Relationship between Antibiotic Resistance, Biofilm Formation, and Biofilm-Specific Resistance in Acinetobacter baumannii

Abstract

In this study, we aimed to examine the relationships between antibiotic resistance, biofilm formation, and biofilm-specific resistance in clinical isolates of Acinetobacter baumannii. The tested 272 isolates were collected from several hospitals in China during 2010-2013. Biofilm-forming capacities were evaluated using the crystal violet staining method. Antibiotic resistance/susceptibility profiles to 21 antibiotics were assessed using VITEK 2 system, broth microdilution method or the Kirby-Bauer disc diffusion method. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) to cefotaxime, imipenem, and ciprofloxacin were evaluated using micro dilution assays. Genetic relatedness of the isolates was also analyzed by pulsed-field gel electrophoresis (PFGE) and plasmid profile. Among all the 272 isolates, 31 were multidrug-resistant (MDR), and 166 were extensively drug-resistant (XDR). PFGE typing revealed 167 pattern types and 103 clusters with a similarity of 80%. MDR and XDR isolates built up the main prevalent genotypes. Most of the non-MDR isolates were distributed in a scattered pattern. Additionally, 249 isolates exhibited biofilm formation, among which 63 were stronger biofilm formers than type strain ATCC19606. Population that exhibited more robust biofilm formation likely contained larger proportion of non-MDR isolates. Isolates with higher level of resistance tended to form weaker biofilms. The MBECs for cefotaxime, imipenem, and ciprofloxacin showed a positive correlation with corresponding MICs, while the enhancement in resistance occurred independent of the quantity of biofilm biomass produced. Results from this study imply that biofilm acts as a mechanism for bacteria to get a better survival, especially in isolates with resistance level not high enough. Moreover, even though biofilms formed by isolates with high level of resistance are always weak, they could still provide similar level of protection for the isolates. Further explorations genetically would improve our understanding of these processes and provide novel insights in the therapeutics and prevention against A. baumannii biofilm-related infections.

Keywords: Acinetobacter baumannii; antibiotic resistance; biofilm; biofilm-specific resistance; pulsed-field gel electrophoresis.

Figure 1

Antibiotic resistance phenotypes of A. baumannii isolates examined in this study. (A) The resistance rate of all strains to 16 of the 21 antibiotics were above 50%. (B) Approximately 72.4% of the isolates exhibited multidrug or extensively drug resistance.

Figure 2

Biofilm formation of A. baumannii isolates examined in this study. OD₅₅₀, optical density at 550 nm, represents biofilm forming capacity. (A) The level of biofilm formation was assessed for each isolate. Strains were divided into three groups according to their antibiotic resistance phenotypes: non-multidrug-resistant (non-MDR), multidrug-resistant (MDR), and extensively drug-resistant (XDR). The red line shows the optical density at 550 nm (OD₅₅₀) of the biofilm biomass produced by the reference strain (ATCC 19606). Larger proportion of non-MDR isolates tended to form stronger biofilms (higher OD₅₅₀ values). (B) Distribution of resistance phenotypes among different biofilm production capacities displayed as a percentage stacked bar graph. Population that exhibited more robust biofilm formation likely contained larger proportion of non-MDR isolates. (C) Distribution of biofilm formation of isolates with different resistance phenotypes. Isolates with higher level of resistance tended to form weaker biofilms.

Figure 3

Relationship between biofilm formation and the resistance of A. baumannii isolates to each of the nine antimicrobial categories. OD₅₅₀, optical density at 550 nm, represents biofilm forming capacity. (A–G): For aminoglycosides, carbapenems, fluoroquinolones, penicillins + β-lactamase inhibitors, folate pathway inhibitors, tetracyclines, and penicillins, susceptible isolates tended to form stronger biofilms (higher OD₅₅₀ values) than non-susceptible ones. (H,I): For cephems and lipopeptides, no significant difference in biofilm formation among susceptible and non-susceptible isolates was observed.

Figure 4

Resistance in A. baumannii to cefotaxime, imipenem, and ciprofloxacin during planktonic and biofilm growth. (A) Cefotaxime. (B) Imipenem. (C) Ciprofloxacin. For these three antibiotics, biofilm-specific resistance (MBEC) was consistently higher in isolates with higher antibiotic resistance of planktonic bacteria (MIC). The enhancement in resistance after biofilm formation occurred independent of biofilm quantity.