Background: Bacterial biofilms pose a challenge in treating implant-associated infections. Biofilms provide bacteria with protection against antimicrobial agents and the immune response and often are invisible to the naked eye. As a biofilm-disclosing agent, methylene blue (MB) has shown promise, but lacks rigorous in vitro evaluation. The purposes of the present study were to assess MB as a biofilm-disclosing agent in vitro for common biofilm-forming organisms and to determine performance characteristics across implant materials and healthy tissue types. Methods: Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 27853) biofilms were grown on culture for 2 days in CDC biofilm reactors on titanium, cobalt chromium, polyethylene, and polyether ether ketone (PEEK) coupons. Biofilms were stained with MB solutions of either 0.005% or 0.01% and then were washed with normal saline solution. Digital photographs were obtained to compare the visual sensitivity of the blue dye at these dilutions. Scanning electron microscopy (SEM) was performed to confirm the absence or presence of biofilm on MB-stained areas. Uninoculated controls were also assessed. Healthy adult sheep tissues were also stained to determine the staining characteristics of the host tissue. ImageJ was used to determine the relative blue intensity of stained implants and tissues compared with standard curves. Results: S. aureus and P. aeruginosa biofilms stained avidly on titanium, cobalt chromium, polyethylene, and PEEK coupons. There was visible dose-dependent staining based on dye concentration. MB was visible only where biofilms were present as confirmed by SEM. MB did not stain uninoculated controls. Articular cartilage and meniscus demonstrated appreciable staining; bone, tendon, muscle, nerve, and fat did not. Bacterial biofilms demonstrated both dose-dependent and species-specific staining. Conclusions: MB is an effective disclosing agent for S. aureus and P. aeruginosa biofilms in vitro. MB did not stain implant materials, nor did it stain most healthy tissues in vitro. MB may allow surgeons to see biofilms and may allow for enhanced debridement once visualized.

Most animal models of infection utilize planktonic bacteria as initial inocula. However, this may not accurately mimic scenarios where bacteria in the biofilm phenotype contaminate a site at the point of injury. We developed a modified CDC biofilm reactor in which biofilms can be grown on the surface of simulated fracture fixation plates. Multiple reactor runs were performed and demonstrated that monomicrobial biofilms of a clinical strain of methicillin-resistant Staphylococcus aureus, S. aureus ATCC 6538, and Pseudomonas aeruginosa ATCC 27853 consistently developed on fixation plates. We also identified a method by which to successfully grow polymicrobial biofilms of S. aureus ATCC 6538 and P. aeruginosa ATCC 27853 on fixation plates. This customized reactor can be used to grow biofilms on simulated fracture fixation plates that can be inoculated in animal models of biofilm implant-related infection that, for example, mimic open fracture scenarios. The reactor provides a method for growing biofilms that can be used as initial inocula and potentially improve the testing and development of antibiofilm technologies.

Clinical preoperative skin preparations (PSPs) do not eradicate skin flora dwelling in the deepest dermal regions. Survivors constitute a persistent infection risk. In search of solutions, we created a porcine model intended for PSP developmental testing. This model employed microbiological techniques sensitive to the deep-dwelling microbial flora as these microorganisms are frequently overlooked when using institutionally-entrenched testing methodologies. Clinical gold-standard PSPs were assessed. Ten Yorkshire pigs were divided into two groups: prepared with either povidone iodine (PVP-I) or chlorhexidine gluconate (CHG) PSP. Bioburdens were calculated on square, 4 cm by 4 cm, full-thickness skin samples homogenized in neutralizing media. Endogenous bioburden of porcine skin (3.3 log₁₀ CFU/cm²) was consistent with natural flora numbers in dry human skin. On-label PSP scrub kits with PVP-I (n = 39) or CHG (n = 40) failed the 2–3 log₁₀-reduction criteria established for PSPs by the Food and Drug Administration (FDA), resulting in a 1.46 log₁₀ and 0.58 log₁₀ reduction, respectively. Porcine dermal microbiota mirrored that of humans, displaying abundant staphylococcal species. Likewise, histological sections showed similarity in hair follicle depths and sebaceous glands (3.2 ± 0.7 mm). These shared characteristics and the considerable fraction of bacteria which survived clinical PSPs make this model useful for developmental work.

Bacteria in natural ecosystems such as soil, dirt, or debris preferentially reside in the biofilm phenotype. When a traumatic injury, such as an open fracture, occurs, these naturally dwelling biofilms and accompanying foreign material can contaminate the injury site. Given their high tolerance of systemic levels of antibiotics that may be administered prophylactically, biofilms may contribute to difficult-to-treat infections. In most animal models, planktonic bacteria are used as initial inocula to cause infection, and this might not accurately mimic clinically relevant contamination and infection scenarios. Further, few approaches and systems utilize the same biofilm and accompanying substrate throughout the experimental continuum. In this study, we designed a unique reactor to grow bacterial biofilms on up to 50 silica beads that modeled environmental wound contaminants. The data obtained indicated that the reactor system repeatably produced mature Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the silica beads, with an average of 5.53 and 6.21 log₁₀ colony-forming units per mm², respectively. The bead substrates are easily manipulable for in vitro or in vivo applications, thus improving translatability. Taken together, the bead biofilm reactor presented herein may be a useful system for repeatably growing established biofilms on silica beads that could be used for susceptibility testing and as initial inocula in future animal models of trauma-related injuries.

Background/Objectives: Accurately determining the bacterial bioburden that survives preoperative skin preparation (PSP) is critical in understanding PSP efficacy and its limitations. Clinical PSP approval relies on a bacterial sampling method described in the American Society for Testing and Materials (ASTM) standard E1173-15. Though common, this technique may overlook deep-dwelling skin bacteria. The objective of this study was to test the hypothesis that deep-dwelling skin flora would survive PSP, and more growth would be detected using a destructive sampling method compared with ASTM E1173-15. Methods: Twelve female participants with a scheduled deep inferior epigastric perforator (DIEP) artery flap procedure at the Huntsman Cancer Institute in Salt Lake City, UT, were enrolled between January and August 2024. PSP was performed using three 26 mL ChloraPrep applicators (2% CHG), and excess tissue was collected. Bacteria in the skin were quantified using a destructive sampling method and ASTM E1173-15, and bioburden outcomes were compared. Two participants were excluded from the quantitative analysis. Results: Bacteria survived PSP in every participant. A greater diversity and more bacteria were quantified with destructive sampling than ASTM E1173-15 (p < 0.01). Generally, anaerobic bioburden values were higher than aerobic bioburden values. Higher bioburden correlated with processing more skin from a participant. Genotypic identification of select isolates identified Staphylococcus epidermidis and Cutibacterium acnes (formerly known as Propionibacterium acnes) as surviving bacteria, among others. Immunofluorescence revealed bacteria in all skin layers. No participant exhibited clinical signs of infection in the abdominal region. Human data corroborated previous porcine data collected using destructive skin sampling after PSP. Conclusions: Clinical PSP application does not create a sterile field. Destructive skin sampling techniques may be more effective than ASTM E1173-15 at resolving bacterial PSP survivors contributing to SSI risk.