Molecular Analysis and Expression of bap Gene in Biofilm-Forming Multi-Drug-Resistant Acinetobacter baumannii

Abstract

Background: Acinetobacter baumannii is commonly resistant to nearly all antibiotics due to presence of antibiotic resistance genes and biofilm formation. In this study we determined the presence of certain antibiotic-resistance genes associated with biofilm production and the influence of low iron concentration on expression of the biofilm-associated protein gene (bap) in development of biofilm among multi-drug-resistant A. baumannii (MDRAB).

Methods: Sixty-five MDRAB isolates from clinical samples were collected. Molecular typing was carried out by random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR). Biofilm formation was assayed by the microtiter method.

Results: The sequence of bap was determined and deposited in the GenBank database (accession no. KR080550.1). Expression of bap in the presence of low iron was analyzed by relative quantitative real time PCR (rqRT-PCR). Nearly half of the isolates belonged to RAPD-types A and B remaining were either small clusters or singleton. The results of biofilm formation revealed that 23 (35.4%), 18 (27.7%), 13 (20%), and 11 (16.9%) of the isolates had strong, moderate, weak, and no biofilm activities, respectively. ompA and csuE genes were detected in all, while bap and bla_PER-1 were detected in 43 (66%) and 42 (64%) of the isolates that showed strong and moderate biofilm activities (p ≤ 0.05), respectively. Analysis of bap expression by rqRT-PCR revealed five isolates with four-fold bap overexpression in the presence of low iron concentration (20 µM).

Conclusion: The results suggest that bap overexpression may influence biofilm formation in presence of low iron concentration.

Keywords: Acinetobacter baumannii; Biofilm; Biofilm-associated Protein (bap); Iron; rqRT-PCR.

Fig. 1

Agarose gels following RAPD-PCR amplification with M13 and DAF4 primers. Fingerprint patterns obtained for A. baumannii isolates A1-A8 from hospital A and B1-B4 from hospital B. M1, 1-Kb DNA ladder. M2, 100-bp DNA ladder, C⁺, positive control consisting of A. baumannii ATCC 19606.

Fig. 2

Dendrogram of 65 A. baumannii isolates based on RAPD-PCR data. The fingerprints show genetic relationships among eleven clusters. Clustering was based on the unweighted pair group method with arithmetic mean. The vertical line is showing 80% similarity cut-off.

Fig. 3

Biofilm quantification by various groupings of the population of 65 clinical A. baumannii isolates. The above results are means of three experiments. SD = standard deviation. The biofilm intensity was determined by microplate assay as described in the text.

Fig. 4

RQ RT-PCR analysis of bap expression in 65 MDRAB isolates recovered from ICU patients in M9 medium containing 20 µM FeCl₃. Each analysis was performed three times.