Bacterial Biofilm on Tissue Expander and Acellular Dermal Graft After Breast Reconstruction.

A 27-year-old female underwent bilateral mastectomy with left axillary dissection and had immediate breast reconstruction with textured silicone implants and acellular dermal graft (ADG) reinforcement of the inferior quadrants. The patient was maintained on oral antibiotics postoperatively and initially did well. However, she subsequently presented with fever, erythema, and tenderness in the left chest and was admitted for intravenous antibiotic therapy. Despite improvement of her symptoms, she ultimately cultured positive for Staphylococcus aureus and had the tissue expander and the ADG material explanted. These explanted specimens were immediately examined with confocal microscopy using Live/Dead staining under hydrated conditions for the presence of bacterial biofilms. Biofilm bacteria were clearly visualized adherent to both the tissue expander shell and also to the ADG surface. This is the first direct demonstration of viable bacteria in biofilm configuration on the surface of a tissue expander and acellular dermal graft after breast reconstruction.

Bioenergetics of simultaneous oxygen and nitrate respiration and nitric oxide production in a Pseudomonas aeruginosa agar colony biofilm.

Pseudomonas aeruginosa is a biofilm forming pathogen commonly associated with infection of the cystic fibrosis (CF) lung, chronic wounds and indwelling medical devices. P. aeruginosa is a facultative aerobe that can use nitrate (NO3-) found in healthy and infected tissues and body fluids to generate energy through denitrification. Further, P. aeruginosa the expression of denitrification genes has been found in specimens from people with CF. The main aim of this study was to determine the relative energy contribution of oxygen (O2) respiration and denitrification in single Pseudomonas aeruginosa PAO1 biofilm colonies under different O2 concentrations to estimate the possible relative importance of these metabolic processes in the context of biofilm infections. We showed that the used strain PAO1 in biofilms denitrified with nitrous oxide (N2O), and not nitrogen (N2), as the end product in our incubations. From simultaneous O2 and N2O microprofiles measured with high spatial resolution by microsensors in agar colony biofilms under air, N2 and pure O2, the rates of aerobic respiration and denitrification were calculated and converted to ATP production rates. Denitrification occurred both in the oxic and anoxic zones, and became increasingly dominant with decreasing O2 concentrations. At O2 concentrations characteristic for tissues and wounds (20-60 µM), denitrification was responsible for 50% of the total energy conservation in the biofilm. In addition the formation of nitric oxide (NO), a precursor of N2O and an important regulator of many cellular processes, was strongly influenced by the local O2 concentrations. NO production was inhibited under pure O2, present under anoxia (∼1 µM) and remarkably high (up to 6 µM) under intermediate O2 levels, which can be found in infected tissues. Possible impacts of such NO levels on both the host and the biofilm bacteria are discussed.

Tobramycin and Vancomycin in an In Vitro Model of Anterior Cruciate Ligament Allograft Decontamination.

