Combining culture and culture-independent methods reveals new microbial composition of halitosis patients' tongue biofilm

BACKGROUND Oral malodor is a very discomforting condition deriving from the presence of volatile sulfur compounds in the expired air. In halitosis of intraoral etiology, the volatile sulfur compounds are metabolic products of the oral microorganisms within the biofilm coating the tongue dorsum as well as other tissues in the oral cavity. The aim of this study was to characterize and compare the microbial composition of tongue biofilm in volunteers suffering from halitosis and healthy volunteers by means of both the culture method and culture-independent cloning technique. RESULTS A high bacterial variety (more than 80 different species) was detected using the combination of both methods. A distinct bacterial composition was revealed in the halitosis-associated biofilms compared with the health-associated biofilms. Actinomyces graevenitzii was shown to be significantly associated with the halitosis condition. The culture method identified 47 species, included Veillonella rogosae, never isolated from the tongue biofilm of halitosis patients so far. In the healthy condition, the culture-dependent method showed that the most frequent species were Streptococcus parasanguinis among the aerobes and Veillonella spp. among the anaerobes. The culture-independent cloning method detected more than 50 species. Streptococci, in particular S. mitis/oralis, S. pseudopneumoniae, and S. infantis as well as Prevotella spp., were found most frequently in halitosis patients. Streptococcus salivarius and Rothia mucilaginosa were found more frequently in the healthy condition. CONCLUSIONS The combination of the culture-dependent and culture-independent cloning techniques allowed for a widespread analysis of the tongue biofilm in halitosis patients. The results can support further pharmacological research for new antimicrobial agents and halitosis therapy strategies