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Using multiplex real-time PCR, 960 fecal samples collected from poultry, cattle, and patients with diarrhea in Bangladesh were screened for diarrheagenic Escherichia coli (DEC). The invasion-related gene virB showed the highest prevalence in human patients (41%) and was shown to be positively correlated first with afaB with regards to diffuse adhesion and second with aggR with regards to aggregative adhesion. These three genes were specific to human patients. In contrast, the Shiga toxin genes stx1 (57%) and stx2 (40%) were prevalent in cattle samples. The eae gene, which is associated with attaching and effacing lesion formation, and the elt and est genes, which are associated with enterotoxins, were detected from all three sample sources. Heat map construction and hierarchical clustering assigned the samples into five different clusters, with the patient samples positive for virB and afaB being placed together in one cluster. Although the detection of virulence genes cannot be a direct indication of the distribution of diarrheagenic organisms, their detection suggests that Shigella spp. or enteroinvasive E. coli are the most prevalent diarrheagenic bacteria in Bangladesh and that diffusely adherent E. coli is concomitantly present with these bacteria. eae-possessing organisms in patients may come from cattle and poultry sources. The small number of stx-positive patients could be explained by the small number of animal samples that were positive for both eae and stx.

Enterotoxigenic Escherichia coli (ETEC) strains that express various fimbrial or nonfimbrial colonisation factors (CFs) and enterotoxins are critical causes of diarrhoeal diseases. Human ETEC serotype O169:H41 (O169) has been a representative of epidemic ETEC worldwide; the organism shows massive adherence to HEp-2 cells similar to enteroaggregative E. coli. Previously, we determined the complete sequence of the unstable virulence plasmid, pEntYN10. The plasmid included a unique set of genes encoding a novel CF resembling K88 (F4) of porcine ETEC, in addition to CS6, a well-known representative CF of human ETEC, and another novel CF similar to CS8 (CFA/III) of human ETEC. In the present study, we focused on K88-like CF (hereafter, K88O169) that may allow the organisms to infect domestic livestock like original K88-harbouring strains that can cause diarrhoea in piglets. Samples were tested for antibodies against recombinant proteins of possible paralogous adhesins, FaeG1 and FaeG2, from K88O169 and the FaeG of typical K88 (F4). The seroepidemiological study using recombinant antigens (two paralogs FaeG1 and FaeG2 from K88O169) showed reactivity of porcine (18.0%) and bovine (17.1%) sera to K88O169 FaeG1 and/or FaeG2 antigens on indirect ELISA tests. These results suggest that E. coli with K88O169 adhesin can infect various hosts, including pigs and cattle. This is the first report of domestic livestock having antibodies to K88O169 of human ETEC. Although human ETEC had been thought to be distinguished from those of domestic animals based on CFs, zoonotic strains may conceal themselves among human ETEC organisms. The concept of One Health should be adopted to intervene in ETEC infections among animals and humans.

Cutibacterium acnes is a human skin-resident bacterium. Although C. acnes maintains skin health by inhibiting invasion from pathogens like Staphylococcus aureus, it also contributes to several diseases, including acne. Studies suggest that differences in genetic background may explain the diverse phenotypes of C. acnes strains. In this study, we investigated the effects of C. acnes strains on the Caenorhabditis elegans life span and observed that some strains shortened the life span, whereas other strains, such as strain HL110PA4, did not alter it. Next, we assessed the effects of C. acnes HL110PA4 on host resistance against S. aureus. The survival time of C. acnes HL110PA4-fed wild-type animals was significantly longer than that of Escherichia coli OP50 control bacterium-fed worms upon infection with S. aureus. Although the survival times of worms harboring mutations at the daf-16/FoxO and skn-1/Nrf2 loci were similar to those of wild-type worms after S. aureus infection, administration of C. acnes failed to improve survival times of tir-1/SARM1, nsy-1/mitogen-activated protein kinase kinase kinase (MAPKKK), sek-1/mitogen-activated protein kinase kinase (MAPKK), and pmk-1/p38 mitogen-activated protein kinase (MAPK) mutants. These results suggest that the TIR-1 and p38 MAPK pathways are involved in conferring host resistance against S. aureus in a C. acnes-mediated manner. IMPORTANCE Cutibacterium acnes is one of the most common bacterial species residing on the human skin. Although the pathogenic properties of C. acnes, such as its association with acne vulgaris, have been widely described, its beneficial aspects have not been well characterized. Our study classifies C. acnes strains based on its pathogenic potential toward the model host C. elegans and reveals that the life span of C. elegans worms fed on C. acnes was consistent with the clinical association of C. acnes ribotypes with acne or nonacne. Furthermore, nonpathogenic C. acnes confers host resistance against the opportunistic pathogen Staphylococcus aureus. Our study provides insights into the impact of C. acnes on the host immune system and its potential roles in the ecosystem of skin microbiota.

