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

Abstract

"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.

Keywords: 16S rRNA gene; infectious disease; uncultured bacterium.

Fig 1

Comparison of the numbers of IOLA-positive specimens detected in each month and each clinic. The numbers of specimen with IOLA-positive PCR result were shown. The upper row represents the results for 2018, while the lower row represents the results for 2019. The left column represents the results for clinic A in Kitakyushu City, and the right column represents the results for clinic B in Fukuoka City. A total of 40 specimens were examined per month at each clinic.

Fig 2

Frequency of IOLA-specific PCR-positive specimens by patient age. The difference between the numbers of IOLA-positive and IOLA-negative patients with respect to patient age was confirmed by Pearson’s χ² test (P < 0.01). Gray boxes indicate the IOLA-positive rate for each age group. The numbers indicate the positive rate and residual analysis results (two-sided P value), and the numbers in parentheses indicate the number of specimens (IOLA-positive/total specimens). Red numbers indicate ages with significantly higher IOLA-positive rates. The number of IOLA-positive and IOLA-negative specimens at each age is also shown.

Fig 3

Phylogenetic analysis of IOLA-16S rRNA gene sequences obtained from nasal discharge of children and from BALF specimens of adult patients with respiratory diseases. (A) ML phylogenetic tree of IOLA-16S rRNA gene sequences. Sample names of nasal discharge obtained from children Kitakyushu City are shown in blue, and samples obtained in Fukuoka City are shown in red. Black letters indicate specimen names obtained from the BALF of adult respiratory disease patients obtained in previous studies. Bootstrap analysis results are shown beside each branch. (B) Sequence similarity profile for each IOLA phylotype. (C) Transition of IOLA-specific PCR results in IOLA-positive patients who visited the hospital multiple times. Numbers indicate the number of months that have passed since the first visit was set to 0. Numbers in parentheses indicate sample numbers. Colored boxes indicate IOLA-positive specimens, and color indicates each phylotype.