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Aim Continuous usage of a ring pessary for pelvic organ prolapse may cause a disturbance of intravaginal microbiota and intravaginal mucosal damage. To avoid the side effects of continuous ring pessary therapy, daily self-replacement of the ring pessary is recommended. The purpose of this study is to evaluate the outcomes of self-replacement versus continuous ring usage, by analysing clinical findings and intravaginal microbiota. Methods Thirty seven patients who managed self-replacement ring pessary therapy and 13 patients with continuous ring pessary therapy participated in this study. The clinical symptoms were checked at 1 month after the initial ring pessary insertion. The changes in the intravaginal microbiota were evaluated by conventional methods, i.e. pH in the vagina, Lactobacillary grade, Nugent score, inflammatory cell counts, and culture-based bacterial detection methods. In addition, our clone library method using 16S rRNA sequencing of vaginal fluid was performed. Results Patients were divided into four groups: self-replacement pre/post-menopause and continuous and pre/post-menopause. Five patients of the self-replacement group (n = 37) and all patients of the continuous use group (n = 13) complained of increased discharge. However, both the conventional methods and the clone library method revealed that the number of the self-replacement group patients who had abnormal intravaginal microbiota were not significantly different from that of the continuous use group. Conclusion Daily self-replacement ring pessary therapy prevented adverse clinical symptoms. However, abnormal intravaginal microbiota was frequently observed during self-replacement of ring pessary therapy as with continuous usage. Regardless, pelvic examinations should be performed routinely.

BackgroundThe Japanese Midwives Association (JMA) collaborated with the Mongolian Midwives Association (MMA) on the twinning project under the guidance of the International Confederation of Midwives (ICM) towards the achievement of the Sustainable Development Goals. The purpose of this study was to evaluate the outcomes of the organisational strengthening of the MMA through the twinning project with the JMA. ICM’s Member Association Capacity Assessment Tool (MACAT) was used as an indicator of MMA’s organisational strengthening—first in the year 2015 and then at the end of 2018—for a comparative analysis. For a comprehensive evaluation, qualitative interviews were conducted with the board members and general members of the MMA in 2018, and the results were compared with the issues identified during the first visit to MMA in 2014. ResultsIn 2015, 27 (28.1%) of the 96 MACAT items were achieved, while in 2018, 66 items (68.8%) were achieved. The average progress rate was 40.7%. Items with a high progress rate corresponded to: Governance (50.0%), Management Practices and Leadership (50.0%), Functions (43.8%), and Financial Resource Management (37.5%). Four themes emerged from the interviews: Professional identity, Strengthening midwifery services, Advocacy for midwives, and Recognition of midwives as autonomous professionals.ConclusionsThe organisational strengthening of the MMA through the twinning project resulted in more positive outcomes than expected. These outcomes were apparent from the evaluation of the MACAT items and the opinions and impressions of the MMA members, as elicited through the qualitative interviews. The MACAT was selected as an easy-to-understand index. However, instead of following the concept of ‘equal participation on the ground level’, a mentorship approach was adopted in line with the concept of equity. Equity and a mentorship approach can serve as key variables of success in international co-operation.

Recent advances in culture-independent microbiological analyses have greatly expanded our understanding of the diversity of unculturable microbes. However, human pathogenic bacteria differing significantly from known taxa have rarely been discovered. Here, we present the complete genome sequence of an uncultured bacterium detected in human respiratory tract named IOLA, which was determined by developing a protocol to selectively amplify extremely AT-rich genomes. The IOLA genome is 303,838 bp in size with a 20.7% GC content, making it the smallest and most AT-rich genome among known human-associated bacterial genomes to our best knowledge and comparable to those of insect endosymbionts. While IOLA belongs to order Rickettsiales (mostly intracellular parasites), the gene content suggests an epicellular parasitic lifestyle. Surveillance of clinical samples provides evidence that IOLA can be predominantly detected in patients with respiratory bacterial infections and can persist for at least 15 months in the respiratory tract, suggesting that IOLA is a human respiratory tract-associated bacterium., Kazumasa Fukuda et al. complete a new genome sequence for an uncultured bacterium detected in human respiratory tract named IOLA. The IOLA genome is found to be among the smallest and most AT-rich of known human-associated bacterial genomes and surveillance of clinical samples indicates that IOLA is in fact a human respiratory tract-associated bacterium.

