Estimation of Minimum Biofilm Eradication Concentration (MBEC) on In Vivo Biofilm on Orthopedic Implants in a Rodent Femoral Infection Model

Abstract

The formation of a biofilm on the implant surface is a major cause of intractable implant-associated infection. To investigate the antibiotic concentration needed to eradicate the bacteria inside a biofilm, the minimum biofilm eradication concentration (MBEC) has been used, mostly against in vitro biofilms on plastic surfaces. To produce a more clinically relevant environment, an MBEC assay against biofilms on stainless-steel implants formed in a rat femoral infection model was developed. The rats were implanted with stainless steel screws contaminated by two Staphylococcus aureus strains (UAMS-1, methicillin-sensitive Staphylococcus aureus; USA300LAC, methicillin-resistant Staphylococcus aureus) and euthanized on days 3 and 14. Implants were harvested, washed, and incubated with various concentrations (64–4096 μg/mL) of gentamicin (GM), vancomycin (VA), or cefazolin (CZ) with or without an accompanying systemic treatment dose of VA (20 μg/mL) or rifampicin (RF) (1.5 μg/mL) for 24 h. The implant was vortexed and sonicated, the biofilm was removed, and the implant was re-incubated to determine bacterial recovery. MBEC on the removed biofilm and implant was defined as in vivo MBEC and in vivo implant MBEC, respectively, and the concentrations of 100% and 60% eradication were defined as MBEC100 and MBEC60, respectively. As for in vivo MBEC, MBEC100 of GM was 256–1024 μg/mL, but that of VA and CZ ranged from 2048–4096 μg/mL. Surprisingly, the in vivo implant MBEC was much higher, ranging from 2048 μg/mL to more than 4096 μg/mL. The addition of RF, not VA, as a secondary antibiotic was effective, and MBEC60 on day 3 USA300LAC biofilm was reduced from 1024 μg/mL with GM alone to 128 μg/mL in combination with RF and the MBEC60 on day 14 USA300LAC biofilm was reduced from 2048 μg/mL in GM alone to 256 μg/mL in combination with RF. In conclusion, a novel MBEC assay for in vivo biofilms on orthopedic implants was developed. GM was the most effective against both methicillin-sensitive and methicillin-resistant Staphylococcus aureus, in in vivo biofilms, and the addition of a systemic concentration of RF reduced MBEC of GM. Early initiation of treatment is desired because the required concentration of antibiotics increases with biofilm maturation.

Extended local release and improved bacterial eradication by adding rifampicin to a biphasic ceramic carrier containing gentamicin or vancomycin

“…VAN also showed a similar trend when tested using a methicillin-resistant S. aureus strain. 54 Taking these findings into consideration, we believe that the preliminary data obtained using a 48-hour-old biofilm provide useful insights about the biofilm destructive action of RIF combinations eluted via CaS/HA carrier. Finally, an analysis of the mechanical properties of CaS/HA alone and in combination with antibiotics was lacking in this study.…”

“… 29 , 44 For biofilm eradication we used a 48-hour-old immature biofilm rather than a 14-day-old biofilm, which is the reported time for biofilm maturation post-infection. 53 However, in a recent study Okae et al 54 reported that the MBEC of GEN and cefazolin obtained on 72-hour-old biofilms was similar to or even twice that of 14-day-old biofilms. VAN also showed a similar trend when tested using a methicillin-resistant S. aureus strain.…”

Dissolvable alginate hydrogel-based biofilm microreactors for antibiotic susceptibility assays

“…In a study of S. aureus biofilm using Calgary biofilm device [ 58 ], the clinical isolate in biofilm tolerated up to 4 × MIC ampicillin. In other studies of S. aureus biofilm in 96-well plate [ 64 ] and on an orthopedic implant surface [ 65 ], it was shown an increased tolerance up to 8000 × MIC and 256 × MIC vancomycin, respectively, after 24 h of treatment (Calculations were based on division of the study-determined MBEC and MIC).

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Rat model of recalcitrant prosthetic joint infection using biofilm inocula

“…[47][48][49] Both in vitro and in vivo generated biofilms show cefazolin tolerance over 250× MIC. 20 Notably, cefazolin and other antibiotics appear to be more effective at targeting biofilm in combination with rifampicin. 21,50 The main limitation of this model was that it still generated significant soft tissue infection and painful inflammation.…”

“…7,17,18 Unfortunately, cefazolin does not kill staphylococcal biofilms on surgical materials at concentrations over 100× its minimum inhibitory concentration (MIC), and S. aureus grown in synovial fluid shows enhanced cefazolin tolerance. 6,19,20 Several rodent studies have demonstrated that systemic cephalosporin treatment fails to eradicate S. aureus bioburden from infected bones or joints. [21][22][23] Overall, the evidence suggests it is not feasible to achieve effective cefazolin concentrations to clear biofilm from the joint, even with maximal systemic clinical dosages.…”