Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry

Abstract

Objectives: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures.

Methods: Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic.

Results: In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays.

Conclusions: This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.

Figure 1

Control growth curves (Ct) and time–kill plots of the effect of cefazolin (CFZ), vancomycin (VAN), dicloxacillin (DCX), tetracycline (TET) and rifampicin (RIF) on S. epidermidis 9142 planktonic cells (a) and biofilm (b). Points represent means and error bars the sem.

Figure 2

Mean fold reduction of cellular viability after 3 h of exposure to antibiotics. The y-axis indicates the difference in the log₁₀ cfu/mL between strains without antibiotic (controls) and strains treated with antibiotics (CFZ, cefazolin; VAN, vancomycin; DCX, dicloxacillin; TET, tetracycline; RIF, rifampicin), in planktonic cells (grey bars) and biofilm (white bars). Different bars represent different strains, from left to right: S. epidermidis 9142, IE186, M129, M187, S. haemolyticus IE246 and M176. *Indicates values that are not significantly different (P > 0.05, paired t-test).

Figure 3

Mean reduction of cellular activity, measured by XTT, after 3 h of exposure to the antibiotics, expressed as the difference in absorbance readings at 490 nm between controls without antibiotic and antibiotic treated strains (CFZ, cefazolin; VAN, vancomycin; DCX, dicloxacillin; TET, tetracycline; RIF, rifampicin) per μg of antibiotic used (Δ/PS), in planktonic cells (grey bars) and biofilm (white bars). Different bars represent different strains, from left to right: S. epidermidis 9142, IE186, M129, M187, S. haemolyticus IE246 and M176. *Indicates values that are not significantly different (P > 0.05, paired t-test).