Surveillance and phylogenetic analysis of a pathogenic bacterium candidate in nasal discharge from children.

"The infectious organism lurking in human airways (IOLA)" is a candidate pathogenic bacterium detected in bronchoalveolar lavage fluid specimens derived from adult patients with chronic lower respiratory tract infections. Genomic analyses of IOLA have revealed that it possesses the smallest and most AT-rich genome among human-derived bacteria. However, its biological properties remain unclear because no culture method has been established for IOLA. Here, we conducted a large-scale IOLA surveillance study of nasal discharge specimens from children in Japan and investigated the correlation between IOLA detection frequency and patient characteristics. We detected IOLA in 5.4% (103 of 1,920) of pediatric nasal discharge samples. No significant differences were observed in the frequency of detection based on the patient's background. However, with respect to age, the frequency of detection tended to be significantly high at 2-3 and 6 years old. Phylogenetic analysis revealed five phylotypes in the IOLA 16S rRNA gene sequences, and the sequences detected in adult patients with respiratory infections in a previous study belonged to one of the five phylotypes. The involvement of IOLA in the symptoms is not clear, but IOLA is detected at a relatively high frequency in pediatric nasal discharge. Many subjects with detected IOLA were not always IOLA positive, and IOLA was detected transiently. Our findings suggest that IOLA is horizontally transmitted through groups in nursery and elementary schools, and there are differences in biological characteristics among the IOLA phylotypes.IMPORTANCE"The infectious organism lurking in human airways (IOLA)" is a candidate pathogenic bacterium strongly suspected to be infectious to the respiratory tracts of humans and animals. However, a culture method for IOLA has not been established yet, and its properties remain unclear. In this study, IOLA was detected at a relatively high frequency in the nasal discharge of children, and five phylotypes of IOLA were identified. One of these phylotypes was found in the bronchoalveolar lavage fluid from adult patients, suggesting lineage-specific differences in the pathogenicity of IOLA. Moreover, it was suggested that IOLA is horizontally transmitted when children gather in groups such as nursery and elementary schools. These findings strongly indicate that IOLAs have been clinically undetected so far but are spreading among children, with one lineage being involved in respiratory diseases in adults. Examining the presence of IOLA in clinical specimens may help to understand the etiology of respiratory diseases with unknown causes.

Non-purulent myositis caused by direct invasion of skeletal muscle tissue by Leptospira in a hamster model.

Myalgia is a common symptom of Leptospira infection in humans. Autopsies have reported that muscle tissue shows degeneration and necrosis of the myofibers and infiltration of inflammatory cells composed mainly of macrophages and lymphocytes. It remains unclear whether Leptospira directly infects the muscle and how the infiltrating inflammatory cells are involved in muscle fiber destruction. This study evaluated the relationship between histopathological changes and leptospiral localization in the muscle tissue of a hamster model. The influence of macrophages in skeletal muscle injury was also investigated, using selective depletion of macrophages by administration of liposomal clodronate. Hamsters infected subcutaneously with Leptospira interrogans serovar Manilae strain UP-MMC-SM showed myositis of the thighs adjacent to the inoculated area beginning at 6 days post-infection. The myositis was non-purulent and showed sporadic degeneration and necrosis of muscle fibers. The degeneration of myofibers was accompanied by aggregations of macrophages. Immunofluorescence staining revealed leptospires surrounding the damaged muscle fibers. Subcutaneous injection of formalin-killed Leptospira or intraperitoneal injection of live Leptospira caused no myositis in hamster thighs. Liposomal clodronate treatment in infected hamsters reduced macrophage infiltration in muscle tissue without impacting bacterial clearance. Muscle necrosis was still observed in the infected hamsters treated with liposomal clodronate, and there was no significant change in serum creatine kinase levels compared to those in animals treated with liposomes alone. Our findings suggest that leptospiral invasion of muscle tissue from an inoculation site leads to the destruction of muscle fibers and causes non-purulent myositis, whereas the infiltrating macrophages contribute less to muscle destruction.

Protective role of stratum corneum in percutaneous Leptospira infection in a hamster model.

