Analysis of the Microflora Associated with Oral Carcinoma

Introduction: Interest in the possible relationships between bacteria and the different stages of cancer development has been rising since the classification by the W.H.O. of Helicobacter pylori as a definite (class 1) carcinogen. The association of bacteria with oral squamous cell carcinoma (OSCC) has yet to be adequately examined. Objectives: The primary aim of the current study was to identify any bacterial species detected within OSCC tumour tissue by standard microbiological culture and a molecular approach. Methods: At the time of surgery 1cm3 specimens were collected by aseptic technique from deep within the tumour. Surface decontamination of tissue specimens was performed by immersion in Betadine and vortexing in PBS. Specimens were subsequently bisected: Half of each was aseptically macerated, resuspended in PBS and cultured under aerobic and anaerobic conditions on non-selective media. The remainder of each specimen was digested with proteinase K, subjected to DNA extraction, and analysed by a nested PCR-DGGE technique using universal primers. Bacteria detected by both methods were identified by 16S rDNA sequence analysis. Results: To date 22 samples have been analysed. Tissues yielded an average of 7 isolates representing over 17 different bacterial groups. PCR-DGGE yielded additional species to the cultural analysis, indicating the advantage of utilising a combined approach. A number of microbial phylotypes detected by PCR showed only low homology to previously cultured species. Negative results from cultural and PCR analysis of washes of the tumour specimens indicated that surface decontamination was successful and that the bacteria detected were from within the tumour tissue. Conclusions: A diversity of bacterial groups including gram positive and negative, facultative and anaerobic species have been isolated from within the tissue of oral squamous carcinoma by culture and molecular means. The significance of these bacteria within the tumour tissue warrants further study.