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Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 43 - 43
1 Oct 2022
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.


The Bone & Joint Journal
Vol. 104-B, Issue 5 | Pages 575 - 580
2 May 2022
Hamad C Chowdhry M Sindeldecker D Bernthal NM Stoodley P McPherson EJ

Periprosthetic joint infection (PJI) is a difficult complication requiring a comprehensive eradication protocol. Cure rates have essentially stalled in the last two decades, using methods of antimicrobial cement joint spacers and parenteral antimicrobial agents. Functional spacers with higher-dose antimicrobial-loaded cement and antimicrobial-loaded calcium sulphate beads have emphasized local antimicrobial delivery on the premise that high-dose local antimicrobial delivery will enhance eradication. However, with increasing antimicrobial pressures, microbiota have responded with adaptive mechanisms beyond traditional antimicrobial resistance genes. In this review we describe adaptive resistance mechanisms that are relevant to the treatment of PJI. Some mechanisms are well known, but others are new. The objective of this review is to inform clinicians of the known adaptive resistance mechanisms of microbes relevant to PJI. We also discuss the implications of these adaptive mechanisms in the future treatment of PJI.

Cite this article: Bone Joint J 2022;104-B(5):575–580.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_17 | Pages 63 - 63
1 Dec 2018
Dusane D Peters C Laycock P Aiken S Stoodley P
Full Access

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
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.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 318 - 318
1 Jul 2011
Stoodley P Kathju S
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Post-operative surgical site infection following total joint arthroplasty occurs at rates between ~ 0.2–5 %, depending on the joint and the surgeon volume, as well as various patient risk factors. Given that an estimated over 700,000 knee and hip arthroplasties are performed in the US each year this translates to thousands of patients that are affected by this serious, costly and traumatic complication. In addition, it is now recognized that clinical culturing underestimates the infection rate and that a number of aseptic loosenings might actually have an infectious etiology. We have used a combination of non-culture based molecular methods to detect bacteria associated with hardware, antimicrobial impregnated cement, reactive tissue and pus collected during revision surgery in a total elbow arthroplasty (TEA) case and a total ankle revision (TAR) case. Confocal microscopy showed live cocci in biofilm cell clusters, and fluorescent in situ hybridization (FISH) demonstrated S. aureus biofilms. Reverse transcriptase (RT)-PCR, and multiplex PCR coupled with electrospray-ionization mass spectrometry (Ibis T5000) to identify S. aureus, S. epidermidis and genes for methicillin resistance. Together our complimentary techniques comprise compelling evidence that viable biofilm bacteria played an important role in the refractory infections in these cases.