Aminoglycoside inhibition of Staphylococcus aureus biofilm formation is nutrient dependent

Abstract

Biofilms represent microbial communities, encased in a self-produced matrix or extracellular polymeric substance. Microbial biofilms are likely responsible for a large proportion of clinically significant infections and the multicellular nature of biofilm existence has been repeatedly associated with antibiotic resistance. Classical in vitro antibiotic-susceptibility testing utilizes artificial growth media and planktonic microbes, but this method may not account for the variability inherent in environments subject to biofilm growth in vivo. Experiments were designed to test the hypothesis that nutrient concentration can modulate the antibiotic susceptibility of Staphylococcus aureus biofilms. Developing S. aureus biofilms initiated on surgical sutures, and in selected experiments planktonic cultures, were incubated for 16 h in 66 % tryptic soy broth, 0.2 % glucose (1× TSBg), supplemented with bactericidal concentrations of gentamicin, streptomycin, ampicillin or vancomycin. In parallel experiments, antibiotics were added to growth medium diluted one-third (1/3× TSBg) or concentrated threefold (3× TSBg). Following incubation, viable bacteria were enumerated from planktonic cultures or suture sonicates, and biofilm biomass was assayed using spectrophotometry. Interestingly, bactericidal concentrations of gentamicin (5 µg gentamicin ml(-1)) and streptomycin (32 µg streptomycin ml(-1)) inhibited biofilm formation in samples incubated in 1/3× or 1× TSBg, but not in samples incubated in 3× TSBg. The nutrient dependence of aminoglycoside susceptibility is not only associated with biofilm formation, as planktonic cultures incubated in 3× TSBg in the presence of gentamicin also showed antibiotic resistance. These findings appeared specific for aminoglycosides because biofilm formation was inhibited in all three growth media supplemented with bactericidal concentrations of the cell wall-active antibiotics, ampicillin and vancomycin. Additional experiments showed that the ability of 3× TSBg to overcome the antibacterial effects of gentamicin was associated with decreased uptake of gentamicin by S. aureus. Uptake is known to be decreased at low pH, and the kinetic change in pH of growth medium from biofilms incubated in 5 µg gentamicin ml(-1) in the presence of 3× TSBg was decreased when compared with pH determinations from biofilms formed in 1/3× or 1× TSBg. These studies underscore the importance of environmental factors, including nutrient concentration and pH, on the antibiotic susceptibility of S. aureus planktonic and biofilm bacteria.

Fig. 1.

Effect of nutrient concentration on gentamicin susceptibility of developing S. aureus biofilms incubated for 16 h. (a) c.f.u. and (b) biofilm biomass following incubation in 3×, 1× and 1/3× TSBg; (c) c.f.u. following incubation in 3×, 1× and 1/3× TSB; (d) direct comparison of c.f.u. numbers after incubation in 3× TSBg versus 3× TSB at each gentamicin concentration. *, Bacteria in 3× medium increased at P<0.01 versus in 1× or 1/3× media; †, bacteria in 3× medium increased at P<0.01 versus in 1/3×; # and ‡, bacteria in TSB decreased at P<0.01 and P<0.05, respectively, compared to that in the corresponding TSBg. Each data point represents six biofilms.

Fig. 2.

(a) The graph shows comparative quantitative uptake of gentamicin–Texas Red by S. aureus incubated for 16 h in 3×, 1× and 1/3× TSBg. *, Decreased at P<0.01 compared with 1× and 1/3× TSBg. Each data point represents the mean of 15 random images. (b, c) Representative fluorescent images (overlaid onto their corresponding differential interference contrast images) showing comparative uptake of gentamicin–Texas Red after incubation for 1 h for S. aureus previously cultivated for 16 h in 3× TSBg (b) or 1/3× TSBg (c). Bars, 2.5 µm.

Fig. 3.

Effect of nutrient concentration on susceptibility of developing suture-associated S. aureus biofilms to streptomycin (a), vancomycin (b) and ampicillin (c) after incubation for 16 h in 3×, 1× and 1/3× TSBg. *, Bacteria in 3× medium increased at P<0.01 versus in 1× or 1/3× media; †, bacteria in 3× medium increased at P<0.01 versus in 1/3× medium; ‡, bacteria in 3× medium increased at P<0.05 versus in 1× and 1/3×. Each data point represents six biofilms.

Fig. 4.

Kinetic change in pH (mean±se) of spent medium from developing S. aureus biofilms incubated up to 16 h in 1/3×, 1× or 3× TSBg additionally supplemented with no gentamicin or with a high bactericidal concentration of 5 µg gentamicin ml⁻¹. pH measurements were collected from three independent experiments with three biofilms representing each time point. * and †, Decreased pH compared with the corresponding 2 h time point at P<0.01 and P<0.05, respectively.