnetic separation (IMS) with anti-Y. enterocolitica O:8 antibody was used. Plating onto cefsulodin-irgasan-novobiocin (CIN) agar and Virulent Yersinia enterocolitica (VYE) agar by means of the agar layer method was found to be effective in isolating the injured cells. The alkali treatment which is generally used for selective detection of Yersinia organism failed to isolate freeze-injured pathogenic Y. enterocolitica O:8 cells. Recovery methods without using the alkali treatment were superior for detecting freeze-injured Y. enterocolitica O:8. Our results demonstrate that the IMS and the agar layer methods should be used to isolate injured pathogenic Yersinia organisms from environmental samples such as water.

From March to December, 2002, a total of 600 rice-field rats (Rattus argentiventer) living in the wild were captured in 5 provinces of the Mekong delta, Vietnam, and examined for the presence of Salmonella. Of the 600 cecal samples from the rats that were examined, 116 (19.3%) were Salmonella-positive. Of 116 Salmonella isolates, 9 serotypes were identified. The most common serotypes of the rats were S. London, S. Weltevreden and S. Derby. These results indicate that the rice-field rat may play an important role as a reservoir for or vehicle for the transmission of Salmonella to the surrounding environment in the Mekong Delta, Vietnam.

In the period from December 2002 to January 2003, 5 of 50 squirrel monkeys (Saimiri sciureus) housed at a Zoological Garden in the Kanto region of Japan died following a few days' history of diarrhea. After this outbreak had ended in the squirrel monkeys, 1 of 2 dark-handed gibbons (Hylobates agilis) died in April of 2003, showing similar clinical signs. We examined the organs of 3 of the dead squirrel monkeys and of the dark-handed gibbon, and Yersinia enterocolitica serovar O:8, which is the most pathogenic serovar of Y. enterocolitica, was isolated. In order to determine the source and the transmission route of infection, 98 fecal samples (45 from squirrel monkeys, 20 from other monkeys of 18 different species, and 33 from black rats captured around the monkey houses) and 7 water samples were collected in the Zoological Garden, and were examined for the prevalence of Y. enterocolitica serovar O:8. Serovar O:8 was isolated from 21 of 65 monkeys (32.3%) and 5 of 33 (15.2%) black rats (Rattus rattus). Furthermore, we examined the 30 isolates using molecular typing methods, pulsed field gel electrophoresis (PFGE), ribotyping using the RiboPrinter system, and restriction endonuclease analysis of virulence plasmid DNA (REAP), and compared the isolates in this outbreak with Japanese O:8 isolates previously identified. Genotyping showed that almost all the isolates were identical, and the genotype of the isolates was highly similar to that from wild rodents captured in Niigata Prefecture. This is the first report of fatal cases of Y. enterocolitica serovar O:8 infection in monkeys anywhere in the world.

We developed a loop-mediated isothermal amplification method able to detect Yersinia pseudotuberculosis strains in 30 min by using six primers designed by targeting the inv gene. This method is more sensitive than PCR and might be a useful tool for detecting and identifying Y. pseudotuberculosis .

1999年4月から2000年5月までに, 千葉および茨城県下に旅鳥や夏鳥として飛来したシギ, チドリ, アジサシなどの水鳥16種343羽におけるVibrio属菌の保有状況を検討し, 以下の成績を得た。1) 供試した水鳥343羽中, Vibrio属菌は103羽 (30.0%) から分離され, このうちV.choleraeは54羽 (15.7%) , V.parahaemolyticusは29羽 (8.5%) から分離された。2) 分離されたV.choleraeはいずれも血清型non-O1で, コレラ毒素を産生し, コレラの主たる原因菌として知られる血清型O1は分離されなかった。分離菌株については, コレラ毒素遺伝子 (ctx) , NAG-ST遺伝子 (stn) , およびエルトール溶血毒遺伝子 (hly A) の有無をPCR法で検索したが, その結果, ctxおよびstnはいずれも陰性であったが, hly Aは分離株の51.8%が陽性で, 供試した水鳥の8.8%から分離された。また, 分離されたV.parahaemolyticusはいずれも腸炎ビブリオの病原因子である耐熱性溶血毒 (TDH) 陰性であった。3) 以上の成績から, 我が国に飛来する水鳥は, 比較的高率にV.choleyaeおよびV.parahaemolyticusを保菌するが, V.choleraeの一部の菌株が環境分離株からもしばしば検出されるhly A遺伝子を保有していた以外は, いずれも非病原性であったことから, これら水鳥は病原性V.choleraeおよびV.parahaemolyticusのレゼルボアではなく, その保菌は水鳥の生息する環境におけるVibrio属菌の分布を反映していると思われた。