Bifidobacterium infantis, a Gram-positive bacterium, is one of the commonly used probiotics. We previously showed that B. infantis modified host defense systems and extended the lifespan of the nematode Caenorhabditis elegans. In the present study, we showed that the lifespan extension caused by B. infantis was enhanced in animals having a mutation in the tol-1 gene that encodes the sole C. elegans homolog of Toll-like receptors (TLRs). Meanwhile, lifespan increased by other probiotic bacteria, such as Bacillus subtilis or Clostridium butyricum, was not affected in the tol-1 mutant animals. A microarray analysis revealed that the expression of innate immune response-related genes was significantly increased in the tol-1 mutant. Worms with the tol-1 mutation exhibited reduced leaving behavior from the B. infantis lawn, while canonical downstream factors trf-1/TRAF and ikb-1/IκB appeared to not be involved. In conclusion, C. elegans tol-1/TLR regulates B. infantis-induced longevity and also regulates behavior against B. infantis.

Enterotoxigenic Escherichia coli (ETEC) strains that express various fimbrial or nonfimbrial colonization factors and enterotoxins are critical causes of diarrheal diseases. Human ETEC serotype O169:H41 (O169) has been a representative of epidemic ETEC worldwide; the organism shows massive adherence to HEp-2 cells similar to enteroaggregative E. coli. Previously, we determined the complete sequence of the unstable virulence plasmid, pEntYN10. The plasmid included a unique set of genes encoding a novel colonization factor (CF) resembling K88 (F4) of porcine ETEC, in addition to CS6, a well-known representative CF of human ETEC, and another novel CF similar to CS8 (CFA/III) of human ETEC. To determine whether the K88-like CF (after this, K88O169) allows the organisms to infect domestic animals like the original K88-harboring strains that can cause diarrhea in piglets, samples were tested for antibodies against recombinant proteins of possible paralogous adhesins, FaeG1 and FaeG2, from K88O169 and the FaeG of typical K88 (F4). The seroepidemiological study using recombinant antigens (two paralogs FaeG1 and FaeG2 from K88O169) showed reactivity of porcine (18.0%) and bovine (17.1%) sera to K88O169 FaeG1 and/or FaeG2 antigens on indirect ELISA tests. These results suggest that E. coli with K88O169 adhesin can infect various hosts, including pigs and cattle. This is the first report of domestic animals having antibodies to K88O169 of human ETEC. Although human ETEC had been thought to be distinguished from those of domestic animals based on colonization factors, zoonotic strains may conceal themselves among human ETEC organisms. The concept of One Health should be adopted to intervene in ETEC infections among animals and humans.

The membrane trafficking events that regulate unicellular tube formation and maintenance are not well understood. Here, using an RNAi screen, we identified the small GTPase ARF1 homolog ARF-1.2 as a regulator of excretory tube formation in Caenorhabditis elegans. RNAi-mediated knockdown and knockout of the arf-1.2 gene resulted in the formation of large intracellular vacuoles at the growth sites (varicosities) of the excretory canals. arf-1.2 mutant animals were sensitive to hyperosmotic conditions. arf-1.2 RNAi affected the localization of the anion transporter SULP-8, which is expressed in the basal plasma membrane of the excretory canals, but did not affect the expression of SULP-4, which is expressed in the apical membrane. The phenotype of arf-1.2 mutants was suppressed by mutation of the small Rho GTPase CDC-42, a regulator of apical/basal traffic balance. These results suggest that ARF-1.2 plays an essential role in basal membrane traffic to regulate the formation of the unicellular excretory tube.

Aims/Introduction Bacterial septicemia has diverse clinical symptoms including severe hypoglycemia. However, sepsis-induced hypoglycemia has not yet been examined in detail. The aim of the present study was to investigate the mechanisms underlying hypoglycemia in sepsis. Materials and Methods We induced endotoxin shock in rats using lipopolysaccharide (LPS). After an intraperitoneal injection of LPS, we measured gluconeogenesis using the pyruvate tolerance test. The effects of LPS on glucose metabolism were investigated in perfused livers and isolated hepatocytes. Furthermore, its effects on the production of inflammatory cytokines were examined in isolated splenocytes. The interaction between splenocytes and hepatocytes in response to LPS was investigated in vitro using a co-culture of splenocytes and hepatocytes. Results In the pyruvate tolerance test, the pretreatment with LPS decreased gluconeogenesis. The in vivo pretreatment of rats with LPS did not inhibit glucose production in perfused livers. The in vitro treatment of isolated hepatocytes with LPS did not decrease hepatic gluconeogenesis. Although LPS increased the production of inflammatory cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1β, interleukin-6 and interleukin-10) and nitric oxide in isolated splenocytes, only nitric oxide significantly inhibited gluconeogenesis in isolated hepatocytes. When splenocytes and hepatocytes were co-cultured in medium containing LPS, the messenger ribonucleic acid expression of glucose-6-phosphatase in hepatocytes was suppressed. Conclusions LPS reduced hepatic gluconeogenesis, at least in part, by stimulating the production of nitric oxide in splenocytes. This effect could contribute to the mechanisms responsible for septicemia-induced hypoglycemia.