Leptospira enters humans and animals through injured skin or mucous membranes by direct or indirect contact with urine excreted from infected reservoirs. Individuals with cut or scratched skin are at high risk of infection and are recommended to be protected from contact with Leptospira, but the risk of infection via skin without apparent wounds is unknown. We hypothesized that the stratum corneum of the epidermis might prevent percutaneous invasion of leptospires. We established a stratum corneum deficient model of hamsters using the tape stripping method. The mortality rate of hamsters lacking stratum corneum that were exposed to Leptospira was higher than that of controls with shaved skin, and was not significantly different from an epidermal wound group. These results indicated that the stratum corneum plays a critical role in protecting the host against leptospiral entry. We also examined the migration of leptospires through the monolayer of HaCaT cells (human keratinocyte cell line) using Transwell. The number of pathogenic leptospires penetrating the HaCaT cell monolayers was higher than that of non-pathogenic leptospires. Furthermore, scanning and transmission electron microscopic observations revealed that the bacteria penetrated the cell monolayers through both intracellular and intercellular routes. This suggested that pathogenic Leptospira can migrate easily through keratinocyte layers and is associated with virulence. Our study further highlights the importance of the stratum corneum as a critical barrier against the invasion of Leptospira found in contaminated soil and water. Hence, preventative measures against contact infection should be taken, even without visible skin wounds.

Increased copy number of 23S ribosomal RNA gene with point mutation in MRSA associated with linezolid resistance in a patient treated with long-term linezolid.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the most difficult infections we have to treat. Linezolid is one of the effective treatment options for refractory MRSA infections. There are cases where we are forced to use long-term linezolid treatment for refractory MRSA infections. OBJECTIVE: To discuss the evolution of Linezolid resistance factors in clinical isolates of MRSA. METHODS: We investigated 16 MRSA isolated from a patient treated with linezolid for a long period of 75 days. We performed antibiotic susceptibility test, 23S rRNA genes sequencing analysis, Pulsed-field gel electrophoresis. RESULTS: MRSA isolates were susceptible to linezolid before the start of treatment, but became less susceptible by prolonged treatment. The 23S rRNA sequencing analysis of linezolid-resistant strains that appeared 17 days after the start of treatment with linezolid revealed that all resistant MRSA had the G2576T substitution (Escherichia coli 23S rRNA gene number). The number of copies of this mutation increased with the use of linezolid. CONCLUSION: Long-term use of linezolid in a patient or reuse of linezolid in a patient who has been previously treated with linezolid can lead to the emerging of linezolid-resistant MRSA in the host.

Measuring the effects of respiratory protective equipment and other protectors in preventing the scattering of vocalization droplets.

This study was conducted to quantitatively examine the effects of respiratory protective equipment (respirators) and various other types of protectors in preventing the scattering of vocalization droplets. Each of 12 adult male volunteers was asked to vocalize intermittently for 1 min at a target intensity of approximately 100 dBA in an experimental room adjusted to a humidity of approximately 60-70%. The subjects vocalized while wearing respirators, other types of protectors, or no protectors at all. The droplet concentration in a particle size range of 0.3 to 10 µm was measured under each experimental condition, and the transmitted particle concentration and penetration were calculated. The concentration and penetration of particles transmitted from the respirators were lower than those transmitted from the other protectors examined. The probability of infection reduction through the use of the protectors was estimated from the data obtained on the effectiveness of the protectors in preventing the scattering of droplets. We concluded that there is no need for additional droplet scattering prevention in various work settings when appropriate respirators are used under optimal conditions.

Differences in the microbiota of oral rinse, lesion, and normal site samples from patients with mucosal abnormalities on the tongue.

