Salmonella contamination and hazard analysis in a storage facility for feed materials in Japan.

AIMS: To reduce Salmonella contamination of animal feeds, Salmonella contamination in a feed materials storage facility was investigated, and hazard analysis was performed to acquire the basic information necessary to establish a control method for Salmonella. The result is that managers of feed materials storage facilities will be able to introduce the Hazard Analysis and Critical Control Point (HACCP) systems to quickly and efficiently. METHODS AND RESULTS: A total of 472 samples, including deposits, adhesions, environment samples and materials, were investigated. The detection rate in the storage facility was 5.1% (24/472) over the three-year investigation period. The serovars of detected Salmonella were Agona, Anatum, Bron (or Agbeni), Chester, Infantis, Mbandaka, Minnesota, Muenster, Sinstorf, Typhimurium, O18:- and O1,3,19:-. As a result of hazard analysis after confirming the multiplication factors and detection rate, important hazards were judged to be the outdoor equipment and dust collector. CONCLUSIONS: The risk of Salmonella contamination in storage facilities was clarified. Important Salmonella hazards in storage facilities are Salmonella-contaminated feed materials, multiplication factors such as rainwater and dew condensation, and Salmonella-accumulated dust in dust collectors for fine-powder recovery. These results suggest that proper control of these hazards could reduce the risk of Salmonella in storage facilities. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing actual Salmonella contamination conditions and risk assessment using the hazard analysis at a feed materials storage facility in Japan. This study will consider implications for the feed industry because the hazard analysis clarified the sites of Salmonella contamination, the tendency of contamination and the points for implementing control measures in the feed materials storage facility.

[Behavior of Staphylococcus aureus and Staphylococcal Enterotoxins A and Q in Scrambled Eggs].

Staphylococcal food poisoning (SFP) is caused by Staphylococcus aureus, and its typical symptom of vomiting is evoked by staphylococcal enterotoxins (SEs). SEs are classified as classical and new types. SEQ is a new-type enterotoxin predicted to have a high potential risk for SFP. To elucidate the correlation between the number of S. aureus cells and the production of SEs as well as classical and new-type enterotoxins in the food environment, the numbers of S. aureus strain cells carrying sea and seq genes and the production of SEA and SEQ protein were examined under 3 pHs values (pH 6.0, 7.0 and 8.0) and 2 NaCl concentrations (0.5 and 1.0%) conditions. The experiments were performed at 25℃, resembling the setting of scrambled eggs at room temperature after cooking. By 24 hr after incubation, the cell number in the scrambled egg was ≥107/10 g, reaching 109/10 g by 48 hr under all conditions. The productions of both SEA and SEQ were detected in the scrambled egg under all conditions by 48 h. SEQ was detected from 24 hr at all 3 pH values in the egg containing 1.0% NaCl, whereas in the egg containing 0.5% NaCl, it was detected from 24 hr at pH 6.0 and from 48 hr at other pHs. The SEQ production was consistently 100-1,000 times less than that of SEA. These results suggest that the new-type enterotoxin SEQ has the potential to evoke symptoms related to SFP following the consumption of egg products cooked under relative lower pH and water activity.

Effects of acupuncture on nutritional state of patients with stable chronic obstructive pulmonary disease (COPD): re-analysis of COPD acupuncture trial, a randomized controlled trial.

BACKGROUND: There are an increasing number of evidences that chronic obstructive pulmonary disease (COPD) is a systemic illness and that bodyweight loss is its prominent manifestation. We focused on the nutritional outcomes to find out the effectiveness of acupuncture on nutritional state of COPD patients and on their prognosis in our previous interventional study. METHODS: The present study is re-analysis of our previous interventional study, COPD Acupuncture Trial (CAT) published in 2012. Data from CAT was re-analyzed in terms of nutritional status, inflammatory biomarkers, and prognostic index. Nutritional states were evaluated by the measurements of body weight, body composition, and muscle strength, and the nutritional hematological examination results (retinol-binding protein (RBP), prealbumin (PA), transferrin (Tf), and hemoglobin (Hb) in serum), and inflammation biomarkers such as carboxyhemoglobin (COHb), High sensitivity C-reactive protein (Hs-CRP), Tumor Necrosis Factor-alpha (TNF-α), Interleukin 6 (IL-6), and Serum Amyloid A (SAA) were measured. The BODE index was measured in terms of prognosis. These measurements were compared between the real acupuncture group (RAG) and the placebo acupuncture group (PAG). All data are presented as mean (SD) or mean (95% CI). The difference between baseline and final volumes was compared using analysis of covariance (ANCOVA). Moreover, correlations between nutritional hematological examination scores and inflammation biomarker parameters were assessed using Spearman's rank correlation coefficient. RESULTS: After 12 weeks, the change in body weight was significantly greater in the RAG compared with the PAG (mean [SD] difference from baseline: 2.5 [0.4] in RAG vs - 0.5 [1.4] in PAG; mean difference between the groups: 3.00, 95% CI, 2.00 to 4.00 with ANCOVA). Patients in RAG also had improvements in the results of nutritional hematological examination (RBP, PA, Tf, Hb), Inflammation biomarkers (TNF-α, IL-6, SAA, Hs-CRP, COHb) and the BODE index. CONCLUSION: This study demonstrated some clear evidences that acupuncture can be a useful adjunctive therapy to improve nutritional state of COPD patients. TRIAL REGISTRATION: UMIN Clinical Trials Registry ( UMIN000001277 ). Retrospectively registered.