BACKGROUND: Approximately 100,000 anterior cruciate ligament (ACL) reconstructions (ACLRs) occur annually in the United States, and postoperative surgical-site infection is a relatively rare but devastating complication, often leading to graft failure or septic arthritis of the knee, necessitating repeat surgery. Wrapping allografts in vancomycin-soaked gauze has been adopted as a common sterilization technique in the operating room to reduce surgical-site infection; however, identifying effective alternatives to vancomycin has not been extensively pursued. HYPOTHESIS: Tobramycin would be as effective as vancomycin in reducing the concentrations of Staphylococcus epidermidis bacteria on tendon allografts. STUDY DESIGN: Controlled laboratory study. METHODS: S. epidermidis strain ATCC 12228 was inoculated onto the human cadaveric gracilis tendon. The tendons were wrapped in sterile gauze saturated with tobramycin or vancomycin at various experimental concentrations. Bacteria remaining on the tendon were dislodged, serially diluted, and plated for colony counting. Statistical analysis was performed utilizing 2-way analysis of variance testing. Results were considered statistically significant when P < .05. RESULTS: Vancomycin (P = .0001) and tobramycin (P < .0001) reduced bacterial concentration. Tobramycin was found to produce a statistically significant reduction in bacterial concentration at concentrations as low as 0.1 mg/mL (P < .0001 and P = .01 at 10 and 20 minutes), while vancomycin produced a statistically significant reduction at a concentration as low as 2.5 mg/mL (P < .0001 at both 10 and 20 minutes). CONCLUSION: This study demonstrates that tobramycin is as effective as vancomycin in bacterial concentration reduction but can achieve this reduction level at lower doses. Further studies clarifying the biomechanical and cytotoxic effects of tobramycin on tendon tissue are indicated to solidify its use as a clinical alternative to vancomycin in ACLR. CLINICAL RELEVANCE: These results will begin establishing tobramycin as an alternative to vancomycin in ACL graft decontamination. Because of relatively frequent shortages of vancomycin, establishing tobramycin as an alternative agent is a useful option for the orthopaedic surgeon.

Editorial Commentary: Low-Grade Infections May Contribute to Anterior Cruciate Ligament Reconstruction Graft Failure.

The etiology of anterior cruciate ligament (ACL) reconstruction failure is often multifactorial, and the role of subclinical bacterial colonization in ACL reconstruction failure has not been fully elucidated. Although the presence of bacterial metabolism in and of itself does not indicate true clinical infection, low-grade infections may contribute to ACL reconstruction graft failure. Bacterial biofilms on soft tissue grafts are shown to change the crimp patterns of collagen and lower graft load to failure. In addition, bacterial DNA has been reported in 80-87% of failed ACL grafts during revision surgery compared to only 20% of primary ACL grafts. Also, higher bacterial DNA concentration is associated with tibial tunnel widening. Further study is needed to establish if any causal relationship between bacterial colonization and ACL graft failure exists. But it does seem that the circumstantial evidence is pointing to such a relationship.

Incidence, Common Pathogens, and Risk Factors for Infection after Primary Anterior Cruciate Ligament Reconstruction: A Systematic Review.

We sought to assess the current literature to present a comprehensive summary of the incidence, common pathogens, and risk factors for infection after anterior cruciate ligament (ACL) reconstruction. PubMed, CINAHL, EMBASE, and Scopus databases were searched for relevant studies reporting on infection after ACL reconstruction. Two reviewers independently screened the extracted studies for adherence to inclusion and exclusion criteria. Studies were selected if they reported on the incidence of infection, pathogens cultured from infected knees, or risk factors for infection after primary ACL reconstruction. Exclusion criteria consisted of studies with fewer than 100 patients or studies that included revision ACL reconstruction. Fifty studies met the inclusion and exclusion criteria, reporting on a total of 316,214 ACL reconstructions. Included studies evaluated between 123 and 104,255 patients. The overall incidence of infection was 0.60% (0.15-2.44%). The most common pathogens were Staphylococcus aureus, S. epidermidis, and coagulase-negative Staphylococci. Five studies reported that the use of hamstring autograft was a statistically significant risk factor for infection after ACL reconstruction, thus making hamstring autograft the most commonly reported risk factor. Other reported risk factors included male sex, use of immuno-suppressive medications or intraarticular steroid injections, prior knee surgery, and diabetes. Systematic review of the literature revealed that infection after ACL reconstruction remains an infrequent event with an incidence of 0.60% (0.15-2.44%). Furthermore, the most common pathogens are from the Staphylococcus genus of bacteria, comprising 84% of all culture-positive infections. Multiple risk factors have been reported for ACL reconstruction; however, statistical significance varied across studies. Together, these findings may help guide physicians in the prevention and treatment of infection after ACL reconstruction.

Influence of Staphylococcus epidermidis on Collagen Crimp Patterns of Soft Tissue Allograft.

