A novel synthetic synovial fluid model for investigating biofilm formation and antibiotic susceptibility in prosthetic joint infections

Abstract

There is growing evidence that bacteria encountered in prosthetic joint infections (PJIs) form surface-attached biofilms on prostheses, as well as biofilm aggregates embedded in synovial fluid and tissues. However, in vitro models allowing the investigation of these biofilms and the assessment of their antimicrobial susceptibility in physiologically relevant conditions are currently lacking. To address this, we developed a synthetic synovial fluid (SSF2) model and validated this model by investigating growth, aggregate formation, and antimicrobial susceptibility using multiple PJI isolates belonging to various microorganisms. In this study, 18 PJI isolates were included belonging to Staphylococcus aureus, coagulase-negative staphylococci, Cutibacterium acnes, Streptococcus spp., Enterococcus spp., Pseudomonas aeruginosa, Escherichia coli, and Candida spp. Growth and aggregate formation in SSF2 were evaluated using light microscopy and confocal laser scanning microscopy. The biofilm preventing concentration (BPC) and minimal biofilm inhibitory concentration (MBIC) of relevant antibiotics were determined using a resazurin-based viability staining. BPC and MBIC values were compared to conventional susceptibility parameters (minimal inhibitory concentration and minimal bactericidal concentration) determined with conventional approaches. The SSF2 medium allowed isolates to grow and form biofilm-like aggregates varying in size and shape between different species. For most isolates cultured in SSF2, a reduced susceptibility to the tested antibiotics was observed when compared to susceptibility data obtained in general media. These data indicate that the in vitro SSF2 model could be a valuable addition to evaluate the antimicrobial susceptibility of biofilm-like aggregates in the context of PJI.

Importance: Infections after joint replacement are rare but can lead to severe complications as they are difficult to treat due to the ability of pathogens to form surface-attached biofilms on the prosthesis as well as biofilm aggregates in the tissue and synovial fluid. This biofilm phenotype, combined with the microenvironment at the infection site, substantially increases antimicrobial tolerance. Conventional in vitro models typically use standard growth media, which do not consider the microenvironment at the site of infection. By replacing these standard growth media with an in vivo-like medium, such as the synthetic synovial fluid medium, we hope to expand our knowledge on the aggregation of pathogens in the context of PJI. In addition, we believe that inclusion of in vivo-like media in antimicrobial susceptibility testing might be able to more accurately predict the in vivo susceptibility, which could ultimately result in a better clinical outcome after antimicrobial treatment.

Keywords: biofilms; prosthetic joint infection; synovial fluid.

Fig 1

Light microscopy images of clinical S. aureus isolate A1 cultured in SSF without Fg (SSF1) and SSF with Fg (SSF2) under aerobic (A), microaerophilic (3% O₂) (B), and anaerobic (C) conditions, evaluated at four different timepoints. The bars represent the average number (n = 3) of CFUs recovered after conventional biofilm disruption for biofilms grown in SSF1 (blue) and SSF2 (light gray) or after biofilm disruption using trypsin (0.25%) after growth in SSF2 (dark gray). Error bars indicate standard deviation; black line indicates the inoculum of 5 × 10⁷ CFU/mL. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant (P ≥ 0.05).

Fig 2

Log CFU per milliliter values of 18 clinical PJI isolates after 24 h (black bars) and 48 h (gray bars) of incubation in SSF2 under microaerophilic conditions (3% O₂), except for C. acnes (anaerobic conditions). Inoculum size: 5 × 10⁵ CFU/mL (red line). Data shown are mean values of biological replicates; error bars represent standard deviations (n = 3).

Fig 3

Light microscopy images of 18 clinical PJI isolates after 24 h of incubation in SSF2 under microaerophilic conditions (3% O₂), except for C. acnes (anaerobic conditions). Scale bars 200 µm.

Fig 4

CLSM (left) and light microscopy (right) images of four of the staphylococci. (A) S. aureus A1, (B) S. aureus SAU060112, (C) S. capitis subsp. capitis CCUG 39451, and (D) S. epidermidis HD05-1 ST2, after 24 h of incubation in SSF2. CLSM: scale bars 50 µm; light microscopy: scale bars 200 µm. The contrast was adjusted to improve visualization. The light microscopy images shown (on the right) are the same as the ones shown in Fig. 3 for the four strains mentioned.

Fig 5

Relation between log CFU per milliliter values and log fluorescence values after resazurin staining. Data are shown for all the replicates tested (n = 3).

Fig 6

BPC:MIC ratios (A), BPC:MBC ratios (B), MBIC:MIC ratios (C), and MBIC:MBC ratios (D) for all antibiotics tested. The solid black line indicates a ratio of 1. Data points for which an exact ratio could not be determined because one or both parameters are outside the testing range are labeled: (1) BPC >testing range, (2) MBC >testing range, (3) MBIC >testing range, and (4) MBIC >testing range and MBC <testing range.