The oral microbiota associated with mucosal diseases, including oral squamous cell carcinoma and oral potentially malignant disorders, have been extensively analyzed at the phylum and genus levels. However, the details of the oral microbiota remain unclear at the species and operational taxonomic unit (OTU) levels. We aimed to determine differences in the microbiota of oral rinse, lesion and normal site swab samples of patients with mucosal abnormalities on the tongues. Oral samples were obtained from 10 patients with oral mucosal abnormalities. Alpha and beta diversity at the OTU and genus levels of the microbiota samples were analyzed using OTUs clustered with 99.6% similarity based on 16S rRNA gene sequences obtained using the Sanger method. At the OTU level, the microbiota of the lesions were the least diverse but were different from those of the normal site and oral rinse samples. The OTUs corresponding to Streptococcus infantis and Haemophilus parainfluenzae were suggested to contribute to the differences between the microbiota of the lesions and normal sites. At the genus level, no significant differences between these microbiota were observed. In conclusion, strict OTU-level microbiota analysis might be able to discriminate lesions from normal sites of patients with mucosal abnormalities.

Helicobacter pylori results in lysis and death after exposure to water.

BACKGROUND: Helicobacter pylori has a high infection rate, and it is possible that more than half of the world's population is infected. The route of transmission of H. pylori has not been completely elucidated yet. The coccoid form of H. pylori is generally considered to be in a VBNC (viable but nonculturable) state, and this form in the environment is thought to play an important role in infection and transmission, but its stability and survivability are still unknown. MATERIALS AND METHODS: In order to promote its changing to coccoid form, the spiral form of H. pylori grown in a culture medium was exposed to sterile distilled water, and we investigated the bacterial cell number and the morphological changes by using fluorescence staining methods and electron microscopic observation. We also examined the dynamics of its growth ability by measuring the colony forming unit on an agar-plate medium. RESULTS: After exposure to sterile distilled water, the H. pylori spiral form rapidly lost its growth ability at 37°C. One day after exposure, approximately 95% of the spiral form disappeared and the proportion of the coccoid form increased. The total number of bacteria also decreased to less than half and continued to decrease over time. Epi-microscopic and electron microscopic observations revealed that deformation of bacterial cells, collapse, and leaking out of cell contents were promoted in exposure to sterile distilled water. CONCLUSION: Helicobacter pylori quickly begins to transform into the coccoid form after exposure to sterile distilled water, rapidly loses its growth ability, and then lyses and dies. Water-exposure is lethal for H. pylori and it is unlikely to survive in the VBNC state in water.

Dissemination of Leptospira into the intestinal tract resulting in fecal excretion in a hamster model of subcutaneous infection with Leptospirainterrogans.

Leptospirosis, caused by pathogenic Leptospira, is one of the most common zoonotic diseases in the world. It is transmitted to humans through the skin and mucous membranes by contact with water or soil contaminated with urine excreted from infected animals. In human infections, gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea have been frequently observed, but there have been no reports analyzing gastrointestinal lesions in leptospirosis, and the pathological mechanism of gastrointestinal symptoms in leptospirosis remains unclear. In this study, we investigated the pathological changes and the distribution of leptospires in the intestinal wall, and the presence of leptospires in the intestinal contents and feces, of hamsters subcutaneously infected with Leptospira interrogans. Results showed that infected hamsters had macroscopic redness in the jejunum and ileum. Submucosal hemorrhage was observed histologically, and there was no infiltration of inflammatory cells such as neutrophils. There were no obvious changes in the colon, either macroscopically or histologically, and the feces were normal (solid stools). Leptospira was isolated from all the intestinal walls from the small intestine to the colon, the intestinal contents, and the feces. These findings suggest that the invasion of leptospires into the intestinal wall and the associated submucosal hemorrhage may be the cause of the gastrointestinal symptoms observed in leptospirosis. Furthermore, not only the urine of infected animals but also the feces could be a source of infection.

Leptospira Is an Environmental Bacterium That Grows in Waterlogged Soil.

