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.

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.

Mapping bacterial biofilms on recovered orthopaedic implants by a novel agar candle dip method.

While the detrimental effects of periprosthetic joint infections (PJIs) are well known, the process of biofilm formation on orthopaedic hardware is unclear. Previous work has shown that encasement of explant hardware in agar can aid in identifying biofilms. This study tested the utility of agar 'candle dip' method in detecting and mapping the location of biofilm on infected orthopedic components. Explant components from 15 patients were rinsed, briefly submerged in agar to create a surface coating, and incubated. Larger components were coated by pipetting agar over them. After incubation, colony outgrowth on the component surface was documented (candle dip status). Data were compared with clinical laboratory results (clinical culture status) and the PJI diagnosis using Musculoskeletal Infection Society criteria (MSIS status). All six patients classified as MSIS and clinical culture positive were also positive with the candle dip technique. Of the nine candle dip negative cases, four were positive and five were negative for both MSIS and clinical culture status. Candle dip may be negative in few cases due to the residual antibiotic eluting from the spacers, limiting the growth of bacterial biofilms on the components. The candle dip method shows promise for biofilm mapping but requires additional testing to evaluate the clinical diagnostic potential.

Reduction in Pseudomonas aeruginosa and Staphylococcus aureus biofilms from implant materials in a diffusion dominated environment.

Antibiotic-loaded calcium sulfate beads (CS-B) are used to treat biofilm-related periprosthetic joint infections (PJI). A previous study has shown that such beads are effective in reducing lawns biofilms grown on agar plates; however, the ability of CS-B to eradicate biofilms grown on solid orthopedic material surfaces has not been investigated. We grew biofilms of bioluminescent strains of Pseudomonas aeruginosa Xen41 and a USA300 MRSA Staphylococcus aureus SAP231 on ultra-high molecular weight polyethylene (PE), hydroxyapatite (HA), and 316L stainless steel (SS) coupons for three days under static growth conditions, with daily nutrient exchange. The coupons were rinsed with sterile phosphate buffered saline (PBS) to remove planktonic bacteria and placed in a petri dish, surrounded by four either antibiotic vancomycin and tobramycin loaded (CS-BV+T ) or unloaded beads (CS-BU ). A thin layer of agar was overlaid to simulate a periprosthetic infection where an implant abuts soft tissue and then incubated for 72 h. The amount of biofilm was measured by bioluminescence imaging (BLI) for activity and viable cell count (CFUs). Coupons exposed to CS-BV+T showed a significant reduction in the amount of biofilm within 24 h, regardless of the bacterial strain or material type. The coupons exposed to control CS-BU had no effect on bacteria over 72 h. Statement of Clinical Significance: Antibiotic-loaded calcium sulfate beads (CS-B) were effective in significantly reducing mature biofilms of P. aeruginosa and S. aureus from orthopedic relevant surfaces in our in vitro agar model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3081-3085, 2018.

Tranexamic acid use during total hip arthroplasty: A single center retrospective analysis.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic agent that has shown promise in reducing blood loss during total hip arthroplasty (THA). Several studies have reported side effects of high-dose TXA administration, including myocardial infarction (MI), thromboembolic events, and seizures. These possible side effects have prevented the widespread adoption of TXA in the surgical community. METHODS: We conducted a retrospective chart review of 564 primary and revision THAs performed at a single academic center. Surgical patients received either no TXA or 1 g IV TXA at the beginning of surgery followed by a second bolus just before the surgical wound closure, at the surgeon's discretion. We analyzed differences in hemoglobin (Hb), hematocrit (Hct), estimated blood loss (EBL), and adverse events in patients receiving TXA versus patients not receiving TXA up to 2 days following surgery. RESULTS: Significantly higher Hb and Hct values were found across all time points among patients undergoing primary posterior or revision THA who had received TXA. In addition, transfusion rates were significantly decreased in both primary posterior THAs and revision THAs when TXA was administered. Patients who received TXA experienced significantly fewer adverse events than those who did not for all surgery types. CONCLUSION: Administration of low-dose intravenous (IV) and intra-articular (IA) TXA does not appear to increase rates of adverse events and may be effective in minimizing blood loss, as reflected by Hb and Hct values following THA.

Antibiotic loaded calcium sulfate bead and pulse lavage eradicates biofilms on metal implant materials in vitro.

Pulse lavage (PL) debridement and antibiotic loaded calcium sulfate beads (CS-B) are both used for the treatment of biofilm related periprosthetic joint infection (PJI). However, the efficacy of these alone and in combination for eradicating biofilm from orthopaedic metal implant surfaces is unclear. The purpose of the study was to understand the efficacy of PL and antibiotic loaded CS-B in eradicating bacterial biofilms on 316L stainless steel (SS) alone and in combination in vitro. Biofilms of bioluminescent strains of Pseudomonas aeruginosa Xen41 and a USA300 MRSA Staphylococcus aureus SAP231 were grown on SS coupons for 3 days. The coupons were either, (i) debrided for 3 s with PL, (ii) exposed to tobramycin (TOB) and vancomycin (VAN) loaded CS-B for 24 h, or (iii) exposed to both. An untreated biofilm served as a control. The amount of biofilm was measured by bioluminescence, viable plate count and confocal microscopy using live/dead staining. PL alone reduced the CFU count of both strains of biofilms by approximately 2 orders of magnitude, from an initial cell count on metal surface of approximately 109 CFU/cm2 . The antibiotic loaded CS-B caused an approximate six log reduction and the combination completely eradicated viable biofilm bacteria. Bioluminescence and confocal imaging corroborated the CFU data. While PL and antibiotic loaded CS-B both significantly reduced biofilm, the combination of two was more effective than alone in removing biofilms from SS implant surfaces. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2349-2354, 2018.

Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection.

Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10× MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1086-1092, 2018.

Biofilms in orthopedic infections: a review of laboratory methods.

Bacterial infection after hardware implantation in orthopedic surgery is a devastating issue as it often necessitates increased hospital costs and stays, multiple revision surgeries, and prolonged use of antibiotics. Because of the nature of hardware implantation into the body, these infections are commonly in the form of attached biofilms. The current literature on a range of methodologies to study clinically explanted infected orthopedic hardware, with potential biofilm, in the laboratory setting is limited. General methods include traditional and advanced culturing techniques, microscopy imaging techniques, and techniques that manipulate genetic material. The future of diagnostic techniques for infected implants, innovative hardware design, and treatment solutions for patients all depend on the successful evaluation and characterization of clinical samples in the laboratory setting. This review provides an overview of current methods to study biofilms associated with orthopedic infections and insight into future directions in the field.

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.

Relationships between varus-valgus laxity of the severely osteoarthritic knee and gait, instability, clinical performance, and function.

Increased varus-valgus laxity has been reported in individuals with knee osteoarthritis (OA) compared to controls. However, the majority of previous investigations may not report truly passive joint laxity, as their tests have been performed on conscious participants who could be guarding against motion with muscle contraction during laxity evaluation. The purpose of this study was to investigate how a measure of passive knee laxity, recorded when the participant is under anesthesia, is related to varus-valgus excursion during gait, clinical measures of performance, perceived instability, and self-reported function in participants with severe knee OA. We assessed passive varus-valgus knee laxity in 29 participants (30 knees) with severe OA, as they underwent total knee arthroplasty (TKA). Participants also completed gait analysis, clinical assessment of performance (6-min walk (6 MW), stair climbing test (SCT), isometric knee strength), and self-reported measures of function (perceived instability, Knee injury, and Osteoarthritis Outcome Score (KOOS) a median of 18 days before the TKA procedure. We observed that greater passive varus-valgus laxity was associated with greater varus-valgus excursion during gait (R2 = 0.34, p = 0.002). Significant associations were also observed between greater laxity and greater isometric knee extension strength (p = 0.014), farther 6 MW distance (p = 0.033) and shorter SCT time (p = 0.046). No relationship was observed between passive varus-valgus laxity and isometric knee flexion strength, perceived instability, or any KOOS subscale. The conflicting associations between laxity, frontal excursion during gait, and functional performance suggest a complex relationship between laxity and knee cartilage health, clinical performance, and self-reported function that merits further study. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1644-1652, 2017.

16S rRNA analysis provides evidence of biofilms on all components of three infected periprosthetic knees including permanent braided suture.

Bacterial biofilms are the main etiological agent of periprosthetic joint infections (PJI); however, it is unclear if biofilms colonize one or multiple components. Because biofilms can colonize a variety of surfaces, we hypothesized that biofilms would be present on all components. 16S ribosomal RNA (rRNA) gene sequencing analysis was used to identify bacteria recovered from individual components and non-absorbable suture material recovered from three PJI total knee revision cases. Bray-Curtis non-metric multidimensional scaling analysis revealed no significant differences in similarity when factoring component, material type, or suture versus non-suture material, but did reveal significant differences in organism profile between patients (P < 0.001) and negative controls (P < 0.001). Confocal microscopy and a novel agar encasement culturing method also confirmed biofilm growth on a subset of components. While 16S sequencing suggested that the microbiology was more complex than revealed by culture contaminating, bacterial DNA generates a risk of false positives. This report highlights that biofilm bacteria may colonize all infected prosthetic components including braided suture material, and provides further evidence that clinical culture can fail to sufficiently identify the full pathogen profile in PJI cases.

Estimating patient-specific soft-tissue properties in a TKA knee.

Surgical technique is one factor that has been identified as critical to success of total knee arthroplasty. Researchers have shown that computer simulations can aid in determining how decisions in the operating room generally affect post-operative outcomes. However, to use simulations to make clinically relevant predictions about knee forces and motions for a specific total knee patient, patient-specific models are needed. This study introduces a methodology for estimating knee soft-tissue properties of an individual total knee patient. A custom surgical navigation system and stability device were used to measure the force-displacement relationship of the knee. Soft-tissue properties were estimated using a parameter optimization that matched simulated tibiofemoral kinematics with experimental tibiofemoral kinematics. Simulations using optimized ligament properties had an average root mean square error of 3.5° across all tests while simulations using generic ligament properties taken from literature had an average root mean square error of 8.4°. Specimens showed large variability among ligament properties regardless of similarities in prosthetic component alignment and measured knee laxity. These results demonstrate the importance of soft-tissue properties in determining knee stability, and suggest that to make clinically relevant predictions of post-operative knee motions and forces using computer simulations, patient-specific soft-tissue properties are needed.