Obtaining precise etiological information regarding causative bacteria is important for the proper use of antimicrobials in hospital-acquired pneumonia (HAP), which is associated with a high rate of mortality. The aim of this study was to comparatively investigate the bacterial diversity in bronchoalveolar lavage fluid (BALF) in Japanese patients with HAP by the clone library method using the 16S rRNA gene. This study included Japanese patients with HAP who were treated at our hospital and referring hospitals. BALF specimens were obtained from pneumonia lesions identified on chest radiographs and/or computed tomography. Sputum specimens were also evaluated in patients with sputum production. Sixty-eight patients were ultimately enrolled. BALF cultivation revealed bacterial positivity in 53 of 68 (77.9%) patients, and Staphylococcus aureus (30.9%) was the most frequently isolated, followed by Pseudomonas aeruginosa (16.2%), and Escherichia coli (10.3%). In contrast, the clone library analysis identified the presence of some bacterial phenotype in 65 of 68 (95.6%) patients, and streptococci (16.2%), Corynebacterium species (11.8%), anaerobes (10.3%) were frequently detected as the predominant phylotypes. Both methods tended to detect S. aureus, Klebsiella pneumoniae, and E. coli in patients with late-onset pneumonia. In addition, the cases that phylotypes of S. aureus and P. aeruginosa were found to account for > 5% of the bacterial flora of each case were 42.9% and 72.7%, respectively. These results indicate that attention should be paid to the roles of gram-positive bacilli such as streptococci, Corynebacterium species and anaerobes, in addition to Gram-negative bacilli, in the pathogenesis of HAP.

Culture-independent methods to detect microorganisms have been developed in parallel with traditional culture-based methods ever since the classification of bacteria based on 16S rRNA gene sequences was advocated in the 1970s. The development and the prevalence of culture-independent molecular technologies have provided revolutionary progress in microbial studies. The development of these technologies contributes significantly to the research of microorganisms that cannot be detected by traditional methods such as culture-dependent methods.Many molecular methods targeting the 16S rRNA gene, such as fluorescence in situ hybridization (FISH), quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), denaturing-gradient gel electrophoresis (DGGE), clone library analysis, and next-generation DNA sequencing (NGS) technologies, have been applied to various microbial studies. Notably, the advent of NGS technologies enabled a large-scale research of the bacterial community. Many recent studies using the NGS technologies have revealed that a larger number of bacteria and taxa than previously thought inhabit various parts of the human body and various places on the earth. The principles and characteristics of each molecular method are different, and each method possesses individual advantages; for example target specificity, comprehensiveness, rapidness, and cost efficiency. Therefore it is important that the methods used in studies are suitable for the objective and materials. Herein, we highlights molecular approaches targeting the 16S rRNA gene in bacterial community analysis, and focuses on the advantages and limitations of each technology.

Background: Appropriate antibiotic use reduces the mortality of patients with lung abscess; however, 40-60% of the bacterial etiologies in these patients have remained unknown with the culture methods. Obligate anaerobes and the Streptococcus anginosus group are common pathogens in lung abscess, but a precise evaluation of these bacteria by ordinary culture methods seems to be difficult due to upper respiratory tract contamination. Objectives: The aim of this study was to assess the microbiota of lung abscess by bronchoalveolar lavage fluid (BALF) using the molecular method in comparison to culture methods. Methods: BALF samples obtained from the affected lesions and sputum samples of 59 patients with lung abscess were evaluated. The microbiota in BALF was analyzed according to the molecular method using the 16S ribosomal RNA gene. Results: Fifty-six of the 59 BALF samples were positive in polymerase chain reaction analysis. Fusobacterium spp. (23.7%) were most frequently detected, followed by the S. anginosus group (15.3%), as the predominant phylotypes. Obligate anaerobes were detected in 42.4% of the BALF specimens as the predominant phylotypes, whereas the detection rate of obligate anaerobes was 13.6% by culture methods using BALF. In addition, the detection rate among those patients in whom the phylotype of obligate anaerobes was detected in >5% of the lung microbiota according to the molecular method was 86.5% in the ‘mixed-bacterial' infection group. Conclusions: The findings by the molecular method suggest that obligate anaerobes play important roles in the pathogenesis of lung abscess and provide additional bacterial information regarding conventional culture methods.

A 23-year-old Japanese woman was admitted to A hospital due to pneumonia. IgM for Mycoplasma pneumoniae was positive, and the patient was treated with imipenem/cilastatin, clindamycin, pazufloxacin and minocycline. However, both the chest radiological findings and the symptoms became exacerbated, and she was therefore transferred to our hospital. The bronchoalveolar lavage fluid was obtained, and a 16S rRNA gene sequencing analysis revealed a monobacterial infection of Mycoplasma pneumoniae. Therefore, corticosteroid treatment in addition to minocycline was administered, and the patients symptoms, laboratory data and chest radiographs improved. Corticosteroid therapy may therefore be considered for patients with refractory M. pneumoniae pneumonia.

