Antibiotic susceptibility of Aggregatibacter actinomycetemcomitans JP2 in a biofilm microenvironment

Objectives: Localized Aggressive Periodontitis (LAgP) is an infectious disease generally affecting young patients, that can result in early tooth loss. Treatment includes mechanical debridement, often associated with empiric antibiotic treatment. The main aetiological agent of LAgP is Aggregatibacter actinomycetemcomitans (Aa). The aims of this study were to develop biofilm models for the growth of Aa, test its antibiotic sensitivity in these biofilm models, and compare it with planktonic growth. Methods: To mimic in-vivo growth of these bacteria in the oral cavity, two biofilm models were developed and used to test the commonly used antibiotics, amoxicillin; amoxicillin+clavulanat; clindamycin; metronidazole; doxycycline and combination of amoxicillin and metronidazole. A 96-well plate static assay was used for screening purposes, and a flow chamber model, which is more similar to the in-vivo microenvironment, was used to examine the effect of the most effective antibiotic on established biofilms. Biofilm biomass was determined after 24h anaerobic incubation by crystal violet staining and absorbance reading in the 96-well plate assay. In the flow cell model biofilms were analyzed by Confocal-Laser Scanning Microscopy and Comstat software analysis program to determine biometric parameters after 30min incubation with either the chosen antibiotic or saline. Statistical analysis was performed using Unpaired student's t-test to compare variables between groups. Results: In the 96-well plate model, Aa biofilms were established that comprised of 3.79±0.22*10⁶cells. Of the antibiotics tested, doxycycline was most efficacious, with a Minimal inhibitory concentration against planktonic cells of 0.13mg/L and Minimal biofilm inhibitory concentration of 9.85mg/L, followed by metronidazole+amoxicillin (13mg/L and 20mg/L, respectively). Addition of 10 mg/L doxycycline to mature Aa biofilms in the flow chamber model resulted in a significant 28% reduction in biofilm thickness, 22.82±3.78µm compared with 31.68±3.36 µm (p=0.04). Conclusions: Out of the commonly used antibiotics examined in this study, doxycycline was the most effective against Aa biofilms.