To examine the contribution of a Resistance-Nodulation-Division (RND) efflux pump to resistance to various antimicrobial agents tested in B. thailandensis and adapt a flow cytometry-assisted susceptibility test (FAST) for the species.

Materials and methods
The mutagenesis experiment comprised several stages. Firstly, flanking regions of the chosen RND efflux pump, BpeAB-OprB, were amplified using PCR, cloned and ligated into a suicide plasmid with a chloramphenicol resistance marker. Once conjugated into B. thailandensis, sucrose counter-selection was used to produce a deletion strain due to allelic exchange. Strains were then screened for chloramphenicol sensitivity and sucrose resistance. The BpeAB-OprB efflux pump mutant was confirmed using a PCR assay. The ability of the efflux pump mutant to resist selected antimicrobial agents (relative to the wild-type) was assessed using the broth microdilution (BMD) susceptibility test method. The wild-type and efflux pump mutant were both exposed to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin and tetracycline at concentrations of 0.25-256µg/mL. The minimum inhibitory concentration (MIC) was recorded after incubation for 24 hours.

To determine the MIC using the the FAST (MICFAST) method, B. thailandensis was incubated overnight. Cells were enumerated by washing an aliquot of bacteria in Hanks buffered saline solution, staining with SYTO® 9 and analysing the stained sample by flow cytometry. A known concentration of bacteria was inoculated into liquid medium and incubated for 3 hours to reach logarithmic phase of growth. Samples were exposed to 0.25-256 µg/mL of meropenem and further incubated for 1 hour. After washing and staining as previously described, cells were analysed using the flow cytometer. Gating methods were applied to the data to obtain the MICFAST. This value was then compared to the MIC value obtained by the BMD method.

Subtle differences in MIC values between the wild-type and efflux pump mutant were observed for ampicillin, ciprofloxacin, tetracycline and chloramphenicol. The FAST method was successfully adapted to wild type B. thailandensis and the MICFAST was within essential agreement of the BMD method for meropenem. Results from this method were obtained within 6-7 hours of the initial incubation step in contrast to the BMD method which requires 18-24 hours.

The contribution of the BpeAB-OprB efflux pump appears to be insignificant for the antibiotics tested. To fully appreciate the role of this efflux system in antimicrobial resistance in B. thailandensis, other factors such as over-lapping substrate specificities and the over-expression of other efflux pump systems need to be excluded. Aspects of the FAST method could be adapted to characterize efflux pump activity. In future studies, a modified FAST method could be applied with efflux pump inhibitors to evaluate the function of BpeAB-OprB and the other efflux pumps of Burkholderia species.