Kuma Bamboo Grass (Sasa veitchii) Extracts Exhibit Protective Effects Against Atypical Aeromonas salmonicida Infection in Goldfish (Carassius auratus).

Atypical Aeromonas salmonicida ( i.e. subsp. achromogenes and subsp. masoucida) are one of the major opportunistic pathogens that cause ulcer diseases in a variety of fishes, in which this pathogen has become a worldwide economic threat in sectors that handle of particular high-priced ornamental fishes like varicolored carp and goldfish due to appearance damages. Here we reported that the kuma bamboo grass (Sasa veitchii) extracts (KBGE) that contained a variety of fatty acids, exhibited antibacterial activity against nine Aeromonas strains including 5 atypical A. salmonicida strains. Experimental challenges with four atypical A. salmonicida strains revealed that supplementation with 375 to 750 µg/ml of the KBGE restored the survival of goldfish in coincidence of inhibition of both bacterial replication and superoxide dismutase (SOD) activity upon infection, compared with those of untreated control. Together, our data demonstrating the antibacterial effects of the plant extracts proposes its possible implication for prevention of Aeromonas infection in the ornamental fish.

Ex vivo proteomics of Campylobacter jejuni 81-176 reveal that FabG affects fatty acid composition to alter bacterial growth fitness in the chicken gut.

Campylobacter jejuni is one of the leading causes of foodborne gastrointestinal illness worldwide. Here we performed ex vivo proteomic analysis of C. jejuni 81-176 in chicken, a main reservoir for human infection. At 0, 1 and 4 weeks post-infection (p.i.) with the GFP-expressing 81-176 strain, inocula were recovered from chicken ceca by cell sorting using flow cytometry. iTRAQ-coupled 2D-LC-MS/MS analyses that detected 55 C. jejuni proteins, among which either 3 (FabG, HydB, CJJ81176_0876) or 7 (MscS, CetB, FlhF, PurH, PglJ, LpxC, Icd) proteins exhibited >1.4-fold-increased expression at 1 or 4 week(s) p.i. compared with those at 0 weeks p.i., respectively. Deletion of the fabG gene clearly decreased the proportion of bacterial unsaturated fatty acids (UFAs) and chicken colonization. The UFA proportion of the parental strain was not altered when grown at 42 °C. These findings suggest that FabG might play a pivotal role in UFA production, linked to bacterial adaptation in the poultry host. To our knowledge, this is the first example of ex vivo C. jejuni proteomics, in which fatty acid metabolism might affect bacterial adaptation to the chicken host.

Evaluation of recombinant forms of the shiga toxin variant Stx2eB subunit and non-toxic mutant Stx2e as vaccine candidates against porcine edema disease.

Porcine edema disease (ED) is a communicable disease of shoats caused by infection with Shiga toxin (Stx)-producing Escherichia coli. Stx2e is classified as a 1A5B-type toxin and is a decisive virulence determinant of ED. The single A subunit of Stx2e possesses enzymatic activity and is accompanied by a pentamer of B subunits, which binds to the host receptor and delivers the A subunit into the cell. In the present study, we used a mouse model to evaluate the immunogenicity of 3 ED vaccine candidates: a non-toxic mutant holotoxin mStx2e and 2 Stx2eB-based fusion proteins, Stx2eA2B-His and Stx2eB-His. Systemic inoculation of mice with mStx2e- and the Stx2eB-derived antigens induced anti-Stx2e IgG responses that were fully and partially capable of neutralizing Stx2e cellular cytotoxicity, respectively. Intranasal immunization with mStx2e protected the mice from subsequent intraperitoneal challenge with a lethal dose of Stx2e, whereas immunization with Stx2eA2B-His and Stx2eB-His afforded partial protection. Analysis of serum cytokines revealed that mStx2e, but not the Stx2eB-based antigens, was capable of inducing a Th2-type immune response. These results suggest that although the Stx2eB-based antigens elicited an immune response to Stx2e, they did so through a different mechanism to the Th2-type response induced by mStx2e.

Membrane topology of Salmonella invasion protein SipB confers osmotolerance.

Salmonella enterica serovar Typhimurium is a major cause of human gastrointestinal illness worldwide. This pathogen can persist in a wide range of environments, making it of great concern to public health. Here, we report that the salmonella pathogenicity island (SPI)-1 effector protein SipB exhibits a membrane topology that confers bacterial osmotolerance. Disruption of the sipB gene or the invG gene (SPI-1 component) significantly reduced the osmotolerance of S. Typhimurium LT2. Biochemical assays showed that NaCl osmolarity increased the membrane topology of SipB, and a neutralising antibody against SipB reduced osmotolerance in the WT strain. The WT strain, but not the sipB mutant, exhibited elevated cyclopropane fatty acid C19:0 during conditions of osmotic stress, correlating with the observed levels of survival and membrane integrity. This result suggests a link between SipB and the altered fatty acid composition induced upon exposure to osmotic stress. Overall, our findings provide the first evidence that the Salmonella virulence translocon SipB affects membrane fluidity and alters bacterial osmotolerance.