Background and objective Recent advances in cultivation-independent molecular biological modalities for detecting bacterial species have indicated that several bacterial species may play a role in the pathogenesis of certain infectious diseases. The aim of this study was to evaluate the role of bacterial flora in the pathogenesis of nontuberculous mycobacteriosis (NTM) using a bacterial floral analysis of bronchoalveolar lavage fluid (BALF) with 16S rRNA gene sequencing in patients with bronchiectasis. Methods Fifty-eight patients with bronchiectasis evaluated using chest computed tomography were enrolled. BALF obtained from the most affected lung lesions was evaluated using culture and culture-independent methodologies. Approximately 600 bp of the bacterial 16S rRNA gene (E341F-E907R) was amplified via polymerase chain reaction using universal primers, and clone libraries were constructed. Nucleotide sequences of 96 randomly chosen clones for each specimen were determined, and the homology was searched using a basic local alignment search tool to determine the bacterial phylotypes and their proportions (bacterial floral analysis) in each specimen. Results Twenty-nine patients with bronchiectasis were diagnosed with NTM based on culture-based methods using Ogawa medium. The molecular method showed a significantly high rate of anaerobes among the patients with NTM compared with that observed in the bronchiectasis patients without NTM. In addition, findings of collapse/consolidation were significantly related to the proportion of Prevotella species in the BALF samples determined using the molecular method (P

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes.

Background Aspiration pneumonia has been a growing interest in an aging population. Anaerobes are important pathogens, however, the etiology of aspiration pneumonia is not fully understood. In addition, the relationship between the patient clinical characteristics and the causative pathogens in pneumonia patients with aspiration risk factors are unclear. To evaluate the relationship between the patient clinical characteristics with risk factors for aspiration and bacterial flora in bronchoalveolar lavage fluid (BALF) in pneumonia patients, the bacterial floral analysis of 16S ribosomal RNA gene was applied in addition to cultivation methods in BALF samples. Methods From April 2010 to February 2014, BALF samples were obtained from the affected lesions of pneumonia via bronchoscopy, and were evaluated by the bacterial floral analysis of 16S rRNA gene in addition to cultivation methods in patients with community-acquired pneumonia (CAP) and healthcare-associated pneumonia (HCAP). Factors associated with aspiration risks in these patients were analyzed. Results A total of 177 (CAP 83, HCAP 94) patients were enrolled. According to the results of the bacterial floral analysis, detection rate of oral streptococci as the most detected bacterial phylotypes in BALF was significantly higher in patients with aspiration risks (31.0 %) than in patients without aspiration risks (14.7 %) (P = 0.009). In addition, the percentages of oral streptococci in each BALF sample were significantly higher in patients with aspiration risks (26.6 ± 32.0 %) than in patients without aspiration risks (13.8 ± 25.3 %) (P = 0.002). A multiple linear regression analysis showed that an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of ≥3, the presence of comorbidities, and a history of pneumonia within a previous year were significantly associated with a detection of oral streptococci in BALF. Conclusions The bacterial floral analysis of 16S rRNA gene revealed that oral streptococci were mostly detected as the most detected bacterial phylotypes in BALF samples in CAP and HCAP patients with aspiration risks, especially in those with a poor ECOG-PS or a history of pneumonia.

Background Determining whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen or reflective of colonization when MRSA is cultured from the respiratory tract remains important in treating patients with pneumonia. Methods We evaluated the bacterial microbiota in bronchoalveolar lavage fluid (BALF) using the clone library method with a 16S ribosomal RNA (rRNA) gene analysis in 42 patients from a pneumonia registry who had MRSA cultured from their sputum or BALF samples. Patients were divided into two groups: those treated with (Group A) or without (Group B) anti-MRSA agents, and their clinical features were compared. Results Among 248 patients with pneumonia, 42 patients who had MRSA cultured from the respiratory tract were analyzed (Group A: 13 patients, Group B: 29 patients). No clones of S. aureus were detected in the BALF of 20 out of 42 patients. Twenty-eight of 29 patients in Group B showed favorable clinical outcomes, indicating that these patients had non-MRSA pneumonia. Using a microflora analysis of the BALF, the S. aureus phylotype was predominant in 5 of 28 (17.9 %) patients among the detected bacterial phylotypes, but a minor population (the percentage of clones ≤ 10 %) in 19 (67.9 %) of 28 patients. A statistical analysis revealed no positive relationship between the percentage of clones of the S. aureus phylotype and risk factors of MRSA pneumonia. Conclusions The molecular method using BALF specimens suggests that conventional cultivation method results may mislead true causative pathogens, especially in patients with MRSA pneumonia. Further studies are necessary to elucidate these clinically important issues.

