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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%).

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.

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.