Rams, Thomas E.; Suzuki, Jon, 1947-; Whitaker, Eugene J. (Temple University. Libraries, 2015)
      Dark-pigmented, gram-negative, anaerobic rods are often recovered in large numbers from the subgingival microbiome of human periodontitis, and are statistically associated with progression of chronic periodontitis. Due to their frequent expression of beta-lactamase enzymes, which hydrolyze and degrade beta-lactam class antibiotics, these species may compromise systemic periodontal antimicrobial chemotherapy involving amoxicillin, which may lead to clinical therapeutic failures in chronic periodontitis therapy. Recent studies using phenotypic methods have identified the in vitro growth of Prevotella intermedia/nigrescens in the presence of therapeutic threshold concentrations of amoxicillin, which is indicative of species antibiotic resistance. Because of uncertainties with their taxonomic classification, only limited information is available on the distribution of amoxicillin-resistant species within the group of dark-pigmented, gram-negative, anaerobic rods that may colonize and inhabit human subgingival sites. 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 in the United States by the United States Food and Drug Administration. This methodology is capable of definitively identifying 4,613 different oral and non-oral microbial species based on mass spectra of their bacterial protein profiles, including many dark-pigmented, gram-negative, anaerobic rods of subgingival origin. However, MALDI-TOF mass spectrometry has yet to be used for species iii identification of dark-pigmented, gram-negative, anaerobic rods recovered from chronic periodontitis lesions, and resistant in vitro to amoxicillin. As a result, the purpose of this study was to use MALDI-TOF mass spectrometry to identify to a species-level the patient distribution of dark-pigmented, gram-negative, anaerobic rods isolated from the subgingival microbiota of chronic periodontitis patients and exhibiting in vitro resistance to therapeutic concentrations of amoxicillin. Methods: 24 chronic periodontitis patients contributed 71 fresh subgingival cultivable isolates (one to 11 isolates per patient) which were presumptively identified by their brown to black colony pigmentation on anaerobically-incubated enriched Brucella blood agar primary isolation plates containing amoxicillin at 8 μg/ml as amoxicillinresistant, dark-pigmented, gram-negative, anaerobic rods. Each of the amoxicillinresistant, dark-pigmented 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 dark-pigmented, gram-negative, anaerobic rods in its reference library of bacterial protein profiles. A MALDI Biotyper log score of ≥ 1.7 was required for reliable taxonomic classification of the clinical isolates. Results: Only 4 (16.7%) of the chronic periodontitis patients yielded two different dark-pigmented species on amoxicillin-supplemented primary isolation plates, while all other study patients had only one amoxicillin-resistant, dark-pigmented species. Amoxicillin-resistant strains of Prevotella nigrescens were identified in 11 (45.8%) patients, Prevotella intermedia in 8 (33.3%) patients, Prevotella denticola and/or Prevotella species in 3 (12.5%) patients, Porphyromonas gingivalis in 2 (8.3%) patients, and Prevotella melaninogenica in one (4.2%) patient. 50 (70.4%) of the amoxicillinresistant clinical isolates exhibited MALDI Biotyper log scores of ≥ 1.7, the threshold for reliable taxonomic classification, whereas 21 (29.6%) had log scores < 1.7, indicating a less reliable species identification. Conclusions: These findings demonstrate that a range of specific amoxicillinresistant bacterial species comprise cultivable isolates of dark-pigmented, gram-negative, anaerobic rods in the human chronic periodontitis subgingival microbiota. P. nigrescens was the most frequently isolated amoxicillin-resistant, dark-pigmented subgingival bacterial species, followed by P. intermedia. Two study patients surprisingly revealed amoxicillin-resistant strains of P. gingivalis. The occurrence of amoxicillin-resistant, dark-pigmented rods in chronic periodontitis lesions may complicate selection, and markedly reduce the potential effectiveness, of systemic periodontal antimicrobial therapies involving beta-lactam antibiotics.