Objectives The aim of this study was to identify bacteria in sludge brought by the 2011 tsunami in Japan to determine the necessary precautions for workers who handle the sludge. Methods Two sludge samples and one water sample were collected from each of two sites in Miyagi Prefecture in June 2011. We also obtained control samples from a paddy field and a dry beach in Fukuoka, Japan. The samples were subjected to physicochemical analyses, conventional cultivation methods, and molecular methods for bacterial flora analysis. The bacterial floras were analyzed using a clone library method employing fragments of the 16S ribosomal RNA gene (rDNA) amplified with universal primers. Results We detected 51-61 genera in sludge samples and 14 and 17 genera in water samples collected in the tsunami-affected areas. In sludge samples collected in the tsunami-affected areas, more genera belonged to Proteobacteria than to Bacteroidetes, but in water samples collected in these areas, more genera belonged to Bacteroidetes than to Proteobacteria. Non-O1, non-O139 V. cholerae (non-agglutinable vibrio) was found at approximately 10(4) cells/m/ near the coast of the tsunami affected area. Sulfate-reducing bacteria were detected in sludge collected from the paddy field, and a relatively high concentration of sulfate ions was found in the water sample (258 mg/l). Conclusions Sludge brought by the tsunami contained some pathogens; therefore, frequent hand washing is recommended for workers who have direct contact with the sludge to minimize their risk of infection. Under the anaerobic conditions of paddy fields, hydrogen sulfide could be produced by sulfate-reducing bacteria metabolizing sulfate ions.

Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC.

Background: The aim of this study is to investigate the reciprocal effect of Staphylococcus aureus colonization and allergic rhinitis in an allergy model of mice. Methods: BALB/c mice with intraperitoneal ovalbumin (OVA) sensitization and/or intranasal S. aureus inoculation were prepared. The following 4 groups were designed: an OVA-sensitized S. aureus-inoculated (AR-SA) group, an OVA-sensitized uninoculated (AR) group, a nonsensitized S. aureus-inoculated (SA) group, and a nonsensitized uninoculated (control) group. After intranasal OVA challenge, nasal lavage fluid, peripheral blood, and nasal mucosa were collected. Polymorphonuclear cells in the nasal lavage fluid were counted, serum OVA-specific IgE and IgG1 were measured by enzyme immunoassays, and IL-4, IL-5, and IFN-γ mRNAs in the nasal mucosa were assessed by quantitative real-time reverse transcription-PCR. The number of S. aureus in the nasal mucosa and lavage fluid was counted. Results: Both eosinophil and neutrophil counts were larger in the AR-SA group than in the other groups. Both IgE and IgG1 levels were higher in the AR and AR-SA groups than in the SA and control groups, and the IgG1 level was higher in the AR-SA group than in the AR group. The expression of IL-4 mRNA was higher in the AR-SA group than in the other groups, and the expression of IL-5 mRNA was higher in the AR-SA group than in the SA group. The AR-SA group showed higher counts of S. aureus in the nasal mucosa than the SA group. Conclusion: These results indicate the mutually potentiating effect of S. aureus colonization and allergic rhinitis.

The purpose of this study was to understand the impact of childbirth and the experience of fatherhood on Japanese men in Japan and a foreign country. Descriptive phenomenology was used to study a total of 14 Japanese men who attended childbirth and experienced parenting in the United States and Japan. The Colaizzi method of data analysis was used to analyze responses to open-ended questions. Responses to these questions showed several similarities between the two groups of men. First, men in both countries felt closer to with their spouses having gone through the experience of childbirth together. Second, both groups nevertheless recognized a strong bond between mother and the baby, leading them to feel at times isolated. Third, both groups were concerned about their wives’ emotional swings during pregnancy and child rearing. Finally, both groups were more focused on their wives and babies than themselves. There were also several interesting differences. Japanese men who were living in Hawaii were more involved in taking care of their children and in helping with household chores than those living in Japan. This was due to living in a more family-oriented society, as well as a result of limited support from their extended families back in Japan. A result of spending more time with their wives and babies was that Japanese men in the United States understood more fully the stress of childcare. On the other hand, due to Japan’s work-oriented society, men in Japan relied more on support from their extended families, leaving them less time with their wives and children. This study clearly shows that social support systems alter gender roles and behavior, leading to significant differences in the experience of parenthood in Japan and a foreign country.

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.