The source and routes of diarrheagenic Escherichia coli (DEC) remain poorly understood. To investigate the involvement of domestic animals in the dissemination of DEC, the prevalence of DEC in foods and fecal specimens from cattle, pigs, chickens, healthy carriers, and patients in Osaka and Hyogo, Japan was investigated using a multiplex real-time Polymerase Chain Reaction assay. The most abundant virulence genes were astA and eae, which had a prevalence 46.8% and 27.4%, respectively. Additionally, stx1 (26.6%) and stx2 (45.9%) were prevalent in cattle feces, while est (8.5%) and elt (7.6%) were prevalent in pig feces. afaB was the second-most prevalent gene in patients and healthy carriers, and it had detection rates of 5.1% and 8.1%, respectively. In contrast, afaB was not detected in animal feces or foods, except for three porcine fecal samples. The aggR gene was more prevalent in humans than in foods or animal feces. Both Shiga toxin-producing E. coli and atypical enteropathogenic E. coli carried by cattle may be sources for diarrheal diseases in humans. Pigs may be a source for human enterotoxigenic E. coli infections, whereas humans are expected to be the reservoir for diffusely adhering E. coli, enteroaggregative E. coli, and enteroinvasive E. coli.

Di-n-dutyl phthalate (DBP), an endocrine disruptor, is one of the most widely used phthalate esters (PAEs) in the world. It can be accumulated in seafood or agricultural products and represents a substantial risk to human health via the food chain. Thus, finding a plant which can remediate DBP but have no effects on growth is the main topic of the development of DBP phytoremediation. This study used garden lettuce (Lactuca sativa L. var. longifolia), which has a significant DBP absorption capability, as a test plant to measure phytoremediation kinetics and proteome changes after being exposed to DBP. The results show that DBP accumulated in different parts of the garden lettuce but the physiological status and morphology showed no significant changes following DBP phytoremediation. The optimal condition for the DBP phytoremediation of garden lettuce is one critical micelle concentration (CMC) of non-ionic surfactant Tween 80 and the half-life (t1/2, days), which calculated by first-order kinetics, was 2.686 days for 5 mg L&minus, 1 of DBP. This result indicated that the addition of 1 CMC of Tween 80 could enhance the efficiency of DBP phytoremediation. In addition, the results of biotoxicity showed that the median effective concentration (EC50) of DBP for Chlorella vulgaris is 4.9 mg L&minus, 1. In this case, the overall toxicity markedly decreased following phytoremediation. In the end, the result of proteome analysis showed six protein spots, revealing significant alterations. According to the information of these proteomes, DBP potentially causes osmotic and oxidative stress in garden lettuce. In addition, since DBP had no significant effects on the morphology and physiological status of garden lettuce, garden lettuce can be recommended for use in the plant anti-DBP toxicity test, and also as the candidate plant for DBP phytoremediation. We hope these findings could provide valuable information for DBP-contaminated water treatment in ecological engineering applications or constructed wetlands.

Atypical enteropathogenic Escherichia coli (aEPEC) strains (36 Japanese and 50 Bangladeshi) obtained from 649 poultry fecal samples were analyzed by molecular epidemiological methods. Clermont’s phylogenetic typing showed that group A was more prevalent (58%, 50/86) than B1 (31%, 27/86). Intimin type β1, which is prevalent among human diarrheal patients, was predominant in both phylogroups B1 (81%, 22/27) and A (70%, 35/50). However, about 95% of B1-β1 strains belonged to virulence group I, and 77% of them were Japanese strains, while 17% (6/35) of A-β1 strains did. Multilocus variable-number tandem-repeat analysis (MLVA) distributed the strains into 52 distinct profiles, with Simpson’s index of diversity (D) at 73%. When the data were combined with those of 142 previous strains from different sources, the minimum spanning tree formed five zones for porcine strains, poultry strains (excluding B1-β1), strains from healthy humans, bovine and human patient strains, and the B1-β1 poultry strains. Antimicrobial resistance to nalidixic acid was most common (74%) among the isolates. Sixty-eight percent of them demonstrated resistance to ≥3 antimicrobial agents, and most of them (91%) were from Bangladesh. The strains were assigned into two groups by hierarchical clustering. Correlation matrix analysis revealed that the virulence genes were negatively associated with antimicrobial resistance. The present study suggested that poultry, particularly Japanese poultry, could be another reservoir of aEPEC (phylogroup B1, virulence group I, and intimin type β1); however, poultry strains seem to be apart from patient strains that were closer to bovine strains. Bangladeshi aEPEC may be less virulent for humans but more resistant to antibiotics. IMPORTANCE Atypical enteropathogenic Escherichia coli (aEPEC) is a diarrheagenic type of E. coli, as it possesses the intimin gene (eae) for attachment and effacement on epithelium. Since aEPEC is ubiquitous even in developed countries, we previously used molecular epidemiological methods to discriminate aEPEC as a human pathogen. The present study assessed poultry as another source of human diarrheagenic aEPEC. Poultry could be the source of aEPEC (phylogroup B1, virulence group I, and intimin type β1) found among patient strains in Japan. However, the minimum spanning tree (MST) suggested that the strains from Japanese poultry were far from Japanese patient strains compared with the distance between bovine and patient strains. Bangladeshi avian strains seemed to be less diarrheagenic but are hazardous as a source of drug resistance genes.