BACKGROUND: Postoperative infections, commonly from Staphylococcus epidermidis, may result in anterior cruciate ligament graft failure and necessitate revision surgery. In biomechanical studies, S. epidermidis has been shown to establish biofilms on tendons and reduce graft strength. PURPOSE/HYPOTHESIS: The goal of this study was to determine the effect of bacterial bioburden on the collagen structure of tendon. It was hypothesized that an increase in S. epidermidis biofilm would compromise tendon crimp, a pattern necessary for mechanical integrity, of soft tissue allografts. STUDY DESIGN: Controlled laboratory study. METHODS: Cultures of S. epidermidis were used to inoculate tibialis anterior cadaveric tendons. Conditions assessed included 5 × 105 colony-forming units or concentrated spent media from culture (no living bacteria). Incubation times of 30 minutes, 3 hours, 6 hours, and 24 hours were utilized. Second-harmonic generation imaging allowed for visualization of collagen autofluorescence. Crimp lengths were determined using ImageJ and compared based on incubation time. RESULTS: Incubation time positively correlated with increasing S. epidermidis bioburden. Both fine and coarse crimp patterns lengthened with increasing incubation time. Significant coarse crimp changes were observed after only 30-minute incubations (P < .029), whereas significant fine crimp lengthening occurred after 6 hours (P < .0001). No changes in crimp length were identified after incubation in media lacking living bacteria. CONCLUSION: The results of this study demonstrate that exposure to S. epidermidis negatively affects collagen crimp structure. Structural alterations at the collagen fiber level occur within 30 minutes of exposure to media containing S. epidermidis. CLINICAL RELEVANCE: Our study highlights the need for antimicrobial precautions to prevent graft colonization and maximize graft mechanical strength.

Bayesian estimation of Pseudomonas aeruginosa viscoelastic properties based on creep responses of wild type, rugose, and mucoid variant biofilms.

Pseudomonas aeruginosa biofilms are relevant for a variety of disease settings, including pulmonary infections in people with cystic fibrosis. Biofilms are initiated by individual bacteria that undergo a phenotypic switch and produce an extracellular polymeric slime (EPS). However, the viscoelastic characteristics of biofilms at different stages of formation and the contributions of different EPS constituents have not been fully explored. For this purpose, we develop and parameterize a mathematical model to study the rheological behavior of three biofilms - P. aeruginosa wild type PAO1, isogenic rugose small colony variant (RSCV), and mucoid variant biofilms against a range of experimental data. Using Bayesian inference to estimate these viscoelastic properties, we quantify the rheological characteristics of the biofilm EPS. We employ a Monte Carlo Markov Chain algorithm to estimate these properties of P. aeruginosa variant biofilms in comparison to those of wild type. This information helps us understand the rheological behavior of biofilms at different stages of their development. The mechanical properties of wild type biofilms change significantly over time and are more sensitive to small changes in their composition than the other two mutants.

Differential metabolism between biofilm and suspended Pseudomonas aeruginosa cultures in bovine synovial fluid by 2D NMR-based metabolomics.

Total joint arthroplasty is a common surgical procedure resulting in improved quality of life; however, a leading cause of surgery failure is infection. Periprosthetic joint infections often involve biofilms, making treatment challenging. The metabolic state of pathogens in the joint space and mechanism of their tolerance to antibiotics and host defenses are not well understood. Thus, there is a critical need for increased understanding of the physiological state of pathogens in the joint space for development of improved treatment strategies toward better patient outcomes. Here, we present a quantitative, untargeted NMR-based metabolomics strategy for Pseudomonas aeruginosa suspended culture and biofilm phenotypes grown in bovine synovial fluid as a model system. Significant differences in metabolic pathways were found between the suspended culture and biofilm phenotypes including creatine, glutathione, alanine, and choline metabolism and the tricarboxylic acid cycle. We also identified 21 unique metabolites with the presence of P. aeruginosa in synovial fluid and one uniquely present with the biofilm phenotype in synovial fluid. If translatable in vivo, these unique metabolite and pathway differences have the potential for further development to serve as targets for P. aeruginosa and biofilm control in synovial fluid.