The aim of this study was to determine the influence of menstruation on the bacterial population of healthy Japanese women's vulvas, especially the labia minora. Labia minora swabs were obtained from 10 premenopausal, nonpregnant Japanese women at premenstruation and on day 2 of menstruation. Vaginal swabs were also obtained from 3 out of the 10 women. No significant difference was found in the average bacterial cell count between the menstruation and premenstruation samples. Molecular analysis using a 16S rRNA gene-based clone library method detected 22 genera from the labia minora swabs (total 20), with the genus Lactobacillus being predominant at both premenstruation and during menstruation in 7 out of the 10 women. Of the other 3 women, 2 showed various kinds of bacterial species, including oral and fecal bacteria, with Atopobium vaginae and Gardnerella vaginalis predominating in the remaining woman's vulva in both conditions. In total, 6 out of 10 cases (60%) showed significantly different microbiota of the labia minora between the two conditions. These results imply that menstruation may promote a distortion of the bacterial flora around the vulva, although it causes no significant increase of the bacterial count.

Intestinal microbiotas of human subjects and effect of antibiotic treatment on them have been reported with cultivation independent methods. However, Japanese fecal microbiotas have not been studied enough. We have constructed a clone library method to obtain results within 3 d. In this study, intestinal microbiotas of 29 healthy Japanese adults, whose fecal samples were collected twice at 5 month intervals from each subject, were analyzed with our clone library method, and using those data as a benchmark effect of antibiotic treatment on intestinal microbiotas was evaluated. The fifty-eight fecal microbiotas were assessed based on percentages at genus level, and the variability was analyzed with a principal component analysis (PCA). PCA showed that the microbiotas divided into three groups depending on the large eigenvectors (genera Ruminococcus, Bacteroides, and Prevotella), and the dual samples from the twenty-two individuals have belonged to the same PCA group. It suggests that almost Japanese adults have own stable intestinal microbiota. The genera Ruminococcus and Bacteroides were present in almost subjects, while the genus Prevotella was found only in nine subjects (approximately 30%) which was preserved with 5 months intervals. Next, the microbiotas before and after antibiotic treatment were evaluated comparing with the 58 healthy adult microbiotas. The results showed that beta-lactams influenced profoundly on intestinal microbiotas and the effect of macrolides depended on the cases. It suggests that our clone library method could show overview of intestinal microbiota and would give us useful information about the effect of antibiotic treatment for daily clinical diagnosis.

The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M. smegmatis strain mc(2)155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli- mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated. In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M. tuberculosis in phagocytic cells; this system may be useful as a screening tool for M. tuberculosis genes.

A new method for procedures using a computer to find out useful candidates for probes discriminating a certain group in higher ranks of bacteria is presented. In order to make the search of the probes systematic, two indices are proposed, i.e., Coincidence Ratio Inside Group (CRIG) and Coincidence Number Outside Group (CNOG), which indicate the rate of matching of probes inside or outside group respectively. Using two indices, allowance grades indicating usefulness of arbitrary sequence as a probe are defined from 9 (5 in species) to 0. Its application to the 16S rRNA gene of 2206 bacterial species selected from the Ribosomal Database Project (RDP-II) (J.R. Cole et al., Nucleic Acids Res. 31: 442-443, 2003) is shown. Small nucleotide sequences of the length L (L = 15, 19, 23) were searched from about 550 bases. As a result of computer calculations, appropriate probes are found in all taxonomic ranks, in addition, it is found that 95% of genera can be identified uniquely. The method is useful for DNA chips or targeted PCR which can select a desirable bacteria set in any taxonomic rank. The method is in principle deterministic, and widely applied to any type of nucleotide sequences.

This study was designed to analyze the diversity of aerobically growing bacteria in a landfill area, compared to those from a forest and a cultivated area at four different depths. The viable cell number of aerobes in the incineration ash layer (3.5(+/-0.4)x10(2)/g) was 10(3)- to 10(4)-fold lower than those in the other areas. On 16S ribosomal DNA similarity analysis of a total of 727 colonies, only the class Bacilli was detected in the incineration ash layer whose pH was extremely high (12.8), while five to seven classes were detected in the forest and the cultivated area. Of the genus Bacillus, B. licheniformis and the recently discovered Bacillus were predominant in the incineration ash layer. These analyses indicate that the incineration ash layer of a landfill might be a source that includes valuable or hitherto unreported Bacillus species.

