Inactivation of human norovirus by chlorous acid water, a novel chlorine-based disinfectant.

INTRODUCTION: Human norovirus (HuNoV) is a leading cause of infectious gastroenteritis. Since HuNoV shows resistance to alcohol, chlorine-based sanitizers are applied to decontaminate the virus on environmental surfaces. Chlorous acid water (CA) has been recently approved as a novel chlorine-based disinfectant categorized as a Type 2 OTC medicine in Japan. In this study, we aimed to evaluate the capability of CA to inactivate HuNoV. METHODS: HuNoV (genogroups GII.2 and GII.4) was exposed to the test disinfectants including CA and sodium hypochlorite (NaClO), and the residual RNA copy was measured by reverse transcription quantitative PCR (RT-qPCR) after pretreatment with RNase. In addition, the log10 reduction of HuNoV RNA copy number by CA and NaClO was compared in the presence of bovine serum albumin (BSA), sheep red blood cells (SRBC), polypeptone, meat extract or amino acids to evaluate the stability of these disinfectants under organic-matter-rich conditions. RESULTS: In the absence of organic substances, CA with 200 ppm free available chlorine provided >3.0 log10 reduction in the HuNoV RNA copy number within 5 min. Even under high organic matter load (0.3% each of BSA and SRBC or 0.5% polypeptone), 200 ppm CA achieved >3.0 log10 reduction in HuNoV RNA copy number while less than 1.0 log10 reduction was observed with 1,000 ppm sodium hypochlorite (NaClO) in the presence of 0.5% polypeptone. CA reacted with only cysteine, histidine and glutathione while NaClO reacted with all of the amino acids tested. CONCLUSIONS: CA is an effective disinfectant to inactivate HuNoV under organic-matter-rich conditions.

Anti-tumor activity of heat-killed Lactobacillus plantarum BF-LP284 on Meth-A tumor cells in BALB/c mice.

Probiotics exert numerous effects on human well-being. Here, heat-killed Lactobacillus plantarum BF-LP284 (H-Lp) was isolated as a potent immuno-modulator among 15 strains of lactobacilli in terms of TNF-α induction ability in peritoneal macrophages. In vitro TNF-α and IFN-γ induction in Peyer's patch (PP) cells was higher when incubated with H-Lp than with live L. plantarum BF-LP284 (L-Lp). Suppression of syngeneic Meth-A tumors in a murine model by oral administration of H-Lp was also greater than that of L-Lp and of controls. H-Lp stimulated IFN-γ production in spleen cells, which displayed inhibited tumor growth in Winn assays when treated with H-Lp. Moreover, H-Lp increased the ratio of CD3(+ )cells among peripheral blood mononuclear cells in Meth-A tumor-bearing mice, suggesting an H-Lp-mediated anti-tumor mechanism whereby immune cells that are activated by H-Lp in PP and acquire anti-tumor activity in the spleen migrate to tumor sites through lymphocyte homing to inhibit tumor growth.

Amounts and species of probiotic lactic acid bacteria affect stimulation of short-chain fatty acid production in fecal batch culture.

The effects of lactate and probiotic lactic acid bacteria (LAB) on intestinal fermentation were analyzed using a fecal batch culture. Lactate was efficiently metabolized to butyrate and propionate by butyrate-utilizing bacteria in fecal fermentation. Probiotic LAB could stimulate butyrate and propionate production through their lactate production in fecal fermentation. It was considered that 109 cfu/g or more of probiotic LAB would be required to stimulate butyrate and propionate production in the large intestine. Due to the low production of lactate, a larger number of heterofermentative LAB than homofermentative LAB would be required for this stimulation.

Effects of non-fermented and fermented soybean milk intake on faecal microbiota and faecal metabolites in humans.

The effects of non-fermented soybean milk (NFSM) and fermented soybean milk (FSM) intake on the faecal microbiota and metabolic activities in 10 healthy volunteers were investigated. Soybean oligosaccharides, raffinose and stachyose were utilized by bifidobacteria except for Bifidobacterium bifidum, but most strains of Escherichia coli and Clostridium perfringens could not use them. During the dietary administration of FSM, the number of bifidobacteria and lactobacilli in the faeces increased (p < 0.05), and clostridia decreased (p < 0.05). Moreover, the concentrations of faecal sulphide were decreased (p < 0.01) in the intake of FSM. During the dietary administration of NFSM, the number of bifidobacteria tended to increase. These results indicate that the consumption of soybean milk, especially FSM, is related to improvement of the intestinal environment.

Effect of apple intake on fecal microbiota and metabolites in humans.

