Biofilm-Induced Antibiotic Resistance in Clinical Acinetobacter baumannii Isolates

Abstract

In order to understand the role of biofilm in the emergence of antibiotic resistance, a total of 104 clinical Acinetobacter baumannii strains were investigated for their biofilm-forming capacities and genes associated with biofilm formation. Selected biofilm-formers were tested for antibiotic susceptibilities when grown in biofilm phase. Reversibility of antibiotic susceptibility in planktonic cells regrown from biofilm were investigated. We found 59.6% of the strains were biofilm-formers, among which, 66.1% were non-multidrug resistant (MDR) strains. Presence of virulence genes bap, csuE, and abaI was significantly associated with biofilm-forming capacities. When strains were grown in biofilm state, the minimum biofilm eradication concentrations were 44, 407, and 364 times higher than the minimum bactericidal concentrations (MBC) for colistin, ciprofloxacin, and imipenem, respectively. Persisters were detected after treating the biofilm at 32-256 times the MBC of planktonic cells. Reversibility test for antibiotic susceptibility showed that biofilm formation induced reversible antibiotic tolerance in the non-MDR strains but a higher level of irreversible resistance in the extensively drug-resistant (XDR) strain. In summary, we showed that the non-MDR strains were strong biofilm-formers. Presence of persisters in biofilm contributed to the reduced antibiotic susceptibilities. Biofilm-grown Acinetobacter baumannii has induced antibiotic tolerance in non-MDR strains and increased resistance levels in XDR strains. To address the regulatory mechanisms of biofilm-specific resistance, thorough investigations at genome and transcription levels are warranted.

Keywords: Acinetobacter baumannii; antibiotic resistance; antibiotic tolerance; biofilm; persister.

Figure 1

CLSM images of A. baumannii ST1894 biofilm treated with bactericidal antibiotics. (A) Untreated biofilm cells, (B) biofilm treated with 512 µg/mL imipenem, (C) biofilm treated with 128 µg/mL colistin, (D) biofilm treated with 512 µg/mL ciprofloxacin. Biofilm was incubated with antibiotics at 37 °C for 48 h, which was followed by costaining with propidium iodide (PI) and SYTO 9, and examined under CLSM. Dead cells were stained with PI and appeared red. Viable cells were stained with SYTO 9 and appeared green in color.

Figure 2

Detection of persisters from biofilm and planktonic cells of A. baumannii ST 1894. The number of viable biofilm and planktonic cells at different time points after treatment with 2048 μg/mL ciprofloxacin. Red solid and dotted lines represent untreated biofilm and planktonic cells, respectively. Blue solid and dotted lines represent biofilm and planktonic cells treated with ciprofloxacin, respectively.

Figure 3

CLSM image of A. baumannii ST1894 biofilm treated with ciprofloxacin at 1024 × MIC. Viable bacterial cells in the biofilm was detected by a Live/Dead Biofilm Viability Kit. Persister cells appear green in color, dead cells appear red in color.