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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%).

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

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

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.