Antibiotic-tolerant Staphylococcus aureus Biofilm Persists on Arthroplasty Materials

The continued presence of biofilm may be one cause of the high risk of failure observed with irrigation and débridement with component retention in acute periprosthetic joint infection (PJI). There is a poor understanding of the role of biofilm antibiotic tolerance in PJI.

Methicillin-sensitive Staphylococcus aureus biofilm was cultured on total knee arthroplasty (TKA) materials and exposed to increasing doses of cefazolin (control, 0.5, 1.0, 10.0, 100.0 μg/mL). Quantitative confocal microscopy and quantitative culture were used to measure viable biofilm cell density. To determine if cefazolin resistance was phenotypic or genotypic, we measured minimum inhibitory concentration (MIC) after exposure to different cefazolin concentrations; changes in MIC would suggest genotypic features, whereas unchanged MIC would suggest phenotypic behavior. Finally, quantitative reverse transcription-polymerase chain reaction was used to quantify expression of yoeB levels between biofilm and planktonic bacteria after exposure to 1 μg/mL cefazolin for 3 hours.

Although live biofilm mass was reduced by exposure to cefazolin when compared with biofilm mass in controls (39.2 × 10³ ± 26.4 × 10³ pixels), where the level after 0.5 µg/mL exposure also showed reduced mass (20.3 × 10³ ± 11.9 × 10³ pixels), no further reduction was seen after higher doses (mass at 1.0 µg/mL: 5.0 × 10³ pixels ± 1.1 × 10³ pixels; at 10.0 µg/mL: 6.4 × 10³ ± 9.6 × 10³ pixels; at 100.0 µg/mL: 6.4 × 10³ ± 3.9 × 10³). At the highest concentration tested (100 µg/mL), residual viable biofilm was present on all three materials, and there were no differences in percent biofilm survival among cobalt-chromium (18.5% ± 15.1%), polymethylmethacrylate (22.8% ± 20.2%), and polyethylene (14.7% ± 10.4%). We found that tolerance was a phenotypic phenomenon, because increasing cefazolin exposure did not result in changes in MIC as compared with controls (MIC in controls: 0.13 ± 0.02; at 0.5 µg/mL: 0.13 ± 0.001, p = 0.96; at 1.0 µg/m: 0.14 ± 0.04, p = 0.95; at 10.0 µg/m: 0.11 ± 0.016, p = 0.47; at 100.0 µg/m: 0.94 ± 0.047, p = 0.47). Expression of yoeB after 1 µg/mL cefazolin for 3 hours in biofilm cells was greater in biofilm but not in planktonic cells (biofilm: 62.3-fold change, planktonic cells: −78.8-fold change, p < 0.001).

Antibiotics are inadequate at complete removal of the biofilm from the surface of TKA materials. Results suggest that bacterial persisters are responsible for this phenotypic behavior allowing biofilm high tolerance to antibiotics.

Antibiotic-tolerant biofilm suggests a mechanism behind the poor results in irrigation and débridement for acute TKA PJI.