Resumen de Microcalorimetric Determination of the Effects of Amoxicillin, Metronidazole, and Their Combination on In Vitro Biofilm
Background: The mechanism of action of adjuvant antibiotic therapy in the treatment of peri-implantitis is not well understood. The aim of this study is to investigate antibiotic susceptibility of an in vitro biofilm by isothermal microcalorimetry (IMC).
Methods: Titanium disks containing a 72-hour three-species biofilm (Streptococcus sanguinis DSM20068, Fusobacterium nucleatum ATCC10953, and Porphyromonas gingivalis DSM20709) were placed in a series of IMC ampoules with nutrient agar supplemented with increasing concentrations of amoxicillin, metronidazole, or their combination and incubated anaerobically for 10 days. Lag time and maximum growth rate were determined from continuous heat-flow recordings of metabolic activity. To validate the IMC biofilm results, adherent S. sanguinis and P. gingivalis were incubated anaerobically in medium supplemented with antibiotics at 37°C for 24 hours, and their vitality was determined by live/dead staining, conventional culturing, and IMC.
Results: In all biofilm samples incubated with antibiotics, a prolonged lag phase was observed compared with controls (P <0.05). Maximum growth rate was significantly lower for samples treated with either amoxicillin or metronidazole compared with controls (P <0.05). Combining the antibiotics did not improve this effect. Concentrations exceeding 10 times the minimum inhibitory concentration completely inhibited the growth of adherent S. sanguinis and P. gingivalis, whereas lower concentrations resulted in only a delay in the lag phase. A poor correlation was observed between live/dead staining and conventional culturing.
Conclusions: IMC gives new evidence about antibiotic effects on oral biofilms and is more informative than conventional culture and live/dead assays. The combination of antibiotics was found to be more efficient than metronidazole alone; however, only minor differences in growth inhibition were detected compared with amoxicillin alone.
The major goal of periodontal therapy is to reduce or eliminate pathogenic species and maintain colonization by host-compatible species in the oral cavity.1,2 The pathogenic microbiota form biofilms on implant surfaces that, when left unattended, lead to inflammatory reactions and progress to peri-implantitis.3 Different therapeutic options have been advocated for the treatment and prevention of periodontal/peri-implant diseases. In particular, the combination of metronidazole and amoxicillin is frequently used and is known to have a synergistic or additive antimicrobial effect and to improve clinical results against periodontal infections.4-8 In surgical peri-implantitis treatment, the use of amoxicillin and clavulanic acid has been recommended owing to the observed presence of Staphylococcus aureus species, while in patients with a history of periodontal disease, the combination of amoxicillin and metronidazole is indicated.9-11 However, it is important to note that bacteria in biofilms have been shown to be 100 to 1,000 times less susceptible to antibiotics than their planktonic counterparts, as the antibiotics have restricted penetration through the biofilm.12 Additionally, microorganisms within biofilms respond to local environmental conditions in several ways, such as altering their gene-expression patterns or undertaking physiologic activities to adapt to a particular condition (i.e., stress through antimicrobials).12-14 Thus, most commonly, mechanical debridement combined with systemic antibiotic treatment is applied in the clinical treatment of peri-implantitis.9,15 Various approaches have been used to quantify the effects of antibiotic treatment on oral microbiota. The common practice is to analyze the microflora via conventional culture methods that have proven to be efficient in testing the susceptibility of bacterial cells to antibiotics.8,16,17 Nevertheless, viable but non-cultivable (VBNC) cells, a population of cells defined by low levels of metabolic activity and the ability to switch to being cultivable again when growth conditions improve, may influence the outcome of conventional tests and predispose patients to (re)infection.14 Fluorescence microscopy combined with live/dead staining has also been used to detect the effects of antibiotics on adherent microorganisms.18-20 Whereas the conventional culture method includes only cultivable bacteria that are able to proliferate, live/dead staining combined with fluorescence microscopy enables the quantification of viable cells based on the intactness of their membranes, including the VBNC population of cells. Combined with confocal laser scanning microscopy, it allows vitality to be assessed throughout biofilms.14 Isothermal microcalorimetry (IMC) is a highly sensitive culture-based technique that allows for the measurement of heat generated by microbial activities. Bacteria replicate in a suitable culture medium, resulting in an exponential increase in the heat production rate that can be recorded in real time by IMC (i.e., heat-flow curve). When using IMC, broth cultures are often preferred for rapid detection of bacterial growth in clinical settings,21-23 but because the method measures heat passively, culture growth can also be evaluated on a solid or viscous medium as well as in matrix.24,25 Heterogeneity of biofilms can also be assessed by this method.26 Because antimicrobials inhibit bacterial growth, IMC has been used to determine antimicrobial susceptibility, as demonstrated with strains of S. aureus, Enterococcus faecalis, and fungi.21,27-30 The mechanism of action of adjuvant antibiotic therapy in the treatment of peri-implantitis is not well understood. The aim of this study is to use IMC to determine the metabolic activity of an in vitro three-species biofilm26 associated with periodontitis and peri-implantitis in the presence of amoxicillin, metronidazole, or their combination.