Legionella thermalis sp. nov., isolated from hot spring water in Tokyo, Japan.

Strain L-47(T) of a novel bacterial species belonging to the genus Legionella was isolated from a sample of hot spring water from Tokyo, Japan. The 16S rRNA gene sequences (1477 bp) of this strain (accession number AB899895) had less than 95.0% identity with other Legionella species. The dominant fatty acids of strain L-47(T) were a15:0 (29.6%) and the major ubiquinone was Q-12 (71.1%). It had a guanine-plus-cytosine content of 41.5 mol%. The taxonomic description of Legionella thermalis sp. nov. is proposed to be type strain L-47(T) (JCM 30970(T) = KCTC 42799(T)).

A galactose-binding lectin isolated from Aplysia kurodai (sea hare) eggs inhibits streptolysin-induced hemolysis.

A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes α-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of d-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth.

Evaluation of Serotyping of Environmental and Clinical Isolates of Legionella pneumophila using MALDI-TOF MS.

Legionella pneumophila (L. pneumophila) is responsible for most Legionnaire's disease cases diagnosed worldwide. The species includes 16 serogroups, but most Legionnaire's disease cases (85.7% in Europe, 87.0% in Japan) are caused by L. pneumophila serogroup 1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify the L. pneumophila serogroup. In this study, we compared three sample preparation methods that are compatible with MALDI-TOF MS: the direct colony transfer method (DCTM), on-target extraction method (OTEM), and in-tube extraction method (ITEM). The aim was to improve the low identification rates for L. pneumophila, and establish and validate a simple, rapid and robust MALDI-TOF MS-based method for routine use in microbiological laboratories for assignment of L. pneumophila isolates to serogroups and identification of reliable peak biomarkers. Using ITEM, 100.0% (29/29) of hot spring water samples and clinical isolates were correctly identified at the species level. Augmented reference spectra correctly identified all 29 strains at the species level and 29 isolates at the serogroup level, displaying sensitivity, specificity and accuracy of 100.0% for serogroup assignment. MALDI-TOF MS is a relatively inexpensive method for assignment of L. pneumophila serogroups that can serve as a first-line tool for rapid prospective typing.

Association between subjective health status and frequency of visits to acupuncture clinic: A cross-sectional study.

OBJECTIVE: There are few studies on the relationship between the frequency of acupuncture use and subjective health status. Therefore, we investigated this relationship using data of a previously performed cross-sectional survey of patients visiting Japanese acupuncture clinics. METHODS: This study used data from a cross-sectional survey conducted in 2011 on patients visiting 180 acupuncture clinics nationwide that were run by members of the alumni association of Meiji University of Integrative Medicine Faculty of Acupuncture and Moxibustion, and did not provide treatment other than acupuncture. We calculated the frequency of visits to acupuncture clinics (< 24 times, 24-47 times, 48-95 times, or ≥ 96 times per year) and the Short Form-36 (SF-36) summary scores (physical, mental, role/social) based on the response of the questionnaire conducted at the time of the survey. Multiple linear regression analysis with multiple imputation was performed with three SF-36 summary scores as the dependent variables, and the frequency of visits to acupuncture clinics as the independent variable. RESULTS: The questionnaire was distributed to 2,379 outpatients of acupuncture clinics, 1,409 of whom met the criteria and were included in the analysis. More frequent visits to acupuncture clinics were associated with lower scores on all three SF-36 summary scores. Compared to those who visited < 24 times a year, those who visited ≥ 96 times a year had unstandardized regression coefficients (95% confidence interval) of -5.6 (-7.8 to -3.3) for the physical, -2.0 (-3.9 to -0.1) for the mental, and -2.9 (-5.4 to -0.4) for the role/social SF-36. CONCLUSIONS: Frequent visits to acupuncture clinics were associated with poor subjective health status, especially physical health.

