Tolerant Small-colony Variants Form Prior to Resistance Within a Staphylococcus aureus Biofilm Based on Antibiotic Selective Pressure.

BACKGROUND: The treatment of periprosthetic joint infection (PJI) is focused on the surgical or chemical removal of biofilm. Antibiotics in isolation are typically ineffective against PJI. Bacteria survive after antibiotic administration because of antibiotic tolerance, resistance, and persistence that arise in the resident bacteria of a biofilm. Small-colony variants are typically slow-growing bacterial subpopulations that arise after antibiotic exposure and are associated with persistent and chronic infections such as PJI. The role of biofilm-mediated antibiotic tolerance in the emergence of antibiotic resistance remains poorly defined experimentally. QUESTIONS/PURPOSES: We asked: (1) Does prior antibiotic exposure affect how Staphylococcus aureus survives within a developing biofilm when exposed to an antibiotic that penetrates biofilm, like rifampicin? (2) Does exposure to an antibiotic with poor biofilm penetration, such as vancomycin, affect how S. aureus survives within a developing biofilm? (3) Do small-colony variants emerge from antibiotic-tolerant or -resistant bacteria in a S. aureus biofilm? METHODS: We used a porous membrane as an in vitro implant model to grow luminescent S. aureus biofilms and simultaneously track microcolony expansion. We evaluated the impact of tolerance on the development of resistance by comparing rifampicin (an antibiotic that penetrates S. aureus biofilm) with vancomycin (an antibiotic that penetrates biofilm poorly). We performed viability counting after membrane dissociation to discriminate among tolerant, resistant, and persistent bacteria. Biofilm quantification and small-colony morphologies were confirmed using scanning electron microscopy. Because of experimental variability induced by the starting bacterial inoculum, relative changes were compared since absolute values may not have been statistically comparable. RESULTS: Antibiotic-naïve S. aureus placed under the selective pressure of rifampicin initially survived within an emerging biofilm by using tolerance given that biofilm resident cell viability revealed 1.0 x 108 CFU, of which 7.5 x 106 CFU were attributed to the emergence of resistance and 9.3 x 107 CFU of which were attributed to the development of tolerance. Previous exposure of S. aureus to rifampicin obviated tolerance-mediate survival when rifampicin resistance was present, since the number of viable biofilm resident cells (9.5 x 109 CFU) nearly equaled the number of rifampicin-resistant bacteria (1.1 x 1010 CFU). Bacteria exposed to an antibiotic with poor biofilm penetration, like vancomycin, survive within an emerging biofilm by using tolerance as well because the biofilm resident cell viability for vancomycin-naïve (1.6 x 1010 CFU) and vancomycin-resistant (1.0 x 1010 CFU) S. aureus could not be accounted for by emergence of resistance. Adding rifampicin to vancomycin resulted in a nearly 500-fold reduction in vancomycin-tolerant bacteria from 1.5 x 1010 CFU to 3.3 x 107 CFU. Small-colony variant S. aureus emerged within the tolerant bacterial population within 24 hours of biofilm-penetrating antibiotic administration. Scanning electron microscopy before membrane dissociation confirmed the presence of small, uniform cells with biofilm-related microstructures when unexposed to rifampicin as well as large, misshapen, lysed cells with a small-colony variant morphology [29, 41, 42, 63] and a lack of biofilm-related microstructures when exposed to rifampicin. This visually confirmed the rapid emergence of small-colony variants within the sessile niche of a developing biofilm when exposed to an antibiotic that exerted selective pressure. CONCLUSION: Tolerance explains why surgical and nonsurgical modalities that rely on antibiotics to "treat" residual microscopic biofilm may fail over time. The differential emergence of resistance based on biofilm penetration may explain why some suppressive antibiotic therapies that do not penetrate biofilm well may rely on bacterial control while limiting the emergence of resistance. However, this strategy fails to address the tolerant bacterial niche that harbors persistent bacteria with a small-colony variant morphology. CLINICAL RELEVANCE: Our work establishes biofilm-mediated antibiotic tolerance as a neglected feature of bacterial communities that prevents the effective treatment of PJI.

Use of Navigation-Enhanced Instrumentation to Mitigate Surgical Outliers During Total Knee Arthroplasty.

Computer-assisted orthopedic surgery improves mechanical alignment and the accuracy of surgical cuts in the context of total knee arthroplasty (TKA). A simplified, navigation-enhanced instrumentation system was assessed to determine whether the same effects could be achieved with a less intrusive system. Two cohorts of surgeons (experienced and trainees) performed a series of TKA cuts using models with and without navigation-enhanced instrumentation. The accuracy of each system was determined via the rate of outliers, measured as any cut that deviated from the planned cut by more than 2° or 2 mm. The effect of experience level was limited, with only the outlier rate for tibial varus or valgus measurement showing a significant difference between user groups with conventional instrumentation (P=.004). The use of navigation-enhanced instrumentation significantly reduced the total outlier rate compared with conventional instrumentation from 35% to 4% for experienced users (P<.001) and from 34% to 10% for trainees (P<.001). These results suggest that navigation-enhanced instrumentation is a viable alternative to conventional instrumentation to reduce outlier rates and improve cut accuracy. This trial also showed that additional experience may not correlate with improved surgical accuracy. Outliers may not reflect individual surgical ability as much as limitations of the instrumentation or other unidentified factors. [Orthopedics. 2021;44(1):54-57.].