Elution of antibiotics from poly(methyl methacrylate) bone cement after extended implantation does not necessarily clear the infection despite susceptibility of the clinical isolates.

Chronic orthopedic infections are commonly caused by bacterial biofilms, which are recalcitrant to antibiotic treatment. In many cases, the revision procedure for periprosthetic joint infection or trauma cases includes the implantation of antibiotic-loaded bone cement to kill infecting bacteria via the elution of a strong local dose of antibiotic(s) at the site. While many studies have addressed the elution kinetics of both non-absorbable and absorbable bone cements both in vitro and in vivo, the potency of ALBC against pathogenic bacteria after extended implantation time is not clear. In this communication, we use two case studies, a Viridans streptococci infected total knee arthroplasty (TKA) and a MRSA-polymicrobial osteomyelitis of a distal tibial traumatic amputation (TA) to demonstrate that an antibiotic-loaded poly(methyl methacrylate) (ALPMMA) coated intermedullary rod implanted for 117 days (TKA) and three ALPMMA suture-strung beads implanted for 210 days (TA) retained killing ability against Pseudomonas aeruginosa and Staphylococcus aureus in vitro, despite different clinical efficacies. The TKA infection resolved and the patient progressed to an uneventful second stage. However, the TA infection only resolved after multiple rounds of debridement, IV vancomycin and removal of the PMMA beads and placement of vancomycin and tobramycin loaded calcium sulfate beads.

Caring for the incarcerated: an orthopedic perspective.

According to the 1976 Supreme Court case of Estelle v Gamble, the government is required to provide prisoners access to the necessary care for known, serious medical needs. Failure to do so violates the Eighth Amendment prohibiting the imposition of "cruel and unusual punishment." Currently, more than 2.5 million Americans are imprisoned, the highest per capita rate in the world. In the current fiscal climate and with burgeoning health care costs for society as a whole, providing care for prisoners poses both moral and logistical challenges. From an orthopedic standpoint, caring for the incarcerated raises uniquely challenging issues due to the nature of this patient population and their types of musculoskeletal conditions.

Is there a gold standard for TKA tibial component rotational alignment?

BACKGROUND: Joint function and durability after TKA depends on many factors, but component alignment is particularly important. Although the transepicondylar axis is regarded as the gold standard for rotationally aligning the femoral component, various techniques exist for tibial component rotational alignment. The impact of this variability on joint kinematics and stability is unknown. QUESTIONS/PURPOSES: We determined how rotationally aligning the tibial component to four different axes changes knee stability and passive tibiofemoral kinematics in a knee after TKA. METHODS: Using a custom surgical navigation system and stability device to measure stability and passive tibiofemoral motion, we tested 10 cadaveric knees from five hemicorpses before TKA and then with the tibial component aligned to four axes using a modified tibial tray. RESULTS: No changes in knee stability or passive kinematics occurred as a result of the four techniques of tibial rotational alignment. TKA produces a 'looser' knee over the native condition by increasing mean laxity by 5.2°, decreasing mean maximum stiffness by 4.5 N·m/°, increasing mean anterior femoral translation during passive flexion by 5.4 mm, and increasing mean internal-external tibial rotation during passive flexion by 4.8°. However, no statistically or clinically important differences occurred between the four TKA conditions. CONCLUSIONS: For all tibial rotations, TKA increased laxity, decreased stiffness, and increased tibiofemoral motion during passive flexion but showed little change based on the tibial alignment. CLINICAL RELEVANCE: Our observations suggest surgeons who align the tibial component to any of the axes we examined are expected to have results consistent with those who may use a different axis.

Lower-extremity soft tissue infections with intra-abdominal sources.

This article describes a series of 3 patients who presented with lower-extremity soft tissue infections. Each patient was treated with prompt debridement by an orthopedic surgeon (J.F.G.) and required at least 1 additional procedure by another surgeon.These infections vary from superficial cellulitis to rapidly advancing necrotizing fasciitis. At times, the source of these infections is clear. Other times, no obvious source of infection exists, in which case the abdomen must be considered as a possible source of infection. A high level of suspicion, complete history and physical examination, and appropriate ancillary studies are required to make an accurate and prompt diagnosis. Options for the treatment of the intra-abdominal source of infection depend on the etiology of the infection and anatomic location of the process. Psoas abscesses can often be decompressed by an interventional radiologist using computed tomography guidance. In the case of bowel involvement, such as suspected carcinoma or diverticulitis, a general surgeon is necessary. When the appropriate diagnosis is made, soft tissue infections of the thigh often respond to appropriate surgical debridement and antibiotic therapy. It is important to remember the whole patient when evaluating soft tissue infections, especially in the thigh. A low threshold for imaging of the abdomen and pelvis is important, especially when the physical examination or medical history reveals the abdomen as a possible source of infection.