We describe here a tool for risk assessment and management of infectious diseases in a workplace. This was constructed as a primary precaution for the prevention of infectious diseases in a workplace, not to be used as a countermeasure of diseases in the time of or after an occurrence. The tool grades risk levels of each of the factors influencing infectious diseases in the workplace and an assessment based on the total point are given. Ordinary workplaces should be chosen, such as factories and office buildings, not places where medical experts or hygienists work, such as hospitals, schools and concessionaries, etc. Three risk factors for infection are pathogens, route and human host. The factor of a pathogen is divided into two groups, spreadable (from human to human) and non-spreadable. The risk of spreadable pathogens is assessed by the ages of workers, CO2 concentration and air volume, and the combination of the existence of common places and collaborative work. The risk of non spreadable pathogens is evaluated by the ages of workers, air current and air volume, and existence of equipment generating aerosol. In cases where the total point is over 7, the risk is assessed as high level (group A) and daily measures must be taken, such as ensuring proper operation of the infection control committee, education, management of working conditions and management of working environments. In cases where the score is 5 or 6, the risk is assessed as intermediate level (group B) and daily measures are recommended, such as ensuring proper operation of the infection control committee and education. In the case of a score less than 4, the risk is assessed as low level (group C) and these daily measures are not necessary. Instead, an infection control committee should be organized and concrete measures should be taken upon an outbreak of an infectious disease.

Appropriate antibiotic use reduces the mortality of patients with lung abscess; however, 40-60% of the bacterial etiologies in these patients have remained unknown with the culture methods. Obligate anaerobes and the Streptococcus anginosus group are common pathogens in lung abscess, but a precise evaluation of these bacteria by ordinary culture methods seems to be difficult due to upper respiratory tract contamination.The aim of this study was to assess the microbiota of lung abscess by bronchoalveolar lavage fluid (BALF) using the molecular method in comparison to culture methods.BALF samples obtained from the affected lesions and sputum samples of 59 patients with lung abscess were evaluated. The microbiota in BALF was analyzed according to the molecular method using the 16S ribosomal RNA gene.Fifty-six of the 59 BALF samples were positive in polymerase chain reaction analysis. Fusobacterium spp. (23.7%) were most frequently detected, followed by the S. anginosus group (15.3%), as the predominant phylotypes. Obligate anaerobes were detected in 42.4% of the BALF specimens as the predominant phylotypes, whereas the detection rate of obligate anaerobes was 13.6% by culture methods using BALF. In addition, the detection rate among those patients in whom the phylotype of obligate anaerobes was detected in5% of the lung microbiota according to the molecular method was 86.5% in the 'mixed-bacterial' infection group.The findings by the molecular method suggest that obligate anaerobes play important roles in the pathogenesis of lung abscess and provide additional bacterial information regarding conventional culture methods.

The purpose of this study is to investigate which kinds of stress women experience during childbirth in a foreign country and to explore whether childbirth in a foreign country influences women's mental health. The study was a quantitative and qualitative mixed study. Forty-five Japanese women, born and raised in Japan and who gave birth in Hawaii, USA, were telephone-interviewed within 1 year after childbirth. The stress factors that emerged were: language barrier, distance from family and friends, different culture, and health-care attitude about childbirth. Half of the participants experienced emotional dysfunction during their pregnancy. All primiparas experienced postpartum depression. The participants who had the maternity blues tended to have postpartum depression. Help from the participants' mothers after childbirth decreased postpartum depression. The importance of mental health for foreign-born primiparas emerges during the perinatal period.

In this study, we tried to isolate legionellae from nine Legionella DNA-positive soil samples collected from four different sites contaminated with industrial wastes in Japan. Using culture methods with or without Acanthamoeba culbertsoni , a total of 22 isolates of legionellae were obtained from five of the nine samples. Identification of species and/or serogroups (SGs), performed by DNA–DNA hybridization and agglutination tests, revealed that the 22 isolates consisted of ten isolates of Legionella pneumophila including five SGs, five Legionella feeleii , and one each of Legionella dumoffii , Legionella longbeachae , and Legionella jamestownensis . The species of the remaining four isolates (strains OA1-1, -2, -3, and -4) could not be determined, suggesting that these isolates may belong to new species. The 16S rDNA sequences (1476–1488 bp) of the isolates had similarities of less than 95.0% compared to other Legionella species. A phylogenetic tree created by analysis of the 16S rRNA (1270 bp) genes demonstrated that the isolates formed distinct clusters within the genus Legionella . Quantitative DNA–DNA hybridization tests on the OA1 strains indicated that OA1-1 should be categorized as a new taxon, whereas OA1-2, -3, and -4 were also genetically independent in another taxon. Based on the evaluated phenotypic and phylogenetic characteristics, it is proposed that one of these isolates from the soils, OA1-1, be classified as a novel species, Legionella impletisoli sp. nov.; the type strain is strain OA1-1 T (=JCM 13919 T =DSMZ 18493 T ). The remaining three isolates belong to another novel Legionella species, Legionella yabuuchiae sp. nov.; the type strain is strain OA1-2 T (=JCM 14148 T =DSMZ 18492 T ). This is the first report on the isolation of legionellae from soils contaminated with industrial wastes.

