Fig 1.
Staphylococcus aureus forms macroscopic biofilm aggregates in the synovial fluid of several different species.
Equine, human or porcine synovial fluid was infected at 1x106 CFU/mL with S. aureus (ATCC25923) and incubated overnight at 37°C in a microaerophilic chamber on a shaker at 120rpm to mimic the joint environment. (A) Macroscopic biofilm aggregates were observed in synovial fluid in all three species and photographed. (B) Aggregates were removed from the synovial fluid, fixed, dehydrated in ethanol, sputter coated and imaged with a scanning electron microscope with a FEI-Tecnai T12 microscope showing bacteria nested within a polymeric cord-like extracellular matrix. (C) Aggregates were stained with wheat germ agglutinin (WGA (blue)) for carbohydrates, SYTO9 for nucleic acids/bacteria (green), and SYPRO (red) for proteinaceous content. Confocal laser scanning microscopy (CLSM) was performed using a 12.5x upright lens on a Leica SP5 Multiphoton Microscope. CLSM images were generated as 3-D reconstructions by sequential Z-stacking and tile scanning with Velocity software.
Fig 2.
Gram-positive and gram-negative arthrotropic clinical isolates form macroscopic biofilm aggregates in equine synovial fluid.
Equine synovial fluid was infected at 1x106 CFU/mL for each clinical isolate (S. aureus, S. zooepidemicus, E. coli, and P. aeruginosa) and incubated overnight at 37°C in a microaerophilic chamber on a shaker at 120rpm to mimic the joint environment. (A) Macroscopic bacterial aggregates were observed in synovial fluid for all four strains and photographed. (B) Aggregates visualized by SEM as in Fig 1. (C) Aggregates visualized by confocal microscopy using WGA, Syto9 and SYPRO as in Fig 1.
Fig 3.
Synovial fluid biofilm aggregates show antimicrobial tolerance against several different classes of antimicrobials.
(A) S. aureus (ATCC25923) biofilm aggregates were allowed to form in equine synovial fluid for 6 hours and this aggregate-containing synovial fluid was treated with a panel of different antimicrobials from several drug classes at 100× the minimum inhibitory concentration (MIC) as determined by in vitro antimicrobial susceptibility testing (Table 1). No concentration of any antimicrobial evaluated in this experiment was able to completely kill S. aureus grown in synovial fluid. (B) The four arthrotropic bacterial isolates from Fig 2 were grown in equine synovial fluid or in tryptic soy broth (TSB). Synovial fluid or TSB was subsequently challenged with 1×, 10× or 100× MIC amikacin, an antibiotic with broad spectrum activity; these isolates were susceptible to amikacin based on our in vitro antimicrobial susceptibility data. (C-E) S. aureus (ATCC25923) were incubated in equine, human or porcine synovial fluid and this bacterial aggregate-containing synovial fluid was subsequently treated with amikacin (C), doxycycline (D) or vancomycin (E) at 1×, 10× or 100× MIC. Bars are means and standard deviations of four biological replicates (i.e. synovial fluid from four individual horses, humans or pigs; n = 4), and significant differences (p<0.05) as determined by ANOVA with Tukey post-hoc are indicated by differing letters.
Table 1.
Median minimum inhibitory concentration of clinical isolates and ATCC25923 measured by antimicrobial susceptibility testing using the Sensititre Complete Automated AST System and the equine (Equine EQUIN1F Vet AST Plate) antimicrobial susceptibility panel.
Fig 4.
Enzymatic dispersal of synovial fluid biofilm aggregates restores antimicrobial efficacy.
(A) S. aureus (ATCC25923) biofilm aggregates in equine synovial fluid were treated with several enzymes in an attempt to breakdown the extracellular matrix and disperse the bacteria: hyaluronidase (1mg/mL), proteinaseK (200μg/mL), trypsin (200μg/mL), endopeptidase or LysC (200μg/mL), DNase (500μg/mL), collagenase type II (750μg/mL), collagenase type IV (750μg/mL), dispase (500μg/mL), DispersinB (1mg/mL), acetylcysteine (8mg/mL) and tissue plasminogen activator or TPA (1mg/mL). Synovial fluid containing biofilm aggregates was treated with the respective enzyme for 1 hour prior to macroscopic imaging. (B) Percent (%) dispersal was evaluated by measuring absorbance (600nm) and calculating a percentage compared to planktonic S. aureus grown in TSB to a similar CFU/mL. (C) After 1 hour of dispersion, amikacin was added at 10× MIC (40μg/mL) and log CFU/mL was measured with serial dilutions and colony counting 8 hours post-antimicrobial challenge. Bars are means and standard deviations of four biological replicates (n = 4), and significant differences (p<0.05) as determined by ANOVA with Tukey post-hoc are indicated by differing letters.
Fig 5.
Tissue plasminogen activator (TPA) disperses S. aureus biofilm aggregates and restores antimicrobial activity in equine, human and porcine synovial fluid.
(A) Equine, human and porcine synovial fluid containing S. aureus (ATCC25923) biofilm aggregates was left untreated or treated with DispersinB (1mg/mL) or TPA (1mg/mL) for 1 hour prior to macroscopic imaging. (B) Percent (%) dispersal was evaluated by measuring absorbance (600nm) and calculating a percentage compared to tryptic soy broth (TSB) containing planktonic S. aureus at a similar CFU/mL. (C) After 1 hour of dispersion, amikacin was added at 10× MIC (40μg/mL) and log CFU/mL was measured with serial dilutions and colony counting 8 hours post-antimicrobial challenge. Bars are means and standard deviations of four biological replicates (n = 4), and significant differences (p<0.05) as determined by ANOVA with Tukey post-hoc are indicated by differing letters.
Fig 6.
TPA disperses synovial fluid biofilm aggregates and restores antimicrobial activity against both gram-negative and gram-positive aggregates.
(A) The four arthrotropic bacterial isolates from Fig 2 were grown in equine synovial fluid. Synovial fluid containing these aggregates bacteria was treated with TPA (1mg/mL) for 1 hour prior to macroscopic imaging. B) Percent (%) dispersal was evaluated by measuring absorbance (600nm) and calculating a percentage compared to planktonic S. aureus grown in TSB to a similar CFU/mL. (C) After 1 hour of dispersion, amikacin was added at 10× MIC (40μg/mL) to the infected synovial fluid containing biofilm aggregates and log CFU/mL was measured with serial dilutions and colony counting 8 hours post-antimicrobial challenge. Bars are means and standard deviations of four biological replicates (n = 4), and significant differences (p<0.05) as determined by ANOVA with Tukey post-hoc are indicated by differing letters.