Leptospirosis is a zoonotic disease caused by infection with pathogenic leptospires. Consistent with recent studies by other groups, leptospires were isolated from 89 out of 110 (80.9%) soil or water samples from varied locations in the Philippines in our surveillance study, indicating that leptospires might have a life cycle that does not involve animal hosts. However, despite previous work, it has not been confirmed whether leptospires multiply in the soil environment under various experimental conditions. Given the fact that the case number of leptospirosis is increased after flood, we hypothesized that waterlogged soil, which mimics the postflooding environment, could be a suitable condition for growing leptospires. To verify this hypothesis, pathogenic and saprophytic leptospires were seeded in the bottles containing 2.5 times as much water as soil, and bacterial counts in the bottles were measured over time. Pathogenic and saprophytic leptospires were found to increase their number in waterlogged soil but not in water or soil alone. In addition, leptospires were reisolated from soil in closed tubes for as long as 379 days. These results indicate that leptospires are in a resting state in the soil and are able to proliferate with increased water content in the environment. This notion is strongly supported by observations that the case number of leptospirosis is significantly higher in rainy seasons and increased after flood. Therefore, we reached the following conclusion: environmental soil is a potential reservoir of leptospires. IMPORTANCE Since research on Leptospira has focused on pathogenic leptospires, which are supposed to multiply only in animal hosts, the life cycle of saprophytic leptospires has long been a mystery. This study demonstrates that both pathogenic and saprophytic leptospires multiply in the waterlogged soil, which mimics the postflooding environment. The present results potentially explain why leptospirosis frequently occurs after floods. Therefore, environmental soil is a potential reservoir of leptospires and leptospirosis is considered an environment-borne as well as a zoonotic disease. This is a significant report to reveal that leptospires multiply under environmental conditions, and this finding leads us to reconsider the ecology of leptospires.

Literature review to integrate information to assist workers to select masks even at workplaces without occupational health personnel.

BACKGROUND: With the COVID-19 pandemic, the idea of universal mask wearing to prevent infecting others when one becomes infected has prevailed among people. In general, any workplace is not exempt and workers are required to wear a mask while working at the sites. OBJECTIVES: This study aims to integrate information to assist workers to select effective protectors for the prevention of droplet infection even at workplaces without occupational health personnel. METHODS: A total of 94 studies were included in this study: 91 studies were identified in MEDLINE, which was used for the literature search, and an additional three studies were identified from other information sources. The studies were checked to eliminate duplication and narrowed down to 31 based on the titles and abstracts. The contents of the 31 studies were read through and then 19 studies were extracted for careful reading. RESULTS AND CONCLUSIONS: Regarding the protectors used at workplaces, it was suggested that (1) workers continue to use respiratory protectors as needed at sites where respiratory protectors such as an N95 respirator had to be used even before the spread of COVID-19 and (2) wear surgical masks, multi-layer cloth masks, or hybrid fabric masks made of several types of fabrics that are recommended in terms of preventing dissemination of droplets and protecting against inhalation of droplets, selected according to the working conditions, taking account of air permeability, breathability, and durability.

A machine learning model of microscopic agglutination test for diagnosis of leptospirosis.

Leptospirosis is a zoonosis caused by the pathogenic bacterium Leptospira. The Microscopic Agglutination Test (MAT) is widely used as the gold standard for diagnosis of leptospirosis. In this method, diluted patient serum is mixed with serotype-determined Leptospires, and the presence or absence of aggregation is determined under a dark-field microscope to calculate the antibody titer. Problems of the current MAT method are 1) a requirement of examining many specimens per sample, and 2) a need of distinguishing contaminants from true aggregates to accurately identify positivity. Therefore, increasing efficiency and accuracy are the key to refine MAT. It is possible to achieve efficiency and standardize accuracy at the same time by automating the decision-making process. In this study, we built an automatic identification algorithm of MAT using a machine learning method to determine agglutination within microscopic images. The machine learned the features from 316 positive and 230 negative MAT images created with sera of Leptospira-infected (positive) and non-infected (negative) hamsters, respectively. In addition to the acquired original images, wavelet-transformed images were also considered as features. We utilized a support vector machine (SVM) as a proposed decision method. We validated the trained SVMs with 210 positive and 154 negative images. When the features were obtained from original or wavelet-transformed images, all negative images were misjudged as positive, and the classification performance was very low with sensitivity of 1 and specificity of 0. In contrast, when the histograms of wavelet coefficients were used as features, the performance was greatly improved with sensitivity of 0.99 and specificity of 0.99. We confirmed that the current algorithm judges the positive or negative of agglutinations in MAT images and gives the further possibility of automatizing MAT procedure.