Intrahost passage alters SigB-dependent acid resistance and host cell-associated kinetics of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that causes gastroenteritis, maternofetal infections and meningoencephalitis in humans. Here we report that an intrahost genome mutation alters bacterial acid resistance and the abilities for replication/invasion in tissue cell culture. Among the L. monocytogenes isolates from the recent outbreak in Japan, we found that one food strain, 668, exhibited the greatest acid resistance, whereas one human clinical strain, 690, sharing identical pulsed-field gel electrophoresis (PFGE) and ribotyping patterns, exhibited an acid-sensitive phenotype. Passage of the 668 food strain through the mouse intestine increased its acid sensitivity without altering the macrogenotypes, indicating intrahost alteration of the bacterial acid-resistant phenotype. Genetic and proteomic analyses revealed a link between acid resistance and SigB (RNA polymerase SigmaB subunit) activity. Compared with the strain 668, the clinical and 4 of 5 mice-passaged strains showed a mutation in the rsbW locus, whose product controls the regulation of SigB activity. Corresponding to the SigB activity, the host-passaged strains had reduced abilities to survive inside macrophages and to invade Caco-2 cells, compared with the food strain 668. Overall, we have demonstrated the first example of a host environment promoting the alteration of SigB-dependent acid resistance and host cell-associated actions of L. monocytogenes. Our study provides new insight into the potential role of intrahost environment in the process of bacterial evolution.

Disinfection methods for spores of Bacillus atrophaeus, B. anthracis, Clostridium tetani, C. botulinum and C. difficile.

To evaluate disinfection methods for environments contaminated with bioterrorism-associated microorganism (Bacillus anthracis), we performed the following experiments. First, the sporicidal effects of sodium hypochlorite on spores of five bacterial species were evaluated. Bacillus atrophaeus was the most resistant to hypochlorite, followed in order by B. anthracis, Clostridium botulinum and Clostridium tetani, and Clostridium difficile. Subsequently, using B. atrophaeus spores that were the most resistant to hypochlorite, the sporicidal effects of hypochlorite at lower pH by adding vinegar were evaluated. Hypochlorite containing vinegar had far more marked sporicidal effects than hypochlorite alone. Cleaning with 0.5% (5000 ppm) hypochlorite containing vinegar inactivated B. atrophaeus spores attached to vinyl chloride and plywood plates within 15 s, while that not containing vinegar did not inactivate spores attached to cement or plywood plates even after 1 h. Therefore, the surfaces of cement or plywood plates were covered with gauze soaked in 0.5% hypochlorite containing vinegar, and the sporicidal effects were evaluated. B. atrophaeus spores attached to plywood plates were not inactivated even after 6 h, but those attached to cement plates were inactivated within 5 min. On the other hand, covering the surfaces of plywood plates with gauze soaked in 0.3% peracetic acid and gauze soaked in 2% glutaral inactivated B. atrophaeus spores within 5 min and 6 h, respectively. These results suggest that hypochlorite containing vinegar is effective for disinfecting vinyl chloride, tile, and cement plates contaminated with B. anthracis, and peracetic acid is effective for disinfecting plywood plates contaminated with such microorganism.

Prevalence of Listeria monocytogenes in raw meats marketed in Bangkok and characterization of the isolates by phenotypic and molecular methods.

Listeria monocytogenes causes listeriosis characterized by septicaemia, encephalitis, and abortion or stillbirth. Regular monitoring of its prevalence in food and characterization of its phenotypes and genotypes are necessary for disease surveillance and tracing the epidemic outbreaks. In this study, the prevalence of L. monocytogenes in raw meats marketed in Bangkok was 15.4%. The bacteria isolated from meat were serotyped and genotyped using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Their virulence-associated genes, antimicrobial susceptibility, and ability to invade intestinal epithelial cells were studied. All 22 L. monocytogenes strains isolated from 104 raw meat samples carried virulence-associated genes, such as actA, flaA, hlyA, lap, inlA, inlB, and prfA. These were serotype 4b, suggesting their pathogenic and epidemic potential. These isolates could be classified into six ERIC-PCR groups: A-E The majority (59.1%) of the isolates belonged to Group A, and three isolates were Group D which was closely related to the Group A. Two isolates each were Group C and E, and one isolate each was group B and F. Although the isolates belonged to the same serotype and genotype and were all equipped with the virulence-associated genes, they showed a different cell invasion capability and antibiotic susceptibility. All the isolates were susceptible to ampicillin, amikacin, chloramphenicol, gentamicin, imipenem, penicillin G, sulphamethoxazole-trimethoprim, and tetracycline. However, one isolate showed only intermediate susceptibility to tetracycline. The data provide the first molecular insight into the L. monocytogenes isolates in Thailand and elucidate a potential risk of people contracting listeriosis.

