Fig 1.
Vancomycin enhances biofilm formation with glucose but reduces propensity to form biofilms without glucose.
(A) A static biofilm assay was performed when VRSA cells (SJC1200) were cultured in BHI medium or medium supplemented with 0.5% (BHIg) or 1.5% (BHIhg) glucose, respectively, in the absence/presence of vancomycin (32 μg/ml). Adherent cells from representative triplicate assays in each condition were stained with safranin O and are shown on the bottom. (B) A time course of the number viable cells was performed when VRSA cells were cultured in BHI or BHIg in the absence (Van0) or presence of vancomycin (32 μg/ml; Van32). The results are presented as the means±sd of the log10 CFU/ml from three separate experiments. (C) A time course of the static biofilm assay was performed when VRSA cells were cultured in different conditions as above. (D) The time course of glucose consumption. (E) A time course of the static biofilm assay was performed as above, but vancomycin was added immediately after the glucose was exhausted in BHI (at 6 h). * P < 0.05 and ** P < 0.005 in this figure and hereafter.
Fig 2.
Vancomycin enhances catheter-associated biofilm formation in diabetic mice infected with vancomycin-non-susceptible S. aureus.
(A) Experimental control of catheter-associated biofilm formation assay in mice. Determination of the number of viable biofilm-bound bacteria in subcutaneous catheters removed from healthy/diabetic mice upon infection with VSSA strain ATCC 12598 in the presence of vancomycin treatment. (B) Subcutaneous catheters removed from healthy/diabetic mice upon infection with VRSA strain SJC1200 in the absence/presence of vancomycin treatment. Two representative catheters in each group are shown in the figure. (C) Catheter-associated biofilm materials were observed under SEM. Photos were taken at the indicated magnifications. (D) Determination of the number of viable biofilm-bound bacteria in the removed catheters, as above. (E) Determination of the number of viable biofilm-bound bacteria in the catheters removed from diabetic mice infected with VISA strain Mu50 following vancomycin treatment. (F) The correlation between the blood glucose concentration and catheter biofilm-forming capacity. The capacity was evaluated by determining the number of viable biofilm-bound bacteria in subcutaneous catheters. The number of mice used in each group is shown.
Fig 3.
Bacterial autolysis triggered by vancomycin plays an important role in the enhancement of biofilm formation.
Determination of the number of viable biofilm-bound bacteria in subcutaneous catheters removed from healthy/diabetic mice (A) infected with VRSA strain SJC1200 (also chloramphenicol resistance) upon chloramphenicol (Cm) treatment or (B) infected with cidA null mutant SJC1201 (autolysis deficient VRSA) upon vancomycin treatment. Six mice were used in each group.
Fig 4.
Vancomycin plays a different role in bladder catheter biofilm formation between healthy and diabetic rats.
Determination of the number of viable biofilm-bound bacteria in the bladder catheters removed from healthy/diabetic rats infected with VRSA SJC1200 in the absence/presence of vancomycin treatment. Six rats were used in each group.
Fig 5.
Investigation of the mechanisms underlying vancomycin-triggered biofilm degradation in the absence of glucose.
(A) Time courses of PIA production by VRSA strain SJC1200 in different media with/without vancomycin treatment. (B) Evaluation of bacteria-secreted DNase activity by visualizing DNA degradation after incubating a DNA product (861 bp) with the supernatant removed from different VRSA culture conditions. 12 hr and 24 hr: supernatant from a 12-h and a 24-h culture system, respectively. M: a 100-bp DNA ladder marker. P and N: positive and negative controls, respectively. (C) Detection of changes in the transcription levels of cidA and agrA in VRSA by qRT-PCR upon different treatments. The fold change of each transcript was compared with vancomycin-untreated samples at T0. (D) Detection of the bacteria-secreted protease activity by the evaluation of the biofilm degradation (suppression) potential using a static biofilm assay in the absence (Non-PI) or presence (PI) of a protease inhibitor cocktail.