Correlation between renal distribution of leptospires during the acute phase and chronic renal dysfunction in a hamster model of infection with Leptospira interrogans.

BACKGROUND: Leptospirosis has been described as a biphasic disease consisting of hematogenous dissemination to major organs in the acute phase and asymptomatic renal colonization in the chronic phase. Several observational studies have suggested an association between leptospirosis and chronic kidney disease (CKD). We investigated the dynamics of leptospires and histopathological changes in the kidney to understand the relationship between them, and also investigated the extent of renal dysfunction in the acute and chronic phases of leptospirosis using a hamster model. FINDINGS: Hamsters (n = 68) were subcutaneously infected with 1 × 104 cells of the Leptospira interrogans serovar Manilae strain UP-MMC-SM. A total of 53 infected hamsters developed fatal acute leptospirosis, and the remaining 15 hamsters recovered from the acute phase, 13 of which showed Leptospira colonization in the kidneys in the chronic phase. Five asymptomatic hamsters also had renal colonization in the chronic phase. Immunofluorescence staining showed that leptospires were locally distributed in the renal interstitium in the early acute phase and then spread continuously into the surrounding interstitium. The kidneys of the surviving hamsters in the chronic phase showed patchy lesions of atrophic tubules, a finding of chronic tubulointerstitial nephritis, which were substantially consistent with the distribution of leptospires in the renal interstitium. The degree of atrophic tubules in kidney sections correlated statistically with the serum creatinine level in the chronic phase (rs = 0.78, p = 0.01). CONCLUSION: Subcutaneous infection with pathogenic leptospires could cause acute death or chronic leptospirosis in hamsters after surviving the acute phase. We suggest that the renal distribution of leptospires during the acute phase probably affected the extent of tubular atrophy, leading to CKD.

Bacterial composition of nasal discharge in children based on highly accurate 16S rRNA gene sequencing analysis.

Nasopharyngeal colonization by bacteria is a prerequisite for progression to respiratory disease and an important source of horizontal spread within communities. We aimed to perform quantitative analysis of the bacterial cells and reveal the microbiota of the nasal discharge in children at the species level based on highly accurate 16S rRNA gene sequencing. This study enrolled 40 pediatric patients with rhinorrhea. The bacterial cells in the nasal discharge were counted by epifluorescence microscopic analysis. The microbiota was analyzed by using the 16S rRNA gene clone library sequencing method. We demonstrated that a high abundance (median 2.2 × 107 cells/mL) of bacteria was contained in the nasal discharge of children. Of the 40 samples, 37 (92.5%) were dominated by OTUs corresponding to Haemophilus aegyptius/influenzae, Moraxella catarrhalis/nonliquefaciens, or Streptococcus pneumoniae. These samples showed higher cell abundance and lower alpha diversity than the remaining three samples in which the other bacteria coexisted. In addition, 12 sequences with low homology to type strains were considered as previously unknown bacterial lineages. In conclusion, the nasal discharge of most young children contains a large amount of respiratory pathogens and several unknown bacteria, which could not only cause endogenous infection but also be a source of transmission to others.

Limitations of Ribotyping as Genotyping Method for Corynebacterium ulcerans.

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate's ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

In vitro characteristics of intravaginal Lactobacilli; why is L. iners detected in abnormal vaginal microbial flora?