Diarrheagenicity of diffusely adherent Escherichia coli (DAEC) remains controversial. Previously, we found that motile DAEC strains isolated from diarrheal patients induced high levels of interleukin 8 (IL-8) secretion via Toll-like receptor 5 (TLR5). However, DAEC strains from healthy carriers hardly induced IL-8 secretion, irrespective of their possessing flagella. In this study, we demonstrated that SK1144, a DAEC strain from a healthy carrier, suppressed IL-8 and IL-6 secretion from human epithelial cell lines. Suppression of IL-8 in human embryonic kidney (HEK293) cells that were transformed to express TLR5 was observed not only upon inflammatory stimulation by flagellin but also in response to tumor necrosis factor alpha (TNF-α) and phorbol myristate acetate (PMA), despite the fact that the TNF-α- and PMA-induced inflammatory pathways reportedly are not TLR5 mediated. SK1144 neither decreased IL-8 transcript accumulation nor increased intracellular retention of IL-8. No suppression was observed when the bacteria were cultured in Transwell cups above the epithelial cells; however, a nonadherent bacterial mutant (lacking the afimbrial adhesin gene) still inhibited IL-8 secretion. Direct contact between the bacteria and epithelial cells was necessary, but diffuse adhesion was dispensable for the inhibitory effects. Infection in the presence of chloramphenicol did not suppress cytokine release by the epithelial cells, suggesting that suppression depended on effectors synthesized de novo Inflammatory suppression was attenuated with infection by a bacterial mutant deleted for hcp (encoding a component of a type VI secretion system). In conclusion, DAEC strains from healthy carriers impede epithelial cell cytokine secretion, possibly by interfering with translation via the type VI secretion system.

Di-n-butyl phthalate (DBP) is an extensively used plasticizer. Most investigations on DBP have been concentrated on its environmental distribution and toxicity to humans. However, information on the effects of plasticizers on algal species is scarce. This study verified the impacts of endocrine disruptor di-n-butyl phthalate ester on microalga Chlorella vulgaris by approaches of proteomics and gene ontology. The algal acute biotoxicity results showed that the 24h-EC50 of DBP for C. vulgaris was 4.95 mg L&minus, 1, which caused a decrease in the chlorophyll a content and an increase in the DBP concentration of C. vulgaris. Proteomic analysis led to the identification of 1257 C. vulgaris proteins. Sixty-one more proteins showed increased expression, compared to proteins with decreased expression. This result illustrates that exposure to DBP generally enhances protein expression in C. vulgaris. GO annotation showed that both acetolactate synthase (ALS) and GDP-L-fucose synthase 2 (GER2) decreased more than 1.5-fold after exposure to DBP. These effects could inhibit both the valine biosynthetic process and the nucleotide-sugar metabolic process in C. vulgaris. The results of this study demonstrate that DBP could inhibit growth and cause significant changes to the biosynthesis-relevant proteins in C. vulgaris.

Clostridium butyricum MIYAIRI 588 (CBM 588), one of the probiotic bacterial strains used for humans and domestic animals, has been reported to exert a variety of beneficial health effects. The effect of this probiotic on lifespan, however, is unknown. In the present study, we investigated the effect of CBM 588 on lifespan and multiple-stress resistance using Caenorhabditis elegans as a model animal. When adult C. elegans were fed a standard diet of Escherichia coli OP50 or CBM 588, the lifespan of the animals fed CBM 588 was significantly longer than that of animals fed OP50. In addition, the animals fed CBM588 exhibited higher locomotion at every age tested. Moreover, the worms fed CBM 588 were more resistant to certain stressors, including infections with pathogenic bacteria, UV irradiation, and the metal stressor Cu2+. CBM 588 failed to extend the lifespan of the daf-2/insulin-like receptor, daf-16/FOXO and skn-1/Nrf2 mutants. In conclusion, CBM 588 extends the lifespan of C. elegans probably through regulation of the insulin/IGF-1 signaling (IIS) pathway and the Nrf2 transcription factor, and CBM 588 improves resistance to several stressors in C. elegans.

Diarrheagenicity of diffusely adherent Escherichia coli (DAEC) remains controversial. Previously, we found that motile DAEC strains isolated from diarrheal patients induced high levels of interleukin 8 (IL-8) secretion via Toll-like receptor 5 (TLR5). However, DAEC strains from healthy carriers hardly induced IL-8 secretion, irrespective of their possessing flagella. In this study, we demonstrated that SK1144, a DAEC strain from a healthy carrier, suppressed IL-8 and IL-6 secretion from human epithelial cell lines. Suppression of IL-8 in human embryonic kidney (HEK293) cells that were transformed to express TLR5 was observed not only upon inflammatory stimulation by flagellin but also in response to tumor necrosis factor alpha (TNF-α) and phorbol myristate acetate (PMA), despite the fact that the TNF-α- and PMA-induced inflammatory pathways reportedly are not TLR5 mediated. SK1144 neither decreased IL-8 transcript accumulation nor increased intracellular retention of IL-8. No suppression was observed when the bacteria were cultured in Transwell cups above the epithelial cells; however, a nonadherent bacterial mutant (lacking the afimbrial adhesin gene) still inhibited IL-8 secretion. Direct contact between the bacteria and epithelial cells was necessary, but diffuse adhesion was dispensable for the inhibitory effects. Infection in the presence of chloramphenicol did not suppress cytokine release by the epithelial cells, suggesting that suppression depended on effectors synthesized de novo. Inflammatory suppression was attenuated with infection by a bacterial mutant deleted for hcp (encoding a component of a type VI secretion system). In conclusion, DAEC strains from healthy carriers impede epithelial cell cytokine secretion, possibly by interfering with translation via the type VI secretion system.

