Reduced ability to detect surface-related biofilm bacteria after antibiotic exposure under in vitro conditions

Abstract

Background and purpose - Antibiotic treatment of patients before specimen collection reduces the ability to detect organisms by culture. We investigated the suppressive effect of antibiotics on the growth of non-adherent, planktonic, and surface-related biofilm bacteria in vitro by using sonication and microcalorimetry methods. Patients and methods - Biofilms of Staphylococcus aureus, S. epidermidis, Escherichia coli, and Propionibacterium acnes were formed on porous glass beads and exposed for 24 h to antibiotic concentrations from 1 to 1,024 times the minimal inhibitory concentration (MIC) of vancomycin, daptomycin, rifampin, flucloxacillin, or ciprofloxacin. The beads were then sonicated to dislodge biofilm, followed by culture and measurement of growth-related heat flow by microcalorimetry of the resulting sonication fluid. Results - Vancomycin did not inhibit the heat flow of staphylococci and P. acnes at concentrations ≤1,024 μg/mL, whereas flucloxacillin at >128 μg/mL inhibited S. aureus. Daptomycin inhibited heat flow of S. aureus, S. epidermidis, and P. acnes at lower concentrations (32-128 times MIC, p < 0.001). Rifampin showed inconsistent results in staphylococci due to random emergence of resistance, which was observed at concentrations ≤1,024 times MIC (i.e. 8 μg/mL). Ciprofloxacin inhibited heat flow of E. coli at ≥4 times MIC (i.e. ≥ 0.06 μg/mL). Interpretation - Whereas time-dependent antibiotics (i.e. vancomycin and flucloxacillin) showed only weak growth suppression, concentration-dependent drugs (i.e. daptomycin and ciprofloxacin) had a strong suppressive effect on bacterial growth and reduced the ability to detect planktonic and biofilm bacteria. Exposure to rifampin rapidly caused emergence of resistance. Our findings indicate that preoperative administration of antibiotics may have heterogeneous effects on the ability to detect biofilm bacteria.

Figure 1.

Heat flow (y-axis, μW) development over time (x-axis, hours) of S. aureus (panels A–D), S. epidermidis (E–G), E. coli (H), and P. acnes (I–K) exposed to different antibiotics. The numbers above each curve indicate the respective antibiotic concentrations. The range for x-axis is different in the case of P. acnes (72 h). Furthermore, y-axis range was changed for non-staphylococci that showed markedly different heat flow levels. The positive controls were biofilms on beads not previously exposed to antibiotics (0 μg/mL). The experiments were performed in triplicate, and a representative experiment is shown.

Figure 2.

Growth inhibition of various antibiotics on S. aureus (panels A–D), S. epidermidis (E–G), E. coli (H), and P. acnes (I–K) in sonication fluid, determined by microcalorimetry and culture. The x-axis shows increasing antibiotic concentrations (μg/mL). The green line represents median bacterial concentration (corresponding to y-axis on right, in CFU/mL) determined by viable count in 3 independent experiments; bars indicate range. The blue line represents median time to detection (corresponding to y-axis on left, in hours) in the microcalorimeter experiment. It demonstrates how significant bacterial heat flow (> 10 μW) is detectable within a few hours. Spontaneous emergence of staphylococcal resistance to rifampin was observed in replicates of experiments C and G.