Hatsumi Taniguchi

Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus

The nucleotide sequences of genes encoding the thermostable direct (TSD) hemolysin and the thermolabile (TL) hemolysin of Vibrio parahaemolyticus were determined. From the nucleotide sequence of the TSD hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 189 amino acids and 165 amino acids, and that the molecular weights were 21.1 kDa or 18.5 kDa, respectively. Our data regarding TSD hemolysin were in complete agreement with previously published data. From the nucleotide sequence of the TL hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 418 amino acids and 398 amino acids, and that the molecular weights were 47.5 kDa and 45.3 kDa, respectively. The GC content of the TSD hemolysin gene was 35.6%, while that of the TL hemolysin gene was 47.6% which is almost the same as that of V. parahaemolyticus genome. Maxicell analysis revealed that the molecular weights of the proteins encoded by the TSD hemolysin gene were 22.0 and 19.5 kDa, and that of the protein encoded by the TL hemolysin gene was 45.5 kDa, and that the promoters of these two hemolysin genes of V. parahaemolyticus were functional in Escherichia coli.

Significance of Anaerobes and Oral Bacteria in Community-Acquired Pneumonia

Background Molecular biological modalities with better detection rates have been applied to identify the bacteria causing infectious diseases. Approximately 10–48% of bacterial pathogens causing community-acquired pneumonia are not identified using conventional cultivation methods. This study evaluated the bacteriological causes of community-acquired pneumonia using a cultivation-independent clone library analysis of the 16S ribosomal RNA gene of bronchoalveolar lavage specimens, and compared the results with those of conventional cultivation methods. Methods Patients with community-acquired pneumonia were enrolled based on their clinical and radiological findings. Bronchoalveolar lavage specimens were collected from pulmonary pathological lesions using bronchoscopy and evaluated by both a culture-independent molecular method and conventional cultivation methods. For the culture-independent molecular method, approximately 600 base pairs of 16S ribosomal RNA genes were amplified using polymerase chain reaction with universal primers, followed by the construction of clone libraries. The nucleotide sequences of 96 clones randomly chosen for each specimen were determined, and bacterial homology was searched. Conventional cultivation methods, including anaerobic cultures, were also performed using the same specimens. Results In addition to known common pathogens of community-acquired pneumonia [Streptococcus pneumoniae (18.8%), Haemophilus influenzae (18.8%), Mycoplasma pneumoniae (17.2%)], molecular analysis of specimens from 64 patients with community-acquired pneumonia showed relatively higher rates of anaerobes (15.6%) and oral bacteria (15.6%) than previous reports. Conclusion Our findings suggest that anaerobes and oral bacteria are more frequently detected in patients with community-acquired pneumonia than previously believed. It is possible that these bacteria may play more important roles in community-acquired pneumonia.

A Higher Significance of Anaerobes: The Clone Library Analysis of Bacterial Pleurisy

BACKGROUND The frequencies of etiologic bacterial agents of intrapleural infections reported until now have been widely varied, largely depending on the implemented detective methods. The aims of this study were to evaluate bacterial etiologies of bacterial pleurisy using a cultivation-independent method. METHODS Pleural fluids were collected from 42 febrile patients with hemipleural effusion. The bacterial flora was analyzed by a clone library method using amplified fragments of the 16S ribosomal RNA gene (rDNA) with universal primers in addition to conventional cultivation methods. RESULTS Forty-two specimens were obtained from 26 patients with bacterial pleurisy, seven with mycobacterial pleurisy, and nine with other pleural effusions. In the 26 bacterial cases, 16 (61.5%) showed positive results for 16S rDNA sequencing analysis, of which 11 (42.3%) were also positive for cultivation method. In seven (43.8%) of the 16 polymerase chain reaction-positive cases, anaerobic phylotypes were predominantly detected. Anaerobic phylotypes (six of these seven cases) were not detected by cultivation method. In nine (34.6%) of the 26 bacterial pleural cases, the results from the clone library methods were not accordant with those of the cultivation method. In seven of these nine cases, the discrepancies between the two detection methods were due to the existence of anaerobes. CONCLUSION The clone library analysis using the 16S rDNA of pleural fluid showed a higher incidence of anaerobic bacteria in infectious pleurisy than that previously expected and provided additional bacterial information for cultivation methods.

