Chemicals and materials
Chlorhexidine digluconate (CHG) aqueous standard 20% solution (C9394), ampicillin (A9518), reserpine (R0875), dimethyl sulphoxide (DMSO) (D5879), 3-(4,5-dimethylthiazolyl)-2, 5-diphenyltetrazolium bromide [MTT, thiazolyl blue] (M2128) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (C2759) used in the study were purchased from Sigma-Aldrich company (Germany). Glucose (G8270), Tryptic soy broth (TSB) (22092) and Tryptic soy agar (TSA) (22091) were also bought from Sigma-Aldrich Company (Germany). Distilled grade water was used for all the experiments.
Bacterial strains
A total of four bacterial isolates were used in the study. The four isolates comprised a single clinical strain of P. aeruginosa and S. aureus along with their respective reference strains; ATCC 27853 and ATCC 9144. Clinical strains used were isolated from patients at Parirenyatwa Hospital (Department of Medical Microbiology, College of Health Sciences, University of Zimbabwe, Harare, Zimbabwe). The ATCC strains were acquired from the Microbiological Section in the Department of Biological Sciences at the University of Botswana (Gaborone, Botswana). All bacteria were kept as glycerol stocks at -35 °C and overnight cultures were used for each assay.
Development of a chlorhexidine accumulation assay protocol
A chlorhexidine accumulation assay protocol was developed using the basic techniques of an accumulation assay established by Mortimer and Piddock [16]. A spectrophotometric method to quantify the CHG accumulated within bacterial cells was developed. A 2800 UNICO UV/VIS spectrophotometer (UNICO United Products and Instruments Inc., Dayton, United States) was used for quantifying the CHG. CHG absorbs optimally at 255 nm [17], thus, the measurement of CHG using a UV/VIS spectrophotometer was carried at a wavelength of 255 nm. The suitability of using the spectrophotometer was validated, by determining the optical density (OD) of a series of the standards of CHG prepared on the day. Standards of 0-12 µM CHG were prepared and analysed in duplicate. A calibration curve was plotted for the absorbances obtained from the series of standard using Graphpad™ version 5 for Windows (Graphpad™ Software Inc., San Diego, California, USA). The goodness-of-fit of linear regression, R2, obtained was used to determine the appropriateness of using a UV/VIS spectrophotometer for measuring the amount of CHG. Sub-inhibitory concentrations of chlorhexidine, CCCP and reserpine used in the accumulation assay were determined using half the MIC value (1/2MIC). A calibration curve was used to interpolate concentrations of chlorhexidine in samples in the accumulation assay.
Determination of MIC
Stock solutions of 5% CHG and 100 µg/ml ampicillin were prepared using sterile distilled water while the stocks of reserpine and CCCP were prepared in DMSO. All two-fold serial dilutions were made in TSB. The plate count method was used to determine the viable cell count of serially diluted overnight culture and appropriate dilutions made to reach 1 × 106 CFU/ml. Microdilution tests of CHG, CCCP and reserpine, as recommended by the Clinical and Laboratory Standards Institute [18] were carried out in quadruplicate using 96-well microtitre plates. The general set-up of the 96-well microtitre plate is as shown in Fig. 1. Each test well was filled with 100 µl of defined chlorhexidine dilution and 100 µl of 2 × 106 CFU/ml test isolate suspension. Wells for the positive control contained 100 µl of media and 100 µl of 2 × 106 CFU/ml to give a final concentration of 106 CFU/ml. After overnight incubation at 37 °C in a humidified atmosphere, bacterial growth was estimated as absorbance for turbidity using a Genios Pro microplate reader (Austria). A separate microtitre plate was set up to determine the MIC of ampicillin against all four isolates. The MIC of CHG in the presence of an EPI: CCCP or reserpine was also determined using sub-inhibitory concentrations (1/2 MIC value for the EPI calculated from MIC values initially obtained in the study). All microplates were incubated overnight at 37 °C in a Lab Companion incubator (Jeio Tech. Co. Ltd, Seoul, Korea) under a closed humidified atmosphere. Bacterial growth was determined as cell density using a Genios Pro microplate reader (Tecan Group Ltd, Grodig, Austria) to measure absorbance at 590 nm prior to and after incubation. The mean absorbance difference of varying concentrations of each bacterium in the presence CHG was compared to that of the media (TSB) that served as the negative control. The MIC was the minimum concentration of chlorhexidine that did not allow the growth of microbes. The MTT assay as outlined by Hansen et al., [19] with minor modifications, was utilised to visually observe bacterial viability. The MTT assay involved the addition of 30 µl of 1 mg/ml MTT to each well and reincubated for 1 h at 37 °C. The yellow colour of the tetrazolium salt would persist if cells were non-viable or be reduced by dehydrogenases and reductases in viable cells to a blue or purple colour. The reduced salt would form an insoluble precipitate, which was dissolved by the addition of 30 µl DMSO at the end of the incubation period. The MIC was determined as the minimum concentration of chlorhexidine in which no colour change in MTT was observed.
