Robert P. Howlin

Antibiotic-Loaded Synthetic Calcium Sulfate Beads for Prevention of Bacterial Colonization and Biofilm Formation in Periprosthetic Infections

ABSTRACT Periprosthetic infection (PI) causes significant morbidity and mortality after fixation and joint arthroplasty and has been extensively linked to the formation of bacterial biofilms. Poly(methyl methacrylate) (PMMA), as a cement or as beads, is commonly used for antibiotic release to the site of infection but displays variable elution kinetics and also represents a potential nidus for infection, therefore requiring surgical removal once antibiotics have eluted. Absorbable cements have shown improved elution of a wider range of antibiotics and, crucially, complete biodegradation, but limited data exist as to their antimicrobial and antibiofilm efficacy. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin, or vancomycin-tobramycin dual treatment (in a 1:0.24 [wt/wt] ratio) were assessed for their abilities to eradicate planktonic methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis relative to that of PMMA beads. The ability of the calcium sulfate beads to prevent biofilm formation over multiple days and to eradicate preformed biofilms was studied using a combination of viable cell counts, confocal microscopy, and scanning electron microscopy of the bead surface. Biofilm bacteria displayed a greater tolerance to the antibiotics than their planktonic counterparts. Antibiotic-loaded beads were able to kill planktonic cultures of 106 CFU/ml, prevent bacterial colonization, and significantly reduce biofilm formation over multiple days. However, established biofilms were harder to eradicate. These data further demonstrate the difficulty in clearing established biofilms; therefore, early preventive measures are key to reducing the risk of PI. Synthetic calcium sulfate loaded with antibiotics has the potential to reduce or eliminate biofilm formation on adjacent periprosthetic tissue and prosthesis material and, thus, to reduce the rates of periprosthetic infection.

Targeting microbial biofilms: current and prospective therapeutic strategies

Biofilm formation is a key virulence factor for a wide range of microorganisms that cause chronic infections. The multifactorial nature of biofilm development and drug tolerance imposes great challenges for the use of conventional antimicrobials and indicates the need for multi-targeted or combinatorial therapies. In this Review, we focus on current therapeutic strategies and those under development that target vital structural and functional traits of microbial biofilms and drug tolerance mechanisms, including the extracellular matrix and dormant cells. We emphasize strategies that are supported by in vivo or ex vivo studies, highlight emerging biofilm-targeting technologies and provide a rationale for multi-targeted therapies aimed at disrupting the complex biofilm microenvironment.

Extracellular DNA Impedes the Transport of Vancomycin in Staphylococcus epidermidis Biofilms Preexposed to Subinhibitory Concentrations of Vancomycin

ABSTRACT Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular d-Ala-d-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

Comparing PMMA and calcium sulfate as carriers for the local delivery of antibiotics to infected surgical sites.

Antibiotic-loaded bone cement is a primary option for treatment of orthopedic infections. Poly(methyl methacrylate) (PMMA) is a widely used cement that, when loaded with antibiotics in spacer or bead form, has been shown to reduce infection rates. However, PMMA is not resorbable and requires a second surgery for removal, while also acting as a potential foreign body for bacterial colonization. Alternatively, resorbable bone cements, such as calcium sulfate, have been proposed and present the advantage of being completely reabsorbed. It is unknown whether the antibiotic elution characteristics of absorbable bone cements are similar to PMMA. This study (1) characterized antibiotic elution from synthetic, highly purified calcium sulfate cement beads of varying sizes against pathogenic bacteria both in liquid culture and seeded on agar plates, (2) tested calcium sulfate beads against PMMA beads loaded with the same antibiotics, and (3) analyzed the structural differences between how PMMA and calcium sulfate bind to antibiotics. In every assay, the calcium sulfate beads performed as well as, or better than, the PMMA beads in inhibition of bacterial growth and elution of vancomycin in vitro with complete elution observed from calcium sulfate within three days. These data suggest that calcium sulfate, functions, as well as PMMA in the patient setting for infection control.

Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic Pseudomonas aeruginosa infection in cystic fibrosis

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases.

Removal of Dental Biofilms with an Ultrasonically Activated Water Stream

Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis. Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and biofilms exposed to the water stream alone without ultrasonic activation (P < 0.05). The water stream alone showed no statistically significant difference in removal compared with the untreated control (P = 0.24). High-speed videography demonstrated a rapid rate (151 mm2 in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a potentially effective method for biofilm removal and improved oral hygiene.

Nitric oxide-mediated dispersal and enhanced antibiotic sensitivity in pseudomonas aeruginosa biofilms from the cystic fibrosis lung

Background and aims Bacterial biofilms present a major challenge in medicine due to their recalcitrance towards antimicrobials, relative to the same bacteria in a planktonic (free living) state. In cystic fibrosis (CF), bacteria colonise the lungs, establishing chronic and persistent infections which can not be eradicated with conventional antibiotic treatments. The effective treatment of CF patients colonised with the opportunistic pathogen Pseudomonas aeruginosa (PA) present a major therapeutic challenge not adequately addressed using current conventional antibiotic regimes (eg, ceftazadime and tobramycin in combination). We have previously demonstrated that biofilm formation by PA is reversed in vitro by nanomolar, non-toxic concentrations of NO by reduction of biofilm antibiotic tolerance. The aim of this project is to investigate nitric oxide as adjunctive therapy to standard antibiotic regimes in CF. Methods Biofilms from clinical isolates of PA were cultured ex vivo from sputum obtained from 20 teenagers and young adults with CF undergoing infective exacerbation. Dispersal, using low dose concentrations of the nitric oxide donor sodium nitroprusside (SNP), was calculated using optical density measurements. The effects of tobramycin and ceftazadime alone, and in combination with SNP, were visualised by Live/Dead fluorescent staining and confocal microscopy. Results The extent of biofilm dispersal was SNP-concentration dependent and could be significantly reduced by the addition of a nitric oxide scavenger. Moreover, biofilm dispersal was accompanied by increased susceptibility of PA to tobramycin and ceftazadime. Preliminary data also demonstrate dispersal and increased antibiotic susceptibility of multi-species biofilms cultured from CF sputum. Conclusions These data demonstrate that NO-mediated dispersal can augment the antibiotic sensitivity of PA biofilms from clinical isolates and have led to an ongoing NIHR Respiratory Biomedical Unit funded proof-of-principle clinical trial to assess the clinical role of nitric oxide as adjunctive therapy in CF teenagers and young adults colonised with PA (EudraCT 2010-023529-39).

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

Abstract 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. Graphical Abstract Figure. No Caption available.

Fluid-driven Interfacial instabilities and turbulence in bacterial biofilms

Biofilms are thin layers of bacteria embedded within a slime matrix that live on surfaces. They are ubiquitous in nature and responsible for many medical and dental infections, industrial fouling and are also evident in ancient fossils. A biofilm structure is shaped by growth, detachment and response to mechanical forces acting on them. The main contribution to biofilm versatility in response to physical forces is the matrix that provides a platform for the bacteria to grow. The interaction between biofilm structure and hydrodynamics remains a fundamental question concerning biofilm dynamics. Here, we document the appearance of ripples and wrinkles in biofilms grown from three species of bacteria when subjected to high-velocity fluid flows. Linear stability analysis suggested that the ripples were Kelvin-Helmholtz Instabilities. The analysis also predicted a strong dependence of the instability formation on biofilm viscosity explaining the different surface corrugations observed. Turbulence through Kelvin-Helmholtz instabilities occurring at the interface demonstrated that the biofilm flows like a viscous liquid under high flow velocities applied within milliseconds. Biofilm fluid-like behavior may have important implications for our understanding of how fluid flow influences biofilm biology since turbulence will likely disrupt metabolite and signal gradients as well as community stratification.

Parallel Evolution in Streptococcus pneumoniae Biofilms

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.