Proteomic comparison of Amoxicillin resistant and susceptible periodontal Provotella Intermedia and Provotella Nigrescens clinical isolation with Matrix-Assisted Laser Disportion Ionization Time-Of-/flight mass spectrometry

Abstract

Objectives: Prevotella intermedia and Prevotella nigrescens are dark-pigmented, non-motile, anaerobic rods regarded as important bacterial pathogens in the etiology and progression of human chronic periodontitis. Because the bacterial species may not be adequately suppressed in the subgingival microbiota of chronic periodontitis lesions by conventional mechanical root debridement when present in high cultivable proportions, systemic and/or local periodontal chemotherapy is often employed to reduce their subgingival numbers to levels compatible with periodontal health. However, many clinical isolates of P. intermedia and P. nigrescens may exhibit resistance to β-lactam antibiotics prescribed in periodontal therapy via expression of β-lactamase enzymes. Conventional methods of in vitro antibiotic susceptibility testing entail cultivation of targeted bacterial species, with evaluations of whether or not the organism is capable of growing in the presence of critical thresholds of test antibiotics, such as β-lactam antibiotics. Because such conventional in vitro antibiotic testing is time-consuming, expensive, and labor-intensive, there is an urgent clinical need for more rapid assays to determine the susceptibility or resistance of important periodontal pathogens, such as P. intermedia and P. nigrescens, to contemplated periodontal antimicrobial chemotherapy. In this regard, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and associated analytic software for the definitive identification P. intermedia and P. nigrescens in clinical specimens, has been approved for clinical microbiology diagnostic use in the United States by the Food and Drug Administration. This methodology relies upon evaluations of bacterial protein profiles for microbial species identification. Since β-lactamase enzymes conferring bacterial resistance to β-lactam antibiotics are proteins, it is possible that MALDI-TOF mass spectrometry may be able to reliably detect characteristic differences in protein profiles of microorganisms elaborating and not elaborating β-lactamase enzymes. Thus, the potential exists that protein profiles generated by MALDI-TOF mass spectrometry may be used to rapidly distinguish between antibiotic-resistant versus susceptible strains of bacterial species without employing time-consuming, expensive, and labor-intensive culture and in vitro drug testing. As a result, this study aimed carry out a proteomic comparison of amoxicillin-resistant and susceptible periodontal P. intermedia and P. nigrescens clinical isolates with MALDI-TOF mass spectrometry. Methods: A total of 10 amoxicillin-resistant and two amoxicillin-susceptible fresh clinical subgingival isolates of P. intermedia, and 10 amoxicillin-resistant and one amoxicillin-susceptible clinical subgingival isolates of P. nigrescens, were recovered by culture and identified to species level with MALDI-TOF mass spectrometry from the subgingival dental plaque biofilms of adults with severe chronic periodontitis. The amoxicillin-resistant clinical isolates of P. intermedia and P. nigrescens exhibited growth on anaerobically-incubated enriched Brucella blood agar primary isolation culture plates supplemented with amoxicillin at 8 μg/ml, whereas no growth on these plates was found with amoxicillin-susceptible strains. Normalized raw mass spectra, as well as normalized peak list spectrum representations, generated by MALDI-TOF mass spectrometry within a routine operating range of 2-20 kDa were visually compared for the amoxicillin-resistant and amoxicillinsusceptible P. intermedia and P. nigrescens clinical isolates, to see if there were consistently reproducible differences in their distribution of mass spectra peaks. Results: Visual comparisons of normalized raw mass spectra, and normalized peak list spectrum, for amoxicillin-susceptible and amoxicillin-resistant subgingival clinical isolates of both P. intermedia and P. nigrescens failed to reveal consistently reproducible differences in their distribution of mass spectra peaks within a routine MALDI-TOF mass spectrometry operating range of 2-20 kDa. Conclusions: Visual examination of raw mass spectra and mass spectra peaks generated by MALDI-TOF mass spectrometry within a routine operating range of 2-20 kDa failed to reveal noteworthy differences between amoxicillin-resistant and amoxicillin-susceptible subgingival clinical isolates of P. intermedia and P. nigrescens. These findings indicate that use of MALDI-TOF mass spectrometry employed within a routine operating range of 2-20 kDa may not provide sufficient differentiation in protein profiles between amoxicillin-resistant and amoxicillin-susceptible Prevotella species to be of rapid diagnostic use for assessing the species in vitro antibiotic susceptibility to β- lactam antibiotics. Additional evaluation of MALDI-TOF mass spectrometry employing higher kDa ranges beyond a routine upper operating range of 20 kDa is warranted to further evaluate its potential to accurately identify antibiotic-resistant versus susceptible strains of pathogenic microorganisms.