Genetic Relationship between Respiratory Pathogens Isolated from Mouth and Lung

OBJECTIVES: The aim of this study was to determine the genetic relationship between oral and respiratory isolates in patients with suspected pneumonia.

CLINICAL SUBJECTS AND SAMPLES: A total of 100 intubated Trauma-Intensive-Care-Unit (TICU) patients were enrolled. Samples were obtained from supragingival plaque and tracheal secretions on the day of admission to the TICU, and every other day. Bronchial alveolar lavage (BAL) was performed on 30 patients with suspected ventilator-associated pneumonia. The suspected respiratory pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and other gram-negative bacilli were selected for study.

METHODS: Genomic DNA profile of respiratory isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Each suspended bacterial sample (OD600=1.0) was incorporated into an agarose plug and genomic DNA was isolated using cell lysis buffer, lysozyme (or lysostaphin), RNAse and proteinase K. In-plug DNA was digested by restriction enzyme such as SmaI, NheI, ApaI or XbaI. The PFGE band patterns were clustered into dendograms to compare the genetic similarity of the strains. Isolates were further analyzed by multilocus sequence typing (MLST) to confirm the genetic relationship. After PCR of seven housekeeping genes of individual isolates, the internal fragment (400-500bp) was sequenced. The sequence type (allelic profile) for each isolate was compared to the database of known alleles in the MLST website.

RESULTS: Isolates recovered from supragingival dental plaque of a single patient were genetically indistinguishable (over 95% similarity) from isolates of the same species from BAL. Inter-patient variation in genetic profiles were noted among the different bacterial pathogens recovered. In the half of cases of Pseudomonas strains, the genetic profiles between patients were identical, suggesting a common source of infection from the environment.

CONCLUSIONS: Dental plaque likely serves as an important reservoir for respiratory pathogens in ventilated intensive care unit patients. This work was funded by the NIDCR grant DE014685.