CULTIVABLE FUSOBACTERIUM SPECIES IN CHRONIC PERIODONTITIS MICROBIOTA IDENTIFIED WITH MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

Abstract

Objectives: Fusobacteria are prominent participants in the maturation of subgingival dental plaque biofilms in humans. A number of various species belonging to the Fusobacterium genus have been recovered from the subgingival microbiota of chronic periodontitis patients. However, conventional Fusobacterium species identification is labor-intensive, time-consuming, and complicated by shortcomings in phenotypic-based classification schemes, where many fusobacteria display overlapping and non-distinguishing morphologic features and biochemical properties. In addition, molecular identification of fusobacteria is plagued with difficulties of validating the specificity of nucleic acid probes and primers to various Fusobacterium species that have closely-related interspecies genetic profiles. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and its associated analytic software, was recently approved for clinical microbiology diagnostic use by the United States Food and Drug Administration. MALDI-TOF mass spectrometry has the potential to rapidly identify cultivable clinical isolates to a species level for 4,613 different bacterial species based on mass spectra of their bacterial protein profiles, including many Fusobacterium species. The purpose of this study was to use MALDI-TOF mass spectrometry to rapidly identify the patient distribution of fusobacteria isolated from the subgingival microbiota of chronic periodontitis patients. Methods: A total of 34 chronic periodontitis patients provided 96 fresh subgingival cultivable fusobacteria isolates (one to seven isolates per patient), which were presumptively identified by their chartreuse-positive colony autofluorescence under long-wave ultraviolet light on anaerobically-incubated, non-selective, enriched Brucella blood agar primary isolation plates. Each of the presumptive fusobacteria clinical isolates were subjected to MALDI-TOF mass spectrometry analysis using a bench top mass spectrometer, Bruker FlexControl 3.0 software, and MALDI Biotyper 3.1 software (Bruker Daltonics, Billerica, MA, USA), which contains mass spectra for a variety of fusobacteria in its reference library of bacterial protein profiles. Each clinical isolate underwent on-target plate formic acid protein extraction, and was taxonomically classified with MALDI-TOF mass spectrometry within an approximately 30-45 minute time period from the point of colony harvesting from primary isolation culture plates. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical fusobacteria isolates. Results: A majority (58.8%) of the chronic periodontitis patients yielded two or three different species of subgingival Fusobacterium on non-selective enriched Brucella blood agar primary isolation plates. Fusobacterium naviforme was identified by MALDI-TOF mass spectrometry analysis in 14 (41.2%) chronic periodontitis study patients, Fusobacterium nucleatum subspecies vincentii in 13 (38.2%) patients, Fusobacterium nucleatum subspecies polymorphum in 9 (26.5%) patients, Fusobacterium nucleatum and Fusobacterium species each in 6 (17.6%) patients, Fusobacterium nucleatum subspecies nucleatum in 4 (11.8%) patients, and Fusobacterium nucleatum subspecies animalis in 3 (8.8%) patients. Three patients additionally yielded subgingival isolates of Fusobacterium canifelinum, normally an inhabitant of the oral cavity of dogs and cats. 52 (54.2%) of the fusobacteria clinical isolates revealed MALDI Biotyper log scores of equal to or larger than 1.7, the threshold for reliable taxonomic classification, while in comparison, 44 (45.8%) had log scores less than 1.7, indicating a less reliable species identification. No other microbial species, other than one of the Fusobacterium species, was listed by the MALDI-TOF mass spectrometry analytic software as the most likely organism for the tested clinical isolates. Conclusions: These findings indicate that a variety of Fusobacterium species may be identified with MALDI-TOF mass spectrometry in the subgingival microbiota of chronic periodontitis patients. F. naviforme and F. nucleatum subspecies vincentii were the most frequently isolated subgingival fusobacteria species in the evaluated study patients. Three chronic periodontitis patients also unexpectedly revealed subgingival isolates of the animal species F. canifelinum, which is normally in the oral cavity of dogs and cats. MALDI-TOF mass spectrometry may facilitate rapid identification of cultivable fusobacteria in human subgingival dental plaque biofilms, and enhance understanding of bacterial community structure in periodontal pockets.