Mapping Bacterial Biofilm on Features of Orthopedic Implants In Vitro.

Implant-associated infection is a major complication of orthopedic surgery. One of the most common organisms identified in periprosthetic joint infections is Staphylococcus aureus, a biofilm-forming pathogen. Orthopedic implants are composed of a variety of materials, such as titanium, polyethylene and stainless steel, which are at risk for colonization by bacterial biofilms. Little is known about how larger surface features of orthopedic hardware (such as ridges, holes, edges, etc.) influence biofilm formation and attachment. To study how biofilms might form on actual components, we submerged multiple orthopedic implants of various shapes, sizes, roughness and material type in brain heart infusion broth inoculated with Staphylococcus aureus SAP231, a bioluminescent USA300 strain. Implants were incubated for 72 h with daily media exchanges. After incubation, implants were imaged using an in vitro imaging system (IVIS) and the metabolic signal produced by biofilms was quantified by image analysis. Scanning electron microscopy was then used to image different areas of the implants to complement the IVIS imaging. Rough surfaces had the greatest luminescence compared to edges or smooth surfaces on a single implant and across all implants when the images were merged. The luminescence of edges was also significantly greater than smooth surfaces. These data suggest implant roughness, as well as large-scale surface features, may be at greater risk of biofilm colonization.

Rapid Aggregation of Staphylococcus aureus in Synovial Fluid Is Influenced by Synovial Fluid Concentration, Viscosity, and Fluid Dynamics, with Evidence of Polymer Bridging.

Early bacterial survival in the postsurgical joint is still a mystery. Recently, synovial fluid-induced aggregation was proposed as a potential mechanism of bacterial protection upon entry into the joint. As synovial fluid is secreted back into the joint cavity following surgery, rapid fluctuations in synovial fluid concentrations, composition, and viscosity occur. These changes, along with fluid movement resulting from postoperative joint motion, modify the environment and potentially affect the kinetics of aggregate formation. Through this work, we sought to evaluate the influence of exposure time, synovial fluid concentration, viscosity, and fluid dynamics on aggregation. Furthermore, we aimed to elucidate the primary mechanism of aggregate formation by assessing the interaction of bacterial adhesins with the synovial fluid polymer fibrinogen. Following incubation under each simulated postoperative joint condition, the aggregates were imaged using confocal microscopy. Our analysis revealed the formation of two distinct aggregate phenotypes, depending on whether the incubation was conducted under static or dynamic conditions. Using a surface adhesin mutant, we have narrowed down the genetic determinants for synovial fluid aggregate formation and identified essential host polymers. We report here that synovial fluid-induced aggregation is influenced by various changes specific to the postsurgical joint environment. While we now have evidence that select synovial fluid polymers facilitate bridging aggregation through essential bacterial adhesins, we suspect that their utility is limited by the increasing viscosity under static conditions. Furthermore, dynamic fluid movement recovers the ability of the bacteria with surface proteins present to aggregate under high-viscosity conditions, yielding large, globular aggregates. IMPORTANCE Infection is a major complication of knee and hip joint replacement surgery, which is used to treat arthritis or joint damage. We have shown that Staphylococcus aureus, a common bacterial pathogen, aggregates upon contact with synovial fluid. Within seconds, the bacterial cells interact with synovial fluid polymers in the joint fluid through their cell wall adhesins. The rapid formation of these aggregates likely aids in early bacterial survival in the joint, potentially contributing to the likelihood of developing an infection. By strengthening our basic understanding of the mechanics of synovial fluid aggregate formation under clinically relevant conditions, we hope to expand the knowledge of how to prevent or disrupt aggregation and reduce and more successfully treat these joint infections.