Genotyping and chlorine-resistance of Methylobacterium aquaticum isolated from water samples in Japan.

For microbial ecological analysis, 14 strains of Methylobacterium aquaticum isolated from water samples were subjected to clustering analysis on the basis of ribotyping and RAPD-PCR tests. The ribopatterns after digestion with EcoRI obtained from 14 strains of M. aquaticum were used to divide the strains into two groups (Groups I and II) with a similarity of 55%. From the analysis of RAPD patterns using primer 208, the 14 strains were divided into 3 groups (A-C) based on a homology of 45% or greater, and from that using primer 272, there were 4 groups (A-D) based on a homology of 50% or greater. The chlorine resistance (99.9% CT values) of these isolates was also experimentally confirmed, and we attempted to define the connection between chlorine resistance and the geno-cluster. The average CT value of group I was 0.89 mg•min/l and the average of group II was 0.69 mg•min/l. No remarkable differences in the CT values for the groups were found.

Pilot Study of Acupuncture's Antispasmodic Effect on Upper Gastrointestinal Tract during Endoscopic Submucosal Dissection for Early Gastric Cancer: Controlled Clinical Trial.

A prospective study was conducted in patients with early-stage gastric cancer to determine the efficacy and safety of acupuncture stimulation as an antispasmodic compared with conventional medication during the procedure of endoscopic submucosal dissection (ESD) of the upper gastrointestinal tract. This study was a prospective single blinded quasi-randomized controlled trial. Seventy-three patients who were scheduled to undergo ESD for gastric cancer at Aizu Medical Center between 19 February 2016 and 30 June 2016 were assessed for eligibility for the study. Sixty out of 73 patients were included in the study and assigned into two intervention groups: medication group (MG) and acupuncture group (AG). Ease of the procedure was evaluated using modified NIWA classification (MNC) by endoscopist considering the frequency and amplitude of the upper gastrointestinal peristalsis. For the statistical analysis, Mann-Whitney test was used to compare the differences of MNC values (baseline and end of procedure) between two groups. The difference of MNC found in the AG (-2.00 (-3.0 to -2.0)) was significantly greater than that in the MG (-1.00 (-2.0 to -1.0), p < 0.0001, Mann-Whitney test). We consider that acupuncture to the abdomen could be an alternative antispasmodic method during upper gastrointestinal endoscopic procedure.

Identification of Legionella londiniensis isolated from hot spring water samples in Shizuoka, Japan, and cytotoxicity of isolates.

As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water and were able to isolate L. londiniensis HYKF-90505 (=JCM 16338), confirming that L. londiniensis inhabits hot spring water in Japan. To investigate the disease potential of L. londiniensis, we examined its ability to grow intracellularly within Acanthamoeba sp. JAC/E1 strain. The isolated HYKF-90505 was able to grow within Acanthamoeba sp. JAC/E1 strain, and we confirmed also that the HYKF-90505 strain showed cytotoxicity for cultured cells such as J774.1 (JCRB0018). However, in a culture of human U937 cells, the bacterial count was not increased by the intracellular growth of the HYKF-90505 strain. Cells infected for 24 h and stained using the Giménez method showed no intracellular growth of the HYKF-90505 strain. Thus, the isolate appears to be weakly pathogenic to humans.

Public status and prevalence of acupuncture in Japan.

Acupuncture originated in China and is widespread throughout Asia. It is expected that a higher utilization of this remedy exists in these countries compared to Western countries. We conducted annual nationwide surveys from 2003 through 2006 on the utilization of acupuncture in Japan. Face-to-face interviews were conducted with 2000 individuals randomly chosen from the resident database. Annual utilization percentages, based on the number of respondents, from 2003 to 2006 were 6.5%, 4.8%, 6.4%, and 6.7%, respectively, while lifetime experiences determined in each surveys were estimated as 26.7, 19.4, 24.4 and 25.4, respectively. Respondents who had utilized acupuncture and/or moxibustion tended to be older than those who had no experience. Acupuncture was mainly used for musculoskeletal symptoms, and a detailed breakdown of the musculoskeletal symptoms identified in the 2005 survey showed 50.9% for low back pain, 35.9% for shoulder stiffness and 12.0% for knee pain. Reasons given for continuing therapy included the effective amelioration of symptoms, comfort of the procedure and low number of side-effects, while those who decided against continuing cited no improvement of symptoms, cost and lack of time for treatment. In conclusion, annual utilization of acupuncture and/or moxibustion was estimated at more than 6%, and the percentage of those with a lifetime experience was ∼25%, thus demonstrating the relatively higher utilization of the remedy in Japan over utilization in western countries. Application of the treatment for musculoskeletal problems and utilization by the older population were specific standouts of the use of acupuncture and/or moxibustion in Japan.