The effects of apple intake on the fecal flora, water content, pH, and metabolic activities in eight healthy volunteers and the utilization of apple pectin in vitro were investigated. Although several isolates of Bifidobacterium, Lactobacillus, Enterococcus, and the Bacteroides fragilis group utilized apple pectin, most isolates of Escherichia coli, Collinsela aerofaciense, Eubacterium limosum, and Clostridium perfringens could not. When fecal samples from healthy adults were incubated in liquid broth with apple pectin present or absent, the numbers of Bifidobacterium and Lactobacillus in the former were higher than those in the later. After the intake of apples (2 apples a day for 2 weeks) by eight healthy adult humans, the number of bifidobacteria in feces increased (p < 0.05 on day 7 and p < 0.01 on day 14 of the intake period), and the numbers of Lactobacillus and Streptococcus including Enterococcus tended to increase. However, lecithinase-positive clostridia, including C. perfringens, decreased (p < 0.05), and Enterobacteriaceae and Pseudomonas tended to decrease. Moreover, the concentrations of fecal acetic acid tended to increase on apple intake. The fecal ammonia concentration showed a tendency to reduce and fecal sulfide decreased (p < 0.05) on apple intake. These findings indicate that apple consumption is related to an improved intestinal environment, and apple pectin is one of the effective apple components improving the fecal environment.

Colonization of segmented filamentous bacteria and its interaction with the luminal IgA level in conventional mice.

Segmented filamentous bacteria (SFB) colonize in the ileum. They promote the development of intraepithelial lymphocytes and immunoglobulin A (IgA)-producing cells in the small intestine. In SFB-monoassociated mice, changes in SFB colonization of the small intestine were related to the level of IgA derived from maternal milk during the suckling period and self-produced in the small intestine after weaning. In this study, we investigated whether or not maternal and neonatal IgA influence the colonization of SFB in conventional mice from 18 to 105 days old. The pups were forcedly weaned at 20 days old. SFB could be detected in the distal small intestine after day 22, and their number rapidly reached a maximum on day 28. Thereafter, they gradually declined to one-fourth of the maximum level. The lowest concentrations of IgA in the small intestinal and cecal contents were detected on day 22. Thereafter, they increased as the age of the mice increased. The expression of the polymeric immunoglobulin receptor gene in the distal small intestine increased after weaning. These results suggested that the colonization of SFB in the pre-weaning and post-weaning periods might be prevented with IgA derived from maternal milk and self-produced IgA, respectively.

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability.

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.

Analysis of Clostridium cluster XI bacteria in human feces.

Six species and one group of Clostridium cluster XI, Clostridium sordellii, Clostridium bifermentans, Clostridium difficile, Clostridium hiranonis, Intestinibacter bartlettii, and Romboutsia lituseburensis and the Terrisporobacter glycolicus group, respectively, in human feces collected from 18 healthy adults were analyzed with real-time PCR. Although individual differences were recognized, the predominant colonization of C. sordellii and I. bartlettii in the human large intestine was identified.

Detection of antibiotic resistance genes in the feces of young adult Japanese.

Antibiotic resistance genes in the feces of healthy young adult Japanese were analyzed with polymerase chain reaction using specific primers. Antibiotic resistance genes against macrolides (ermB, ermF, ermX, and mefA/E), tetracyclines (tetW, tetQ, tetO, and tetX), ß-lactam antibiotics (blaTEM ), and streptomycin (aadE) were detected in more than 50% of subjects. These antibiotic resistance genes are likely widespread in the large intestinal bacteria of young adult Japanese.

The effect of soymilk intake on the fecal microbiota, particularly Bifidobacterium species, and intestinal environment of healthy adults: a pilot study.

The influence of soymilk on the fecal microbiota, particularly Bifidobacterium species, and metabolic activities were investigated in eight healthy adult humans. During the soymilk intake period, the number of bifidobacteria in feces was significantly higher (p<0.05) on day 14 of the soymilk intake period than before the intake period, whereas that of Enterobacteriaceae was significantly lower (p<0.05) on days 7 and 14 of the soymilk intake period than before the intake period. In an investigation of Bifidobacterium at the species or group level, the numbers of all species and groups studied slightly increased during the soymilk intake period. These results show that the intake of soymilk may contribute to improving the intestinal environment.

Transition of the intestinal microbiota of cats with age.

The transition of intestinal microbiota with age has been well described in humans. However, the age-related changes in intestinal microbiota of cats have not been well studied. In the present study, we investigated the composition of intestinal microbiota of cats in 5 different age groups (pre-weanling, weanling, young, aged, senile) with a culture-based method. For lactobacilli and bifidobacteria, we also quantified with molecular-based method, real-time PCR. The results suggested that the composition of the feline intestinal microbiota changes with age, while the changes were different from those of humans and dogs. Bifidobacteria which are predominant in human intestine or lactobacilli which are predominant in dog intestine, did not appear to be important in cat intestines. Enterococci, instead, seem to be major lactic acid producing bacteria in cats. We also identified lactobacilli and bifidobacteria at the species level based on 16S rRNA gene sequences and found that the species composition of Lactobacillus also changed with age.

