Emergence of Antibiotic-Resistant Porphyromonas gingivalis in United States Periodontitis Patients.

Antibiotic resistance patterns of the major human periodontal pathogen Porphyromonas gingivalis were assessed over a 20-year period in the United States. Subgingival P. gingivalis was cultured pre-treatment from 2193 severe periodontitis patients during three time periods: 1999-2000 (936 patients), 2009-2010 (685 patients), and 2019-2020 (572 patients). The clinical isolates were tested for in vitro resistance to 4 mg/L for clindamycin and doxycycline, 8 mg/L for amoxicillin, and 16 mg/L for metronidazole, with a post hoc combination of data for metronidazole plus amoxicillin. Clindamycin-resistant P. gingivalis was significantly more prevalent in 2009-2010 (9.1% of patients) and 2019-2020 (9.3%; 15-fold increase) as compared to 1999-2000 (0.6%). P. gingivalis resistance to amoxicillin also significantly increased from 0.1% of patients in 1999-2000 to 1.3% in 2009-2010 and 2.8% (28-fold increase) in 2019-2020. P. gingivalis resistance to metronidazole, metronidazole plus amoxicillin, and doxycycline was low (≤0.5% prevalence), and statistically unchanged, over the 20-year period. These findings are the first to reveal marked increases over 20 years in clindamycin-resistant and amoxicillin-resistant P. gingivalis in United States periodontitis patients. Increased antibiotic resistance of P. gingivalis and other periodontitis-associated bacteria threatens the efficacy of periodontal antimicrobial chemotherapy.

Evaluation of a Rapid Biological Spore Test for Dental Instrument Sterilization.

AIM: This study evaluated the reliability of a new rapid biological spore test (BST) for determining the sterilization efficacy of dental steam autoclaves within 20 minutes, as compared to a conventional BST requiring 2 days of incubation after autoclave exposure. MATERIALS AND METHODS: A total of 177 pairs of BST, each composed of a rapid test (Celerity™ 20 Steam Biologic Indicator, Steris) and a conventional BST (Attest™ 1262 Biological Indicator, 3M), both containing Geobacillus stearothermophilus spores, were placed into steam autoclaves loaded with instruments, and subjected to either sterilizing (157 pairs) or non-sterilizing conditions (20 pairs). Celerity™ BST was then incubated for 20 minutes at 57°C, with the growth medium evaluated spectrophotometrically for fluorescent α-glucosidase signal changes (no change with successful sterilization; increased fluorescence after failed sterilization). Attest™ BST was incubated for 48 hours at 57°C, after which a pH-based color change in the culture broth was visually assessed (no change in purple color with successful sterilization; change to yellow color with failed sterilization). RESULTS: Celerity™ and Attest™ BST both accurately identified successful sterilization, with no G. stearothermophilus spore growth from either BST after exposure to sterilizing steam autoclave conditions (100% agreement between 157 pairs of each BST). Both BST also accurately detected unsuccessful sterilization, with all tested ampoules positive for G. stearothermophilus spore germination after non-sterilizing steam autoclave time periods. Both BST exhibited 100% sensitivity, specificity, and accuracy for detection of sterilizing steam autoclave conditions. CONCLUSION: Celerity™ BST, after only 20 minutes incubation, performed equally as well as a BST requiring 48 hours incubation in determining the sterilization efficacy of dental steam autoclaves. CLINICAL SIGNIFICANCE: Rapid BST offer earlier detection of sterilization failure before potentially contaminated dental instruments are used in clinical patient care.

Molecular Iodine Mouthrinse Antimicrobial Activity Against Periodontopathic Bacteria.