Salmonella enterica serovar Typhimurium pulsed-field gel electrophoresis cluster VII has been isolated from cattle populations in Japan since the mid-2000s. Some cluster VII isolates exhibited extended-spectrum cephalosporin resistance defined by the bla CMY-2 gene located in a chromosomal genomic island, GI-VII-6. We determined the whole-genome sequence of strain L-3553 as the reference strain.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- (S. 4,[5]12:i:-) is believed to be a monophasic variant of S. enterica serovar Typhimurium (S. Typhimurium). This study was conducted to corroborate this hypothesis and to identify the molecular and phenotypic characteristics of the S. 4,[5]12:i:- isolates in Japan. A total of 51 S. 4,[5]12:i:- isolates derived from humans, cattle, swine, chickens, birds, meat (pork), and river water in 15 prefectures in Japan between 2000 and 2010 were analyzed. All the S. 4,[5],12:i:- isolates were identified as S. Typhimurium by two different polymerase chain reactions (PCR) for identification of S. Typhimurium. Of the 51 S. 4,[5],12:i:- isolates, 39 (76.5%) harbored a 94-kb virulence plasmid, which is known to be specific for S. Typhimurium. These data suggest that the S. 4,[5],12:i:- isolates are monophasic variants of S. Typhimurium. The flagellar phase variation is induced by three adjacent genes (fljA, fljB, and hin) in the chromosome. The results of PCR mapping of this region and comparative genomic hybridization analysis suggested that the deletion of the fljAB operon and its flanking region was the major genetic basis of the monophasic phenotype of S. 4,[5],12:i:-. The fljAB operon and hin gene were detectable in eight of the S. 4,[5],12:i:- isolates with common amino acid substitutions of A46T in FljA and R140L in Hin. The introduction of these mutations into S. Typhimurium isolates led to the loss of selectability of isolates expressing the phase 2 H antigen. These data suggested that a point mutation was the genetic basis, at least in part, of the S. 4,[5],12:i:- isolates. The results of phenotypic analysis suggested that the S. 4,[5],12:i:- isolates in Japan consist of multiple distinct clones. This is the first detailed characterization of the S. 4,[5],12:i:- isolates derived from various sources across Japan.

A captive male Bolivian squirrel monkey (Saimiri boliviensis) of less than 1 year of age died following diarrhea and debilitation on the day of death. At necropsy, necrotizing enteritis accompanied with enlarged Peyer's patches, solitary lymphatic follicles and mesenteric lymph nodes, and multiple yellowish-white nodules in the spleen and liver were found. Histopathologically, these lesions were necrotizing inflammation containing Gram-negative bacilli. Yersinia pseudotuberculosis serotype 7 was isolated from the spleen and liver. The virF gene, which is an essential virulent plasimid (pYV) in pathogenic Y. pseudotuberculosis isolates, and the ypmA gene, which is a superantigenic toxin, were detected in the isolates. This is the first report of a fatal case of Yersinia pseudotuberculosis 7 infection in the world.

