Search

Group A streptococcus is a bacterium that resides in the throat and skin and causes respiratory infection and occasionally glomerulonephritis and rheumatic fever. Streptolysin O (SLO) produced by Streptococcus pyogenes (S. pyogenes) binds to the cell membrane, particularly to that of white and red blood cells, and is toxic to the cells and tissue. In this study, we evaluated the inhibitory activity of water-soluble polyphenols in olives (Olea europaea) against SLO-induced hemolysis. Hydroxytyrosol inhibited SLO-induced hemolytic activity, and the amount required for 50% inhibition of hemolysis was 1.30 µg. These findings suggest that the water-soluble polyphenols contained in olives have inhibitory activity against SLO-induced hemolysis.

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.

Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC90) showed the strongest tolerance to ampicillin (ABPC) at a concentration of > 128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively. Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant to ABPC and KM, ABPC and SM, and ABPC and FOM, respectively. In addition, 128 strains were resistant to the single antibiotic of ABPC alone. Of the 140 strains demonstrating antibiotic resistance, 137 (97.9%) retained the conjugative R-plasmid transfer factor, excluding three strains of S. marcescens. All transfer factors were ABPC and retained by a high proportion of the bacterial groups, with one strain (100%) being resistant to three antibiotics, nine (81.8%) of the 11 strains being resistant to two antibiotics, and 127 (99.2%) of the 128 strains being resistant to a single antibiotic. In addition, we examined ESBL productivity in the 140 strains of bacteria demonstrating drug tolerance; however, no strains exhibited this characteristic. Therefore, further observation is required to ascertain trends in antibiotic-tolerant bacteria.

For a microbial ecological analysis, 20 strains of Legionella pneumophila isolated from both unchlorinated Noyu (unattended natural hot spring) samples and chlorinated hot spring bath water samples collected throughout Japan were subjected to a clustering analysis on the basis of a Pulsed-Field Gel Electrophoresis (PFGE) pattern analysis. The PFGE patterns obtained from 19 strains of L. pneumophila after digestion with SfiI were used to divide the strains into two groups (Groups A and B), although the similarity level was very low among the groups. Group A consisted of 8 strains, and all of these strains were isolated from hot spring bath water samples. Group B consisted of 11 strains, and all but two of these strains were isolated from Noyu samples. The chlorine resistance (99.9% CT values) of these isolates was experimentally confirmed, and we attempted to define the relationship between chlorine resistance and the geno-cluster. The average CT value of group A (8 strains from hot spring bath water) was 0.49 mg･min/l and the average of group B (9 strains from Noyu samples) was 0.29 mg･min/l. No remarkable differences in the CT values for the groups were found. A chlorine-sensitive Noyu strain (0.14 mg･min/l) and a chlorine-resistant strain (0.62 mg･min/l) from hot spring bath water were then compared to identify any differences in their lipid composition. There was no notable difference in the ratio of saturated to unsaturated fatty acids between the chlorine-sensitive and chlorine-resistant bacteria. However, the chlorine-sensitive and chlorine-resistant bacteria demonstrated differences in the relative percentages of cell wall and cell membrane fatty acids.

Strain K-20(T), a Gram-negative, nonmotile, nonspore-forming and strictly aerobic coccobacillus, which produces a pale pink pigment (R2A agar medium, 30℃, seven days) was isolated from a sample of biofilm obtained from a cooling tower in Tokyo, Japan. A phylogenetic analysis of the 16S rRNA partial gene sequences (1,439 bp) showed that the strain (accession number: AB297501) was related to Roseomonas frigidaquae CW67(T) and Roseomonas stagni HS-69(T) with 97.4% and 96.9% sequence similarity, respectively. Strain K-20(T) formed a distinct cluster with Roseomonas frigidaquae CW67(T) in the phylogenetic tree at a high bootstrap value (93%); however, distance was recognized between the strains. In addition, the DNA-DNA hybridization level between strain K-20(T) and Roseomonas frigidaquae JCM 15073(T) was 33%. The taxonomic data indicate that K-20(T) (=JCM 14634(T) =KCTC 32152(T)) should be classified in the genus Roseomonas as the type strain of a novel species, Roseomonas tokyonensis sp. nov.