AIM: This study compared two molecular iodine mouthrinses for their in vitro bactericidal effects against subgingival biofilm bacteria from severe periodontitis patients. MATERIALS AND METHODS: In a subgingival biofilm eradication assay, dilution aliquots of subgingival microbial specimens from 32 adults with severe periodontitis were mixed in vitro with either a mouthrinse containing 100 parts per million (ppm) molecular iodine (Iorinse®) or one containing 150 ppm molecular iodine (iClean®), followed by mouthrinse neutralization after 60 seconds with 3% sodium thiosulfate. The mixtures, along with unexposed subgingival biofilm aliquots, were inoculated onto enriched Brucella blood agar and incubated anaerobically for 7 days to quantitate total viable bacterial counts and selected red/orange complex periodontal pathogens (Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Campylobacter rectus, and Fusobacterium nucleatum). RESULTS: Both molecular iodine mouthrinses significantly reduced total viable bacterial counts in the subgingival biofilm samples, with iClean® providing significantly greater in vitro suppression than Iorinse®. Both molecular iodine mouthrinses also significantly reduced total red/orange complex periodontal pathogens, with significantly greater suppression also exhibited by iClean®. CONCLUSION: The molecular iodine mouthrinses exerted marked bactericidal activity in vitro against human subgingival biofilm microbial species, including red/orange complex periodontal pathogens associated with severe periodontitis, with iClean® providing significantly better antimicrobial activity than Iorinse®. CLINICAL SIGNIFICANCE: These findings suggest potential value of molecular iodine mouthrinses in the treatment and prevention of periodontal diseases.

Antibiotic Resistance of Human Periodontal Pathogen Parvimonas micra Over 10 Years.

Changes were evaluated over 10 years in the in vitro resistance of human periodontopathic strains of Parvimonas micra to four antibiotics. Subgingival biofilms culture positive for P. micra from 300 United States adults with severe periodontitis in 2006, and from a similar group of 300 patients in 2016, were plated onto anaerobically incubated enriched Brucella blood agar alone, or supplemented with either doxycycline (4 mg/L), clindamycin (4 mg/L), amoxicillin (8 mg/L), or metronidazole (16 mg/L). P. micra growth on antibiotic-supplemented media indicated in vitro resistance to the evaluated antibiotic concentration. P. micra resistance was significantly more frequent among patients in 2016, as compared to 2006, for doxycycline (11.3% vs. 0.3% patients; 37.7-fold increase), and clindamycin (47.3% vs. 2.0% patients; 23.7-fold increase) (both p < 0.001), whereas resistance to amoxicillin (2.3% vs. 1.0% patients) and metronidazole (0% vs. 0.3% patients) remained low and statistically unchanged between the two patient groups (p-values > 0.05). No P. micra isolates in 2006 or 2016 were jointly resistant in vitro to both amoxicillin and metronidazole. The alarming increases in subgingival P. micra resistance to doxycycline and clindamycin raise serious questions about the empiric use of these antibiotics, either locally or systemically, in the treatment of United States periodontitis patients harboring subgingival P. micra.

Antimicrobial activity of silver diamine fluoride on human periodontitis microbiota.

Silver diamine fluoride (SDF) has been used in management of dentinal hypersensitivity and dental caries. This in vitro study evaluated the antimicrobial effects of SDF on subgingival microorganisms from severe human periodontitis lesions. Subgingival biofilm specimens from 24 adults with severe periodontitis were mixed in vitro with 19% or 38% SDF or left untreated (n = 24 per group) and then inoculated on enriched Brucella blood agar with anaerobic incubation. Selected red- and orange-complex periodontal pathogens were phenotypically identified in the subgingival specimens, including Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Campylobacter rectus, Fusobacterium nucleatum, and Streptococcus constellatus. Other microbial species recovered from SDF-treated specimens were identified using matrix-assisted laser desorption- ionization time-of-flight mass spectrometry. The SDF-treated specimens yielded significantly lower mean total viable counts and significantly lower mean total cultivable proportional levels of red- and orange-complex periodontal pathogens (0.5%-0.6%) than did untreated specimens (25.9%) (P < 0.001). The only red- and orange-complex species recovered from SDF-treated specimens were P micra (3 patients) and S constellatus (1 patient). The predominant cultivable isolates from SDF-treated specimens were Streptococcus oralis and other streptococci of relatively low periodontopathic and cariogenic potential. No statistically significant in vitro antimicrobial differences were found between 19% and 38% SDF against subgingival biofilm specimens. In this experiment, SDF exhibited substantial in vitro antimicrobial activity against putative periodontal pathogens from severe periodontitis lesions. The suppression of red- and orange-complex periodontal pathogens in subgingival biofilms by SDF treatment, along with the selection of SDF-resistant Streptococcus species that are associated with periodontal health, suggests a potential new therapeutic use for SDF in the management of human periodontal infections.

