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.

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.

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.

Autoantibodies against citrullinated histone H3 in rheumatoid arthritis and periodontitis patients.

AIM: To determine the presence of citrullinated histones in inflamed periodontal tissue and to determine the presence of anti-citrullinated histone autoantibodies in sera from patients with rheumatoid arthritis (RA) and periodontitis (PD) patients. METHODS: The presence of citrullinated histone H3, PAD4 and CD68 was determined in 15 periodontal tissue biopsies from PD patients by immunohistochemistry. Sera from 36 healthy controls (HC), 113 PD patients and 84 patients with RA were assessed on presence of autoantibodies against citrullinated histones by Western blot and against citrullinated histone H3 by ELISA. RESULTS: Citrullinated histone H3, PAD4 and CD68 were present in periodontal tissue from nine (60%), 14 (93%) and 13 (87%) PD patients, respectively. Anti-citrullinated histone H3 autoantibodies were found in 33 (39%) patients with RA compared to three (8%) HC and 11 (10%) PD patients. Anti-citrullinated histone H3 levels were higher in anti-cyclic citrullinated peptide (anti-CCP)-positive compared to anti-CCP-negative patients with RA (p = .0008) and correlated moderately with anti-CCP levels (ρ = .22). No associations were found between anti-citrullinated histone H3 levels and periodontal status or smoking behaviour of patients with RA. CONCLUSION: PD patients are exposed to citrullinated histone H3 in inflamed periodontal tissue. Citrullinated histone H3 is targeted by autoantibodies present in RA sera. This supports a role for periodontitis in generation of antigens targeted by autoantibodies directed against citrullinated proteins.

Introduction to Clinical Microbiology for the General Dentist.

Clinical oral microbiology may help dental professionals identify infecting pathogenic species and evaluate their in vitro antimicrobial susceptibility. Saliva, dental plaque biofilms, mucosal smears, abscess aspirates, and soft tissue biopsies are sources of microorganisms for laboratory testing. Microbial-based treatment end points may help clinicians better identify patients in need of additional or altered dental therapies before the onset of clinical treatment failure, and help improve patient oral health outcomes. Microbiological testing appears particularly helpful in periodontal disease treatment planning. Further research and technological advances are likely to increase the availability and clinical utility of microbiological analysis in modern dental practice.

Head and neck intensity modulated radiation therapy leads to an increase of opportunistic oral pathogens.

OBJECTIVES: The introduction of intensity modulated radiation therapy (IMRT) has led to new possibilities in the treatment of head and neck cancer (HNC). Limited information is available on how this more advanced radiation technique affects the oral microflora. In a prospective study we assessed the effects of various advanced treatments for HNC on the oral microflora, as well as the effects of elimination of oral foci of infection. MATERIALS AND METHODS: All consecutive dentate patients >18years, diagnosed with a primary oral or oropharynx carcinoma and seen for a pre-treatment dental screening (May 2011-May 2013) were included. Patients were grouped by oncologic treatment: surgery (SURG), IMRT (IMRT) or IMRT+chemotherapy (CHIMRT). Dental screening data, demographic data, subgingival biofilm samples, oral lavages and whole saliva samples were obtained to microbiologically analyze the effects of cancer treatments (1-year follow-up). RESULTS: This study included 82 patients (29 SURG, 26 IMRT and 27 CHIMRT). The trends in changes in prevalence and proportions of microorganisms were comparable in the IMRT and CHIMRT group. However, relative to the SURG group, increased prevalence of enteric rods, staphylococci and Candida species was observed in the IMRT and CHIMRT groups. In these groups, elimination of oral foci decreased the frequency of detection of pathogens such as Porphyromonas gingivalis, Tannerella forsythia and Streptococcus mutans. CONCLUSION: Different treatments in HNC patients result in different changes in the oral microflora. Opportunistic pathogens such as staphylococci, enteric rods and Candida sp. tend to increase in prevalence after IMRT with or without chemotherapy, but not after surgical intervention.

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.

Non-oral gram-negative facultative rods in chronic periodontitis microbiota.

