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.

Environmental factors related to differences in the microbiota in the upper respiratory tract in young children: Focusing on the impact of early nursery attendance.

Background: Microbial colonization of the upper respiratory tract (URT) during the first years of life differs significantly according to environmental factors. We investigated the association between early nursery attendance, URT infection (URTI) and drugs used for its treatment and the differences in the URT microbiota. Methods: This prospective study included 33 young children (11 and 22 with and without nursery attendance during their infancy, respectively). URT secretions were collected from the nasopharynx of these children at 2, 4, 6, 12, 18 and 24 months old. Clinical information after the latest sampling, including histories of URTI and the uses of antibiotics or cold medicines, was collected from all children. URT bacteria were identified by a clone library analysis of the 16S rRNA gene. Results: In the diversity of URT microbiota using the Shannon index, we did not detect any associations between variations in the URT microbiota and environmental factors (nursery attendance, development of URTIs, or the uses of antibiotics or cold medicines). However, in a clustering analysis, the proportion of the samples classified as Corynebacterium propinquum-dominant cluster was significantly lower in children ≥6 months old with nursery attendance than in those without nursery attendance. In addition, the URT microbiota was significantly different between samples from children ≥6 months old with and without a history of ≥3 URTI episodes after the first sampling. Furthermore, the URT microbiota was also significantly different between samples from these children with and without antibiotic use between the previous and present samplings. Conclusion: Early nursery attendance and its related factors, including the frequency of URTI and antibiotic treatment, may be associated with the differences in the URT flora in young children.

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.

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.

The optimal duration of antimicrobial therapy for lower respiratory tract infection in patients with neuromuscular disorders based on a clone library analysis of the bacterial 16S rRNA gene sequence.

OBJECTIVES: The aim of this study is to determine the optimal duration of antimicrobial therapy for lower respiratory tract infection (LRTI) in neuromuscular disorder (NMD) patients. METHODS: This prospective study included 13 episodes from 9 NMD patients hospitalized for bacterial LRTI. Sputum samples were collected from these patients during the three consecutive days after their admission. Bacterial cell counts and the proportion of the most predominant bacterium identified by a clone library analysis of the bacterial 16S rRNA gene sequence in the samples obtained before antimicrobial therapy were serially investigated. RESULTS: All episodes were initially treated with ampicillin/sulbactam. In 12 episodes with a therapeutic effect, the bacterial cell counts in the samples obtained on the third day of therapy were significantly lower than those before therapy (P = 0.0013). In most of these episodes, the most predominant bacterium in the sample obtained before therapy was undetectable by the third day of therapy. In the one patient without a therapeutic effect, neither the bacterial cell counts nor the proportion of the most predominant bacterium in the sample obtained before therapy decrease after therapy. CONCLUSION: Short-term antimicrobial therapy is sufficiently effective for LRTI in NMD patients if the initial therapy is effective.

Sex Differences in the Prevalence and Severity of Exercise-Induced Bronchoconstriction in Kindergarteners in Japan.

BACKGROUND: Exercise-induced bronchoconstriction (EIB), a strong positive predictor of asthma, becomes progressively less frequent with age. Although asthma tends to become less common only in boys during adolescence, sex differences in EIB, especially in preschoolers, remain unclear. To find EIB for early diagnosis and intervention asthma, mass-screening tests considering sex differences in preschoolers are needed. In this study, we investigated whether sex differences influence the prevalence and severity of EIB in prepubertal children aged 5-6 years. METHODS: Fifty-one children aged 5-6 years who were attending a kindergarten in Matsuyama City, Ehime, Japan, were enrolled in this cross-sectional study. The children underwent a 6-minute free-running test in 2015. The peak expiratory flow rate (PEFR) was measured before exercise and 0, 3, 10, and 20 minutes after exercise. The severity of EIB was classified according to the reduction in PEFR, measured as the difference between the postexercise PEFR and the highest pre-exercise PEFR. RESULTS: Of the 51 children (23 boys and 28 girls) enrolled, the prevalence of EIB defined as three criteria: a ≥15%, ≥20%, or ≥25% decrease was 54.9% (28/51), 41.2% (21/51), and 25.5% (13/51), respectively. The prevalence of EIB defined as ≥25% decrease was significantly higher in girls than in boys (39.3% vs 8.7%, P = .013). In girls, the mean percentage change in PEFR was significantly higher 20 minutes than 10 minutes postexercise (P = .043). CONCLUSIONS: Sex difference in the prevalence and severity of EIB should be considered when evaluating EIB, even in young, prepubertal children.

