Killing of a Multispecies Biofilm Using Gram-Negative and Gram-Positive Targeted Antibiotic Released from High Purity Calcium Sulfate Beads.

BACKGROUND: Multispecies biofilm orthopedic infections are more challenging to treat than mono-species infections. In this in-vitro study, we aimed to determine if a multispecies biofilm, consisting of Gram positive and negative species with different antibiotic susceptibilities could be treated more effectively using high purity antibiotic-loaded calcium sulfate beads (HP-ALCSB) containing vancomycin (VAN) and tobramycin (TOB) in combination than alone. METHODS: Three sets of species pairs from bioluminescent strains of Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) and clinical isolates, Enterococcus faecalis (EF) and Enterobacter cloacae were screened for compatibility. PA + EF developed intermixed biofilms with similar cell concentrations and so were grown on 316L stainless steel coupons for 72 h or as 24 h agar lawn biofilms and then treated with HP-ALCSBs with single or combination antibiotics and assessed by viable count or bioluminescence and light imaging to distinguish each species. Replica plating was used to assess viability. RESULTS: The VAN + TOB bead significantly reduced the PA + EF biofilm CFU and reduced the concentration of surviving antibiotic tolerant variants by 50% compared to single antibiotics. CONCLUSIONS: The combination of Gram-negative and positive targeted antibiotics released from HP-ALCSBs may be more effective in treating multispecies biofilms than monotherapy alone.

Antifungal-Loaded Calcium Sulfate Beads as a Potential Therapeutic in Combating Candida auris.

Candida auris provides a substantial global nosocomial threat clinically. With the recent emergence that the organism can readily colonize skin niches, it will likely continue to pose a risk in health care units, particularly to patients undergoing surgery. The purpose of this study was to investigate the efficacy of antifungal-loaded calcium sulfate (CS) beads in combatting C. auris infection. We demonstrate that the CS-packed beads have the potential to interfere with planktonic and sessile C. auris.

Pseudomonas aeruginosa biofilm killing beyond the spacer by antibiotic-loaded calcium sulfate beads: an in vitro study.

Introduction: Bacterial biofilms are an important virulence factor in chronic periprosthetic joint infection (PJI) and other orthopedic infection since they are highly tolerant to antibiotics and host immunity. Antibiotics are mixed into carriers such as bone cement and calcium sulfate bone void fillers to achieve sustained high concentrations of antibiotics required to more effectively manage biofilm infections through local release. The effect of antibiotic diffusion from antibiotic-loaded calcium sulfate beads (ALCS-B) in combination with PMMA bone cement spacers on the spread and killing of Pseudomonas aeruginosa Xen41 (PA-Xen41) biofilm was investigated using a "large agar plate" model scaled for clinical relevance. Methods: Bioluminescent PA-Xen41 biofilms grown on discs of various orthopedic materials were placed within a large agar plate containing a PMMA full-size mock "spacer" unloaded or loaded with vancomycin and tobramycin, with or without ALCS-B. The amount of biofilm spread and log reduction on discs at varying distances from the spacer was assessed by bioluminescent imaging and viable cell counts. Results: For the unloaded spacer control, PA-Xen41 spread from the biofilm to cover the entire plate. The loaded spacer generated a 3 cm zone of inhibition and significantly reduced biofilm bacteria on the discs immediately adjacent to the spacer but low or zero reductions on those further away. The combination of ALCS-B and a loaded PMMA spacer greatly reduced bacterial spread and resulted in significantly greater biofilm reductions on discs at all distances from the spacer. Discussion: The addition of ALCS-B to an antibiotic-loaded spacer mimic increased the area of antibiotic coverage and efficacy against biofilm, suggesting that a combination of these depots may provide greater physical antibiotic coverage and more effective dead space management, particularly in zones where the spread of antibiotic is limited by diffusion (zones with little or no fluid motion).

Elution Kinetics from Antibiotic-Loaded Calcium Sulfate Beads, Antibiotic-Loaded Polymethacrylate Spacers, and a Powdered Antibiotic Bolus for Surgical Site Infections in a Novel In Vitro Draining Knee Model.

Antibiotic-tolerant bacterial biofilms are notorious in causing PJI. Antibiotic loaded calcium sulfate bead (CSB) bone void fillers and PMMA cement and powdered vancomycin (VP) have been used to achieve high local antibiotic concentrations; however, the effect of drainage on concentration is poorly understood. We designed an in vitro flow reactor which provides post-surgical drainage rates after knee revision surgery to determine antibiotic concentration profiles. Tobramycin and vancomycin concentrations were determined using LCMS, zones of inhibition confirmed potency and the area under the concentration-time curve (AUC) at various time points was used to compare applications. Concentrations of antibiotcs from the PMMA and CSB initially increased then decreased before increasing after 2 to 3 h, correlating with decreased drainage, demonstrating that concentration was controlled by both release and flow rates. VP achieved the greatest AUC after 2 h, but rapidly dropped below inhibitory levels. CSB combined with PMMA achieved the greatest AUC after 2 h. The combination of PMMA and CSB may present an effective combination for killing biofilm bacteria; however, cytotoxicity and appropriate antibiotic stewardship should be considered. The model may be useful in comparing antibiotic concentration profiles when varying fluid exchange is important. However, further studies are required to assess its utility for predicting clinical efficacy.