The semi-solidified nutrition supplemented with soluble dietary fiber, xanthan gum (XG), inhibited postprandial glycemia in rats. The purpose of the present study is to examine whether XG exerts the same effects in humans. Subjects fasted for 12 h and then ingested the enteral nutrient, Meibalance with or without XG at 9 AM. Blood glucose levels were measured 0, 20, 40, 60, and 120 min after its ingestion. Postprandial blood glucose levels were lower in the XG group than in the control group. At 20 min, postprandial blood glucose levels were significantly lower in the XG group (84±5.3 mg/dL) than in the control group (107±7.8 mg/dL) (p

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen in developing countries, and causes foodborne infections through contaminated vegetables and water. ETEC also caused some foodborne infections in developed countries, though the vehicles are often unclear. We analyzed ETEC foodborne outbreaks in Japan based on the National Food Poisoning Statistics. Vegetables and private well water accounted for 50% and 22.2% of vehicles, respectively. The main vehicles were similar to those in developing countries. Serogroups of ETEC were also analyzed, and O6, O25, O27, O148, O153, O159, and O169 were the seven major O-serogroups. We investigated suitable detection methods for the pathogen (O148) in food samples associated with an outbreak of ETEC in Japan in 2011. We show that ETEC O148 could be effectively detected in cut leeks by means of a two-step enrichment and real-time PCR assay targeting heat-stable enterotoxin gene. Our survey of the vehicles and the major O-serogroups of ETEC outbreaks in Japan indicates that ETEC survives in the environment in Japan.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 has been an extremely destructive epidemic ETEC type worldwide. The strain harbors a large unstable plasmid that is regarded as responsible for its virulence, although its etiology has remained unknown. To examine its genetic background specifically on the unstable retention and responsibility in the unique adherence to epithelial cells and enterotoxin production, the complete sequence of a plasmid, pEntYN10, purified from the serotype strain was determined. The length is 145,082 bp; its GC content is 46.15%. It contains 182 CDSs, which include 3 colonization factors (CFs), an enterotoxin, and large number of insertion sequences. The repertory of plasmid stability genes was extraordinarily scant. Uniquely, results showed that 3 CFs, CS6, CS8 (CFA/III)-like, and K88 (F4)-like were encoded redundantly in the plasmid with unique variations among previously known subtypes. These three CFs preserved their respective gene structures similarly to those of other ETEC strains reported previously with unique sequence variations respectively. It is particularly interesting that the K88-like gene cluster of pEntYN10 had 2 paralogous copies of faeG, which encodes the major component of fimbrial structure. It remains to be verified how the unique variations found in the CFs respectively affect the affinity to infected cells, host range, and virulence of the ETEC strain.

The aim of this study was to evaluate the effect of lactic acid (LA) with and without organic material at various post-treatment recovery times on the heat resistance of Listeria monocytogenes (Lm). LA decreased Lm numbers; however, the effect was remarkably attenuated by the presence of organic matter. Five strains of Lm were treated with LA and the listericidal effects were compared. The effect of LA varied depending on the strain, with ≥3.0% (w/w) LA required to kill the Lm strains in a short time. The heat resistance of Lm treated with LA was examined with respect to the time interval between the acid treatment and the subsequent manufacturing step. The heat resistance of Lm was shown to significantly increase during the post-treatment period. Heat tolerance (D value) increased up to 3.4-fold compared with the non-treated control bacteria. RNA sequencing and RT-PCR analyses suggested that several stress chaperones, proteins controlled by RecA and associated with high-temperature survival, were involved in the mechanism of enhanced heat resistance. These results are applicable to manufacturers when LA and heat treatment methods are utilized for the effective control of Lm in foods.

Nutritional control has been proposed as a potential therapy for slowing the senescence of immune function and decreasing mortality. This study investigated whether sesamin could modify host defense systems and extend the lifespan of the nematode Caenorhabditis elegans.Nematodes were fed standard food (the bacterium Escherichia coli strain OP50) supplemented with various doses of sesamin/γ-cyclodextrin inclusion compounds starting from young adulthood. The mean lifespan, muscle function, lipofuscin accumulation, protein carbonyl content, and stress resistance of the worms were examined. Then, C. elegans mutants harboring loss-of-function lesions in longevity- and host defense-related signaling pathways were supplemented with sesamin to identify the genes involved in the longevity effects.Worms supplemented with sesamin displayed higher locomotion and prolongevity and produced offspring at levels similar to unsupplemented control animals. The growth curves of nematodes were similar to those of controls, suggesting that sesamin did not induce prolongevity effects through dietary restriction. Notably, sesamin made the worms more resistant to infection by Legionella pneumophila and more resistant to oxidative stressors such as paraquat and hydrogen peroxide and prolonged the lifespan of a mev-1 mutant that produces abundant superoxide anions. However, the accumulation of protein carbonyls and lipofuscin was similar in sesamin-exposed and control worms, suggesting that sesamin is unlikely to work simply as an antioxidant. Sesamin supplementation failed to extend the lifespan of loss-of-function mutants of daf-2, daf-16, pmk-1, and skn-1.Sesamin enhances the host defense of C. elegans and increases the average lifespan via activation of both skn-1 (encoding a component of the p38 MAPK pathway) and daf-16 (encoding a component of the IGF-1 pathway).

