The In Vitro Efficacy of Doxycycline over Vancomycin and Penicillin in the Elimination of Cutibacterium Acnes Biofilm

Abstract

Cutibacterium acnes (formerly Propionibacterium acnes) is a significant pathogen in periprosthetic joint infections (PJIs) in total shoulder arthroplasty. Poor outcomes seen in PJIs are due to the established C. acnes bacterial biofilms. The prolonged nature of C. acnes infections makes them difficult to treat with antibiotics. The goal of this study was to determine the relative efficacy of vancomycin compared with penicillin and doxycycline against planktonic and mature biofilms. Clinical isolates from PJI patients as well as a laboratory strain of C. acnes were tested. Planktonic minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were obtained using modified clinical laboratory standard index assays. Biofilm MICs and MBCs were also obtained. The MIC was determined for both using the PrestoBlue viability stain. The MBC was determined using differential reinforced clostridial medium agar plates for colony-forming unit analysis. Using the PrestoBlue viability reagent, the planktonic MIC values for vancomycin were significantly higher than doxycycline. Across 10 strains of C. acnes, all three antibiotics had decreased efficacy when comparing planktonic and biofilm cultures. Although effective antibiotic doses ranged from 1 to 1,000 μg/mL, only doxycycline achieved inhibitory and bactericidal concentrations in all tested strains. Penicillin failed to achieve the minimum biofilm inhibitory concentration (MBIC) in 60% of tested strains, whereas vancomycin failed in 80% of tested strains. Penicillin, doxycycline, and vancomycin have similar abilities in inhibiting C. acnes growth planktonically. The MBIC for doxycycline was within the clinical dosing range, suggesting C. acnes biofilm offers minimal tolerance to these antibiotics. The MBIC for penicillin was within clinical dosing ranges in only 60% of trials, suggesting the relative tolerance of C. acnes to penicillin. The minimum biofilm bactericidal concentration (MBBC) of doxycycline showed efficacy in 90% of trials, whereas penicillin and vancomycin achieved MBBC in 15% of samples.

Keywords: Cutibacterium acnes; antibiotic tolerance; arthroplasty; biofilm; periprosthetic joint infection; planktonic.

FIG. 1

Variability in antibiotic effectiveness between planktonic and biofilm states of the four C. acnes samples. Across sampled isolates, C. acnes demonstrated varying resistances in both planktonic (blue) and increased resistance to antibiotics in biofilm (red) cultures. MICs were determined using a PrestoBlue fluorescence assay. To determine the MBC, a CFU analysis was performed using DRCM plates. The differences between the MBIC of doxycycline and penicillin, as well as the MBBC of doxycycline and vancomycin were statistically significant. The dashed line represents the maximum tested dose (1,000 μg/mL).

FIG. 2

C. acnes biofilm demonstrates increased antibiotic tolerance. Individual isolates were compared against themselves between planktonic and biofilm sensitivities. Across all but two tested isolates, biofilm MIC was greater than planktonic MIC (A). All tested isolates demonstrated greater biofilm MBC than planktonic MBC (B).

FIG. 3

C. acnes biofilms show increased tolerance to killing by doxycycline, vancomycin, and penicillin. Sensitivity to antibiotics was compared between C. acnes strains grown planktonically compared to mature biofilms. Doxycycline, vancomycin, and penicillin had similar planktonic MIC and planktonic MBCs. A Mann-Whitney test showed statistically significant differences between MIC and MBIC for all antibiotics, as well as between MBC and MBBC for all tested antibiotics, as denoted by the asterisk (*p > 0.05). In all cases, biofilms demonstrated significantly increased tolerance to the antibiotics tested compared with their planktonic counterpart.