This study was conducted to investigate the distribution and diversity of extended-spectrum cephalosporin (ESC) resistance determinants in Salmonella enterica and Escherichia coli obtained from the same cecal samples and to provide evidence of transmission of the resistance determinants among these bacteria in broiler farms in southern Japan. Salmonella enterica and E. coli were characterized by serotyping and multilocus sequence typing, respectively. An antimicrobial susceptibility test, plasmid analysis, and identification and localization of resistance genes were performed to determine the relatedness of ESC resistance determinants among the isolates. Of 48 flocks examined, 14 had S. enterica. In total, 57 S. enterica isolates were obtained, 45 of which showed ESC resistance. Extended-spectrum cephalosporin-resistant E. coli were also obtained from all of these ESC-resistant Salmonella-positive samples. β-Lactamase genes, blaTEM-52 (38 isolates), blaCTX-M-14 (1 isolate), and blaCMY-2 (6 isolates), were carried by conjugative untypable or IncP plasmids detected in the S. enterica serovars Infantis and Manhattan. The β-lactamase genes blaCTX-M-14 (3 isolates), blaCTX-M-15 (3 isolates), blaSHV-2 (1 isolate), blaSHV-12 (2 isolates), and blaCMY-2 (32 isolates) associated with IncI1-Iγ, IncFIB, IncFIC, IncK, IncB/O, and IncY plasmids were detected in E. coli co-isolates. Restriction mapping revealed similar plasmids in Salmonella Infantis and Salmonella Manhattan and in different sequence types of E. coli. Intraspecies transmission of plasmids was suggested within S. enterica and E. coli populations, whereas interspecies transmission was not observed. This study highlights the importance of plasmids as carriers of ESC resistance determinants.

Multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates with four different antimicrobial resistance patterns obtained from a beef cattle farm were characterized to determine their clonality. Macrorestriction analysis of genomic DNA revealed that these four isolates are closely related to each other and can be classified as a newly emerged pulsed-field gel electrophoresis type among cattle: cluster VII. Three of the four isolates showed resistance to extended-spectrum cephalosporins (ESCs), and this resistance was mediated by AmpC β-lactamase encoded by the bla(CMY-2) gene in a 190-kbp IncA/C plasmid. Results of restriction analysis and IncA/C backbone PCR suggest that the three 190-kbp plasmids are identical and that a 70-kbp IncA/C plasmid of the ESC-susceptible isolate is derived from the 190-kbp plasmid by a deletion event. Three isolates harboured a virulence-resistance plasmid (165 or 180 kbp), and restriction analysis revealed that these plasmids were identical or closely related to each other. These results suggest that the four S. Typhimurium cluster VII isolates originate from a common ancestor that probably invaded the farm prior to the salmonellosis outbreak. Antimicrobial resistance patterns may not necessarily reflect the relationships of the isolates.

Since 2004, extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium ( S . Typhimurium) isolates have been detected from cattle in the northern major island of Japan, Hokkaido. Resistance to ESCs was found to be mediated by CMY-2 type β-lactamase among 22 epidemiologically unrelated isolates showing indistinguishable pulsed-field gel electrophoresis patterns. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that the CMY-2 β-lactamase gene ( bla CMY-2 ) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S . Typhimurium isolate L-3553 indicated that bla CMY-2 was located on a unique 125-kb genomic island, GI-VII-6, which consists of 140 open reading frames. Pairwise alignment of GI-VII-6 and Escherichia coli plasmid pAR060302 (size, 167 kb) revealed that a large proportion of GI-VII-6 (99%) shows a high sequence similarity (>99%) with pAR060302. GI-VII-6 contains 11 antimicrobial resistance genes including sul1 , qacE Δ 1 , aadA2 , and dfrA12 in the aadA2 region; sugE1 and bla CMY-2 in the bla CMY-2 region; and sul2 , strA , strB , tet (A), and floR in the floR region. Two directly repeated IS 26 copies were present at both ends of GI-VII-6. Junction regions of GI-VII-6 were flanked by an 8-bp direct repeat, indicating that GI-VII-6 was acquired by transposition involving IS 26 transposase. PCR scanning revealed that the overall structure of GI-VII-6 was almost identical in the 22 isolates. Phylogenetic analysis suggested that S . Typhimurium isolates harboring GI-VII-6 belong to a different genomic lineage than other whole-genome-sequenced S . Typhimurium strains. These data indicate that a particular clone of S . Typhimurium harboring GI-VII-6 has spread among the cattle population in Hokkaido, Japan.