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.

Ultrastructure imaging of Pseudomonas aeruginosa lawn biofilms and eradication of the tobramycin-resistant variants under in vitro electroceutical treatment.

Electrochemically generated bactericidal compounds have been shown to eradicate bacterial lawn biofilms through electroceutical treatment. However, the ultrastructure of biofilms exposed to these species has not been studied. Moreover, it is unknown if the efficacy of electroceutical treatment extends to antibiotic-resistant variants that emerge in lawn biofilms after antibiotic treatment. In this report, the efficacy of the in vitro electroceutical treatment of Pseudomonas aeruginosa biofilms is demonstrated both at room temperature and in an incubator, with a ~4 log decrease (p < 0.01) in the biofilm viability observed over the anode at both conditions. The ultrastructure changes in the lawn biofilms imaged using transmission electron microscopy demonstrate significant bacterial cell damage over the anode after 24 h of electroceutical treatment. A mix of both damaged and undamaged cells was observed over the cathode. Finally, both eradication and prevention of the emergence of tobramycin-resistant variants were demonstrated by combining antibiotic treatment with electroceutical treatment on the lawn biofilms.

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.

Electroceutical Treatment of Pseudomonas aeruginosa Biofilms.

Electroceutical wound dressings, especially those involving current flow with silver based electrodes, show promise for treating biofilm infections. However, their mechanism of action is poorly understood. We have developed an in vitro agar based model using a bioluminescent strain of Pseudomonas aeruginosa to measure loss of activity and killing when direct current was applied. Silver electrodes were overlaid with agar and lawn biofilms grown for 24 h. A 6 V battery with 1 kΩ ballast resistor was used to treat the biofilms for 1 h or 24 h. Loss of bioluminescence and a 4-log reduction in viable cells was achieved over the anode. Scanning electron microscopy showed damaged cells and disrupted biofilm architecture. The antimicrobial activity continued to spread from the anode for at least 2 days, even after turning off the current. Based on possible electrochemical ractions of silver electrodes in chlorine containing medium; pH measurements of the medium post treatment; the time delay between initiation of treatment and observed bactericidal effects; and the presence of chlorotyrosine in the cell lysates, hypochlorous acid is hypothesized to be the chemical agent responsible for the observed (destruction/killing/eradication) of these biofilm forming bacteria. Similar killing was obtained with gels containing only bovine synovial fluid or human serum. These results suggest that our in vitro model could serve as a platform for fundamental studies to explore the effects of electrochemical treatment on biofilms, complementing clinical studies with electroceutical dressings.

Effects of loading concentration, blood and synovial fluid on antibiotic release and anti-biofilm activity of bone cement beads.

Antibiotic loaded cement beads are commonly used for the treatment of biofilm related orthopaedic periprosthetic infections; however the effects of antibiotic loading and exposure of beads to body fluids on release kinetics are unclear. The purpose of this study was to determine the effects of (i) antibiotic loading density (ii) loading amount (iii) material type and (iv) exposure to body fluids (blood or synovial fluid) on release kinetics and efficacy of antibiotics against planktonic and lawn biofilm bacteria. Short-term release into an agar gel was evaluated using a fluorescent tracer (fluorescein) incorporated in the carrier materials calcium sulfate (CaSO4) and poly methyl methacrylate (PMMA). Different fluorescein concentrations in CaSO4 beads were evaluated. Mechanical properties of fluorescein-incorporated beads were analyzed. Efficacy of the antibiotics vancomycin (VAN) or tobramycin (TOB) alone and in combination was evaluated against lawn biofilms of bioluminescent strains of Staphylococcus aureus and Pseudomonas aeruginosa. Zones of inhibition of cultures (ZOI) were measured visually and using an in-vivo imaging system (IVIS). The influence of body fluids on release was assessed using CaSO4 beads that contained fluorescein or antibiotics and were pre-coated with human blood or synovial fluid. The spread from the beads followed a square root of time relationship in all cases. The loading concentration had no influence on short-term fluorescein release and pre-coating of beads with body fluids did not affect short-term release or antibacterial activity. Compared to PMMA, CaSO4 had a more rapid short term rate of elution and activity against planktonic and lawn biofilms. This study highlights the importance of considering antibiotic loading and packing density when investigating the clinical application of bone cements for infection management.