Tight junctions contribute to the formation and establishment of intestinal epithelial barriers against microbial infections. However, a variety of enteric pathogens have developed strategies to adhere to and invade epithelial cells and disrupt epithelial integrity. The aim of this study was to ascertain if enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) can cause deterioration of epithelial barrier integrity. Since STa shows amino acid similarity to guanylin, we evaluated the effects of both of these molecules on T84 epithelial cells. T84 epithelial monolayers were grown on 24-well Transwell filters and barrier integrity was assayed by measurement of transepithelial electrical resistance (TER). Macromolecular permeability of the monolayers was determined by measuring the paracellular passage of FITC-labeled dextran 4000 from apical to basolateral compartments of the Transwell filter culture. Treatment of T84 monolayers with either ST or guanylin did not increase paracellular permeability to FITC-dextran. However, although guanylin, which is a physiological guanylate cyclase activator, did not change TER in polarized T84 monolayers, ST did elicit a reduction in TER within 2h, at concentrations above 4μM. These data suggest that STa causes not only induction of water secretion but also intestinal barrier dysfunction.

Lactobacilli and bifidobacteria are probiotic bacteria that modify host defense systems and have the ability to extend the lifespan of the nematode Caenorhabditis elegans. Here, we attempted to elucidate the mechanism by which bifidobacteria prolong the lifespan of C. elegans. When the nematode was fed Bifidobacterium infantis (BI) mixed at various ratios with the standard food bacterium Escherichia coli strain OP50 (OP), the mean lifespan of worms was extended in a dose-dependent manner. Worms fed BI displayed higher locomotion and produced more offspring than control worms. The growth curves of nematodes were similar regardless of the amount of BI mixed with OP, suggesting that BI did not induce prolongevity effects through caloric restriction. Notably, feeding worms the cell wall fraction of BI alone was sufficient to promote prolongevity. The accumulation of protein carbonyls and lipofuscin, a biochemical marker of aging, was also lower in worms fed BI; however, the worms displayed similar susceptibility to heat, hydrogen peroxide, and paraquat, an inducer of free radicals, as the control worms. As a result of BI feeding, loss-of-function mutants of daf-16, jnk-1, aak-2, tol-1, and tir-1 exhibited a longer lifespan than OP-fed control worms, but BI failed to extend the lifespan of pmk-1, skn-1, and vhp-1 mutants. As skn-1 induces phase 2 detoxification enzymes, our findings suggest that cell wall components of bifidobacteria increase the average lifespan of C. elegans via activation of skn-1, regulated by the p38 MAPK pathway, but not by general activation of the host defense system via DAF-16.

Diarrheagenic E. coli (DEC) isolates were recovered from local retail markets and the Osaka Municipal Central Wholesale Market in Japan. Retail food samples were collected for analysis in Osaka Japan from 2005 to 2008 and consisted of 32 beef, 28 pork, 20 poultry, 136 fish, 66 fruits and vegetables and 51 ready-to-eat (RTE) food samples. A total of 82 DEC strains were recovered from 64 (19%) food samples with the highest prevalence in poultry (100%, 20/20), followed by pork (54%, 15/28), beef (28%, 9/32), fruits and vegetables (12%, 8/66), fish (6.6%, 9/136) and RTE foods (5.9%, 3/51). Most of the strains belonged to E. coli possessing the enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) gene (EAST1EC; n=62, P0.0001) and enteropathogenic E. coli (EPEC; n=16, P0.01), whereas only 1 strain belonged to Shiga toxin-producing E. coli (STEC), 1 to EAEC and 2 to enterotoxigenic E. coli (ETEC) strains. Of the 82 DEC isolates, 22 O and 13H serogroups were detected, including some specific serogroups (O91, O103, O115, O119, O126, and O157) which have been associated with human diarrheal infections. Phylogenetic group A and B1 were predominant among the DEC isolates. Antimicrobial resistance to tetracycline was most common (49%), followed by nalidixic acid (28%), ampicillin (24%), sulfamethoxazole/trimethoprim (20%), and cephalothin (18%). All isolates were susceptible to aztreonam. Of the resistant strains, 44% (22/50) demonstrated resistance to3 antimicrobial agents. Isolates resistant to5 antimicrobials were only found in the meat samples, while isolates from the fruits and vegetables as well as RTE foods showed resistance to only 1 or 2 antimicrobial agents. Sixty one percent of EAST1EC, 56% of EPEC and all of the EAEC and ETEC were resistant to at least 1 antimicrobial agent. Multiple-locus variable-number tandem repeat analysis (MLVA) was used in this study for genotyping of DEC. The 82 isolates collected for this study showed 77 distinct MLVA profiles located among 3 branches. The Simpson's Index of Diversity (D) was 99.9% at its highest. The high diversity of these food strains would suggest their originating from a variety of sources and environments. In conclusion, retail food samples in Japan were contaminated with DEC; EAST1EC, a putative DEC, were detected at high rates in poultry, pork and beef. Isolates resistant to3 antimicrobials were found only in raw meat and fish. Food animals may act as the reservoir for multi-resistant bacteria. Due to the finding that nearly 1/3 of EAST1EC strains were resistant to3 antimicrobials, additional surveillance for EAST1EC should be initiated.

