Toll-like receptor homolog TOL-1 regulates Bifidobacterium infantis-elicited longevity and behavior in Caenorhabditis elegans.

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.

Structure of a sialo-oligosaccharide from glycophorin in carp red blood cell membranes.

We isolated a high-purity carp glycophorin from carp erythrocyte membranes and prepared the oligosaccharide fraction from glycophorin by ß-elimination [1]. The oligosaccharide fraction was separated into two components (P-1 and P-2) using a Glyco-Pak DEAE column. These O-linked oligosaccharides (P-1 and P-2) were composed of glucose, galactose, fucose, N-acetylgalactosamine and N-glycolylneuraminic acid (NeuGc). The P-1 and P-2 contained one and two NeuGc residues, respectively, and the P-1 exhibited bacteriostatic activity [1]. Using NMR and GC-MS, we determined that the structure of the bacteriostatic P-1 was NeuGcα2→6 (Fucα1→4) (Glcα1→3) Galß1→4GalNAc-ol. This O-linked oligosaccharide was unique for a vertebrate with respect to the hexosamine and hexose linkages and its non-chain structure.

Isolation and characterization of glycophorin from carp red blood cell membranes.

We isolated a high-purity carp glycophorin from carp erythrocyte membranes following extraction using the lithium diiodosalicylate (LIS)-phenol method and streptomycin treatment. The main carp glycophorin was observed to locate at the position of the carp and human band-3 proteins on an SDS-polyacrylamide gel. Only the N-glycolylneuraminic acid (NeuGc) form of sialic acid was detected in the carp glycophorin. The oligosaccharide fraction was separated into two components (P-1 and P-2) using a Glyco-Pak DEAE column. We observed bacteriostatic activity against five strains of bacteria, including two known fish pathogens. Fractions from the carp erythrocyte membrane, the glycophorin oligosaccharide and the P-1 also exhibited bacteriostatic activity; whereas the glycolipid fraction and the glycophorin fraction without sialic acid did not show the activity. The carp glycophorin molecules attach to the flagellum of V. anguillarum or the cell surface of M. luteus and inhibited bacterial growth.

Comparison of two possible routes of pathogen contamination of spinach leaves in a hydroponic cultivation system.

The route of pathogen contamination (from roots versus from leaves) of spinach leaves was investigated with a hydroponic cultivation system. Three major bacterial pathogens, Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes, were inoculated into the hydroponic solution, in which the spinach was grown to give concentrations of 106 and 10³ CFU/ml. In parallel, the pathogens were inoculated onto the growing leaf surface by pipetting, to give concentrations of 106 and 10³ CFU per leaf. Although contamination was observed at a high rate through the root system by the higher inoculum (106 CFU) for all the pathogens tested, the contamination was rare when the lower inoculum (10³ CFU) was applied. In contrast, contamination through the leaf occurred at a very low rate, even when the inoculum level was high. For all the pathogens tested in the present study, the probability of contamination was promoted through the roots and with higher inoculum levels. The probability of contamination was analyzed with logistic regression. The logistic regression model showed that the odds ratio of contamination from the roots versus from the leaves was 6.93, which suggested that the risk of contamination from the roots was 6.93 times higher than the risk of contamination from the leaves. In addition, the risk of contamination by L. monocytogenes was about 0.3 times that of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis and E. coli O157:H7. The results of the present study indicate that the principal route of pathogen contamination of growing spinach leaves in a hydroponic system is from the plant's roots, rather than from leaf contamination itself.

A survey of iceberg lettuce for the presence of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in Japan.

No information has been available on the prevalence of pathogens in fresh produce in Japan. In the present study, information was collected on the occurrence of contamination by Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in iceberg lettuce in a Japanese retail store. A total of 419 samples of lettuce that had been harvested in different districts and/or by different producers from July 2008 to March 2009 were examined. A multiplex PCR method was used to simultaneously identify the three bacterial pathogens. No pathogenic bacteria, including Salmonella, E. coli O157:H7, and L. monocytogenes, were detected from any of the samples with this highly sensitive and validated procedure. The aerobic bacteria plate counts and coliform bacteria counts in lettuce throughout the examination period did not show any seasonal trends, and the numbers were comparable to those reported by others from around the world. Based on the results of this study, we concluded that none of the three major pathogens were present in this limited survey of iceberg lettuce sold by a retailer in Japan.

Modeling of pathogen survival during simulated gastric digestion.

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.

[Analysis of factors affecting long-term administration of anti-methicillin resistant Staphylococcus aureus (MRSA) drugs].

In this study, we aimed to determine an index of anti-MRSA drugs for long-term treatment. We examined adult patients to whom the anti-MRSA drugs arbekacin sulfate (ABK), vancomycin hydrochloride (VCM), and teicoplanine (TEIC) had been administered in the St. Marianna University School of Medicine, Yokohama City Seibu Hospital, for 1, year. The number of patients treated for>or==14 days was 22 (31%) among 71 patients. Immunosuppressive agent positivity (p=0.07), albumin (ALB) level of or==10 mg/dl (p=0.01),%STAB >or==15% (p=0.11), and the period until the blood drug level is measured (p=0.06) were analyzed with respect to the differences between both groups by univariate analysis. An ALB level of

Isolation of Bacillus and Paenibacillus bacterial strains that produce large molecules of cyclic isomaltooligosaccharides.