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone.

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.

Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease.

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

Isolation and characterization of Listeria monocytogenes from commercial asazuke (Japanese light pickles).

Asazuke is a ready-to-eat Japanese light pickle, mainly made of vegetables which are known to be one of the sources of Listeria monocytogenes contamination. Although asazuke is a popular side-dish in Japan, the hazard of bacterial contamination has not been evaluated yet. In this study, we investigated the prevalence of L. monocytogenes, Salmonella spp., verotoxigenic E. coli (VTEC) and coliforms in 108 asazuke samples that randomly collected from supermarkets in Obihiro (Hokkaido prefecture, Japan) during the period of June to November 2007. Twelve (11.11%) L. monocytogenes were isolated with predominant serotype 4b (seven isolates) followed by 1/2a (two isolates), 1/2b, 3b and 4c (one isolate each) while Salmonella spp., VTEC and coliforms were not detected. All L. monocytogenes isolates demonstrated hemolytic activity by CAMP test and possessed all the virulence-associated genes (prfA, actA, mpl, inlA, inlC, plcA, plcB, hly, iap, clpC and opuCA) as resulted in PCR, thus revealed their potential pathogenicity. Moreover, 7 out of 12 isolates were from asazuke samples produced by the same factory and their pulsed-field gel electrophoresis (PFGE) profiles suggested that 6 of them were indistinguishable and one was different. L. monocytogenes contamination in the asazuke factory environment was further investigated and 23 out of 60 environmental swabs (38.33%) contained the bacterium. Comparison of PFGE profiles showed relatedness between food and environmental isolates indicating that contamination probably occurred in the asazuke factory during manufacturing. Interestingly, after HACCP training course conducted to the factory workers, 20 samples collected during the period of November to December 2008 were negative to L. monocytogenes revealing that the hygienic status has improved.

A V H H that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type A.

The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/V(H)H) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinum toxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/V(H)H. Soluble VH/V(H)H were produced and purified from 10 VH/V(H)H phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/V(H)H-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel V(H)H, i.e. (F/Y)(42)E(49)R(50)(G/F)(52). The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V(42)G(49)L(50)W(52). V(H)H of one clone (V(H)H17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of V(H)H17 matched with the 194-206 amino acid residues of BoTxA/LC, which are located near the S'1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the V(H)H17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the V(H)H17 should be due to the V(H)H insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism.

Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD.

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB - encoded by a prophage in S. Typhimurium DT104 - are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [(32)P]NAD and ArtA, and the label was released by HgCl(2), which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA(6Arg-Ala) and ArtA(115Glu-Ala), in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

Detection of Salmonella spp. Isolates from specimens due to pork production Chains in Hue City, Vietnam.

From August 2007 until March 2008, we perfomed a detection and epidemiological analysis for Salmonella spp. in specimens collected from pork production chains to improve the quality of meat hygiene conditions in Hue, Vietnam. A total of 306 specimens were examined for Salmonella spp., aerobic bacterial counts and coliform. Seven serovars of Salmonella spp. were detected in retail pork, slaughterhouse carcasses and environmental specimens with the following detection rates: 32.8% of retail pork, 15.5% of slaughterhouse carcasses, 47.4% of floors, 38.1% of weighing bowls, 28.6% of cooking boards and 16.7% of tank water samples. Based on these results, we recommend that exhaustive sterilization, washing, routine bacteriological examinations and treatments at low temperature are performed in slaughterhouses, transportation facilities and retail stores.

Hazard analysis of Listeria monocytogenes contaminations in processing of salted roe from walleye pollock (Theragra chalcogramma) in Hokkaido, Japan.

Hazard analysis of Listeria monocytogenes contamination during processing of salted walleye pollock (Theragra chalcogramma) roe was performed for a seafood plant in Japan from December 2005 to February 2006. As a result, L. monocytogenes number was detected on the pallet used for transport of barrels in the salting process and one of the rollers of the roller conveyor, which rotates while in contact with the bottoms of the barrels, but was not detected in any raw materials, interim products or final products. Thus, we believe that the pallet contamination initially occurred because of insufficient washing, that it was passed on to the bottoms of the barrels and that it was then passed on the roller of the roller conveyor by cross-contamination. Therefore, it is possible that interim and final products may become contaminated by processing devices and machinery. In addition, we conducted an inoculation study designed at the 1/20 actual factory scale using interim products with or without artificial color and seeded with L. monocytogenes to observe changes in its growth. In the inoculation study, multiplication of L. monocytogenes during the salting process was not confirmed in the samples with artificial color.