Accumulation assay
The amount of CHG that accumulated in P. aeruginosa and S. aureus in the presence of standard efflux pump inhibitors, either CCCP or reserpine was studied using the protocol developed based on the procedure outlined by Mortimer and Piddock [16]. All tests were carried out in duplicate. A volume of 200 µl of an overnight culture was transferred into 200 ml TSB and incubated overnight at 37 °C with shaking (120 rpm). Cells were collected from the broth by centrifuging at 3000 rpm for 15 min using a Rotafix Centrifuge (Taufkirchen, Germany), washed two times using phosphate buffer solution (PBS), pH 7.4. The washed cells were suspended in PBS and centrifuged for 15 min. The pellet was resuspended in PBS containing sodium azide to give a final cell concentration of 40 mg/ml. A final concentration of 1/2MIC of CHG was added to the cells. The mixture was incubated at 37 °C for 30 min with shaking (120 rpm). Cells were collected by centrifuging at 4000 rpm for 15 min and distributed into 5 ml aliquots of tubes A to D. Tube A was the negative control containing no glucose thus no source of energy. Tube B was the positive control containing 1.0 M glucose being the source of ATP. Tubes C contained 1.0 M glucose and a final concentration of 1/2MIC of CCCP (calculated from the MIC value CCCP). Tube D contained 1.0 M glucose and a final concentration of 1/2MIC of reserpine. All tubes were incubated at 37 °C with agitation (120 rpm) for 1 hour in a Lab Companion incubator. Cells were collected by centrifuging at 4000 rpm for 15 min and the supernatant stored for CHG efflux quantification. The pellet obtained from each tube was resuspended in 0.1 M glycine–HCl pH 3, was mixed using a vortex mixer and incubated overnight to allow cell lysis. The tubes with lysed cells were centrifuged at 4000 rpm for 15 min and the supernatant retained for quantification of CHG. The quantification of CHG of the supernatants was performed using a 2800 UNICO UV/VIS spectrophotometer (UNICO United Products and Instruments Inc., Dayton, United States) at a wavelength of 255 nm. Sample concentrations were interpolated from a standard curve initially plotted using known standards of CHG. Sample quantities obtained from interpolated values were expressed as percentage chlorhexidine accumulated (% CHX accumulated) in relation to possible accumulation in the presence of energy in the form of ATP from glucose, calculated as:
$$\frac{{[{\text{chlorhexidine}}_{\text{sample}} ]}}{{[{\text{chlorhexidine}}_{\text{control}} ]}} \times 100$$
where, [chlorhexidinesample] is concentration of chlorhexidine under different treatments and [Chlorhexidinecontrol] is concentration of chlorhexidine of control (glucose only).
Statistical analyses
Graphpad™ for Windows Version 5 (Graphpad™ Software Inc., San Diego, California, USA) was used for statistical analyses of the results. One way analysis of variance (ANOVA) and a Dunnet post-test was used to compare results obtained for the positive control against that of test samples a P value of 0.05 or less were considered significant.