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

Bacteriological Assessment of Healthcare-Associated Pneumonia Using a Clone Library Analysis

<div><p>Background</p><p>The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a true causative pathogen of HCAP.</p><p>Methods</p><p>We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed.</p><p>Results</p><p>Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. <i>Staphylococcus aureus</i> was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method.</p><p>Conclusions</p><p>The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.</p></div

Clinical and laboratory features of the 82 patients with HCAP.

<p>Data are presented as n (%) or mean ± SD unless otherwise stated.</p><p><i>Definition of abbreviations</i>: HCAP, healthcare-associated pneumonia; BMI, body mass index; BP, blood pressure; SpO₂, pulse oximetric saturation; PaO₂, partial pressure of arterial oxygen; BUN, blood urea nitrogen; PSI, pneumonia severity index; SD, standard deviation</p><p>^§1BMI was evaluated in 59 patients</p><p>^§2Respiratory rate was evaluated in 68 patients</p><p>^§3Arterial blood gas analysis was performed in 51 patients</p><p>^§4 0, can be active without any problems or limitations, daily life the same as before the onset; 1, intense activity limited, but can walk and perform light work or work while sitting; 2, can walk and perform all personal care, but cannot work; more than 50% of daytime hours out of bed; 3, can only do limited personal care; more than 50% of daytime hours spent in bed or chair; 4, cannot move at all or perform personal care, all day spent in bed or chair</p><p>Clinical and laboratory features of the 82 patients with HCAP.</p

Blast search results of the CDSs on IOLA genomic fragment.

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

Comparison of the predominat phylotype and the rates of phylotypes of streptococci or anaerobes.

<p>A) With respect to streptococci, the rate of a predominant phylotype was significantly higher in the patients equal to or older than 75 years of age (the rates of streptococci in the patients younger than 75 years old and equal to or older than 75 years of age were 12.1% and 31.9%, respectively; P<0.05); B) The rates of the phylotypes in each microbiota were significantly higher in the patients equal to or older than 75 years of age (<75 vs ≥75 were 11.0 ± 27.0% and 29.5 ± 34.1%, respectively; P<0.05); C-D) Regarding anaerobes, the rate of the predominant phylotype (<75 vs ≥75 were 11.8% vs 8.7%, respectively; P = 0.46) and the phylotypes in each microbiota (<75 vs ≥75 were 12.7 ± 26.5% vs 10.2 ± 23.1%, respectively; P = 0.66) were not significantly different between the two age groups.</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

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

Additional file 1: Table S1. of The clinical features of respiratory infections caused by the Streptococcus anginosus group

The clinical and laboratory features of patients with pneumonia/lung abscess with pleural effusion and bacterial pleurisy only. Table S2. The clinical and laboratory features of patients with Streptococcus anginosus group infections. (DOCX 28 kb

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