The microbiological characteristics of lower respiratory tract infection in patients with neuromuscular disorders: An investigation based on a multiplex polymerase chain reaction to detect viruses and a clone library analysis of the bacterial 16S rRNA gene sequence in sputum samples.

We performed gene amplification methods for the detections of bacteria and viruses using sputum samples to clarify the microbiological characteristics of lower respiratory tract infection in patients with neuromuscular disorders. The tendencies of higher proportion of respiratory virus detection and lower diversity of bacteria in sputum were observed.

Utility of the Performance of Lumbar Puncture for Febrile Children With Status Epilepticus in the Bacterial Conjugate Vaccine Era.

BACKGROUND: The necessity of lumbar puncture for the differentiation of central nervous system infection in children with febrile status epilepticus (FSE) remains controversial. The aim of the present study is to investigate the diagnostic necessity of lumbar puncture for children with FSE after the introduction of bacterial conjugate vaccines into the infant immunization program. METHODS: Our retrospective cohort study was performed in children 6 to 60 months of age who were admitted to the pediatric ward at Kitakyushu General Hospital from January 1, 2011, to December 31, 2015, for seizures with fever. Of these patients, we compared the clinical characteristics between the patients initially diagnosed as having FSE and complex febrile seizures excluding FSE (non-FSE). RESULTS: Of the eligible 333 children, 98 and 235 children were classified into FSE and non-FSE groups, respectively. The proportion of the children who underwent lumbar puncture in FSE group was significantly higher than that in non-FSE group (P < 0.0001). On the other hand, no significant difference in the proportion of the patients with abnormal cerebrospinal fluid findings was seen between the 2 groups. There was no patient with bacterial meningitis. The proportion of patients with encephalitis/encephalopathy in FSE group (n = 7, 7.1%) was significantly higher than that in non-FSE group (n = 0, P = 0.0003). However, only 1 of 6 patients with encephalitis/encephalopathy who underwent lumber puncture showed pleocytosis in cerebrospinal fluid. CONCLUSIONS: Routine lumbar puncture for the differentiation of severe central nervous system infection cannot be recommended in children with FSE.

An infant case with hydrocephalus as the initial manifestation of Mycoplasma hominis-associated meningitis.

We report an infant with hydrocephalus as the initial manifestation of Mycoplasma hominis-associated meningitis, who recovered without appropriate antimicrobial treatment. The analysis of the 16S rRNA gene by polymerase chain reaction amplification using universal primers and pathogen-specific primers was useful for the diagnosis and the investigation of serial detection status of the pathogen. This method may be helpful for the assessment of the frequency and the prediction of severity in M. hominis-associated central nervous system infection in infants, and investigating the association between M. hominis and the development of hydrocephalus.

Difference between Japanese Secondary and Tertiary Medical Facilities Regarding Changes in the Hospitalization of Children for Pneumonia after the Introduction of the Pneumococcal Conjugate Vaccine.

This study aimed to compare hospitalization of children for pneumonia between secondary and tertiary medical facilities, which hospitalize many children without and with underlying diseases, respectively, after the introduction of the pneumococcal conjugate vaccine (PCV). Our retrospective study included children admitted to the Department of Pediatrics at Kitakyushu General Hospital, a secondary medical facility, and the Hospital of the University of Occupational and Environment Health, Japan, a tertiary medical facility, from 2009 to 2013 for pneumonia. We compared the change in the rate of hospitalization for pneumonia after the introduction of the 7-valent PCV between the secondary and tertiary medical facilities. Hospitalization of patients with pneumonia declined by 28.8% in our secondary medical facility. In particular, hospitalization for pneumonia other than confirmed mycoplasmal or viral pneumonia was significantly reduced by 49.2%. In contrast, hospitalization of patients with pneumonia did not decline in our tertiary medical facility. After the introduction of PCV, hospitalization of children for pneumonia was not reduced at the tertiary medical facility. Various other pathogens besides pneumococcus may be associated with the development of pneumonia in children with underlying diseases.