Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms

Abstract

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions of biofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.

FIG. 1.

Amounts of biofilm formed by S. epidermidis M187 and S. haemolyticus M176 in the absence of dicloxacillin (dark gray bars) and in the presence of a sub-MIC concentration of dicloxacillin (light gray bars), as evaluated by the safranin colorimetric assay. The error bars indicate standard deviations. ABS 490nm, absorbance at 490 nm.

FIG. 2.

Three-dimensional representations of the biofilms using confocal microscopy analysis. Green represents PNAG, which was asymmetrically distributed in the biofilm, and red represents the bacterial cells. (A1) M187 control, lateral view; (A2) M187 control, top view; (B1) M187 in the presence of dicloxacillin, lateral view; (B2) M187 in the presence of dicloxacillin, top view; (C) M176 control; (D) M176 in the presence of dicloxacillin. The images were adjusted with Paint Shop Pro 6.

FIG. 3.

Immunological detection and relative quantification of PNAG extracted from the biofilm matrix. Lane A, M187 control; lane B, M187 in the presence of dicloxacillin; lane C, M176 in the presence of dicloxacillin; lane D, M176 control. Rows from top to bottom represent serial fourfold dilutions of cell surface extract. The image was adjusted with Paint Shop Pro 6.

FIG. 4.

Shifts in the MIC ranges for dicloxacillin, tetracycline, and rifampin for biofilm cells formed in the presence (DCX) or in the absence (CT) of a sub-MIC concentration of dicloxacillin. The image was created with Paint Shop Pro 6.