An Unclassified Microorganism: Novel Pathogen Candidate Lurking in Human Airways

<div><p>During the assessments of the correlation of the diseases and the microbiota of various clinical specimens, unique 16S ribosomal RNA (rRNA) gene sequences (less than 80% similarity to known bacterial type strains) were predominantly detected in a bronchoalveolar lavage fluid (BALF) specimen from a patient with chronic lower respiratory tract infection. The origin of this unique sequence is suspected to be the causative agent of the infection. We temporarily named the owner organism of this sequence “IOLA” (Infectious Organism Lurking in Airways). In order to evaluate the significance of IOLA in human lung disorders, we performed several experiments. IOLA-16S rRNA genes were detected in 6 of 386 clone libraries constructed from clinical specimens of patients with respiratory diseases (in our study series). The gene sequences (1,427 bp) are identical, and no significantly similar sequence was found in public databases (using NCBI blastn) except for the 8 shorter sequences detected from patients with respiratory diseases in other studies from 2 other countries. Phylogenetic analyses revealed that the 16S rRNA gene of IOLA is more closely related to eukaryotic mitochondria than bacteria. However, the size and shape of IOLA seen by fluorescent <i>in-situ</i> hybridization are similar to small bacteria (approximately 1 µm with a spherical shape). Furthermore, features of both bacteria and mitochondria were observed in the genomic fragment (about 19 kb) of IOLA, and the GC ratio of the sequence was extremely low (20.5%). Two main conclusions were reached: (1) IOLA is a novel bacteria-like microorganism that, interestingly, possesses features of eukaryotic mitochondria. (2) IOLA is a novel pathogen candidate, and it may be the causative agent of human lung or airway disease. IOLA exists in BALF specimens from patients with remarkable symptoms; this information is an important piece for helping solve the elusive etiology of chronic respiratory disorders.</p></div

Patient characteristics.

a<p>WBC; white blood cell,</p>b<p>CRP; C-reactive protein,</p>c<p>CT; computed tomography,</p>d<p>GRNX; Garenoxacin,</p>e<p>MEPM; Meropenem,</p>f<p>N/A; not applicable.</p

Blast search results of the CDSs on IOLA genomic fragment.

<p>Blast search results of the CDSs on IOLA genomic fragment.</p

GC content and annotation of IOLA genomic fragment (18,834 bp).

<p>High GC contents per 500-base pairs (kbp). The black box represents a 16S rRNA gene. The other boxes represent ORFs. The shaded boxes show similarities (amino-acid sequence) with known bacterial proteins. Annotation results of the ORFs are indicated via arrows. White boxes represent products of ORFs showing extremely low similarities with known proteins.</p

Bacterial cell numbers and compositions in the specimens from patients, detected IOLA in clone library analysis.

<p><b>A,</b> Results of bacterial cell counts of the specimens from patient A using an epifluorescent staining method with ethidium bromid. Open circles indicate numbers of bacterial cells per ml of each the specimens. <b>B,</b> Percentage of the detected bacteria in the specimens with the clone library analysis of 16S rRNA gene. The percentages of IOLA-clones (orange box) in each of the clone libraries are shown in parentheses.</p

Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of bacteria and mitochondria of eukaryota.

<p>The phylogenetic tree was calculated with MEGA5.2.2 using the maximum likelihood method. The tree is based on the alignment of the 71 sequences (16S rRNA gene sequences). The percentages of bootstrap values for 1,000 replications are shown at the branching points (values less than 50% were ignored). The scale bar indicates substitutions per site. A sequence of chloroplast of <i>Zea mays</i> was used as an outgroup. Accession numbers of each sequence are shown in parentheses.</p

Size estimation of the IOLA cells by filtration and assessment of 18S rRNA gene.

<p>In order to estimate the size of IOLA, the PCR examinations were conducted by extracting and purifying the DNAs from the filtrates series of the BALF (A3). <b>A,</b> PCR results using a bacterial universal primer set (E341F and E907R). <b>B,</b> PCR results using an IOLA-specific primer set (IOLA-F1N and IOLA-R0N). <b>C,</b> PCR results using a primer set (18S_0067a_deg and NSR 399) for 18S rRNA genes of various eukaryotic groups. <b>D,</b> PCR results using a primer set (Human-P and NSR 399) for human 18S rRNA gene. “Control” indicates no template control reaction. The DNA extracted from <i>Candida albicans</i> was used as a positive/negative reaction control of the PCRs for 18S rRNA gene.</p