Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp

Abstract

Background: Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.

Methodology/principal findings: Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C(12). The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown.

Conclusions/significance: These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.

Figure 1. Biofilm formation among the 50 isolates of Acinetobacter spp. in microtiter plates.

Strain numbers are designated in x axis. About 60% of the isolates significantly forms biofilms when incubated for prolonged hours. Data are pooled from triplicate experiments. The results are expressed as the mean and the error bars indicate the standard deviation. The P value was less than 0.05 (P<0.05).

Figure 2. Agar plate assays for screening the production of acylated homoserine lactone (AHL) in Acinetobacter spp. clinical isolates using Chromobacterium violaceum CV026 (Inhibition) as monitor system.

All the seven isolates that tested positive for long-chain AHLs are shown.

Figure 3

(A) Chromobacterium violaceum CV026 well-diffusion assay in LB agar. N-hexanoyl homoserine lactone (HHL) was added to wells in agar containing Chromobacterium violaceum CV026. Zones of HHL-induced violacin production is seen surrounding the wells. Increasing concentration of AHL shows the increase in zone diameters. Standard curve showing the relationship between concentration of acylated homoserine lactone (N-hexanoyl homoserine lactone (HHL) & N-Decanoyl homoserine lactone (DHL)) and resulting diameters of induced/inhibited zones in Chromobacterium violaceum CV026 monitor system. (B) Well-diffusion assay for the tested isolates. Extraction efficiencies were tested on supernatants from Acinetobacter strains. Yields obtained were in the range of 1×10⁻⁹ moles to 6×10⁻⁹ moles of AHLs.

Figure 4. MS profiles of AHL signals.

An Agilent 6520 LC-ESI-QTOF in full scanning positive ion mode or auto-MS/MS mode in a range of 50–1600 m/z was used. TIC trace of each sample (1, 11, 53, 54, 93, 102, 117) with overlaid common precursor ions are respectively shown in figures 1, 2, 3, 4, 5, 6 and 7. Each of the peaks was then analyzed by MS to identify the parent ion, and the full MS profile for each ion was established to identify the exact chain lengths of the AHLs.

Figure 5. The electrophoretic banding patterns of the PCR products using the primers for the abaI gene.

Lane 1–100bp DNA ladder, lane 2, 3, 4, 5, 6, 7, 8– amplicons from isolates S1, S11, S53, S54, S93, S102 and S117 respectively, lane 9– Negative control.

Figure 6. Biofilm formation of the wild type abaI and abaI::Tc mutant.

(A) The microtiter plate was employed to evaluate the biofilm formation among the wild type abaI and abaI::Tc mutant over time. There is considerable inhibition of biofilm formation in the abaI::Tc mutant compared to the wild type. (B) Ethyl-acetate (EtAc) extracts of wild type and the abaI::Tc mutant from the culture supernatants were added for the complementation experiments.