IN VITRO ANTIMICROBIAL ACTIVITY OF POVIDONE-IODINE AGAINST SELECTED HUMAN RED AND ORANGE COMPLEX PERIODONTAL PATHOGENS.

Abstract

Objectives: Successful treatment of severe human periodontitis lesions has been shown to be highly dependent upon adequate suppression or eradication of key bacteria, most often species classified as red and orange complex periodontal pathogens, in the subgingival microbiome of diseased periodontal sites. Multiple clinical studies have reported superior therapeutic outcomes when conventional mechanical root debridement of severe periodontitis lesions is supplemented with professional periodontal pocket delivery of a povidone-iodine antiseptic solution, which offers antimicrobial effects against red and orange complex periodontal pathogens. However, many questions involving the clinical application of povidone-iodine in periodontal therapy remain unresolved, such as which concentration of povidone-iodine is preferred, and how long of a contact time period is needed between povidone-iodine and targeted bacteria in periodontal pockets. Previous in vitro studies on the effects of povidone-iodine on periodontal bacterial pathogens employed 5-minute or longer contact times and most often tested laboratory stock strains of microorganisms, even though inflamed periodontal pockets undergo a rapid washout of introduced fluids, and bacterial stock collections frequently develop altered properties and decreased virulence in comparison to freshly-recovered wild-type clinical isolates. To address some of these issues, the objective of this study was to further explore the in vitro effects of povidone-iodine on periodontal bacterial pathogens by employing a subgingival biofilm species eradication assay to test the antimicrobial effects of a 60- second in vitro exposure of 5% and 10% povidone-iodine on freshly-isolated red and orange complex periodontal pathogens from severe human periodontitis lesions. Methods: Paper point subgingival biofilm samples from 22 adults with severe periodontitis that were to be discarded after microbiological analysis at the Oral Microbiology Testing Service Laboratory at Temple University School of Dentistry were secondarily employed in this study. Dilution aliquots from each subgingival specimen were mixed with either 10% or 5% povidone-iodine for a 60-second in vitro contact time period, and then neutralized with 3% sodium thiosulfate. The mixtures were then inoculated onto enriched Brucella blood agar plates, and incubated anaerobically for 7 days at 37°C. Bacterial species growing subsequent to the 60-second povidone-iodine contact time were considered to be resistant to that concentration of povidone-iodine. Total viable counts in povidone-iodine exposed subgingival specimens were quantitated, and established phenotypic criteria employed to identify the following red and orange complex periodontal pathogens: Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Campylobacter rectus, Fusobacterium nucleatum group species, and Streptococcus constellatus. Other cultivable isolates recovered from povidone-iodine exposed subgingival specimens were identified using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and Bruker MALDI Biotyper analytic software. Subgingival sample dilution aliquots not exposed to povidone-iodine were similarly processed as controls for comparison with povidone-iodine exposed specimens, and were additionally inoculated onto enriched Brucella blood agar plates supplemented with either metronidazole at 16 mg/L, doxycycline at 4 mg/L, amoxicillin at 8 mg/L, or clindamycin at 4 mg/L, which represent recognized non-susceptible drug breakpoint concentrations for each of the antibiotics, followed by anaerobic incubation for 7 days at 37°C. In vitro antibiotic resistance was noted when any of the evaluated red and orange complex periodontal pathogens displayed growth on one or more of the antibiotic- supplemented enriched Brucella blood agar plates. Paired t-tests compared mean total subgingival viable counts, and mean total subgingival proportions of the evaluated anaerobic red and orange complex periodontal pathogens per patient, between subgingival biofilm samples exposed and not exposed in vitro to 10% or 5% povidone-iodine, with a P-value of < 0.05 required for statistical significance. Comparisons were also made between antibiotic-resistance and susceptible strains of the evaluated red and orange complex periodontal pathogens to assess their in vitro sensitivity to 10% and 5% povidone-iodine. Results: Subgingival specimens exposed in vitro for 60-seconds to either 10% or 5% povidone-iodine yielded significantly lower total subgingival viable counts per patient than those not exposed to povidone-iodine (P < 0.001, paired t-test), with no statistically significant differences found between the two povidone-iodine concentrations (P = 0.125, paired t-test). All evaluated red and orange complex periodontal pathogens were suppressed below detection levels after 10% povidone-iodine in vitro exposure, except for one F. nucleatum strain in one patient specimen. As a result, 21 (95.5%) of the patient subgingival biofilm samples were culture-negative for red and orange complex periodontal pathogens after 10% povidone-iodine in vitro exposure. A similar suppression of red and orange complex periodontal pathogens was found following 5% povidone iodine in vitro exposure, where only one patient sample yielded P. intermedia/nigrescens, and two other samples F. nucleatum. This resulted in 19 (86.4%) of patient subgingival biofilm samples being devoid of red and orange complex periodontal pathogens after 5% povidone-iodine in vitro exposure. Total subgingival proportions of red and orange complex periodontal pathogens averaged 0.5% per patient in subgingival specimens exposed in vitro to 10% povidone-iodine, and 0.7% per patient in those exposed to 5% povidone-iodine, which were both significantly lower than 14.8% mean proportions detected in subgingival biofilms not exposed to povidone-iodine (P < 0.0001, paired t-test). No statistically significant differences were found between 10% and 5% povidone-iodine relative to suppression of total red and orange complex periodontal pathogen proportions (P = 0.743, paired t-test). Both povidone-iodine concentrations were similarly active against red and orange complex periodontal pathogens which were resistant in vitro to breakpoint levels of doxycycline, amoxicillin, or clindamycin, as compared to antibiotic-susceptible clinical isolates. Various Streptococcus species, particularly Streptococcus oralis, were the most frequently recovered microorganisms in subgingival biofilm specimens after 60-second in vitro exposure to povidone-iodine, indicative of their in vitro resistance to both 10% and 5% povidone-iodine. Conclusions: Povidone-iodine exerted marked antimicrobial effects against both antibiotic-resistant and susceptible fresh clinical isolates of red and orange complex periodontal pathogens, and total viable counts, in subgingival biofilm specimens from severe periodontitis patients after only a brief (60-second) in vitro contact time, with no statistically significant differences found between 10% and 5% concentrations of povidone-iodine. The profound suppression of red and orange complex periodontal pathogens, and concurrent predominance of periodontal health-associated Streptococcus species, that occurs in subgingival biofilm specimens following in vitro exposure to povidone-iodine, further supports the clinical use of povidone-iodine in periodontal therapy as an adjunct to mechanical root debridement in altering pathogenic subgingival microbial populations towards one compatible with periodontal health.