Aim This study assessed whether multi-locus variable-number tandem repeat analysis (MLVA) and antimicrobial susceptibility testing discriminated diarrhoeagenic atypical enteropathogenic Escherichia coli (aEPEC) from aEPEC indigenous to domestic animals or healthy people. Methods and Results MLVA genotyping of 142 aEPEC strains isolated from foods and faecal samples of domestic animals and humans revealed 126 distinct MLVA profiles that distributed to four clusters, yielding a Simpson's index of diversity (D) of 99.8%. Cluster 2 included 87% of cattle isolates and 67% of patient isolates. The plurality (15/34, 44%) of strains from healthy humans mapped to Cluster 1, while half (18/41, 44%) of the swine strains belonged to Cluster 4. Testing for antimicrobial susceptibility revealed that 52 strains (37%) of aEPEC were resistant to one or more agents; only 10 strains (7%) exhibited resistance to more than three agents. Strains isolated from swine or food exhibited a wider variety of resistance phenotypes than bovine or human strains. Conclusions MLVA assigned the aEPEC isolates from cattle and patients to Cluster 2, distinct from aEPEC from other sources. Hog yards may be a larger source of drug-resistant strains than are cattle ranches. Significance and Impact of the Study MLVA suggests that human diarrhoeagenic aEPEC are derived from cattle and are distinct from strains carried by healthy people and other animals. Cattle appear to be reservoirs of human diarrhoeagenic aEPEC. This article is protected by copyright. All rights reserved.

Aims: The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria. Methods and Results: Digoxigenin-labelled DNA probes were designed to detect seven pathotypes of DEC based on type-specific genes. A total of 615 meat, food and faeces samples identified as DEC-positive by multiple real-time PCR for the virulence genes (eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid-membrane filters. DEC were isolated from 72·5% (446/615) of samples by the colony hybridization method but were only detected in 26·3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low-level contaminants, such as enterotoxigenic and Shiga toxin-producing E. coli, which were isolated from 51·8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4·5% (5/112) of samples analysed by the conventional method. Conclusions: The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes. Significance and Impact of the Study: The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC.

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL-8 secretion in cultures of human carcinoma epithelial cells (HEp-2, Caco-2), is enterovirulent. In the present study, we examined whether IL-8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL-8 high-inducing strains were highly motile in swarming tests. Partially purified flagella induced IL-8 in a dose-dependent manner. However, IL-8 induction was inhibited by small-interfering RNA against TLR5 or by treating flagella with disialoganglioside-GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL-8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL-8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL-8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL-8 secretion.

Aims: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. Methods and Results: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0·99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7·1 × 102 to 1·1 × 104 CFU ml−1, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g−1) were found to be positive by the method. Conclusions: The present system allows for the efficient and simultaneous determination of various DEC pathotypes. Significance and Impact of the Study: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

Colicins are proteins produced by and active against several strains of Escherichia coli . Previously we reported that colicinogenic bacteria seemed beneficial in preventing the clinical manifestations of infectious disease caused by enterohemorrhagic E. coli O157 in humans. The inhibitory effects could be due to a decrease in O157 levels and/or pathogenicity. This study investigated the effects of colicinogenic E. coli on the production of Shiga toxin (Stx) by O157. Standard strains of colicinogenic bacteria carrying plasmids for each type of colicin (E3/5/8/9) were used for the study. The O157 strains were cultured in the presence of colicinogenic bacteria or extracted colicins. Compared with results for controls, DNase colicins (E8/9) facilitated an 8- to 64-fold increase in production of Stx2, while RNase colicins (E3/5) suppressed Stx production in only two strains. Stx prophages were induced in synchrony with Stx production. Semiquantitative real-time reverse transcription-PCR (RT-PCR) was then performed to examine SOS gene expression. The RT-PCR results clearly indicated a marked increase in mRNA levels of SOS reaction-associated genes after the addition of DNase colicins. We believe that Stx prophages are induced by the SOS response to DNA damage caused by DNase colicins, thus leading to higher Stx production. These findings suggest that while colicinogenic bacteria can be antagonistic to O157 infection, DNase colicins may enhance Stx production. Thus, colicinogenic flora is likely to be involved in the complex pathogenic pathways of O157 infection, and further investigation should be performed before the use of colicinogenic bacteria as an intervention method.

The role of diffusely adherent Escherichia coli (DAEC) in diarrheal disease has been controversial. However, DAEC strains were recently implicated in diarrheal disease in developing countries. To clarify whether DAEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, E. coli strains isolated between July 1997 and March 2000 during diarrheagenic E. coli (DEC) investigation were retrospectively examined. DAEC strains were recognized among 41 (4.4%) of 924 patients and formed the biggest subgroup of DEC. Previously, we reported that some DAEC strains caused epithelial cells to secrete as much IL-8 as enteroaggregative E. coli strains did. In this study, we attempted to evaluate epidemiologically whether the ability of DAEC to induce IL-8 was involved in the pathogenesis. Relationship among patient age, symptoms, Afa adhesins, season and IL-8 induction were examined. The subgroup of DAEC that possessed Afa genes and/or induced a high level of IL-8 was significantly prevalent among patients age 1 to 4 years; however total DAEC was not significantly high among the children compared to other age group. IL-8 inducing DAEC seems to play a role in causing sporadic diarrheal illnesses, particularly in pediatric fields. Investigations highlighting the relationship between IL-8 induction and enteropathogenicity are clearly necessary to confirm the role of DAEC in infectious enteritis.

