PLoS One. 2013 May 6;8(5):e63103.

Significance of anaerobes and oral bacteria in community-acquired pneumonia.

Yamasaki K, Kawanami T, Yatera K, Fukuda K, Noguchi S, Nagata S, Nishida C, Kido T, Ishimoto H, Taniguchi H, Mukae H.

Department of Respiratory Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan.

 

ABSTRACT:

Background: Molecular biological modalities with better detection rates have been applied to identify the bacteria causing infectious diseases. Approximately 10–48% of bacterial pathogens causing community-acquired pneumonia are not identified using conventional cultivation methods. This study evaluated the bacteriological causes of community-acquired pneumonia using a cultivation-independent clone library analysis of the 16S ribosomal RNA gene of bronchoalveolar lavage specimens, and compared the results with those of conventional cultivation methods.

Methods: Patients with community-acquired pneumonia were enrolled based on their clinical and radiological findings. Bronchoalveolar lavage specimens were collected from pulmonary pathological lesions using bronchoscopy and evaluated by both a culture-independent molecular method and conventional cultivation methods. For the culture-independent molecular method, approximately 600 base pairs of 16S ribosomal RNA genes were amplified using polymerase chain reaction with universal primers, followed by the construction of clone libraries. The nucleotide sequences of 96 clones randomly chosen for each specimen were determined, and bacterial homology was searched. Conventional cultivation methods, including anaerobic cultures, were also performed using the same specimens.

Results: In addition to known common pathogens of community-acquired pneumonia [Streptococcus pneumoniae (18.8%), Haemophilus influenzae (18.8%), Mycoplasma pneumoniae (17.2%)], molecular analysis of specimens from 64 patients with community-acquired pneumonia showed relatively higher rates of anaerobes (15.6%) and oral bacteria (15.6%) than previous reports.

Conclusion: Our findings suggest that anaerobes and oral bacteria are more frequently detected in patients with community-acquired pneumonia than previously believed. It is possible that these bacteria may play more important roles in community-acquired pneumonia.

 

SUPPLEMENT:

Recent molecular techniques have been applied to identify pathologic bacteria in cases of infectious disease, with higher detection rates compare to that achieved with ordinary culture methods. Evaluations of the sequences of 16S ribosomal RNA belonging to bacteria only can be used to detect causative bacteria independent of bacterial cultures. The utility of these methods have been reported in patients with cystic fibrosis1 and ventilator-associated pneumonia 2 and other respiratory infections, indicating that cultivating anaerobes as causative bacteria is usually difficult in patients with these respiratory diseases, compared to that observed in previous reports.

Using this clone library method, we previously reported the identification of patients with pneumonia caused by Legionella pneumophila serogroup 8 3 and Leptotrichia 4 by analyzing bronchoalveolar lavage fluid and cases of bacterial pleuritis using pleural effusion 5. Assessing causative bacteria using this culture-independent molecular method is very important for identifying the true causative bacteria in infectious diseases. We herein show the utility of this molecular method, with descriptions of several cases of community acquired pneumonia, based on an analysis of the bacterial phylotypes in BALF.

Materials and Methods (comprehensive bacterial floral analysis)

After destroying bacterial bodies by shaking the mixture with glass beads, the bacterial 16S rRNA genes in each specimen were amplified using a universal primer pair. Then, each clone was transfected into E. coli, and the sequences of amplified DNA (approximately 580 bp) of 96 randomly chosen clones were determined. These sequences were analyzed using an in-house database with the Basic Local Alignment Search Tool (BLAST), and the phylotype of each sequence was determined. Subsequently, the number of assumed phylotypes in each sample was evaluated (with the construction of a clone library).

Example case of community-acquired pneumonia

An 80-year-old female with chronic obstructive pulmonary disease had a productive cough for one week. A chest X-ray film and computed tomography scan revealed infiltration in the left lower lobe. Upon admission, the peripheral white blood cell count was 15,600/µL (neutrophils: 88.0%) and the serum level of C-reactive protein was elevated to 5.33 mg/dL. Gram staining of bronchoalveolar lavage fluid obtained from the left S8 showed Gram-negative small rods and cocci (Figure 1), and the results of the BALF culture revealed Moraxella catarrhalis, Haemophilus influenzae and a small amount of alpha-Streptococcus. An analysis of the 16S rRNA of the same sample identified M. catarrhalis (30/46 clones, 65%) and H. influenzae (10/46 clones, 22%) (Figure 2). The results of the molecular method used in the present case are consistent with those of ordinary cultivation methods.

Summary of the present study

A total of 64 bronchoalveolar lavage fluid samples from patients with community-acquired pneumonia were evaluated using both the molecular method of 16S rRNA gene analysis and conventional cultivation methods. The molecular method used in the present study identified assumed causative bacterial phylotypes in all patients. In contrast to the molecular method, the ordinary cultivation methods identified 81.2% of assumed causative bacteria, while 18.8% remained unknown, even when using bronchoalveolar lavage fluid. In addition, the molecular method detected common known pathogens of pneumonia, such as S. pneumoniae, H. influenzae, Mycoplasma pneumoniae, anaerobes and oral streptococci, more frequently than the ordinary cultivation methods.

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FIG. 2

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