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.

In August, 2010, strain HYMO-6 was isolated from a sample of hot spring water in Aomori, Japan. The 16S rDNA sequences (1,496bp) of this strain (accession number: AB597175) had a similarity of less than 96.6% to other Legionella species, prompting us to hypothesize that this strain might be a novel species belonging to the genus Legionella. However, in March of 2011, it was became clear that the HYMO-6 strain (=JCM 17450 =KCTC 23560 =DSM 24727) was Legionella nagasakiensis CDC-1796-JAP-E(T) (=ATCC BAA-1557(T) =JCM 15315(T)). When this strain was cultured on BCYEα agar at 36°C for 7 d, no long cells were observed. The dominant fatty acids of strain HYMO-6 were 16:1ω7c (32.4%), and the DNA G+C content was 42.0 mol%.

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.

Forty-five strains of Pseudomonas aeruginosa isolated from cockroaches captured in hospitals were investigated with regard to their biofilm-forming ability and resistance to various disinfectants in various cellular states. The hydrophobicity value varied among the test strains from 0.001 to 0.241, and the mean +/- standard deviation (SD) was 0.101 +/- 0.074. The relative viscosity of the extracellular product was measured. All test strains produced a mucous substance, and the value was 1.05-1.30 (mean +/- SD: 1.11 +/- 0.06), showing that all test strains had a biofilm-forming ability. Bactericidal experiments were performed using 6 disinfectants: ethanol, Hibitane, Isojin, Osvan, Tego 51, and Welpas. When bacteria in suspension were exposed to the disinfectants for 1 min (suspension test), 5 of the 6 disinfectants excluding Tego 51 completely killed all test strains, showing high bactericidal effects. In contrast, when adherent bacteria were exposed to the disinfectants for 1 min (adhesion test), the killing rate by Welpas was 100%, but those by Isojin and ethanol were lower (88.9 and 60.0%, respectively), that by Osvan was only 4.4%, and no bacteria were killed by Hibitane or Tego 51. These findings showed that the bactericidal effects markedly decreased in the case of adherent P. aeruginosa.

Pseudomonas aeruginosa strains were isolated from 45 of 370 (12.2%) cockroaches captured in hospitals. By cockroach species, the bacterial strains were isolated from 39 of 181 (21.5%) Periplaneta fuliginosa and 6 of 183 (3.3%) Blattella germanica, showing a significant difference (p

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.