A Scoping Review of Neuromuscular Electrical Stimulation to Improve Gait in Cerebral Palsy: The Arc of Progress and Future Strategies.

Background: Neuromuscular deficits of children with spastic cerebral palsy (CP) limits mobility, due to muscle weakness, short muscle-tendon unit, spasticity, and impaired selective motor control. Surgical and pharmaceutical strategies have been partially effective but often cause further weakness. Neuromuscular electrical stimulation (NMES) is an evolving technology that can improve neuromuscular physiology, strength, and mobility. This review aims to identify gaps in knowledge to motivate future NMES research. Methods: Research publications from 1990- July 20th 2019 that investigated gait-specific NMES in CP were reviewed using the PubMed and Google Scholar databases. Results were filtered by the National Institute of Neurological Disorder and Stroke common data elements guidelines for CP. The Oxford Centre for Evidence Based Medicine guidelines were used to determine levels of evidence for each outcome. Gait-specific NMES research protocols and trends are described, with implications for future research. Results: Eighteen studies met inclusion criteria, reporting on 212 participants, 162 of whom received NMES while walking, average age of 9.8 years, GMFCS levels I-III. Studies included 4 randomized control trials, 9 cohort studies and 5 case studies. A historical trend emerged that began with experimental multi-channel NMES device development, followed by the commercial development of single-channel devices with inertial sensor-based gait event detection to facilitate ankle dorsiflexion in swing phase. This research reported strong evidence demonstrating improved ankle dorsiflexion kinematics in swing and at initial contact. Improved walking speed, step length, and muscle volume were also reported. However, improvements in global walking scores were not consistently found, motivating a recent return to investigating multi-channel gait-specific NMES applications. Conclusions: Research on single-channel gait-specific NMES found that it improved ankle motion in swing but was insufficient to address more complex gait abnormalities common in CP, such as flexed-knee and stiff-knee gait. Early evidence indicates that multi-channel gait-specific NMES may improve gait patterns in CP, however significantly more research is needed. The conclusions of this review are highly limited by the low level of evidence of the studies available. This review provides a historical record of past work and a technical context, with implications for future research on gait-specific NMES to improve walking patterns and mobility in CP.

Effect of trabecular metal on the elution of gentamicin from Palacos cement.

Periprosthetic joint infections continues to be a common complication in total joint arthroplasty, resulting in significant morbidity, mortality, and additional cost. Trabecular metal implants with an internal cemented interface may be customizable drug delivery devices with an ingrowth interface. Thirty-six acetabular implants were assembled in vitro, half with a trabecular metal shell and half without. The antibiotic loaded bone cement was prepared via three different mixing techniques and at two different mixing times. Mixing time had a significant effect on the total amount of gentamicin eluted. The long mixing protocol eluted up to 126% (p = 0.001) more gentamicin than the short mixing protocol at 4 h and 192% (p < 0.001) more at 7 days. Hand or mechanical mixing technique had no significant effect on elution at 4 h. At 7 days, the mechanical mixing system under vacuum eluted over 50% (p = 0.031) more gentamicin than without a vacuum and nearly 60% (p = 0.040) more gentamicin than hand mixing. The use of a trabecular metal shell had no significant effect on the bulk elution of gentamicin at 4 h (p > 0.05) but significantly reduced total gentamicin elution under certain mixing protocols at 7 days. A possible optimization strategy to improve elution kinetics would be to use a long mixing time with a mechanical mixing system under vacuum. The establishment of trabecular metal as an effective delivery vehicle for antibiotics makes possible an entirely new class of drug eluting device designs. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Periprosthetic bacterial biofilm and quorum sensing.

Periprosthetic joint infection (PJI) is a common complication after total joint arthroplasty leading to severe morbidity and mortality. With an aging population and increasing prevalence of total joint replacement procedures, the burden of PJI will be felt not only by individual patients, but in increased healthcare costs. Current treatment of PJI is inadequate resulting in incredibly high failure rates. This is believed to be largely mediated by the presence of bacterial biofilms. These polymicrobial bacterial colonies form within secreted extracellular matrices, adhering to the implant surface and local tissue. The biofilm architecture is believed to play a complex and critical role in a variety of bacterial processes including nutrient supplementation, metabolism, waste management, and antibiotic and immune resistance. The establishment of these biofilms relies heavily on the quorum sensing communication systems utilized by bacteria. Early stage research into disrupting bacterial communication by targeting quorum sensing show promise for future clinical applications. However, prevention of the biofilm formation via early forced induction of the biofilm forming process remains yet unexplored. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2331-2339, 2018.