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Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 43 - 43
1 Oct 2022
KILLING OF A MULTISPECIES BIOFILM USING A GRAM-NEGATIVE AND GRAM-POSITIVE TARGETED ANTIBIOTIC RELEASED FROM HIGH-PURITY CALCIUM SULFATE
Moore K Li A Gupta N Price B Delury C Laycock P Aiken S Stoodley P
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Aim

Multispecies biofilms are associated with difficult periprosthetic joint infections (PJI), particularly if they have different antibiotic sensitivities. We aimed to determine if we could generate and kill a multispecies biofilm consisting of a Gram negative and Gram positive pathogen in-vitro with antibiotic loaded calcium sulfate beads containing single or combination antibiotics.

Methods

To establish whether we could co-culture mixed species biofilms various combinations of Pseudomonas aeruginosa (PA), Enterococcus faecalis (EF), Staphylococcus aureus (SA) and Enterobacter faecalis (EF) were grown together on 316L stainless steel coupons and agar plates. Based on this screen we focused on PA + EF and challenged them with high purity calcium sulfate beads (Stimulan Rapid Cure) loaded with vancomycin (V), alone tobramycin (T) alone or vancomycin and tobramycin in combination (V+T). Bioluminescence, light imaging, plate count, confocal microscopy and scanning electron microscopy were used to quantify growth.

Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 63 - 63
1 Dec 2018
BIOFILM PREVENTION OF CARBAPENEM-RESISTANT ENTEROBACTERIACEAE AND VANCOMYCIN-RESISTANT ENTEROCOCCI BY ANTIBIOTIC-LOADED CALCIUM SULFATE BEADS IN VITRO
Dusane D Peters C Laycock P Aiken S Stoodley P
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Aim

Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin resistant Enterococci (VRE) have emerged as multi-drug resistant Gram-negative pathogens associated with Periprosthetic Joint Infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ABLCB) to inhibit bacterial growth, biofilm formation and eradicate preformed biofilms of K. pneumoniae and E. faecalis.

Method

Three strains of K. pneumoniae (carbapenem resistant BAA1705, New Delhi metallo-beta-lactamase producing BAA2146 [NDM-1], a carbapenemase producing BAA2524) and a vancomycin resistant strain of E. faecalis (ATCC51299) were used. 4.8mm diameter ABLCBs (Stimulan Rapid Cure, Biocomposites) were loaded with vancomycin (VAN) & gentamicin (GEN) at 500 and 240 mg/10cc pack or VAN & rifampicin (RIF) at 1000 and 600 mg/10cc pack respectively and placed onto tryptic soy agar (TSA) plates spread with each of the four strains independently and incubated for 24 hours at 37°C. The beads were transferred daily onto fresh TSA medium spread with the test cultures. The zone of inhibition was recorded until no inhibition was observed. Biofilm prevention efficacy was investigated in 6 well plates. Bacterial cells (5×105 CFU/mL in tryptic soy broth) were treated with ABLCBs. Media was removed and challenged with bacteria daily for 7 days. CFU counts were taken after 1, 2, 3 and 7 days. For biofilm killing, ABLCB were added to 3 day formed biofilms in 6 well plates. CFU counts were estimated at 1, 3 and 7 days with daily media exchange.

Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_22 | Pages 59 - 59
1 Dec 2017
ANTIBIOFILM EFFICACY OF ANTIBIOTIC-LOADED SYNTHETIC CALCIUM SULPHATE BEADS IN A P. AERUGINOSA/S. AUREUS CO-CULTURE MODEL FOR PROSTHETIC INFECTIONS
Frapwell C Duignan C Webb J Aiken S Cooper J Stoodley P Howlin R
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Aim

Bacterial biofilms play a key role in prosthetic infection (PI) pathogenesis. Establishment of the biofilm phenotype confers the bacteria with significant tolerance to systemic antibiotics and the host immune system meaning thorough debridement and prosthesis removal often remain the only possible course of treatment. Protection of the prosthesis and dead-space management may be achieved through the use of antibiotic loaded cements and beads to release high concentrations of antibiotics at the surgical site. The antibacterial and antibiofilm efficacy of these materials is poorly understood in the context of mixed species models, such as are often encountered clinically.

Methods

A P. aeruginosa and S. aureus in vitro co-culture biofilm model was grown using 1/5th BHI supplemented with 20 µM hemin. The ability of beads made from a synthetic calcium sulfate (CaSO4) loaded with vancomycin, tobramycin and vancomycin & tobramycin in combination to prevent biofilm formation and kill established co-culture biofilms were assessed using viable cell counts and confocal scanning laser microscopy (CSLM) over a 7 day time course. To assay for genetic changes to the individual species as a result of their presence together within a biofilm, mutation rates were measured using fluctuation analysis following growth as planktonic and biofilm cultures, alone or in co-culture. Mutants were determined based on their ability to grow on agar plates containing an inhibitory concentration of rifampicin. Mutation rates were calculated using the Ma-Sandri-Sarkar Maximum Likelihood Estimator and 94% confidence intervals compared for significance.

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