Microscopic and spectroscopic analyses of chlorhexidine tolerance in delftia acidovorans biofilms

Abstract

The physicochemical responses of Delftia acidovorans bio films exposed to the commonly used antimicrobial chlorhexidine (CHX) were examined in this study. A CHX-sensitive mutant (MIC, 1.0 μg ml-1) was derived from a CHX-tolerant (MIC, 15.0 μg ml-1) D. acidovorans parent strain using transposen mutagenesis. D. acidovorans mutant (MT51) and wild-type (WT15) strain bio films were cultivated in flow cells and then treated with CHX at sub-MIC and inhibitory concentrations and examined by confocal laser scanning microscopy (CLSM), scanning transmission X-ray microscopy (STXM), and infrared (IR) spectroscopy. Specific morphological, structural, and chemical compositional differences between the CHX-treated and -untreated biofilms of both strains were observed. Apart from architectural differences, CLSM revealed a negative effect of CHX on bio film thickness in the CHX-sensitive MT51 biofilms relative to those of the WT15 strain. STXM analyses showed that the WT15 bio -films contained two morphochemical cell variants, whereas only one type was detected in the MT51 bio films. The cells in the MT51 bio films bioaccumulated CHX to a similar extent as one of the cell types found in the WT15 biofilms, whereas the other cell type in the WT15 biofilms did not bioaccumulate CHX. STXM and IR spectral analyses revealed that CHX-sensitive MT51 cells accumulated the highest levels of CHX. Pretreating bio films with EDTA promoted the accumulation of CHX in all cells. Thus, it is suggested that a subpopulation of cells that do not accumulate CHX appear to be responsible for greater CHX resistance in D. acidovorans WT15 bio film in conjunction with the possible involvement of bacterial membrane stability. Copyright