OBJECTIVE: The subgingival prevalence of gram-negative facultative rods not usually inhabiting or indigenous to the oral cavity (non-oral GNFR), as well as selected periodontal bacterial pathogens, were evaluated by culture in untreated and treated chronic periodontitis patients. METHODS: Subgingival biofilm specimens from 102 untreated and 101 recently treated adults with chronic periodontitis in the Netherlands were plated onto MacConkey III and Dentaid selective media with air-5% CO2 incubation for isolation of non-oral GNFR, and onto enriched Oxoid blood agar with anaerobic incubation for recovery of selected periodontal bacterial pathogens. Suspected non-oral GNFR clinical isolates were identified to a species level with the VITEK 2 automated system. RESULTS: A total of 87 (42.9%) out of 203 patients yielded subgingival non-oral GNFR. Patients recently treated with periodontal mechanical debridement therapy demonstrated a greater prevalence of non-oral GNFR (57.4% vs 28.4%, P < 0.0001), and a greater number of different non-oral GNFR species (23 vs 14 different species), than untreated patients. Sphingomonas paucimobilis was the most frequently isolated subgingival non-oral GNFR species. Several GNFR species normally found in animals and human zoonotic infections, and not previously detected in human subgingival biofilms, were recovered from some patients, including Bordetella bronchispetica, Pasteurella canis, Pasteurella pneumotropica and Neisseria zoodegmatis. Porphyromonas gingivalis and Tannerella forsythia were significantly associated with the presence of subgingival non-oral GNFR. CONCLUSIONS: A surprisingly high proportion of Dutch chronic periodontitis patients yielded cultivable non-oral GNFR in periodontal pockets, particularly among those recently treated with periodontal mechanical debridement therapy. Since non-oral GNFR species may resist mechanical debridement from periodontal pockets, and are often not susceptible to many antibiotics frequently used in periodontal practice, their subgingival presence may complicate periodontal treatment in species-positive patients and increase risk of potentially dangerous GNFR infections developing at other body sites.

Centipeda periodontii in human periodontitis.

This study assessed the subgingival occurrence of the flagellated, Gram-negative, anaerobic rod Centipeda periodontii in chronic periodontitis and periodontal health/gingivitis with species-specific nucleic acid probes, and evaluated the in vitro resistance of subgingival isolates to therapeutic levels of amoxicillin, metronidazole, and doxycycline. Subgingival plaque biofilm specimens from 307 adults with chronic periodontitis, and 48 adults with periodontal health/localized gingivitis, were evaluated with digoxigenin-labeled, whole-chromosomal, DNA probes to C. periodontii ATCC 35019 possessing a 10(4) cell detection threshold. Fifty-two C. periodontii subgingival culture isolates were assessed on antibiotic-supplemented enriched Brucella blood agar for in vitro resistance to either amoxicillin at 2 µg/ml, metronidazole at 4 µg/ml, or doxycycline at 2 µg/ml. A significantly greater subgingival occurrence of C. periodontii was found in chronic periodontitis subjects as compared to individuals with periodontal health/gingivitis (13.4 vs. 0 %, P < 0.003), although high subgingival counts of the organism (≥ 10(6) cells) were rarely detected (1.3 % of chronic periodontitis subjects). In vitro resistance was not found to amoxicillin or metronidazole, and to doxycycline in only 2 (3.9 %) of the 52 C. periodontii clinical isolates studied. These findings indicate that C. periodontii is not a major constituent of the subgingival microbiome in chronic periodontitis or periodontal health/gingivitis. The potential contribution of C. periodontii to periodontal breakdown in the few chronic periodontitis subjects who yielded high subgingival levels of the organism remains to be delineated. C. periodontii clinical isolates were susceptible in vitro to therapeutic concentrations of three antibiotics frequently used in treatment of human periodontitis.

Antibiotic susceptibility of periodontal Streptococcus constellatus and Streptococcus intermedius clinical isolates.