In this study, Listeria monocytogenes contamination in a cold-smoked fish processing plant in Osaka, Japan, was examined from 2002 to 2004. A total of 430 samples were collected and divided into five categories: raw fish, materials during processing, processing equipment, environment, and finished products. A total of 59 finished products were examined throughout this study. L. monocytogenes was isolated from four of these samples during summer and autumn but was not found during winter or spring. During the warmer seasons, L. monocytogenes was more prevalent on processing equipment, especially slicing machines (8 of 54 samples in summer and autumn versus 1 of 50 samples in winter and spring). L. monocytogenes was not detected on whole skins removed from 23 frozen raw fish. L. monocytogenes strains isolated from 56 samples were characterized by serotyping, pulsed-field gel electrophoresis, and three PCR-based methods. Seventy-seven L. monocytogenes strains were recognized as contaminants of the samples: 2 distinguishable strains were identified in each of 13 samples, 3 strains were identified in 2 samples, 5 strains were identified in 1 sample, and the other 40 strains were identified in 40 samples. Combining the results from these techniques, 77 strains were classified into 13 different types. Three of these types prevailed throughout the plant, and two of the three were also isolated from final products. The DNA subtype found in the product was also found on the slicing machines. Our findings suggest that the slicing machines at this plant were the source of the product contamination. Implementing an appropriate cleaning regime for the slicing machines was effective in preventing contamination.

Escherichia coli that sparsely adhere to human epithelial cells are known as diffusely adherent E. coli (DAEC), and the role of the Afa/Dr family of adhesins is now understood. Strains that do not possess Afa/Dr, however, comprise another group of DAEC, of which the pathogenicity remains unknown. The ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells might be a feature of enterovirulent bacteria. We previously found that some Afa/Dr DAEC strains induce IL-8 by stimulating epithelial cells with flagella. The present study examines whether non-Afa/Dr DAEC can induce IL-8 in epithelial cells (HEp-2, INT407, and T84). Among 21 strains, 11 (52%; 11/21) induced as much IL-8 as high inducer strains of Afa/Dr DAEC. Adhesion did not significantly differ between high and low inducers; therefore diffuse adhesion alone is probably insufficient to induce IL-8. It was shown that IL-8 induction and the number of intracellular bacteria directly correlated. Wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase, reduced both intracellular bacteria and IL-8 secretion. Motile strains were significantly more prevalent among high (10/11) than low (4/10) inducers. However, 4 low invasive strains hardly induced IL-8 despite their motility. In conclusion, some non-Afa/Dr DAEC invoke the induction of high levels of inflammatory cytokines. Unlike Afa/Dr DAEC, however, non-Afa/Dr strains may require invasion to cause strong induction. These non-Afa/Dr high inducers can be enteropathogenic for the cytokine-inducing properties.

Aims: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. Methods and Results: A total of 220 E. coli strains belonging to 60 different ‘O’ serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx1, stx2, eaeA and hlyA genes. Twenty-eight E. coli (75·6%) strains from local and nine (24·3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16·47%) (belonging to 13 different serogroups) from local goats carried stx1 gene alone or in combination with stx2 gene, while as only one strain (2%) from migratory goats possessed stx2 gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx1 gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14·7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. Conclusions: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. Significance and Impact of the Study: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.

Elucidation of the function and mechanism of the innate immune system has shown that natural killer (NK) cells play an important role, particularly for protection against viral infection and cancer; they also seem to participate in maintaining the health of senescent people and their longevity. The fundamental aspects of innate immunity and NK cells is explained first in this review. Bibliographic information on effective nutrient composition, food ingredients, and life style for activating NK cells is then presented and explained.

Meat samples and fecal specimens from adult cattle were collected in Changchun, China and were examined for presence of Shiga toxin-producing Escherichia coli (STEC) serogroup O157. STEC O157 strains were isolated from 2 (5%) of 40 beef, 1 (3.3%) of 30 pork, and 3 (1.7%) of 176 adult cattle fecal samples. The strains belonged to phage types (PT) 4, 8, or 47. Two beef strains and a strain previously isolated from a patient in Shandong, China, were PT-4 and showed a similar PFGE pattern, suggesting the possibility of food-borne transmission. It is suggested that cattle are a reservoir of STEC O157:H7 and meat products are contaminated by this pathogen in Changchun, China as well as in other countries.

In previous studies of human information processing, the visual separate system and the auditory separate system have been frequently studied. However, human related characteristic between visual and auditory systems has not been investigated substantially. If the mechanisms of human visual and auditory parallel information processing are elucidated, the finding will contribute to improving technical systems. In our previous studies, human related characteristic between visual search and speech perception is studied. The results showed the mutual effect between visual information processing and auditory information processing in the visual and the auditory parallel information processing. However, to clarify the cause of many car accidents lies in the use of car-phone on driving, it is necessary to use the same kind of visual and auditory stimuli in psychological experiments. In this paper, characteristic of reaction time in parallel information processing of human visual and auditory system is measured by using the same kind of stimuli. The experimental results show the factors that influence the visual and the auditory reaction time are the difficulty of the visual and the auditory stimuli and the timing of the visual and the auditory inputs. The experimental results also suggest that one of the causes of car accidents (to use car-phone while driving) is that the human visual information processing is affected by the auditory information.