PURPOSE: Our purpose is to investigate the reasons why Lactobacillus iners is detected in abnormal vaginal microbial flora. METHODS: In this study, in vitro characteristics of four type strains (L. crispatus, L. iners, L. gasseri, and L. jensenii) were examined by measuring the growth speed by OD660, and acid resistance, with gram stain and Live/Dead stain. RESULTS: The growth speed was L. gasseri > L. jensenii > L. crispatus > L. iners. Bacterial counts of all Lactobacilli in MRS medium began to decrease at the middle of the log-phase of the growth curve. In addition, L. iners grew to 106 CFU/mL and the others grew to 108 CFU/mL. L. iners was mostly Gram-negative with very short rod, while the others were mostly Gram-positive rods. L. iners was completely killed in the pH 3 medium, however, the others grew (in pH 3 medium) in 1/100 order compared with those in the pH 6 medium. CONCLUSION: L. iners was not a typical gram-positive long rod Lactobacilli and presented weak acid-resistance. The reasons why L. iners is detected in abnormal vaginal microbial flora were presumed to be due to the unique morphologic and microbiologic characteristics.

Coinfection With Multiple Nontuberculous Mycobacteria as a Possible Exacerbating Factor in Pulmonary Nontuberculous Mycobacteriosis: Clone Library Analysis Using the 16S Ribosomal RNA Gene.

BACKGROUND: Mycobacterial culture is the gold standard for the diagnosis of nontuberculous Mycobacterium (NTM) infections. However, this method is not suitable for detection of coinfection with different NTMs. RESEARCH QUESTION: The goal of this study was to determine if clone library analysis of BAL fluid (BALF) was useful for detection of NTM phylotypes, including multiple NTM phylotypes, in pulmonary NTM infections. STUDY DESIGN AND METHODS: BALF samples obtained from 120 patients with suspected pulmonary NTM infections were retrospectively evaluated by using the mycobacterial culture and clone library methods between July 2010 and August 2016. RESULTS: In total, 55 (45.8%) patients were diagnosed as NTM positive according to results of mycobacterial culture, and 52 patients were NTM positive as determined by using the clone library method. Furthermore, 45 (86.5%) and seven (13.5%) patients exhibited a single phylotype (mono-phylotype group) and multiple phylotypes of NTM (multi-phylotype group), respectively. Compared with the mono-phylotype group, the multi-phylotype group had a significantly higher incidence of adverse chest CT findings (P = .048). In addition, 11 patients who were NTM negative according to results of BALF mycobacterial culture were determined to be NTM positive according to the clone library method. Six of these 11 patients were eventually diagnosed as NTM positive by using mycobacterial culture results within 6.2 ± 2.1 months following the initial sample collection. INTERPRETATION: Coinfection multiple phylotypes could be associated with adverse clinical findings. In addition, patients who test positive for NTM genes but negative for mycobacterial culture may be diagnosed with NTM lung infection within 1 year of the initial sample collection. Further follow-up of these patients may facilitate early detection of NTM species.

Acquisition of genetic mutations in Group A Streptococci at infection site and subsequent systemic dissemination of the mutants with lethal mutations in a streptococcal toxic shock syndrome mouse model.

Streptococcal toxic shock syndrome (STSS) is caused mainly by Streptococcus pyogenes (Group A Streptococci, GAS), and it has a fatality rate of 25%. Mutations in CsrRS and RopB, which suppress the transcription of many virulence factors, were recently found in clinical isolates from STSS patients, but it is not fully understood when and where GAS acquires the mutations in the host. To resolve this question, we used our mouse model of human STSS to recover GAS strains from injections sites, spleens and blood of moribund mice with STSS-like symptoms, and analyzed the sequence of the covR/covS genes and ropB gene that encode CsrRS and RopB. Fifteen out of twenty mice that were inoculated transdermally into muscles with GAS organisms became moribund with STSS-like symptoms after more than 20 days after inoculation. We found that all the disseminated GAS strains recovered from the blood and spleens of the moribund mice had mutations in either the covR genes or the covS genes. The mutation sites in the GAS strains recovered from the blood and spleen were identical in each mouse, whereas the strains recovered from the muscles included a mix of disseminated strains, other mutant strains, and the parent strain. The mutant strains killed mice significantly earlier than the parent strain. Our data indicated that GAS organisms remained at the injection site, and various mutants appeared there, among which the strain that acquires the mutation in the covR/S gene is expected to overexpress various virulence factors simultaneously and cause systemic infection such as STSS.

