Objective: To observe the antimicrobial activity of topical agents commonly used for burns on Acinetobacter baumannii (AB) in both free and biofilm states, and their synergistic effect with ambroxol on AB within biofilm.
Methods: Eleven AB strains were isolated from wound excretion, respiratory tract, and blood of patients hospitalized in our hospital from August 2005 to April 2007. (1) The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of mafenide acetate and chlorhexidine acetate to free AB (including drug-resistant, drug-sensitive, and standard strains) were determined by dilution method. (2) AB was cultured with LB or TSB medium for 12, 24, and 48 h to form biofilm, and it was treated with above-mentioned two topical agents in MBC (mafenide group and chlorhexidine group) for 30 min. Biofilm not treated by topical agent was used as control group. The biofilm thickness was determined with confocal laser scanning microscope. The proportion of living bacteria in biofilm was calculated. AB biofilm in each topical agent group was mixed and inoculated into LB culture dish to observe the growth of bacteria. (3) AB was cultured with LB medium for 48 h to form biofilm, which was respectively treated by above-mentioned two topical agents in MBC (mafenide group and chlorhexidine group) and combination of each topical agent with 3.75 mg/mL ambroxol solution (ambroxol + mafenide group and ambroxol + chlorhexidine group) for 30 min. Biofilm not treated by topical agents was used as control group. Growth of bacteria in biofilm was detected with MTT method (denoted as absorbance value). Data were processed with one-way analysis of variance and LSD-t test.
Results: (1) MIC of mafenide acetate and chlorhexidine acetate for free AB was respectively 25.00 mg/mL and 0.03 mg/mL. MBC of both agents for free AB was the same as their MIC. (2) Among three groups, the thickness of biofilm of sensitive AB was thicker than that of drug-resistant bacteria at most of the time points. Compared with those in control group, biofilm thickness and proportion of living bacteria in biofilm were slightly decreased in mafenide and chlorhexidine groups. The growth of bacteria was abundant in each group. (3) Absorbance value of drug-resistant bacteria in control, mafenide, and chlorhexidine groups was respectively 0.776 ± 0.071, 0.625 ± 0.063, and 0.420 ± 0.068. Absorbance value of drug-resistant bacteria in ambroxol + mafenide group (0.174 ± 0.089) was significantly lower than that of control group (t = 11.823, P = 0.000) and mafenide group (t = 9.248, P < 0.01). Absorbance value of ambroxol + chlorhexidine group (0.178 ± 0.044) was significantly lower than that of control group (t = 16.009, P = 0.000) and chlorhexidine group (t = 6.681, P < 0.01).
Conclusions: Drug-resistant AB forms biofilm readily, which prevents topical agents from killing the bacteria inside. Combined use of ambroxol with topical agents gives synergistic effect on killing AB in biofilm in the wound.