Synovial Fluid-Induced Aggregation Occurs across Staphylococcus aureus Clinical Isolates and is Mechanistically Independent of Attached Biofilm Formation.

Rapid synovial fluid-induced aggregation of Staphylococcus aureus is currently being investigated as an important factor in the establishment of periprosthetic joint infections (PJIs). Pathogenic advantages of aggregate formation have been well documented in vitro, including recalcitrance to antibiotics and protection from host immune defenses. The objective of the present work was to determine the strain dependency of synovial fluid-induced aggregation by measuring the degree of aggregation of 21 clinical S. aureus isolates cultured from either PJI or bloodstream infections using imaging and flow cytometry. Furthermore, by measuring attached bacterial biomass using a conventional crystal violet assay, we assessed whether there is a correlation between the aggregative phenotype and surface-associated biofilm formation. While all of the isolates were stimulated to aggregate upon exposure to bovine synovial fluid (BSF) and human serum (HS), the extent of aggregation was highly variable between individual strains. Interestingly, the PJI isolates aggregated significantly more upon BSF exposure than those isolated from bloodstream infections. While we were able to stimulate biofilm formation with all of the isolates in growth medium, supplementation with either synovial fluid or human serum inhibited bacterial surface attachment over a 24 h incubation. Surprisingly, there was no correlation between the degree of synovial fluid-induced aggregation and quantity of surface-associated biofilm as measured by a conventional biofilm assay without host fluid supplementation. Taken together, our findings suggest that synovial fluid-induced aggregation appears to be widespread among S. aureus strains and mechanistically independent of biofilm formation. IMPORTANCE Bacterial infections of hip and knee implants are rare but devastating complications of orthopedic surgery. Despite a widespread appreciation of the considerable financial, physical, and emotional burden associated with the development of a prosthetic joint infection, the establishment of bacteria in the synovial joint remains poorly understood. It has been shown that immediately upon exposure to synovial fluid, the viscous fluid in the joint, Staphylococcus aureus rapidly forms aggregates which are resistant to antibiotics and host immune cell clearance. The bacterial virulence associated with aggregate formation is likely a step in the establishment of prosthetic joint infection, and as such, it has the potential to be a potent target of prevention. We hope that this work contributes to the future development of therapeutics targeting synovial fluid-induced aggregation to better prevent and treat these infections.

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.

Mycobacterium abscessus biofilms have viscoelastic properties which may contribute to their recalcitrance in chronic pulmonary infections.

Mycobacterium abscessus is emerging as a cause of recalcitrant chronic pulmonary infections, particularly in people with cystic fibrosis (CF). Biofilm formation has been implicated in the pathology of this organism, however the role of biofilm formation in infection is unclear. Two colony-variants of M. abscessus are routinely isolated from CF samples, smooth (MaSm) and rough (MaRg). These two variants display distinct colony morphologies due to the presence (MaSm) or absence (MaRg) of cell wall glycopeptidolipids (GPLs). We hypothesized that MaSm and MaRg variant biofilms might have different mechanical properties. To test this hypothesis, we performed uniaxial mechanical indentation, and shear rheometry on MaSm and MaRg colony-biofilms. We identified that MaRg biofilms were significantly stiffer than MaSm under a normal force, while MaSm biofilms were more pliant compared to MaRg, under both normal and shear forces. Furthermore, using theoretical indices of mucociliary and cough clearance, we identified that M. abscessus biofilms may be more resistant to mechanical forms of clearance from the lung, compared to another common pulmonary pathogen, Pseudomonas aeruginosa. Thus, the mechanical properties of M. abscessus biofilms may contribute to the persistent nature of pulmonary infections caused by this organism.

High Number of Door Openings Increases the Bacterial Load of the Operating Room.

