Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

Abstract

Chronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency of Acinetobacter baumannii and the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolated A. baumannii strains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designated A. baumannii ABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI, adeB, adeG, adeJ, carO, and ompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation of adeG correlated with biofilm induction. The consistent upregulation of adeG and abaI was detected in A-III-type A. baumannii in response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused by A. baumannii. This study provides useful information for the development of antibiofilm strategies.

FIG 1

ERIC-PCR fingerprinting (top) and dendrogram illustration (bottom). The dendrogram was produced using the squared Euclidean distance measurement and average linkage clustering method with the program IBM SPSS Statistics 19. The dissimilarity units are arbitrary and are based on the sum of the squared Euclidian distance measurements.

FIG 2

Bacterial biofilms formed by A. baumannii ABR2 (A and B), ABR11 (C and D), and ABS17 (E and F) in the presence of subinhibitory concentrations of levofloxacin and meropenem, respectively. The error bars represent the standard deviations. *, significant difference at a P value of 0.05.

FIG 3

Fold changes in the expression of the abaI, adeB, adeG, adeJ, carO, and ompA genes in A. baumannii ABR2 (A and B), ABR11 (C and D), and ABS17 (E and F) in the presence of subinhibitory concentrations of levofloxacin and meropenem, respectively. The error bars represent the standard deviations. *, significant difference at a P value of 0.05.

FIG 4

Potential role of the AdeFGH efflux pump in the transport of quorum-sensing molecules (AHLs) in A. baumannii during biofilm formation. Antibiotics induce the overexpression of the AdeFGH efflux pump (step 1), leading to a coefflux of AHLs together with antibiotics (step 2). Consequently, an increase in the density of AHLs in the extracellular environment accelerates the entrance of AHLs into the intracellular environment to form AbaR-AHL complexes (step 3). The formation of AbaR-AHL activates AbaI via AbaR quorum-sensing regulation, which induces the synthesis of AHL (step 4). The synthesized AHLs are transferred to the extracellular environment by the activated AdeFGH efflux pump and sensed by nearby A. baumannii cells. The enhancement in cell interactions through increased AHL sensing results in the augmentation of biofilm formation (steps 5 and 6).