Resumen de Validation of Antibiotic Efficacy on In Vitro Subgingival Biofilms
Background: Systemic antibiotics are often used as adjunctive treatment modalities for periodontal diseases. Testing of antimicrobial efficacy can be relevant only if the bacteria are in the form of biofilms rather than the planktonic state, and at concentrations of physiologic relevance, i.e., reachable in the periodontal pocket. The aim of the present study is to test the antimicrobial efficacy of five common antibiotic schemes at physiologically relevant concentrations on a multispecies in vitro biofilm model.
Methods: A 10-species in vitro subgingival biofilm model was exposed to metronidazole (15 µg/mL), amoxicillin (15 µg/mL), metronidazole and amoxicillin in combination, doxycycline (2 µg/mL), and azithromycin (10 µg/mL) over 24 hours. Species-specific bacterial numbers were determined by culture on selective agar media or by epifluorescence microscopy.
Results: Metronidazole alone did not affect biofilm composition. Total bacterial counts were significantly reduced by doxycycline, azithromycin, and amoxicillin alone or in combination with metronidazole, albeit by less than 1 log. On the species-specific level, these regimens significantly reduced the numbers of Streptococcus anginosus, Porphyromonas gingivalis, and Fusobacterium nucleatum, as well as Campylobacter rectus (except for amoxicillin alone). The strongest effects were displayed by the combination of amoxicillin and metronidazole.
Conclusion: Antibiotics at concentrations detectable in gingival crevicular fluid do not dramatically reduce total bacterial loads in this in vitro biofilm model, but cause species-specific reductions, which may disrupt the biofilm unity.
Antibiotics are frequently used as adjunctive therapies in the treatment of periodontitis, since the recognition of the infectious etiology of the disease.1 The decision whether to administer antibiotics is based on the severity of untreated disease or the recurrence of prior disease activity. Systemic antibiotics are potentially more efficient compared with locally delivered antibiotics.1 They may reach bacteria invading from the periodontal pocket into the gingival tissue or bacteria residing at distant niches of the oral cavity with the potential to eventually colonize subgingival biofilms.2,3 Common antibiotics used as adjuncts in periodontal treatment are amoxicillin and metronidazole (alone or in combination), doxycycline, and azithromycin.4 After oral administration, the concentration of various antibiotics in gingival crevicular fluid (GCF) can rise to a few micrograms per milliliter. Three days after administration of 500 mg, amoxicillin was found at 14 µg/mL in GCF.5 The same amount of metronidazole leads to approximately 13 µg/mL in GCF after at least 2 days of administration.6 Administration of 100 mg doxycycline hyclate resulted in approximately 2 µg/mL in GCF 2 hours after a single-dose administration.7 Administration of 500 mg azithromycin, followed by 250 mg 24 hours later, resulted in 7.5 and 2.5 µg/mL in GCF after 2 and 15 days, respectively.8 Prior microbiologic testing of the periodontal pockets can be useful in directing the clinical decision to administer a systemic antibiotic regimen as adjunctive periodontal treatment. Detection of high numbers of the red-complex species (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) would be a suitable indication for the administration of metronidazole alone or (preferably) in combination with amoxicillin, a regimen that is also effective against Aggregatibacter actinomycetemcomitans and Gram-positive pathogens.9,10 Indeed, the systemic co-administration of amoxicillin and metronidazole has demonstrated superiority over other antibiotic regimens in the treatment of clinically or microbiologically defined variants of periodontal disease.11,12 The minimum inhibitory concentration (MIC) for a number of putative periodontal pathogens is shown to be below the antibiotic concentrations achievable in GCF.13 Nevertheless, studies on the efficacy of antibiotics have traditionally used planktonic bacterial cultures of periodontal pathogens, and in many cases, at concentrations that are not of physiologic relevance. Importantly, the targeted bacteria within the periodontal pocket constitute part of complex biofilm communities, which makes them more virulent and more impenetrable to antibiotics.14 It is therefore well established that biofilm-associated periodontal diseases cannot be treated by chemical agents (such as antibiotics) alone. Hence, to be effective within the environment of the periodontal pocket, the antibiotic concentration in GCF should be considerably higher than the MICs indicated by their in vitro efficacy.15 Therefore, the aim of the present study is to validate the efficacy of five antibiotic schemes commonly used in periodontal practice on a well-defined 10-species in vitro subgingival biofilm model, at concentrations reachable in GCF.