Background: Operating room (OR) traffic and door openings have emerged as potential modifiable risk factors for the development of surgical site infections. Methods: This study compared the microbial load of a Control OR without traffic versus a Simulated OR with the traffic in a typical orthopedic surgery case. Air particle counts and colony forming units (CFUs) were measured. A novel iOS app was developed to provide real-time door counts. Results: There were 1,862 particles >5.0 mcm in the Simulated OR compared with 56 in the Control OR. The CFUs from plates in the Simulated OR ranged from 4-22 (on brain heart infusion [BHI] agar), 2-266 (on mannitol salt agar [MSA]), and 1-19 (on Pseudomonas isolation agar [PIA]), while all plates in the Control OR grew 0-1 CFUs. Conclusions: High number of door openings leads to more airborne bacteria in the OR and viable bacterial on OR surfaces. The increased bacterial load throughout the OR was independent of distance from the door.

Evaluation of Peptide-Based Probes toward In Vivo Diagnostic Imaging of Bacterial Biofilm-Associated Infections.

The clinical management of bacterial biofilm infections represents an enormous challenge in today's healthcare setting. The NIH estimates that 65% of bacterial infections are biofilm-related, and therapeutic outcomes are positively correlated with early intervention. Currently, there is no reliable imaging technique to detect biofilm infections in vivo, and current clinical protocols for accurate and direct biofilm identification are nonexistent. In orthopedic implant-associated biofilm infections, for example, current detection methods are based on nonspecific X-ray or radiolabeled white blood cell imaging, coupled with peri-prosthetic tissue or fluid samples taken invasively, and must be cultured. This approach is time-consuming and often fails to detect biofilm bacteria due to sampling errors and a lack of sensitivity. The ability to quantify bacterial biofilms by real-time noninvasive imaging is an urgent unmet clinical need that would revolutionize the management and treatment of these devastating types of infections. In the present study, we assembled a collection of fluorescently labeled peptide candidates to specifically explore their biofilm targeting properties. We evaluated these fluorescently labeled peptides using various in vitro assays for their ability to specifically and nondestructively target biofilms produced by model bacterial pathogen Pseudomonas aeruginosa. The lead candidate that emerged, 4Iphf-HN17, demonstrated rapid biofilm labeling kinetics, a lack of bactericidal activity, and biofilm targeting specificity in human cell infection models. In vivo fluorescently labeled 4Iphf-HN17 showed enhanced accumulation in biofilm-infected wounds, thus warranting further study.

Staphylococcus aureus Aggregates on Orthopedic Materials under Varying Levels of Shear Stress.

Periprosthetic joint infection (PJI) occurring after artificial joint replacement is a major clinical issue requiring multiple surgeries and antibiotic interventions. Staphylococcus aureus is the bacterium most commonly responsible for PJI. Recent in vitro research has shown that staphylococcal strains rapidly form aggregates in the presence of synovial fluid (SF). We hypothesize that these aggregates provide early protection to bacteria entering the wound site, allowing them time to attach to the implant surface, leading to biofilm formation. Thus, understanding the attachment kinetics of these aggregates is critical in understanding their adhesion to various biomaterial surfaces. In this study, the number, size, and surface area coverage of aggregates as well as of single cells of S. aureus were quantified under various conditions on different orthopedic materials relevant to orthopedic surgery: stainless steel (316L), titanium (Ti), hydroxyapatite (HA), and polyethylene (PE). It was observed that, regardless of the material type, SF-induced aggregation resulted in reduced aggregate surface attachment and greater aggregate size than the single-cell populations under various shear stresses. Additionally, the surface area coverage of bacterial aggregates on PE was relatively high compared to that on other materials, which could potentially be due to the rougher surface of PE. Furthermore, increasing shear stress to 78 mPa decreased aggregate attachment to Ti and HA while increasing the aggregates' average size. Therefore, this study demonstrates that SF induced inhibition of aggregate attachment to all materials, suggesting that biofilm formation is initiated by lodging of aggregates on the surface features of implants and host tissues.IMPORTANCE Periprosthetic joint infection occurring after artificial joint replacement is a major clinical issue that require repeated surgeries and antibiotic interventions. Unfortunately, 26% of patients die within 5 years of developing these infections. Staphylococcus aureus is the bacterium most commonly responsible for this problem and can form biofilms to provide protection from antibiotics as well as the immune system. Although biofilms are evident on the infected implants, it is unclear how these are attached to the surface in the first place. Recent in vitro investigations have shown that staphylococcal strains rapidly form aggregates in the presence of synovial fluid and provide protection to bacteria, thus allowing them time to attach to the implant surface, leading to biofilm formation. In this study, we investigated the attachment kinetics of Staphylococcus aureus aggregates on different orthopedic materials. The information presented in this article will be useful in surgical management and implant design.