Clinical characteristics of patients with bacterial pleuritis in the presence of Streptococcus anginosus group and obligate anaerobes detected by clone library analysis.

INTRODUCTION: Bacterial pleuritis is one of the most important pleural and respiratory infectious diseases, in addition, there have been no reports describing the clinical characteristics of patients with bacterial pleuritis according to molecular methods. An accurate understanding of the clinical characteristics and etiology of bacterial pleuritis is an issue that must be addressed. OBJECTIVES: The aim of this study was to clarify the clinical characteristics of the bacterial species in bacterial pleuritis. METHODS: Pleural effusion samples were obtained from 29 patients with bacterial pleuritis. The microbiota of pleural effusion samples was analyzed by clone library analysis using the 16S ribosomal RNA gene. RESULTS: The phylotypes of Fusobacterium spp. (24.1%) were most frequently the predominant phylotypes, followed by those of Streptococcus anginosus group (SAG) (20.7%) and S. aureus (17.2%). The predominant phylotypes of obligate anaerobes, including the Fusobacterium spp., were detected in 11 of 29 patients (37.9%). Patients in the SAG group were significantly older and presented lower serum albumin levels than those in the obligate anaerobe and other bacterial groups. Patients from the obligate anaerobe group took longer to present symptoms, and therefore the diagnosis of pleuritis was also delayed, in comparison to patients in the other bacterial groups. CONCLUSIONS: Our results demonstrated that there were characteristic differences between patients in SAG, obligate anaerobe and other bacterial groups. Physicians may need to consider treatment strategy options based on the clinical characteristics of patients with bacterial pleuritis.

Dynamics of microbiota during mechanical ventilation in aspiration pneumonia.

BACKGROUND: The emergence of multi-drug resistant pathogens is an urgent health-related problem, and the appropriate use of antibiotics is imperative. It is often difficult to identify the causative bacteria in patients with aspiration pneumonia because tracheal aspirate contains contaminants of oral bacteria. We investigated the dynamics of microbiota in mechanically ventilated patients with aspiration pneumonia to develop a treatment strategy. METHODS: Twenty-two intubated patients with aspiration pneumonia were recruited. Saliva and tracheal aspirate of the subjects were collected at three time points: (A) within 2 h after intubation, (B) just before administration of antibiotics, and (C) 48-72 h after administration of antibiotics. The microbiota in each specimen was analyzed by using the 16S rRNA gene clone library sequencing method. Bacterial floras of the samples were analyzed by principal component analysis. RESULTS: Principal component analysis based on the composition of genus revealed that although the changes of microbiota in the saliva from (A) to (B) were not clear, the composition of anaerobes in the tracheal aspirate (B) was lower than (A). In fact, the reduction of anaerobes, not in the saliva but in the tracheal aspirate from (A) to (B), was confirmed by incident rate ratios estimated by a multilevel Poisson regression model (p < 0.001). The extent of decrease in anaerobes was fully dependent on the time difference between the sampling of tracheal aspirate (A) and (B)-in particular, over 3 h of mechanical ventilation. This indicates that the alterations of microbiota (involving the reduction of anaerobes in the lower respiratory tract) occurred during mechanical ventilation prior to the administration of antibiotics. After the administration of antibiotics, Enterobacter spp., Corynebacterium spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Granulicatera adiacens were predominantly detected in the tracheal aspirate (C). CONCLUSION: The microbiota of the lower respiratory tract changes dynamically during mechanical ventilation and during the administration of antibiotics in intubated patients with aspiration pneumonia. Antibiotics should be selected on the premise that dynamic changes in microbiota (involved in the reduction of anaerobes) may occur during the mechanical ventilation in these patients.

Mutation and Diversity of Diphtheria Toxin in Corynebacterium ulcerans.

Corynebacterium ulcerans infection is emerging in humans. We conducted phylogenetic analyses of C. ulcerans and C. diptheriae, which revealed diverse diphtheria toxin in C. ulcerans. Diphtheria toxin diversification could decrease effectiveness of diphtheria toxoid vaccine and diphtheria antitoxin for preventing and treating illnesses caused by this bacterium.