BACKGROUND: Streptococcus constellatus and Streptococcus intermedius in subgingival dental plaque biofilms may contribute to forms of periodontitis that resist treatment with conventional mechanical root debridement/surgical procedures and may additionally participate in some extraoral infections. Because systemic antibiotics are often used in these clinical situations, and little is known of the antibiotic susceptibility of subgingival isolates of these two bacterial species, this study determined the in vitro susceptibility to six antibiotics of fresh S. constellatus and S. intermedius clinical isolates from human periodontitis lesions. METHODS: A total of 33 S. constellatus and 17 S. intermedius subgingival strains, each recovered from separate patients with severe chronic periodontitis (n = 50) before treatment, were subjected to antibiotic gradient strip susceptibility testing with amoxicillin, azithromycin, clindamycin, ciprofloxacin, and doxycycline on blood-supplemented Mueller-Hinton agar and to the inhibitory effects of metronidazole at 16 mg/L in an enriched Brucella blood agar dilution assay. Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing interpretative standards were used to assess the results. RESULTS: Clindamycin was the most active antibiotic against S. constellatus (minimum inhibitory concentration at 90% [MIC90] 0.25 mg/L), and amoxicillin was most active against S. intermedius (MIC90 0.125 mg/L). A total of 30% of the S. constellatus and S. intermedius clinical isolates were resistant in vitro to doxycycline, 98% were only intermediate in susceptibility to ciprofloxacin, and 90% were resistant to metronidazole at 16 mg/L. CONCLUSION: Subgingival S. constellatus and S. intermedius exhibited variable antibiotic susceptibility profiles, potentially complicating empirical selection of periodontitis antibiotic therapy in patients who are species positive.

Acute focal infections of dental origin.

This article describes the most important pus-producing acute oral infections (dental infections) that can spread extra-orally. Most of these infections are spread by bacteria entering the bloodstream. However, dental infections have a number of other pathways for dissemination. By forming abscesses or phlegmon they can reach facial spaces that communicate with each other and then spread downwards to the mediastinum or upwards to the brain. In such cases dental infections can become, if not properly treated, life-threatening. It seems that early diagnosis and treatment are imperative, and potentially infectious foci should be traced and eliminated. Dental hygiene and prophylaxis to prevent dental biofilm formation are important measures to reduce the risk of these calamities. The more compromised the host defense is, the more importance should be put on these measures. Although commensal bacteria are often involved in these infections, attention should also be paid to specific periodontal pathogens, and a proper microbial diagnosis, obtained using molecular methods plus bacterial sensitivity testing, can provide the patient with optimal care. Drainage of pus must be established where possible so that the optimal effect of antibiotics can be achieved. Penicillin is still the drug of first choice in settings where suspicion of methicillin-resistant Staphylococcus aureus is low.

Antibiotic resistance in human peri-implantitis microbiota.

OBJECTIVES: Because antimicrobial therapy is often employed in the treatment of infectious dental implant complications, this study determined the occurrence of in vitro antibiotic resistance among putative peri-implantitis bacterial pathogens. METHODS: Submucosal biofilm specimens were cultured from 160 dental implants with peri-implantitis in 120 adults, with isolated putative pathogens identified to species level, and tested in vitro for susceptibility to 4 mg/l of doxycycline, 8 mg/l of amoxicillin, 16 mg/l of metronidazole, and 4 mg/l of clindamycin. Findings for amoxicillin and metronidazole were combined post-hoc to identify peri-implantitis species resistant to both antibiotics. Gram-negative enteric rods/pseudomonads were subjected to ciprofloxacin disk diffusion testing. RESULTS: One or more cultivable submucosal bacterial pathogens, most often Prevotella intermedia/nigrescens or Streptococcus constellatus, were resistant in vitro to clindamycin, amoxicillin, doxycycline, or metronidazole in 46.7%, 39.2%, 25%, and 21.7% of the peri-implantitis subjects, respectively. Only 6.7% subjects revealed submucosal test species resistant in vitro to both amoxicillin and metronidazole, which were either S. constellatus (one subject) or ciprofloxacin-susceptible strains of gram-negative enteric rods/pseudomonads (seven subjects). Overall, 71.7% of the 120 peri-implantitis subjects exhibited submucosal bacterial pathogens resistant in vitro to one or more of the tested antibiotics. CONCLUSIONS: Peri-implantitis patients frequently yielded submucosal bacterial pathogens resistant in vitro to individual therapeutic concentrations of clindamycin, amoxicillin, doxycycline, or metronidazole, but only rarely to both amoxicillin and metronidazole. Due to the wide variation in observed drug resistance patterns, antibiotic susceptibility testing of cultivable submucosal bacterial pathogens may aid in the selection of antimicrobial therapy for peri-implantitis patients.