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods.

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.

Evaluation of sorbitol-salicin MacConkey medium containing cefixime and tellurite (CT-SSMAC medium) for isolation of Escherichia coli O157:H7 from raw vegetables.

The utility of CT-SSMAC medium (sorbitol-salicin MacConkey medium containing cefixime and tellurite) for the isolation of Escherichia coli O157:H7 from raw vegetables was investigated. The colonies of all E. coli O157:H7 and O157:NM strains tested were colorless and beta-galactosidase-positive on CT-SSMAC medium. Furthermore, the number of colorless colonies on the CT-SSMAC medium was less than that on the sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) from several raw vegetable samples. All colorless colonies grown on CT-SSMAC medium from raw vegetable samples were beta-galactosidase-negative. These findings suggest that the CT-SSMAC medium is useful for the isolation of E. coli O157:H7 from raw vegetable samples.

Characterization of Campylobacter lanienae from pig feces.

To isolate Campylobacter spp., the feces of healthy cattle, pigs, and broilers were examined between June 1999 and January 2000. Campylobacter lanienae strains were isolated from the feces of healthy pigs, but not from the feces of cattle or broilers. In six C. lanienae isolates, there was only 21-38% DNA-DNA homology to Campylobacter hyointestinalis subsp. lawsonii strain NCTC 12901. Thus, the primary host of C. lanienae is likely to be the pig and C. lanienae appears to be a species distinct from C. hyointestinalis subsp. lawsonii. In addition, an intervening sequence of 226 bp in the 16S rRNA gene was found in four isolates.

[Presence of the genes regarding adherence factors of Escherichia coli isolates and a consideration of the procedure for detection of diarrheagenic strain].

Diarrheagenic Escherichia coli are differentiated from non-pathogenic members with enterotoxin production, enteroinvasiveness and serotyping. However, the serotypic members are rarely sufficient to reliably identify a strain as diarrheagenic on E. coli. Recently, there are many definite articles which the adhesive E. coli strain against intestinal epithelial cells is enterovirulent. In this study, 1,748 E. coli isolates of diarrheagenic and non-diarrheagenic categories which belonged to EHEC, ETEC, EIEC EPEC and non-EPEC were examinated by PCR method for the presence of eaeA, aggR and bfpA regarding adherence factor genes, and astA of EAST1. The strains examined were recognized to variable carrying geno-patterns, and a large number of EHEC, EPEC and non-EPEC had carried either eaeA or aggR genes. In EHEC isolates, a carrying pattern with the most high frequency was only eaeA, and this type was recognized in the isolates of serotype O157, O26 and O111. EPEC and non-EPEC isolates were recognized eaeA or aggR which harboring with astA or not. Of 508 EPEC isolates from human, a total of 137 isolates (27.0%) carried aggR, and a total of 74 isolates (14.6%) had eaeA, while of the 91 isolates from non-human were recognized aggR and eaeA with 2.2% (2 isolates) and 12.1% (11 isolates), respectively. Also, of 266 non-EPEC isolates from human, a total of 16 isolates (6.0%) carried aggR, and a total of 58 isolates (21.8%) had eaeA. On the other hand, 22 (7.0%) of 316 isolates examined from non-human had eaeA, however no isolate had aggR. Thirteen isolates of EIEC and 218 ETEC isolates were screened, and only 6 ETEC isolates had either eaeA or aggR. The astA gene was recognized in the isolates of all categories, and ETEC strains had more frequently. The bfpA gene was recognized with more frequently in a serotype O157: H45, which is obtained from human with diarrhea, however, this strain was not recognized a member of the EPEC serotype. There is no diagnostic system for the strain of E. coli that cause diarrheal diseases, therefore more laboratories are unable to identify them. The authors had confirmed which PCR technique is a useful simple and rapid method for the detection of adherence factor genes on E. coli strains. From the these results, we showed a differentiation method using PCR technique which have relation with adherence factor, enterotoxin-production and invasiveness, and we firmly believe that application of the procedure is a reasonable and useful method for the identification of diarrheagenic E. coli.

[Influence of the conditions of storage and cooking on growth, invasion and survival of Salmonella enteritidis in eggs].