Bacterial contamination of stock solutions in storage cases for contact lens, and the disinfectant-resistance of isolates.

To investigate the bacterial contamination of preservative solutions for contact lenses, contact lens cases were swabbed and the swabs were cultured. Various bacteria were isolated from 26 of 100 samples (26.0%). By preservative solution, the bacterial presence was low (16.7%) in the 30 samples from cases using ReNu, which was the most frequently employed solution, and 27.3% in 11 samples from cases using Complete, which was the second most frequently employed solution. Of 34 strains isolated and identified, gram-negative bacilli-glucose non-fermenting bacteria were the most frequently isolated, accounting for 61.8% (21 strains); particularly, 10 isolates were Stenotrophomonas maltophilia, accounting for 29.4%. The biofilm-forming ability of these isolates was investigated by staining. The mean absorbance of the gram-negative rods was 0.369, which was about 3 times higher than that (0.107) of the Staphylococcus group, confirming marked biofilm-forming ability. In addition, the bactericidal effects of the preservative solutions on the isolates were investigated. The effect varied among the preservative solutions in the suspension experiment, but the highest disinfection rate, which was achieved by Complete, was 99.9->99.99%, showing a favorable bactericidal effect. In contrast, none of the 3 test preservative solutions showed any bactericidal effect in the adhesion experiment.

Characterization of heterotrophic bacteria isolated from the biofilm of a kitchen sink.

Heterotrophic bacteria constituting the biofilm produced in a kitchen sink drain were analyzed, and the biofilm formation abilities and the hydrophobicity of the cell surface layer were measured for the isolates. When the biofilm sample was cultured at 36 degrees C and 25 degrees C for 7 days, there were about 10 times more colonies on oligotrophic R2A agar medium than on eutrophic BHI agar medium. From isolates from the biofilm sample, 13 bacterial species were detected. To examine the biofilm formation ability of these strains, we measured the absorbance (OD570) by crystal violet staining. The absorbance of Brevibacterium casei 7-R-36-1 was the highest (3.029). In the comparison of the absorbance values between genera, Brevibacterium spp. (4 strains) showed the highest absorbance (mean: 2.056), followed by K. pneumoniae (4 strains) with a mean of 1.111. Regarding the hydrophobicity of the isolates, the values ranged from 0.002 for P. nitroreducens (strain 1-B-36-2) to 0.096 for M. lacticum (strain 5-R-25-2). The hydrophobicity values were generally low, and the cell surface layer of all tested strains was highly hydrophilic. The diversity of species of bacteria in the biofilm sample produced in the kitchen sink drain was recognized, and all the isolates had biofilm formation abilities.

The sensitivity of commercial kits in detecting the genes of pathogenic bacteria in venison.

The expansion of the wild deer population is a major problem for the Japanese farm and forestry industries because their damage to farm products and vegetation results in huge economic loss. To promote game meat consumption, hygiene inspections should be performed to detect main bacterial pathogens before products are shipped. In this study, we aimed to evaluate the ability of commercial test kits to genetically detect EHEC, Salmonella and Listeria monocytogenes in venison. Our results demonstrated that the kits for three pathogens could be useful for venison as well as other domestic meat products. Our comparative study showed that the LAMP kits were more sensitive than the RT-qPCR kits in the detection of all of these pathogens.

Relief of low back pain immediately after acupuncture treatment--a randomised, placebo controlled trial.

BACKGROUND: The purpose of this study was to examine the immediate effect of single acupuncture stimulation to the most painful point in patients with low back pain. METHOD: A randomised, evaluator-blinded, sham controlled clinical trial was conducted in which 31 patients with low back pain were randomly allocated to either an acupuncture group (n = 15) or a sham acupuncture group (n = 16). Both acupuncture and sham acupuncture were performed at the most painful point on the lower back of the subjects. For the acupuncture group, a stainless steel needle was inserted to a depth of 20 mm and manually stimulated (sparrow pecking method) for 20 seconds, while for the sham treatment a guide tube without a needle was placed at the point and tapped on the skin. Changes in low back pain were evaluated with a visual analogue scale (VAS) and the Schober test. Participants were also asked if they felt the needling sensation or not. The therapy and the evaluation were independently performed by two different acupuncturists. RESULTS: VAS score and the Schober test score showed significant improvement after treatment as compared with the sham group (P = 0.02, 0.001, respectively). There were no significant differences in the needling sensation between the acupuncture and sham group. CONCLUSION: These results suggest that acupuncture at the most painful point gives immediate relief of low back pain.

[Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo].

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.

Isolation, Identification and Antibacterial Susceptibility of Staphylococcus spp. Associated with the Mobile Phones of University Students.

From May 2014 to February 2015, 319 university students (male, n=173; female n=146) of 18 to 24 years of age who carried mobile phones or computer tablets were selected as subjects. Staphylococcus spp. were detected in 101 of 319 samples (31.7%). In the present study, 11 strains of S. aureus were isolated and identified, not all of which were methicillin-resistant Staphylococcus aureus (MRSA). Overall, 14 species were identified, with 11 strains (10.9%) of S. xylosus being isolated at the highest frequency. Following this were eight strains (7.9%) of S. cohnii and seven strains (6.9%) each of S. capitis and S. haemolyticus. Staphylococcus spp. isolation was performed with bacterial samples obtained from the mobile phones of 22 specific subjects (males, n=12; females, n=10). Staphylococcus spp. isolation was performed on days -1, 7 and 30 of the experiment. Staphylococcus spp. were positively detected one or more times in 12 subjects (54.5%). In one subject (8.3%), all three tests were positive. Furthermore, two tests were positive in three (25.0%). In the eight remaining subjects (66.7%) Staphylococcus spp. were detected only once. For the three abovementioned tests, we investigated the pulsed-field gel electrophoresis (PFGE) patterns of the strains derived from the mobile phone and from the fingers of three subjects in whom the same bacterial species were isolated twice. From the cases with similarities between strains derived from the fingers and the mobile phones and cases, with consistency in the strains derived from the mobile phone at different times, commonality was observed in the strains derived from the fingers and mobile phones along with chronological uniformity in the strains derived from the mobile phones. A total of 101 Staphylococcus spp. strains were isolated from mobile phones. According to drug susceptibility tests, 99 strains (98.0%) were found to have some degree of resistance to drugs (excluding one strain each of S. aureus and S. haemolyticus). Among these, the strain that showed the highest level of drug resistance was one strain (1.0%) of Staphylococcus spp., which showed resistance to nine drugs. The strain that showed the second highest level of drug resistance was one strain (1.0%) of S. caprea, which showed resistance to seven drugs. In this manner, the drug-resistant tendencies of Staphylococcus spp. isolated from mobile phones were observed.

[Investigation of Coxiella burnetii contamination in commercial milk and PCR method for the detection of C. burnetii in egg].

A total of 244 milk samples collected from supermarkets in Tokyo were examined for contamination with Coxiella burnetii. C. burnetii DNA was detected in 131 (53.7%) of the samples by nested PCR. PCR-positive samples were injected into immunosuppressed A/J strain mice. Of the 22 PCR-positive milk samples tested, none resulted in isolation of C. burnetii from the mice. Heat-treatment was sufficient to inactivate C. burnetii in commercial milk. In addition, a PCR detection method for C. burnetii in chicken egg was developed. Egg yolk was added to an equal volume of 1 mol/L of NaCl phosphate buffer and homogenized for removal of protein and lipid. After centrifugal separation, the supernatant was removed, and template DNA in the precipitate was extracted using SDS, proteinase K and NaI. Using such prepared samples, 3.2 x 10(1) C. burnetii particles in 1 g of egg yolk could be detected by nested PCR. All of 200 chicken egg samples collected from supermarkets in Tokyo were negative for C. burnetii by the nested PCR method.

Bacterial contamination in cold water samples obtained from water dispensers.

We carried out a basic study in order to evaluate the bacterial contamination in water dispensers. Water samples were obtained from water dispensers from October 2012 to November 2013, and standard plate counts (at 36˚C, 24 h) of the samples, as well as heterotrophic plate counts (at 25˚C, 7 d), were estimated with the standard methods for the examination of drinking water in Japan. Standard plate counts exceeding the water-quality standard (1.0×10(2) CFU/ml) were observed in 42 of the 140 samples (30.0%), with a maximum detected bacterial count of 2.1×10(5) CFU/ml. The rate of the standard plate counts exceeding the water quality standard tended to be higher when using a one-way type method or water dispensers with natural water. Ralstonia spp. was most commonly isolated, and Pseudomonas aeruginosa was isolated in a few cases. Some opportunistic pathogens were also isolated, suggesting that we should be more concerned about bacterial contamination in cold water supplied from water dispensers.