Differences in peri-implant microflora between fully and partially edentulous patients: a systematic review.

BACKGROUND: The current evidence suggests that the oral microflora differs between individuals who are fully edentulous (FES) and those who are partially edentulous (PES). It is unknown whether this leads to differences in peri-implant microflora when implants are installed. The aim of the study is to compare the submucosal peri-implant microflora between FES and PES. METHODS: A systematic review was conducted. The MEDLINE, Embase, and Cochrane databases were searched for publications up to September 1, 2012. To reduce methodologic variations, only studies reporting in the same article about the submucosal peri-implant microflora of FES and PES were selected. RESULTS: Eleven publications describing 10 studies were selected. Because of numerous differences among the selected studies, no meta-analysis could be performed. Six of 10 studies showed a significant difference in the composition of the submucosal peri-implant microflora in healthy and peri-implant mucositis conditions between FES and PES, with the latter showing a potentially more pathogenic composition. However, microbiologic results were not unanimous among the studies. CONCLUSIONS: In healthy and peri-implant mucositis conditions, PES harbor a potentially more pathogenic peri-implant microflora than FES. The current data are insufficient for a clear conclusion regarding peri-implantitis cases. Overall, because of the lack of a meta-analysis, the variability in microbiologic outcomes and the limited number of studies available, the current evidence seems not to be robust.

Antibiotic resistance in human chronic periodontitis microbiota.

BACKGROUND: Patients with chronic periodontitis (CP) may yield multiple species of putative periodontal bacterial pathogens that vary in their antibiotic drug susceptibility. This study determines the occurrence of in vitro antibiotic resistance among selected subgingival periodontal pathogens in patients with CP. METHODS: Subgingival biofilm specimens from inflamed deep periodontal pockets were removed before treatment from 400 adults with CP in the United States. The samples were cultured, and selected periodontal pathogens were tested in vitro for susceptibility to amoxicillin at 8 mg/L, clindamycin at 4 mg/L, doxycycline at 4 mg/L, and metronidazole at 16 mg/L, with a post hoc combination of data for amoxicillin and metronidazole. Gram-negative enteric rods/pseudomonads were subjected to ciprofloxacin disk-diffusion testing. RESULTS: Overall, 74.2% of the patients with CP revealed subgingival periodontal pathogens resistant to at least one of the test antibiotics. One or more test species, most often Prevotella intermedia/nigrescens, Streptococcus constellatus, or Aggregatibacter actinomycetemcomitans, were resistant in vitro to doxycycline, amoxicillin, metronidazole, or clindamycin, in 55%, 43.3%, 30.3%, and 26.5% of the patients with CP, respectively. Fifteen percent of patients harbored subgingival periodontal pathogens resistant to both amoxicillin and metronidazole, which were mostly either S. constellatus (45 individuals) or ciprofloxacin-susceptible strains of Gram-negative enteric rods/pseudomonads (nine individuals). CONCLUSIONS: Patients with CP in the United States frequently yielded subgingival periodontal pathogens resistant in vitro to therapeutic concentrations of antibiotics commonly used in clinical periodontal practice. The wide variability found in periodontal pathogen antibiotic-resistance patterns should concern clinicians empirically selecting antibiotic treatment regimens for patients with CP.

Antibiotic susceptibility of periodontal Enterococcus faecalis.

