Matrix assisted Laser Desorption/Ionization time of flight Mass spectrometry validation of a Periodontal Prevotella intermedia/Nigrescens identification scheme

Abstract

Objectives: Prevotella intermedia and Prevotella nigrescens are two genetically distinct, gram-negative, anaerobic rods associated with the subgingival microbiome of human periodontitis. The two species are frequently isolated from subgingival dental plaque biofilms in chronic periodontitis patients clinically experiencing progressive destructive disease activity. In anaerobically-incubated liquid or solid culture media, P. intermedia and P. nigrescens exhibit nearly identical phenotypic properties, with regard to their colony morphology features and biochemical properties, which differ from other subgingival Prevotella and non-Prevotella microbial species. As a result, rapid differentiation and identification of P. intermedia/nigrescens group organisms from other bacterial species in anaerobically-cultivated subgingival dental plaque biofilms has been based upon examination of culture isolates for a dark-pigmented colony appearance, presence of brick-red autofluorescence of colonies to long-wave ultraviolet light exposure, and biochemical testing demonstrating a lack of colony lactose fermentation. However, the accuracy of this phenotypic-based identification scheme for periodontal P. intermedia/nigrescens group species has yet to be validated with a broad-based reference method that encompasses testing for a wide array of microbial species. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and associated analytic software, is recently approved for clinical microbiology diagnostic use in the United States by the Food and Drug Administration, and is capable of definitively identifying 4,613 different microbial species based on mass spectra of their bacterial proteins. To date, no performance evaluation has been carried out comparing the phenotypic-based identification scheme for periodontal P. intermedia/ nigrescens group species with definitive MALDI-TOF mass spectrometry identification of the organisms. As a result, the purpose of this study was to assess with MALDI-TOF mass spectrometry the accuracy of the rapid phenotypic-based periodontal P. intermedia/ nigrescens group species identification scheme widely utilized since 1986 by clinical periodontal microbiology laboratories and periodontal microbiology culture-based research studies. Methods: 84 fresh subgingival cultivable isolates from 23 chronic periodontitis patients were presumptively identified on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens group species based on their dark-pigmented colony morphology, presence of brick-red autofluorescence under long-wave ultraviolet light, and a negative MUG fluorescence test for lactose fermentation activity. Each of the putative P. intermedia/nigrescens 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 P. intermedia and P. nigrescens in its reference library of bacterial protein profiles. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical isolates, with scores of equal to or larger than 2.0 representing more definitive species identification. Results: A total of 60 (71.4%) of the putative P. intermedia/nigrescens clinical isolates were reliably identified with MALDI-TOF mass spectrometry as either P. intermedia (25 isolates, with eight isolates exhibiting MALDI Biotyper log scores of equal to or larger than 2.0), or P. nigrescens (35 isolates, with nine isolates exhibiting MALDI Biotyper log scores of equal to or larger than 2.0). Among the 24 putative P. intermedia/nigrescens clinical isolates generating MALDI Biotyper log scores < 1.7, indicating a less reliable species identification, only P. intermedia (14 isolates) or P. nigrescens (10 isolates) were listed by the analytic software as the first choice among the most likely bacterial species. No other bacterial species other than P. intermedia or P. nigrescens were identified by MALDI-TOF mass spectrometry for any of the 84 tested putative P. intermedia/ nigrescens clinical isolates. Conclusions: These findings document, for the first time with MALDI-TOF mass spectrometry, the relative accuracy of a rapid phenotypic-based periodontal P. intermedia/nigrescens identification scheme based on dark-pigmented colony morphology, presence of brick-red long-wave ultraviolet light autofluorescence, and a negative MUG test for lactose fermentation activity. 100% of the 84 presumptive P. intermedia/nigrescens clinical isolates tested were identified with MALDI-TOF mass spectrometry, with varying levels of reliability, as being only either P. intermedia or P. nigrescens. These findings provide validation for the continued use of this rapid phenotypic identification scheme for periodontal P. intermedia/nigrescens.