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other maritime Asian countries where seafood is frequently consumed. A total of 535 strains of V. parahaemolyticus were recovered mostly (97%) from clinical samples obtained in Taiwan or in 14 other countries. These strains were typed by pulsed-field gel electrophoresis following SfiI digestion and a typing scheme was generated. The 115 different patterns identified were grouped into 13 types with dissimilarity values less than 15, plus 16 miscellaneous patterns not grouped into any of the types. Types I, A, D and J contained the most patterns, with the numbers of patterns being 17, 13, 12, and 11, respectively. However, types I, B, D, A, H and C contained the most strains, with the numbers of strains being 204, 73, 71, 54, 29 and 25, respectively. Type I consisted exclusively of the pandemic O3:K6 strains and genetically closely related strains. This PFGE typing scheme for V. parahaemolyticus could be used for the characterization of pathogenic isolates.

In order to evaluate the risk of bacterial infection to workers from the sludge in a sewage pit of UOEH Hospital, a bacterial flora analysis based on the molecular-technique using the 16S rRNA gene was performed. Sludge samples taken from five different points in the pit were used. Bacterial numbers of each sludge sample were detected in the range of 1.9 X 10(7) cells/g to 4.4 x 10(8) cells/g. The results of bacterial flora analysis showed that the proportion of known bacteria (similarities of 16S rDNA sequences to that of type strain being 97% or more) and that of unclassified bacteria in the sludge samples were about 20 and 80%, respectively. Regarding the pathogenic bacteria, two Legionella species were detected in two samples, and one Mycobacterium species was detected in one sample. Since the frequency of pathogenic bacteria was equivalent to normal soil, it was concluded that the risk of bacterial infection from the sludge was low.

The bacterial flora of 8 sediment samples taken from Doukai Bay in Kitakyushu City in May, 2006 was analyzed by using the culture-independent molecular method. The total bacterial numbers determined by epifluorescence microscopic method were in the range of 2.6 x 10(8) to 6.4 x 10(8) cells/g. The results of bacterial flora analysis suggested that unknown bacterium comprised over 80% of total bacterial flora in the sea sediment samples. Regarding the pathogenic bacteria, although 8 of the Vibrio species, 1 of the Staphylococcus species, and 2 of the Mycobacterium species were detected in the sediment samples, their frequency was low. A large proportion of bacteria related to sulfur circulation, such as sulfur-oxidizing bacteria (10.9 to approximately 30.5%) and sulfate-reducing bacteria (19.6 to approximately 25.1%), was detected in all of the samples from sea sediment. A remarkable difference in bacterial flora between ground soil and sea sediment was clarified by this experiment.

Bioaerosols cause sick building syndrome (SBS) and allergy. Many kinds of bioaerosol impactors are used for measurement of airborne microorganism concentrations in Japan. However, because the impactors are set on agar plates, some microorganisms cannot make colonies on the plates because of their lower viability or demands of nutrition. On the other hand, by double staining using ethidium bromide (EtBr) and carboxyfluorescein diacetate (CFDA), both total cells and cells with esterase activities can be detected without incubation. In this study, we calculated total cell concentrations and percentages of cells with esterase activities by the combination of filter sampling and double staining (EtBr and CFDA) from air of a laboratory, a conference room and outdoors. Temperature and humidity in the laboratory were constantly kept by an air conditioner, but in the conference room, an air conditioner was only operated sometimes because of its low frequency of use. There were no significant differences between total cell concentrations and humidity in both rooms, but increase of the percentages of cells with esterase activities depended on rainfall before the samplings (n=15, p

Background and Objectives: Chlamydophila pneumoniae has been recognized as one of common causes in pneumonia and acute respiratory tract infection. Serological analysis is commonly used but it is not enough accurate and convenient for diagnosing C. pneumoniae pneumonia in the real-world clinical setting. Therefore, the true prevalence of C. pneumoniae pneumonia seems to be unclear. In addition, no C. pneumoniae were detected in 16S rRNA gene analysis in our previous study. The aim of this study was to detect C. pneumoniae using two specific PCR methods using bronchoalveolar lavage fluid (BALF) samples in patients with pneumonia. Methods: BALF samples from 147 patients with bacterial pneumonia were retrospectively evaluated using two C. pneumoniae -specific PCR method. Results: None of them was positive for specific PCR for C. pneumoniae using two sets of specific primers and 16S rRNA gene analysis. On the contrary, serological analysis revealed that 1 of 38 (2.6%) patients was presumptive as acute infection of C. pneumonia e and 15 of the 94 (16.0%) patients were considered as doubt of acute C. pneumoniae . Cultivation and/or 16S rRNA gene sequencing revealed that streptococci, H. influenzae , and M. pneumoniae were detected in presumptive/doubted cases according to serological method. Conclusions: No C. pneumoniae DNA was detected in BALF samples from patients with pneumonia, even though molecular modalities were applied. These results may suggest that C. pneumoniae may be extremely few in the lower respiratory tract in patients with pneumonia, and physicians may consider low possibility of C. pneumoniae as causative pathogen.