Drug susceptibility testing was carried out using 14 antibiotics in order to identify trends in the antibiotic tolerance of 142 strains of Enterobacteriaceae isolated from horsemeat commercially available for raw consumption (basashi). A comparison of the sensitivity to the 14 antibiotics using the 90% MIC (minimum inhibitory concentration) values (MIC90) showed the strongest tolerance to ampicillin (ABPC) at a concentration of128 μg/mL, followed by that to fosfomycin (FOM) at a concentration of 128 μg/mL. When the sensitivity to these antibiotics was examined for each individual genus of tested bacteria, Hafnia spp. exhibited relative tolerance to ceftazidime (CAZ) and ceftriaxone (CTRX) at a concentration of 4 μg/mL and 2 μg/mL, respectively, which was high in comparison to that observed for the other strains. Furthermore, Raoultella spp. and Serratia spp. were found to be highly resistant to tetracycline (TC) at a concentration of 128 μg/mL and 64 μg/mL, respectively. Of the 142 strains of test bacteria, 140 (98.6%) demonstrated resistance to ABPC, with the exception of Hafnia alvei and Klebsiella pneumonia. In addition, a total of eight strains (5.6%), seven Serratia marcescens strains and one Raoultella terrigena strain, were found to be resistant to TC. Furthermore, one strain of Citrobacter freundii exhibited resistance to nalidixic acid (NA), while another displayed resistance to ofloxacin (OFLX) (0.7% each), and one strain (0.7%) each of Enterobacter cloacae, Serratia marcescens and Citrobacter youngae demonstrated resistance to fosfomycin (FOM), streptomycin (SM) and kanamycin (KM), respectively. A single strain of C. freundii was found to be resistant to three antibiotics, ABPC, NA and OFLX. Resistance to two antibiotics was confirmed in 11 strains, including seven strains of S. marcescens and one strain of R. terrigena (a total of eight strains) resistant to ABPC and TC, and one strain each of C. youngae, S. marcescens and E. cloacae resistant to ABPC and KM, ABPC and SM, and ABPC and FOM, respectively. In addition, 128 strains were resistant to the single antibiotic of ABPC alone. Of the 140 strains demonstrating antibiotic resistance, 137 (97.9%) retained the conjugative R-plasmid transfer factor, excluding three strains of S. marcescens. All transfer factors were ABPC and retained by a high proportion of the bacterial groups, with one strain (100%) being resistant to three antibiotics, nine (81.8%) of the 11 strains being resistant to two antibiotics, and 127 (99.2%) of the 128 strains being resistant to a single antibiotic. In addition, we examined ESBL productivity in the 140 strains of bacteria demonstrating drug tolerance; however, no strains exhibited this characteristic. Therefore, further observation is required to ascertain trends in antibiotic-tolerant bacteria.

The colony hybridization (CH) method as a rapid method for detecting Legionellaspp. was compared with the conventional culture method. In the comparison of colony count in which a suspension ofL. pneumophila was examined, a positive correlation was shown between the results by both methods. The regression line was Y=0.85+1.34Xand the correlation coefficient was 0.95. The maximum number of detectable colonies on the plate was 144 colony forming units, although there was a tendency for the CH data to give slightly lower counts than those of the culture method. The detection time in the CH method was half of that in the culture method, and the final result was obtained within 4 or 5 d. In addition the results by the CH method were rarely influenced by other contaminating microorganisms, since the transfer of colonies was performed at the initial stage of culture.

In May 2012, strain HNN-6 (=JCM 18566) , a Gram-negative, non-spore-forming, motile and strictly aerobic rod, which produces a pale orange pigment, was isolated from a hot spring water sample obtained in Kagoshima, Japan, by a plating method using R2A medium at 30°C for 7 d. The 16S rRNA gene sequences (1,437bp) of this strain (accession number: AB731137) had a close similarity (99.1%) to Hydrotalea sandarakina AF-51T (JF739858) . Growth occurred at 25-45°C and pH 5.0-8.0, with optimal growth at 40°C and pH 6.0-7.0. Growth did not occur in the presence of ≧2% NaCl. The API 20NE identification system gave positive results for nitrate, aesculin, gelatin, 4-nitrophenyl-β-D-galactopyranoside, D-glucose, D-mannose, maltose and oxidase (API code number 1472204) . The dominant cellular fatty acids of strain HNN-6 were iso-C15:0 (32.6%) , iso-C17:0 3-OH (24.2%) and iso-C16:0 (8.4%) . The guanine-plus-cytosine (G+C) content of DNA was 36.2 mol%. This article is the first report to describe the characteristics of an orange-pigmented bacterium isolated from a hot spring water sample 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.

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.

As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water samples, and were able to isolate Legionella micdadei from 3 (5.5%) of 55 samples. All of these isolates were able to grow within Acanthamoeba sp., suggesting that the isolates will be pathogens. We also confirmed that the K-2 strain from hot spring water grew in guinea pig monocytes. Sensitivity tests using 10 drugs showed that the isolates were most sensitive to imipenem, with the MIC90 of 0.032 microg/ml, were least sensitive to minocycline, with the MIC90 of 4 microg/ml, and were not sensitive to low amounts of other drugs.