Computational and Experimental Investigation of Biofilm Disruption Dynamics Induced by High-Velocity Gas Jet Impingement.

Experimental data showed that high-speed microsprays can effectively disrupt biofilms on their support substratum, producing a variety of dynamic reactions such as elongation, displacement, ripple formation, and fluidization. However, the mechanics underlying the impact of high-speed turbulent flows on biofilm structure is complex under such extreme conditions, since direct measurements of viscosity at these high shear rates are not possible using dynamic testing instruments. Here, we used computational fluid dynamics simulations to assess the complex fluid interactions of ripple patterning produced by high-speed turbulent air jets impacting perpendicular to the surface of Streptococcus mutans biofilms, a dental pathogen causing caries, captured by high-speed imaging. The numerical model involved a two-phase flow of air over a non-Newtonian biofilm, whose viscosity as a function of shear rate was estimated using the Herschel-Bulkley model. The simulation suggested that inertial, shear, and interfacial tension forces governed biofilm disruption by the air jet. Additionally, the high shear rates generated by the jet impacts coupled with shear-thinning biofilm property resulted in rapid liquefaction (within milliseconds) of the biofilm, followed by surface instability and traveling waves from the impact site. Our findings suggest that rapid shear thinning under very high shear flows causes the biofilm to behave like a fluid and elasticity can be neglected. A parametric sensitivity study confirmed that both applied force intensity (i.e., high jet nozzle air velocity) and biofilm properties (i.e., low viscosity and low air-biofilm surface tension and thickness) intensify biofilm disruption by generating large interfacial instabilities.IMPORTANCE Knowledge of mechanisms promoting disruption though mechanical forces is essential in optimizing biofilm control strategies which rely on fluid shear. Our results provide insight into how biofilm disruption dynamics is governed by applied forces and fluid properties, revealing a mechanism for ripple formation and fluid-biofilm mixing. These findings have important implications for the rational design of new biofilm cleaning strategies with fluid jets, such as determining optimal parameters (e.g., jet velocity and position) to remove the biofilm from a certain zone (e.g., in dental hygiene or debridement of surgical site infections) or using antimicrobial agents which could increase the interfacial area available for exchange, as well as causing internal mixing within the biofilm matrix, thus disrupting the localized microenvironment which is associated with antimicrobial tolerance. The developed model also has potential application in predicting drag and pressure drop caused by biofilms on bioreactor, pipeline, and ship hull surfaces.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

Bacterial DNA is associated with tunnel widening in failed ACL reconstructions.