Assessing the Bioactive Profile of Antifungal-Loaded Calcium Sulfate against Fungal Biofilms.

Calcium sulfate (CS) has been used clinically as a bone- or void-filling biomaterial, and its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm-inhibitory doses. A panel of different fungal species (n = 15) were selected for planktonic broth microdilution testing with fluconazole (FLZ), amphotericin B (AMB), and caspofungin (CSP). After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n = 5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semidry material, to mimic active infection in vivo In general, antifungals released from loaded CS beads were all effective at inhibiting the pathogenic fungi over 7 days within standard MIC ranges for these fungi. We observed a significant reduction of pregrown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal-loaded CS beads produce a sustained antimicrobial effect that inhibits and kills clinically relevant fungal species in vitro as planktonic and biofilm cells.

Antibiotic loaded ß-tricalcium phosphate/calcium sulfate for antimicrobial potency, prevention and killing efficacy of Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

This study investigated the efficacy of a biphasic synthetic ß-tricalcium phosphate/calcium sulfate (ß-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded ß-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. ß-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded ß-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of ß-TCP/CS in the management of orthopaedic infections.

Prevention and Killing Efficacy of Carbapenem Resistant Enterobacteriaceae (CRE) and Vancomycin Resistant Enterococci (VRE) Biofilms by Antibiotic-Loaded Calcium Sulfate Beads.

Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE) have emerged as multidrug-resistant (MDR) pathogens associated with periprosthetic joint infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ALCSB) in inhibiting bacterial growth, encouraging biofilm formation and killing preformed biofilms of CRE and VRE. Three strains of Klebsiella pneumoniae (KP) and a strain of Enterococcus faecalis (EF) were used. ALCSB of 4.8-mm diameter were loaded with vancomycin (V) and gentamicin (G), V and rifampicin (R), V and tobramycin (T) or R and meropenem (M), and placed onto tryptic soy agar (TSA), spread with one of the test strains and incubated for 24 h at 37 °C. Beads were transferred daily onto fresh TSA spread plates and the zone of inhibition (ZOI) was recorded until no inhibition was observed. ALCSB containing R + M or R + V produced the most extensive ZOI up to 5 weeks. Biofilm prevention efficacy was investigated by challenging ALCSB daily with 5 × 105 CFU/mL bacterial cells and analyzing for biofilm formation at challenges 1, 2 and 3. In the biofilm killing experiments, ALCSB were added to pre-grown 3-day biofilms of KP and EF strains, which were then analyzed at days 1 and 3 post-exposure. The CFU counts and confocal images of the attached cells showed that ALCSB treatment reduced colonization and biofilm formation significantly (5-7 logs) with combinations of R + M or R + V, compared to unloaded beads. This study provides evidence that the local release of antibiotics from ALCSB may be useful in treating the biofilms of multidrug-resistant strains of CRE and VRE.

Complete Killing of Agar Lawn Biofilms by Systematic Spacing of Antibiotic-Loaded Calcium Sulfate Beads.

Background: Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are the major causative agents of acute and chronic infections. Antibiotic-loaded calcium sulfate beads (ALCSB) are used in the management of musculoskeletal infections such as periprosthetic joint infections (PJI). Methods: To determine whether the number and spatial distribution of ALCSB are important factors to totally eradicate biofilms, ALCSBs containing vancomycin and tobramycin were placed on 24 h agar lawn biofilms as a single bead in the center, or as 16 beads placed as four clusters of four, a ring around the edge and as a group in the center or 19 beads evenly across the plate. Bioluminescence was used to assess spatial metabolic activity in real time. Replica plating was used to assess viability. Results: For both strains antibiotics released from the beads completely killed biofilm bacteria in a zone immediately adjacent to each bead. However, for PA extended incubation revealed the emergence of resistant colony phenotypes between the zone of eradication and the background lawn. The rate of biofilm clearing was greater when the beads were distributed evenly over the plate. Conclusions: Both number and distribution pattern of ALCSB are important to ensure adequate coverage of antibiotics required to eradicate biofilms.

In Vitro Efficacy of Antibiotics Released from Calcium Sulfate Bone Void Filler Beads.

15 different antibiotics were individually mixed with commercially available calcium sulfate bone void filler beads. The antibiotics were: amikacin, ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, colistamethate sodium, daptomycin, gentamicin, imipenem/cilastatin, meropenem, nafcillin, rifampicin, teicoplanin, tobramycin and vancomycin. The efficacy of specific released antibiotics was validated by zone of inhibition (ZOI) testing using a modified Kirby⁻Bauer disk diffusion method against common periprosthetic joint infection pathogens. With a subset of experiments (daptomycin, rifampin, vancomycin alone and rifampin and vancomycin in combination), we investigated how release varied over 15 days using a repeated ZOI assay. We also tested the ability of these beads to kill biofilms formed by Staphylococcus epidermidis 35984, a prolific biofilm former. The results suggested that certain antibiotics could be combined and released from calcium sulfate with retained antibacterial efficacy. The daptomycin and rifampin plus vancomycin beads showed antimicrobial efficacy for the full 15 days of testing and vancomycin in combination with rifampin prevented resistant mutants. In the biofilm killing assay, all of the antibiotic combinations showed a significant reduction in biofilm bacteria after 24 h. The exposure time was an important factor in the amount of killing, and varied among the antibiotics.