Differential expression of the outer membrane protein W (OmpW) stress response in enterohemorrhagic Escherichia coli O157:H7 corresponds to the viable but non-culturable state.

During an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157, we showed previously that food isolates were resistant to oxidative stress, while patient isolates were sensitive to it. Because food isolates increased stress-sensitivity after mouse passage, this change most likely occurred during passage through patients. Here we demonstrate that the phenotypic change occurring during mouse passage correlates with the stress response of outer membrane protein W (OmpW) in EHEC O157 strains. Upon induction of oxidative stress, OmpW was highly expressed only in the stress-sensitive MP37 strain, obtained by mouse passage of food strain F2, but not in the F2 strain. Western blotting confirmed that expression of OmpW was induced in the viable but non-culturable (VBNC) state. Deletion of ompW in the MP37 strain increased recovery from dormancy, while overexpression of OmpW in the F2 strain decreased recovery when exposed to oxidative stress, suggesting that high levels of OmpW sensitize the bacteria to stress. DNA alignment revealed that the class I integron (int1I) fragments flanking the ompW gene are oriented in opposite directions between stress-resistant and -sensitive strains. All stress-sensitive strains induced ompW under stress. We propose that the different stress response of OmpW was introduced by genetic alteration during in vivo passage.

Proteomic characterization of enterohemorrhagic escherichia coli O157:H7 in the oxidation-induced viable but non-culturable state.

Enterohemorrhagic Escherichia coli (EHEC) O157 strain F2, a food isolate of an outbreak, is resistant to oxidative stress, but has increased stress-sensitivity after passage through mice. The stress-sensitive variant of F2 (designated MP37) has decreased culturability, but retains membrane integrity under stress conditions, indicating that the cells enter a viable but non-culturable (VBNC) state. Proteomic analyses revealed that MP37 in the VBNC state had decreased levels of some oxidation-responsive factors (AhpCF, AceF), but it markedly increased levels of outer membrane protein W (OmpW). Because F2 expressed higher levels of some ribosome-associated proteins (RaiA, S6, Bcp) than MP37, the effect of animal passage on the induction of the VBNC state in the EHEC O157 cells might be due to ribosomal activity.

Epsilon-polylysine microparticle adjuvant drives cytokine production to Th1 profile.

Epsilon-polylysine micro particles (SGEPL) and polyethyleneimine micro particles (SGPEI) were developed by the addition of a hydrophobic group and the immunological characterization of these micro particles and aluminum hydroxide (ALUM) was investigated. BALB/c mice were injected intraperitoneally with ovalbumin (OVA) as an antigen and SGEPL, SGPEI or ALUM as an adjuvant. The results showed that the mice injected with SGEPL produced a significant portion of anti-OVA antibody subclass IgG2a in the sera and suppressed interleukin (IL)-4 and IL-5, but enhanced IL-12 and interferon-gamma (IFN-gamma) from the spleen cells. Similar results relating to cytokines were also obtained, even without OVA. Direct stimulation with SGEPL to naïve BALB/c mouse spleen cells induced IL-12 and IFN-gamma. Both spleen and purified B cells produced IgG1 and IgE after stimulation with IL-4 and the anti-CD40 monoclonal antibody. With the addition of SGEPL, the IgE production from the cells was suppressed as a result of enhanced IFN-gamma production. Furthermore, IgE production was also suppressed in the purified B cells without the influence of IFN-gamma or IL-12. Thus, we suggest SGEPL drives cytokine production to Th1 profile. It will be a novel promising adjuvant based on this viewpoint.

Rapid multiplex immunofluorescent assay to detect antibodies against Burkholderia pseudomallei and taxonomically closely related nonfermenters.

An indirect immunofluorescent assay to detect antibodies against the lipopolysaccharide (LPS) of Burkholderia pseudomallei and taxonomically closely related species was developed with the Luminex system. LPSs of Pseudomonas aeruginosa, Burkholderia cepacia, Burkholderia thailandensis, Burkholderia vietnamiensis, B. pseudomallei, and Burkholderia mallei were successfully conjugated to Luminex microspheres. Antibodies measured against the LPS of B. pseudomallei-conjugated Luminex beads only cross-reacted with those of two genetically closely related species, B. mallei and B. thailandensis (previously classified as non-pathogenic arabinose-negative B. pseudomallei). However, this system could distinguish other closely related species from B. pseudomallei. This assay is able to detect significantly high levels of anti-LPS antibodies of B. pseudomallei in serum from patients with culture-proven melioidosis.