Comparative In Vitro Resistance of Human Periodontal Bacterial Pathogens to Tinidazole and Four Other Antibiotics.

The in vitro resistance of selected red/orange complex periodontal pathogens to tinidazole was compared with four other antibiotics. Subgingival biofilm samples from 88 adults with severe periodontitis were anaerobically incubated on enriched Brucella blood agar with and without supplementation with tinidazole (16 mg/L), metronidazole (16 mg/L), amoxicillin (8 mg/L), doxycycline (4 mg/L), or clindamycin (4 mg/L). Growth of Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Fusobacterium nucleatum, Streptococcus constellatus, or Campylobacter rectus on antibiotic-supplemented plates indicated their in vitro antibiotic resistance. Tinidazole inhibited all test species, except P. intermedia/nigrescens, P. micra, and S. constellatus in 3.8%, 10.2%, and 88.9% of species-positive patients, respectively. Significantly fewer patients yielded tinidazole-resistant test species, and had significantly lower subgingival proportions of tinidazole-resistant organisms, than patients with amoxicillin, doxycycline, or clindamycin-resistant species, but not those with metronidazole-resistant strains. Joint in vitro species resistance to tinidazole and amoxicillin, or metronidazole and amoxicillin, was rare. Tinidazole performed in vitro similar to metronidazole, and markedly better than amoxicillin, doxycycline, or clindamycin, against fresh clinical isolates of red/orange complex periodontal pathogens. As a result of its similar antimicrobial spectrum, and more convenient once-a-day oral dosing, tinidazole should be considered in place of metronidazole for systemic periodontitis drug therapy.

Phenotypic identification of periodontal Prevotella intermedia/nigrescens group isolates validated by MALDI-TOF mass spectrometry.

The accuracy of a phenotypic scheme to recognize periodontal Prevotella intermedia/nigrescens group clinical isolates on primary isolation culture plates was assessed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 84 fresh subgingival isolates from 23 chronic periodontitis patients were presumptively recognized on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens based on their dark-pigmented colony morphology, brick-red autofluorescence under long-wave ultraviolet light, and a negative fluorescence test for lactose production. The presumptive P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF MS analysis using Bruker MALDI Biotyper analytic software containing mass spectra for P. intermedia and Prevotella nigrescens in its reference library of bacterial protein profiles. Using a ≥1.7 log score agreement threshold, 60 (71.4%) of the presumptive P. intermedia/nigrescens clinical isolates were confirmed as either P. intermedia (25 isolates) or P. nigrescens (35 isolates). All isolates with a <1.7 log score were also identified as P. intermedia or P. nigrescens from the top choice designated on the MALDI Biotyper most likely species identification list. These MALDI-TOF MS findings document the ability of the phenotypic scheme to correctly recognize most periodontal P. intermedia/nigrescens group clinical isolates on primary isolation culture plates.