An Epidemiological Study of Work-related Violence Experienced by Physicians who Graduated from a Medical School in Japan

An Epidemiological Study of Work‐related Violence Experienced by Physicians who Graduated from a Medical School in Japan: Mayuri Arimatsu, et al. Department of Work Systems and Health, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan—Healthcare workers are at high risk of being victim of verbal and physical violence perpetrated by patients. There are only a few studies on work related violence among physicians. The aim of this study was to determine how prevalent work‐related violence against physicians is and whether gender, age, specializations and workplaces are associated with verbal and physical violence against physicians in Japan. A questionnaire was mailed to all the 1,705 physicians who had graduated from one medical school in Japan and had practiced for a minimum of 3 yr by the time of this study. The verbal and physical violence experienced by physicians at the hands of their patients and/or clients in the last 6 months preceding this study were collected. We defined ‘verbal violence’ as ‘any threatening statement or complaint’ while ‘physical violence’ referred to ‘the attempted or actual exercise by persons of any physical force so as to cause injury to a physician’. Multiple logistic regression analysis was used to determine the independent contribution of each factor with violence. A total of 540 men and 158 women responded. The adjusted response rate was 41.8%. Among the participants, 168 (24.1%) physicians had experienced verbal violence and 15 (2.1%) physicians had experienced physical violence in the prior 6 months. Verbal violence was positively associated with physicians who were under 30 yr old (odds ratio [OR] = 2.1; 95% confidence interval [CI], 1.0–4.1 for 27–29 yr old) and, psychiatry (OR, 2.4; 95% CI, 1.1–5.4). Physical violence was significantly associated with women (OR, 3.8; 95% CI, 1.1–13.5), specializations such as emergency and anesthesiology (OR, 18.9; 95% CI, 2.8–126.1), and psychiatry (OR, 7.6; 95% CI, 1.6–35.4). There was a considerable number of physicians exposed to violence. Younger physicians and psychiatrists are likely to be exposed to verbal violence. Female physicians, psychiatrists, and emergency physicians are likely to be exposed to physical violence. Education on avoiding from violence should be provided for physicians early in their career.

Commercially Distributed Meat as a Potential Vehicle for Community-Acquired Methicillin-Resistant Staphylococcus aureus

ABSTRACT The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has been increasing; however, the sources of infection remain unclear. Therefore, we investigated the involvement of meat as a possible mediator of CA-MRSA infection. We examined the distribution of MRSA strains in commercially distributed raw meat samples (n = 197) and diarrheal stool samples of outpatients (n = 1,287) that were collected in Oita Prefecture, Japan, between 2003 and 2009 for routine legal inspections. Fourteen MRSA strains were isolated from three meat and 11 stool samples. Among these, seven isolates from three meat and four stool samples exhibited the same epidemiological marker profiles [coagulase type III, staphylococcal enterotoxin C, staphylococcal chromosomal cassette mec (SCCmec) type IV, ST8, spa type 606 (t1767), and toxic shock syndrome toxin-1 (TSST-1) producing type]. Furthermore, of the seven strains, three isolates from two meat samples and one stool sample collected in 2007 exhibited completely identical characteristics with respect to phage open reading frame (ORF) typing, pulsed-field gel electrophoresis, and drug susceptibility profiles. The results suggest that commercially distributed meat could play a role in the prevalence of CA-MRSA in the community.

Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan

In order to study the diversity and community of genus Mycobacterium in polluted soils, we tried to isolate mycobacteria from 11 soil samples collected from an illegal dumping site and 3 landfills in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 19 isolates of mycobacteria were obtained from 5 soil samples and 3 of them were isolated only by the co‐culture method with the amoeba. Conventional biochemical tests and sequencing of the hsp65, rpoB, and 16S rRNA genes were performed for species identification of 17 of the 19 isolates. Among the 17 isolates, there was one isolate each of Mycobacterium vanbaalenii, Mycobacterium mageritense, Mycobacterium frederiksbergense, M. vanbaalenii or Mycobacterium austroafricanum, and Mycobacterium chubuense or Mycobacterium chlorophenolicum. The remaining 12 isolates could not be precisely identified at the species level. A phylogenic tree based on the hsp65 sequences indicated that 2 of the 12 isolates were novel species. In addition, 4 isolates were phylogenically close to species that degrade polycyclic aromatic hydrocarbons, which induce some cancers in humans. These results demonstrated that there were many hitherto‐unreported mycobacteria in the polluted soils, and suggested that some mycobacteria might play roles in the natural attenuation and engineered bioremediation of contaminated sites with other microorganisms.

Physician Job Satisfaction and Working Conditions in Japan

Physician Job Satisfaction and Working Conditions in Japan: Koji Wada, et al. Department of Preventive Medicine and Public Health, Kitasato University School of Medicine

Virulence Conversion of Legionella Pneumophila by Conjugal Transfer of Chromosomal DNA

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x 10(9) CFU) and Chicago-2S (approximately 8.9 x 10(9) CFU) were mated typically yielded 10(3) Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.

Original ResearchDisorders of the PleuraA Higher Significance of Anaerobes: The Clone Library Analysis of Bacterial Pleurisy

BACKGROUND The frequencies of etiologic bacterial agents of intrapleural infections reported until now have been widely varied, largely depending on the implemented detective methods. The aims of this study were to evaluate bacterial etiologies of bacterial pleurisy using a cultivation-independent method. METHODS Pleural fluids were collected from 42 febrile patients with hemipleural effusion. The bacterial flora was analyzed by a clone library method using amplified fragments of the 16S ribosomal RNA gene (rDNA) with universal primers in addition to conventional cultivation methods. RESULTS Forty-two specimens were obtained from 26 patients with bacterial pleurisy, seven with mycobacterial pleurisy, and nine with other pleural effusions. In the 26 bacterial cases, 16 (61.5%) showed positive results for 16S rDNA sequencing analysis, of which 11 (42.3%) were also positive for cultivation method. In seven (43.8%) of the 16 polymerase chain reaction-positive cases, anaerobic phylotypes were predominantly detected. Anaerobic phylotypes (six of these seven cases) were not detected by cultivation method. In nine (34.6%) of the 26 bacterial pleural cases, the results from the clone library methods were not accordant with those of the cultivation method. In seven of these nine cases, the discrepancies between the two detection methods were due to the existence of anaerobes. CONCLUSION The clone library analysis using the 16S rDNA of pleural fluid showed a higher incidence of anaerobic bacteria in infectious pleurisy than that previously expected and provided additional bacterial information for cultivation methods.

A revised biosynthetic pathway for phosphatidylinositol in Mycobacteria

For the last decade, it has been believed that phosphatidylinositol (PI) in mycobacteria is synthesized from free inositol and CDP-diacylglycerol by PI synthase in the presence of ATP. The role of ATP in this process, however, is not understood. Additionally, the PI synthase activity is extremely low compared with the PI synthase activity of yeast. When CDP-diacylglycerol and [(14)C]1L-myo-inositol 1-phosphate were incubated with the cell wall components of Mycobacterium smegmatis, both phosphatidylinositol phosphate (PIP) and PI were formed, as identified by fast atom bombardment-mass spectrometry and thin-layer chromatography. PI was formed from PIP by incubation with the cell wall components. Thus, mycobacterial PI was synthesized from CDP-diacylglycerol and myo-inositol 1-phosphate via PIP, which was dephosphorylated to PI. The gene-encoding PIP synthase from four species of mycobacteria was cloned and expressed in Escherichia coli, and PIP synthase activity was confirmed. A very low, but significant level of free [(3)H]inositol was incorporated into PI in mycobacterial cell wall preparations, but not in recombinant E. coli cell homogenates. This activity could be explained by the presence of two minor PI metabolic pathways: PI/inositol exchange reaction and phosphorylation of inositol by ATP prior to entering the PIP synthase pathway.