Background: The reported common pathogens of lung abscess are anaerobes, Streptococcus species and mixed infection. However, it is frequently difficult to detect anaerobes by sputum or bronchoalveolar lavage fluid (BALF) culture. The aim of the present study was to evaluate the bacteriological incidence of lung abscess in Japanese patients by means of bacterial floral analysis of the BALF specimens using clone library analysis of 16S rRNA gene. Patients and Methods: A total of 39 Japanese patients with lung abscess were enrolled, and the BALF specimens taken from the affected lesions were evaluated by both culture-independent molecular method and conventional cultivation methods. The molecular method was conducted as follows: approximately 600 base pairs of 16S ribosomal RNA genes were amplified by polymerase chain reaction using universal primers, then clone libraries were constructed. Nucleotide sequences of randomly chosen 96 clones for each specimen were determined and the homology with in-house database was searched to estimate the bacterial species. Results: The molecular method revealed that relatively high rate of anaerobes (54%) and S. anginosus group (18%) were detected. Mixed bacterial infection was observed in 76.9% of the patients, and 90.0% of anaerobes were found in patients with mixed infection. Conclusion: Our data suggests that anaerobes and S. anginosus group might play important roles in the pathogenesis of lung abscess.

Rapidly growing non-tuberculous mycobacteria (RGM) are usually detected in blood cultures after 4-5 days of incubation, so it is important to differentiate RGM from contamination of commensal organisms on human skin. We report an unusual case of Mycobacterium mageritense bacteremia and infection of an implantable cardioverter defibrillator originally misidentified as Corynebacterium spp. or Nocardia spp. in gram-stained smears. 16S rRNA gene sequencing had utility in the definitive identification of isolates. We should be aware that RGM infection may exist in repeated implantable device infections.

The purpose of this paper is to report our evaluation of changes in intravaginal microbial flora after ring pessary therapy for pelvic organ prolapse (POP) using conventional and our clone library method. Thirteen patients with POP who were fitted with a ring pessary participated in this longitudinal study that incorporates data from before and 1 month after beginning ring pessary therapy. Changes in intravaginal microbial flora were evaluated by conventional methods, i.e., vaginal pH, lactobacillary grade (LAC grade), Nugent score, and culture-based bacterial detection methods. In addition, we performed our clone library method using 16S ribosomal RNA (rRNA) sequencing of vaginal fluid. Conventional methods revealed that most patients had abnormal intravaginal microbial flora. Mean numbers of detected bacterial species by the culture-based and our clone library method were 3.1 (1–6) and 11.8 (1–25), respectively. Our clone library method showed that Lactobacillus spp. increased in four and decreased in two cases after ring pessary therapy but reappeared after therapy in two cases; no Lactobacillus spp. were detected in five cases. Our study showed that ring pessary therapy did not always disturb intravaginal microbial flora, especially for patients with Lactobacillus spp. prior to ring pessary insertion. Anaerobic circumstances in the vagina after therapy seem to have induced the growth of anaerobic bacteria.

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc(2)155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc(2)155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc(2)155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

Bioaerosols including bacteria and fungi have been almost unrecognized as pollutants of work environments in Japan. The combination of filter sampling and DNA staining by ethidium bromide (EtBr) was examined for the detection and evaluation of total numbers of bioaerosols, including viable and dead microorganisms, for risk assessment in work environments. With direct counting of microorganisms by EtBr concentrations of total cells were obtained in a shorter time than plate culture, the traditional method for detection of microorganisms. Total cell concentrations (cells/m3) were about 100 times greater than colonized cell concentrations (CFU/m3) in all samples. In some microscopic fields, macro conidia produced from some kinds of fungi were observed, but they were not detected by plate culture. Airborne bacterial 16S rDNA amplified by PCR were determined for their base sequences by DNA sequence analysis and classified by sequence-based homologies. Base sequences from 2 samples each contained 6 common groups of phylum. The combination of filter sampling and direct counting by EtBr staining was shown to be a better method for detecting and evaluating total cell concentrations in the risk assessment of sick building syndrome and allergy.