PURPOSE: To determine if tunnel widening, defined as change in maximal tunnel diameter from the time of initial bone tunnel drilling to revision surgery is associated with bacterial deoxyribonucleic acid (DNA) presence and concentration in torn graft tissue from failed anterior cruciate ligament reconstructions (ACLRs). METHODS: Thirty-four consecutive revision ACLRs were included (mean age 27.3 years SD 10.9; median time to failure 4.9 years range 105 days-20 years). Graft selection of the failed reconstruction was 68% autograft, 26% allograft, and 6% autograft/allograft hybrid with a mean drilled tunnel diameter of 8.4 mm SD 0.8. Maximal tunnel diameters prior to revision were measured on pre-operative three-dimensional imaging and compared to drilled tunnel diameters at the time of the previous reconstruction. Tissue biopsies of the failed graft were obtained from tibial, femoral, and intraarticular segments. Sterile water left open to air during revision ACLRs and tissue from primary ACLRs were used as negative controls. Clinical cultures were obtained on all revision ACLRs and PCR with universal bacterial primer on all cases and negative controls. Fluorescence microscopy was used to confirm the presence and location of biofilms in two patients with retrieved torn graft tissue and fixation material. Amount of tunnel widening was compared to bacterial DNA presence as well as bacterial DNA concentration via Welch ANOVA. RESULTS: Bacterial DNA was present in 29/34 (85%) revision ACLRs, 1/5 (20%) of primary ACLR controls and 0/3 (0%) sterile water controls. Cultures were positive (coagulase negative Staphylococcus sp.) in one case, which also had the greatest degree of tunnel widening. Femoral widening was greater in cases with detectable bacterial DNA (mean widening 2.6 mm SD 3.0) versus without (mean 0.3 mm SD 0.6) (p = 0.003) but was unaffected by bacterial DNA concentration (p = 0.44). Tibial widening was not associated with the presence of bacterial DNA (n.s.); however, higher bacterial DNA concentrations were observed in cases with tibial widening ≥ 3.0 mm (median 2.47 ng bacterial DNA/µg total DNA) versus widening < 3.0 mm (median 0.97 ng bacterial DNA/µg total DNA) (p = 0.046). Tunnel widening was not associated with time to failure, graft selection, or number of prior surgeries (n.s., all comparisons). Fluorescence microscopy confirmed the presence of biofilms on ruptured tendon graft as well as fixation material in 2/2 cases. CONCLUSION: Bacterial DNA is commonly encountered on failed ACLR grafts and can form biofilms. Bacterial DNA does not cause clinically apparent infection symptoms but is associated with tunnel widening. Further research is needed to determine whether graft decontamination protocols can reduce graft bacterial colonization rates, ACLR tunnel widening or ACLR failure risk. LEVEL OF EVIDENCE: Therapeutic III.

Bacterial Deoxyribonucleic Acid Is Often Present in Failed Revision Anterior Cruciate Ligament Reconstructions.

PURPOSE: To determine whether bacterial DNA will be detectable by polymerase chain reaction (PCR) in torn graft tissue at the time of revision anterior cruciate ligament reconstruction (ACLR). METHODS: A total of 31 consecutive revision ACLR cases from 1 center from 2014-2016 were recruited. No patients had clinical signs of infection on presentation. Torn graft tissue was obtained in revision cases and subjected to clinical culture and PCR analysis with a universal bacterial primer. Fluorescence microscopy was used to confirm the presence of a biofilm. We obtained negative control samples of water open to air on the field and excess primary ACLR graft tissue, as well as torn native ligament, to evaluate for PCR positivity due to environmental contamination. RESULTS: Clinical cultures were positive (coagulase-negative Staphylococcus) in 1 revision case (3%, 1 of 31). Bacterial DNA was detectable in most revision ACLR cases (87.0%, 27 of 31), and there was a low rate of PCR positivity in negative control samples of water open to air (0%, 0 of 3), excess primary ACLR graft tissue after passage (20%, 1 of 5), or native torn ligament (20%, 1 of 5). Bacterial biofilm presence on failed graft tissue as well as monofilament suture was visually confirmed with fluorescence microscopy. CONCLUSIONS: Bacterial DNA is frequently present in failed ACLR grafts, with high rates of DNA detection by PCR but low culture positivity. LEVEL OF EVIDENCE: Level IV, case series.