BACKGROUND: Enterococcus faecalis may contribute to periodontal breakdown in heavily infected subgingival sites, particularly in patients responding poorly to mechanical forms of periodontal therapy. Because only limited data are available on the antimicrobial sensitivity of enterococci of subgingival origin, this study evaluates the in vitro antibiotic susceptibility of E. faecalis isolated from periodontitis patients in the United States. METHODS: Pure cultures of 47 subgingival E. faecalis clinical isolates were each inoculated onto specially prepared broth microdilution susceptibility panels containing vancomycin, teicoplanin, and six oral antibiotics of potential use in periodontal therapy. After incubation in ambient air for 18 to 20 hours, minimal inhibitory drug concentrations were determined using applicable Clinical and Laboratory Standards Institute criteria and interpretative guidelines. The organisms were additionally evaluated for in vitro resistance to metronidazole at 4 µg/mL. RESULTS: Periodontal E. faecalis exhibited substantial in vitro resistance to tetracycline (53.2% resistant), erythromycin (80.8% resistant or intermediate resistant), clindamycin (100% resistant to 2 µg/mL), and metronidazole (100% resistant to 4 µg/mL). In comparison, the clinical isolates were generally sensitive to ciprofloxacin (89.4% susceptible; 10.6% intermediate resistant) and 100% susceptible in vitro to ampicillin, amoxicillin/clavulanate, vancomycin, and teicoplanin. CONCLUSIONS: Tetracycline, erythromycin, clindamycin, and metronidazole revealed poor in vitro activity against human subgingival E. faecalis clinical isolates, and would likely be ineffective therapeutic agents against these species in periodontal pockets. Among orally administered antibiotics, ampicillin, amoxicillin/clavulanate, and ciprofloxacin exhibited marked in vitro inhibitory activity against periodontal E. faecalis, and may be clinically useful in treatment of periodontal infections involving enterococci.

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.

Phylogenetic variation of Aggregatibacter actinomycetemcomitans serotype e reveals an aberrant distinct evolutionary stable lineage.

The periodontal pathogen Aggregatibacter actinomycetemcomitans that comprises six serotypes (a-f), is often identified by PCR-based techniques targeting the 16S rRNA gene. In this study, 16S rRNA gene sequence analysis revealed an aberrant cluster of 19 strains within serotype e, denoted as serotype e'. The 16S rRNA gene sequence similarities found between serotype e' strains ranged from 99.7% to 100.0%, whereas 96.8-97.5% sequence similarity was obtained with members of the other serotypes, indicating that the serotype e' strains might not be true members of A. actinomycetemcomitans. However, DNA-DNA hybridizations between a representative serotype e' strain and representative strains of serotypes b, d and e of A. actinomycetemcomitans revealed 68-75% DNA-DNA relatedness, demonstrating that the serotype e' strains do belong to the species A. actinomycetemcomitans. AFLP analysis of 33 A. actinomycetemcomitans strains, representing all serotypes (a-f), but mainly serotype e' strains, showed that the latter form a distinct cluster, demonstrating that these strains are also closely related on the whole genome level. Moreover, the serotype e' strains were unable to ferment starch and glycogen in contrast to almost all other A. actinomycetemcomitans strains tested. Overall, the data obtained in this study suggest that the serotype e' strains form an evolutionary relatively stable distinct subgroup within A. actinomycetemcomitans.

The capsule of Porphyromonas gingivalis reduces the immune response of human gingival fibroblasts.

BACKGROUND: Periodontitis is a bacterial infection of the periodontal tissues. The Gram-negative anaerobic bacterium Porphyromonas gingivalis is considered a major causative agent. One of the virulence factors of P. gingivalis is capsular polysaccharide (CPS). Non-encapsulated strains have been shown to be less virulent in mouse models than encapsulated strains. RESULTS: To examine the role of the CPS in host-pathogen interactions we constructed an insertional isogenic P. gingivalis knockout in the epimerase-coding gene epsC that is located at the end of the CPS biosynthesis locus. This mutant was subsequently shown to be non-encapsulated. K1 capsule biosynthesis could be restored by in trans expression of an intact epsC gene. We used the epsC mutant, the W83 wild type strain and the complemented mutant to challenge human gingival fibroblasts to examine the immune response by quantification of IL-1beta, IL-6 and IL-8 transcription levels. For each of the cytokines significantly higher expression levels were found when fibroblasts were challenged with the epsC mutant compared to those challenged with the W83 wild type, ranging from two times higher for IL-1beta to five times higher for IL-8. CONCLUSIONS: These experiments provide the first evidence that P. gingivalis CPS acts as an interface between the pathogen and the host that may reduce the host's pro-inflammatory immune response. The higher virulence of encapsulated strains may be caused by this phenomenon which enables the bacteria to evade the immune system.