Phenotypic identification of Porphyromonas gingivalis validated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

OBJECTIVE: Porphyromonas gingivalis is a major bacterial pathogen in human periodontitis. This study used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to assess the accuracy of a rapid phenotypic identification scheme for detection of cultivable P. gingivalis in human subgingival plaque biofilms. METHODS: A total of 314 fresh cultivable subgingival isolates from 38 adults with chronic periodontitis were presumptively identified on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. gingivalis based on dark-pigmented colony morphology, lack of a brick-red autofluorescence reaction under long-wave ultraviolet light, and a positive CAAM fluorescence test for trypsin-like enzyme activity. Each presumptive P. gingivalis isolate, and a panel of other human subgingival bacterial species, were subjected to MALDI-TOF mass spectrometry analysis using a benchtop mass spectrometer equipped with software containing mass spectra for P. gingivalis in its reference library of bacterial protein profiles. A MALDI-TOF mass spectrometry log score of ≥1.7 was required for species identification of the subgingival isolates. RESULTS: All 314 (100%) presumptive P. gingivalis subgingival isolates were confirmed as P. gingivalis with MALDI-TOF mass spectrometry analysis (Cohen's kappa coefficient = 1.0). MALDI-TOF mass spectrometry log scores between 1.7 and 1.9, and ≥2.0, were found for 92 (29.3%) and 222 (70.7%), respectively, of the presumptive P. gingivalis clinical isolates. No other tested bacterial species was identified as P. gingivalis by MALDI-TOF mass spectrometry. CONCLUSIONS: Rapid phenotypic identification of cultivable P. gingivalis in human subgingival biofilm specimens was found to be 100% accurate with MALDI-TOF mass spectrometry. These findings provide validation for the continued use of P. gingivalis research data based on this species identification methodology.

Spiramycin resistance in human periodontitis microbiota.

PURPOSE: The occurrence of in vitro resistance to therapeutic concentrations of spiramycin, amoxicillin, and metronidazole was determined for putative periodontal pathogens isolated in the United States. MATERIALS AND METHODS: Subgingival plaque specimens from 37 consecutive adults with untreated severe periodontitis were anaerobically cultured, and isolated putative periodontal pathogens were identified to a species level. In vitro resistance to spiramycin at 4 µg/ml, amoxicillin at 8 µg/ml, and/or metronidazole at 16 µg/ml was noted when putative periodontal pathogen growth was noted on the respective antibiotic-supplemented primary culture plates. RESULTS: A total of 18 (48.7%) subjects yielded antibiotic-resistant putative periodontal pathogens with spiramycin at 4 µg/ml in drug-supplemented primary isolation plates, as compared to 23 (62.2%) subjects with amoxicillin at 8 µg/ml, and 10 (27.0%) subjects with metronidazole at 16 µg/ml. Spiramycin in vitro resistance occurred among species of Fusobacterium nucleatum (44.4% of organism-positive subjects), Prevotella intermedia/nigrescens (11.1%), Parvimonas micra (10.8%), Streptococcus constellatus (10%), Streptococcus intermedius (10%), Porphyromonas gingivalis (6.7%), and Tannerella forsythia (5.3%). Amoxicillin in vitro resistance was found in P. intermedia/nigrescens (55.5%), T. forsythia (15.8%), S. constellatus (10%), F. nucleatum (5.6%), and P. micra (2.7%). Only S. constellatus (70%) and S. intermedius (40%) exhibited in vitro resistance to metronidazole. When subject-based resistance data for spiramycin and metronidazole were jointly considered, all isolated putative periodontal pathogens were inhibited in vitro by one or the other of the antibiotic concentrations, except for one strain each of S. constellatus and S. intermedius from one study subject. Similarly, either amoxicillin or metronidazole at the drug concentrations tested inhibited in vitro all recovered putative periodontal pathogens, except S. constellatus in one subject. CONCLUSIONS: In vitro spiramycin resistance among putative periodontal pathogens of United States origin occurred in approximately one-half of severe periodontitis patients evaluated, particularly among subgingival F. nucleatum species. In vitro resistance patterns also suggest that therapeutic concentrations of spiramycin plus metronidazole may have potential antimicrobial efficacy in non-Aggregatibacter actinomycetemcomitans-associated periodontitis similar to amoxicillin plus metronidazole, which may be beneficial, where spiramycin is clinically available, for patients hypersensitive to amoxicillin or other beta-lactam antibiotics.