Perspective on the clone library method for infectious diseases

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.