Basal studies for the confirmation of sanitary rules in the kitchen were performed, focusing on preventing an outbreak of food poisoning due to eggs contaminated with Salmonella Enteritidis (SE), using hen and quail eggs. SE did not grow at 5 degrees C but grew markedly at 25 degrees C in eggs. The invasion and growth of SE were marked under very humid conditions regardless of whether the eggshell was damaged. The invasion of SE into egg also occurred when eggs were taken in and out of the refrigerator. Moreover, SE was spread immediately to all non-contaminated eggs when SE-contaminated eggs were cracked into a bowl with non-contaminated eggs. In homemade mayonnaise containing 15% vinegar, sterilization took several hours to occur. On a stainless-steel bowl, SE survived for 2 weeks or more. These findings suggest that it is necessary to pay attention to secondary contamination.

[Comparison of intestinal bacteria composition identified by various analytical methods].

Many different kinds of bacteria are normally found in the intestines of healthy humans and animals. To study the ecology and function of these intestinal bacteria, the culture method was fundamental until recent years, and suitable agar plates such as non-selective agar plates and several selective agar plates have been developed. Furthermore, the roll-tube, glove box, and plate-in-bottle methods have also been developed for the cultivation of fastidious anaerobes that predominantly colonize the intestine. Until recently, the evaluation of functional foods such as pre- and probiotics was mainly done using culture methods, and many valuable data were produced. On the other hand, genomic analysis such as the fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), clone-library, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), terminal-restriction fragment length polymorphism (T-RFLP) methods, and metagenome analysis have been used for the investigation of intestinal microbiota in recent years. The identification of bacteria is done by investigation of the phenotypic characteristics in culture methods, while rRNA genes are used as targets in genomic analysis. Here, I compare the fecal bacteria identified by various analytical methods.

Effect of nisin (Nisaplin) on the growth of Listeria monocytogenes in Karashi-mentaiko (Red-pepper Seasoned Cod Roe).

The influence of Nisaplin, which contains 2.5% nisin, on the growth of Listeria monocytogenes in Karashi-mentaiko (red-pepper seasoned cod roe) was investigated. The MICs of Nisaplin for L. monocytogenes (10(8) CFU/mL) were measured; seven isolates showed a value of 1,600 microg/mL and one isolate showed a value of 800 microg/mL. All L. monocytogenes isolates had a MIC of 800 microg/mL at 10(6) CFU/mL. The number of L. monocytogenes in Karashi-mentaiko stored at 4 degrees C was decreased by Nisaplin added at 60 and 600 microg/g. These results indicated that Nisaplin effectively inhibits the growth of L. monocytogenes in Karashi-mentaiko.

A taxonomic study on erysipelothrix by DNA-DNA hybridization experiments with numerous strains isolated from extensive origins.

The purpose of this study was to clarify the taxonomic relationship between all the serovars and species of the genus Erysipelothrix by performing DNA-DNA hybridization experiments, the customary criterion for separation of bacterial genospecies. A total of 93 strains were isolated from a wide variety of sources, including pigs affected with acute or chronic erysipelas, other diseased animals, healthy animals, fish, retail meats, and environmental materials from throughout the world during the period 1958 to 1996. The present data on phenotypic characterization and DNA relatedness values demonstrate that 24 strains (96%) of E. tonsillarum are avirulent for swine, whereas 39 strains (66%) of genomic E. rhusiopathiae induced generalized or local urticarial lesion in swine after intradermal inoculation. This observation suggests that genomic E. tonsillarum has little etiological significance. Three minor groups contained several strains which exhibited minimal association with each type strain of E. rhusiopathiae and E. tonsillarum. In conclusion, it was confirmed that members of the E. rhusiopathiae and E. tonsillarum groups resemble each other in regard to many phenotypic characteristics, but differ in their ability to produce acid from saccharose and in their pathogenicity for swine. The genus Erysipelothrix certainly contains two main species: E. rhusiopathiae and E. tonsillarum.

Lactobacillus apodemi sp. nov., a tannase-producing species isolated from wild mouse faeces.

A Gram-positive, rod-shaped, non-endospore-forming bacterium, strain ASB1(T), able to degrade tannin, was isolated from faeces of the Japanese large wood mouse, Apodemus speciosus. Comparative analysis of the 16S rRNA gene sequence revealed that the strain could be assigned as a member of the genus Lactobacillus. The nearest phylogenetic neighbours were determined as Lactobacillus animalis DSM 20602(T) (98.9 % 16S rRNA gene sequence similarity) and Lactobacillus murinus ASF 361 (98.9 %). Subsequent polyphasic analysis, including automated ribotyping and DNA-DNA hybridization experiments, confirmed that the isolate represents a novel species, for which the name Lactobacillus apodemi sp. nov. is proposed. The DNA G+C content of the novel strain is 38.5 mol%. The cell-wall peptidoglycan is of type A4alpha L-lys-D-asp. The type strain is ASB1(T) (=DSM 16634(T)=CIP 108913(T)).