Cyclic isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.

Use of mild-heat treatment following high-pressure processing to prevent recovery of pressure-injured Listeria monocytogenes in milk.

This study examined the inactivation of Listeria monocytogenes in milk by high-pressure processing (HPP) and bacterial recovery during storage after HPP. We developed a technique to inhibit the bacterial recovery during storage after HPP (550 MPa for 5 min) using a mild-heat treatment (30-50 degrees C). Various mild-heat treatments were conducted following HPP to investigate the condition on which the bacterial recovery was prevented. Immediately after HPP of 550 MPa at 25 degrees C for 5 min, no L. monocytogenes cells were detected in milk regardless of the inoculum levels (3, 5, and 7 log(10)CFU/ml). However, the number of L. monocytogenes cells increased by >8 log(10)CFU/ml regardless of the inoculum levels after 28 days of storage at 4 degrees C. Significant recovery was observed during storage at 25 degrees C; the bacterial number increased by >8 log(10)CFU/ml after 3 days of storage in the case of an initial inoculum level of 7 and 5 log(10)CFU/ml. Even in the case of an initial inoculum level of 3 log(10)CFU/ml, the bacterial number reached the level of 8 log(10)CFU/ml after 7 days of storage. No bacterial recovery was observed with storage at 37 degrees C for 28 days. Milk samples were treated by various mild-heat treatments (30-50 degrees C for 5-240 min) following HPP of 550 MPa at 25 degrees C for 5 min, and then stored at 25 degrees C for 70 days. The mild-heat treatment (e.g., 37 degrees C for 240 min or 50 degrees C for 10 min) inhibited the recovery of L. monocytogenes in milk after HPP. No recovery of L. monocytogenes in milk was observed during 70-day storage at 25 degrees C in samples that received mild-heat treatments such as mentioned above following HPP (550 MPa for 5 min). Moreover, the mild-heat treatment conditions (temperature and holding time) required to inhibit the recovery of L. monocytogenes in milk was modelled using a logistic regression procedure. The predicted interface of recovery/no recovery can be used to calculate the mild-heat treatment condition to control bacterial recovery during storage at 25 degrees C after HPP (550 MPa for 5 min). The results in this study would contribute to enhance the safety of high-pressure-processed milk.

Predictive modelling of the recovery of Listeria monocytogenes on sliced cooked ham after high pressure processing.

This study examined bacterial recovery on sliced cooked ham that was inoculated with Listeria monocytogenes, treated by high pressure processing (HPP) and then stored at 10 degrees C for 70 days. The number of L. monocytogenes on the ham inoculated with 5 log(10) CFU/g was initially reduced by HPP at 500 MPa for 10 min to below the detectable level (10 CFU/g). However, the bacterial count gradually increased during storage, and exceeded the initial inoculum level at the end of the 70-day period, having risen by 7-8 log(10) CFU/g. A novel predictive model was therefore developed to estimate the recovery of L. monocytogenes during storage after HPP. Recovery of L. monocytogenes was defined as the detection of >10(2) CFU/g bacteria, in view of the relevant food safety objectives of L. monocytogenes. At each 14-day sampling session, the ham was scored as either 1 or 0 indicating bacterial recovery or no bacterial recovery, respectively. The data were then subjected to a simple linear logistic regression model, which provided a good fit as indicated by the performance statistics. Using this model, we estimated the minimum HPP conditions necessary for the required storage periods. Additionally, as the developed model was based on logistic regression, the probability of the recovery of L. monocytogenes during storage after HPP was estimated. Our model not only calculated the appropriate shelf life and process conditions, but also provided a method for evaluating the risk of the recovery of pathogenic bacteria during storage.

A novel cyclic isomaltooligosaccharide (cycloisomaltodecaose, CI-10) produced by Bacillus circulans T-3040 displays remarkable inclusion ability compared with cyclodextrins.

Cyclodextrans (CIs) are cyclic isomaltooligosaccharides and only CI-7, CI-8, and CI-9 were known. CI-7, CI-8, and CI-9, consisting of seven, eight, and nine glucoses, respectively, bound by alpha-(1-->6) linkages, are known to be produced by T-3040 strain of Bacillus circulans. However, we have found, using 13C NMR and mass spectrometry, that this strain also produces CI-10, CI-11 and CI-12. These large CIs are very soluble in water and inhibit the glucan synthesis of glucansucrases to the same degree as do the smaller CIs. The CIs were thought to be poor at forming inclusion complexes with chemical compounds, due to their flexible alpha-(1-->6)-glucosidic structure. Among these six CIs, CI-10 was much better at forming an inclusion complex, and it ability to do so was as good as cyclodextrins, as determined by its ability to stabilize the color of Victoria blue B. Therefore, CI-10 may be the most commercially useful CI.

Cysteine synthase of an extremely thermophilic bacterium, Thermus thermophilus HB8.

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.