Supplementary Material for: The Effect of Citric and Ascorbic Acids as Anti-Biofilm and Anti-Capsular Agents on Multidrug Resistant Acinetobacter baumannii

Objective: Acinetobacter baumannii (A. baumannii) is an opportunistic bacterium with multiple virulence factors, including capsule and biofilm, and is known for its high drug resistance. Anti-virulence natural substances have been suggested as novel alternatives to conventional antibiotics. We aimed to evaluate the effect of citric acid and ascorbic acid as anti-biofilm and anti-capsular agents against multidrug resistant (MDR) A. baumannii clinical isolates. Materials and Methods: Twenty-eight A. baumannii MDR isolates were collected from different clinical sources. The minimum inhibitory concentration (MIC) of each agent was estimated. Biofilm formation and capsule were investigated phenotypically in the absence and presence both agents at ½ and ¼ MICs. The presence of 14 adhesive and non-adhesive virulence genes was investigated. Results: Phenotypically, all the isolates were biofilm producers and were capsulated. The MIC of citric acid ranged from 1.25 to 2.5 mg/ml while that of ascorbic acid was 3 mg/ml for all isolates. Both agents showed significant reduction in biofilm and capsular thinning. Ascorbic acid showed a dose-dependent effect in both biofilm reduction and capsule thinning unlike citric acid. Four genes, papG23, sfa1, fyuA and cvaC, were absent among all isolates, while iutA was present in 100% of isolates. Other genes showed different distributions among the isolates. These virulence genes were not correlated to the antibiofilm effect of both agents. Ascorbic acid was observed to have a better effect than citric acid. This can provide a clue for a better treatment regimen including ascorbic acid against MDR A. baumannii infections.