Synergistic Effects of Pulsed Lavage and Antimicrobial Therapy Against Staphylococcus aureus Biofilms in an in-vitro Model

Abstract

Background: Prosthetic joint infections (PJI) are difficult to treat complications of joint arthroplasty. Debridement with implant retention is a common treatment strategy and frequently involves the use of pulsed lavage (PL). However, PL effects on biofilms and antibiotic activity have been scarcely studied in-vitro. We report the effects of PL, vancomycin or flucloxacillin used independently or in combination against Staphylococcus aureus biofilms. Methods: Biofilms of 3 methicillin-susceptible (MSSA) and of 3 methicillin-resistant (MRSA) S. aureus were grown on Ti6Al4V coupons in TGN (TSB + 1%glucose + 2%NaCl). After 24 h, PL was applied to half of the samples (50 mL saline from 5 cm). Samples were either reincubated for 24 h in TGN or TGN + flucloxacillin or vancomycin. Analyses included CFUs counts, biomass assays or fluorescence microscopy. Results: PL transiently reduced bacterial counts by 3-4 Log 10 CFU/coupon, but bacterial regrowth to baseline levels was seen after 24 h. At 20 mg/L, flucloxacillin reduced both the CFU counts (3 Log 10 CFU/coupon) and biomass (−70%) in one MSSA only, while vancomycin had no effects against MRSA. PL combined with a 24 h reincubation with vancomycin or flucloxacillin at 20 mg/L was synergistic (−5 to 6.5 Log 10 CFU/coupon; 81-100% biomass reduction). Fluorescence microscopy confirmed that PL removed most of the biofilm and that subsequent antibiotic treatment partially killed bacteria. Conclusions: While PL only transiently reduces the bacterial load and antibiotics at clinically relevant concentrations show no or limited activity on biofilms, their combination is synergistic against MRSA and MSSA biofilms. These results highlight the need for thorough PL before antibiotic administration in PJI.

Direct-Contact Low-Frequency Ultrasound and Pulse Lavage Eradicates Biofilms on Implant Materials In Vitro

“…CLSM also showed that the biofilm had been reduced to a monolayer of cells on the surface, and the remaining cells appeared to be mostly viable after PL. These findings are consistent with previous studies that reported approximately two-log reduction in cell numbers following PL [ 20 , 26 ]. Knecht et al [ 20 ] have demonstrated that PL reduced the CFU count of strain of biofilms by approximately two orders of magnitude, from an initial cell count on the metal surface of approximately 10 9 CFU/cm 2 .…”

In VitroStudy of the Synergistic Effect of an Enzyme Cocktail and Antibiotics against Biofilms in a Prosthetic Joint Infection Model

“…The plates were incubated for 24 h (ATCC 33591 and ATCC 35984) or 48 h (ATCC 47076) at 37°C, with a constant orbital shaking of 50 rpm. In the case of strain ATCC 47076 biofilms, the medium was changed after 24 h. These culture times were selected as they allowed the biofilms to reach steady states over the study period and were determined in previous growth kinetic experiments for S. aureus (14). The culture duration period for S. epidermidis was derived from these results.…”

Recent Strategies to Combat Infections from Biofilm-Forming Bacteria on Orthopaedic Implants

“…Another novel strategy is the use of engineered cationic amphipathic peptide, WLBU2, as an irrigation adjunct to be used during the surgical debridement of BRII; WLBU2 has been previously shown to eliminate biofilms and create culture negative implants in 30 min, and in a recent in vitro and in vivo murine infection model, WLBU2 solution was able to markedly reduce S. aureus biofilm on implant material [128]. The use of pulsed-lavage irrigation fluid in combination with physiological concentrations of antibiotic agents, vancomycin and flucloxacillin, was demonstrated in vitro to reduce the bacterial load of biofilms adhered to implant materials but did not demonstrate complete eradication [14].…”

“…Bacterial biofilms display emergent properties: that is, properties that are not predictable from study of the planktonic cells from which they emerge [12]. These properties give the biofilms a significant survival advantage, making them extremely resilient to host immune or conventional anti-microbial therapies; they have been shown to be up to 1000× more resistant to antibiotic eradication, which ultimately results in the recalcitrance and recurrence of biofilm-related implant infection [13][14][15][16].…”

“…Biphasic Ceramic Carrier Another novel strategy uses a biphasic ceramic-based drug delivery system, a biphasic nanohydroxyapatite/calcium sulphate carrier; a recent paper combining in vitro analysis and computer modelling demonstrated that this void filler not only has the potential to promote bone regeneration, it also demonstrating desirable, controlled, and sustained drug release, which would make it an excellent treatment strategy for bone and joint tuberculosis [14].…”