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AIMS: We present a dynamic typodont biofilm model (DTBM) incorporating (1) human dentition anatomy, (2) fluid flow over intermittently fluid bathed tooth surfaces and (3) an oxic headspace to allow aerobic and anaerobic niches to develop naturally, as a screening tool to assess the effect of stannous fluoride (SnF2 ) toothpaste against a simulated human plaque biofilm (SPB). METHODS AND RESULTS: First, hydroxyapatite (HA) coupons were inoculated with human saliva/plaque and cultured at 37°C under air. Selected species representative of common commensal and anaerobic pathogens were quantified for relative abundance changes over 4 days by PCR densitometry to confirm the culture conditions allowed the proliferation of these species. A continuous culture DTBM reactor on a rocker table was inoculated with saliva/plaque and incubated at 37°C for 24 h. Tooth shear stress was estimated by particle tracking. A SnF2 toothpaste solution, or a sham rise was administered twice daily for 3 days to mimic routine oral hygiene. SPB biomass was assessed by total bacterial DNA and methylene blue (MB) staining. Early colonizer aerobes and late colonizer anaerobes species were detected in the HA and DTBM, and the trends in changing abundance were consistent with those seen clinically. CONCLUSIONS: Treatment with the SnF2 solution showed significant reductions of 53.05% and 54.4% in the SPB by MB staining and DNA, respectively. SIGNIFICANCE AND IMPACT OF STUDY: The model has potential for assessing dentition anatomy and fluid flow on the efficacy of antimicrobial efficacy against localized SPB and may be amenable to the plaque index clinical evaluation.
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Fluoruros de Estaño , Pastas de Dientes , Biopelículas , Humanos , Saliva , Fluoruros de Estaño/uso terapéutico , Pastas de Dientes/farmacología , Pastas de Dientes/uso terapéuticoRESUMEN
Pseudomonas aeruginosa is an opportunistic bacterial pathogen and is known to produce biofilms. We previously showed the emergence of colony variants in the presence of tobramycin-loaded calcium sulfate beads. In this study, we characterized the variant colonies, which survived the antibiotic treatment, and identified three distinct phenotypes-classically resistant colonies, viable but nonculturable colonies (VBNC), and phoenix colonies. Phoenix colonies, described here for the first time, grow out of the zone of clearance of antibiotic-loaded beads from lawn biofilms while there are still very high concentrations of antibiotic present, suggesting an antibiotic-resistant phenotype. However, upon subculturing of these isolates, phoenix colonies return to wild-type levels of antibiotic susceptibility. Compared with the wild type, phoenix colonies are morphologically similar aside from a deficiency in green pigmentation. Phoenix colonies do not recapitulate the phenotype of any previously described mechanisms of resistance, tolerance, or persistence and, thus, form a novel group with their own phenotype. Growth under anaerobic conditions suggests that an alternative metabolism could lead to the formation of phoenix colonies. These findings suggest that phoenix colonies could emerge in response to antibiotic therapies and lead to recurrent or persistent infections, particularly within biofilms where microaerobic or anaerobic environments are present.
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Infecciones por Pseudomonas , Pseudomonas aeruginosa , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Biopelículas , Farmacorresistencia Bacteriana/genética , Humanos , Pseudomonas aeruginosa/genética , Tobramicina/farmacologíaRESUMEN
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.
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Equipo Ortopédico/microbiología , Resistencia al Corte , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/fisiología , Estrés Mecánico , Líquido Sinovial/microbiologíaRESUMEN
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.
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Reconstrucción del Ligamento Cruzado Anterior/métodos , ADN Bacteriano/análisis , Fémur/microbiología , Fémur/cirugía , Tibia/microbiología , Tibia/cirugía , Adolescente , Adulto , Lesiones del Ligamento Cruzado Anterior/cirugía , Femenino , Humanos , Masculino , Reoperación , Trasplante Autólogo , Trasplante Homólogo , Adulto JovenRESUMEN
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.
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Lesiones del Ligamento Cruzado Anterior/cirugía , Reconstrucción del Ligamento Cruzado Anterior/efectos adversos , Bacterias/metabolismo , ADN Bacteriano/análisis , ADN/análisis , Infección de la Herida Quirúrgica/microbiología , Líquido Sinovial/química , Adulto , Bacterias/genética , Biopelículas , Femenino , Humanos , Masculino , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Reoperación , Infección de la Herida Quirúrgica/metabolismo , Líquido Sinovial/microbiologíaRESUMEN
Chromobacterium violaceum is an opportunistic pathogen that causes infections that are difficult to treat. The goal of this research was to evaluate the effect of selected tannins (tannic acid (TA) and gallic acid (GA)) on bacterial growth, motility, antibiotic (carbenicillin, tetracycline) susceptibility, and biofilm formation. Both tannins, particularly TA, impaired bacterial growth levels and swimming motilities at sub-minimum inhibitory concentrations (sub-MICs). In combination with tannins, antibiotics showed increased MICs, suggesting that tannins interfered with antibacterial activity. Sub-MICs of tetracycline or TA alone enhanced biofilm formation of C. violaceum; however, in combination, these compounds inhibited biofilm formation. In contrast, carbenicillin at sub-MICs was effective in inhibiting C. violaceum biofilm formation; however, in combination with lower concentrations of TA or GA, biofilms were enhanced. These results provide insights into the effects of tannins on C. violaceum growth and their varying interaction with antibiotics used to target C. violaceum infections.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Carbenicilina/farmacología , Chromobacterium/efectos de los fármacos , Ácido Gálico/farmacología , Taninos/farmacología , Tetraciclina/farmacología , Biopelículas/crecimiento & desarrollo , Chromobacterium/citología , Chromobacterium/crecimiento & desarrollo , Sinergismo Farmacológico , Pruebas de Sensibilidad MicrobianaAsunto(s)
Biopelículas , Staphylococcus , ADN , Infecciones Estafilocócicas , Staphylococcus epidermidisRESUMEN
A tropical marine bacterium isolated from the hard coral, Symphyllia sp. was identified as Serratia marcescens on the basis of morphological, biochemical and 16S rDNA analysis. The bacterium showed antimicrobial activity towards the pathogens Candida albicans and Pseudomonas aeruginosa and the marine biofouling bacterium Bacillus pumilus. S. marcescens displayed biosurfactant activity as evidenced by drop collapse, blood hemolysis and surface tension reduction (52.0-27 mN m(-1)). The active compound was purified by solvent extraction and silicic acid chromatography. Characterization was by thin layer chromatography, gas chromatography mass spectroscopy (GC-MS), Fourier transform infrared (FTIR) spectroscopy and (1)H as well as (13)C nuclear magnetic resonance (NMR) analysis. The surfactant was found to be a glycolipid composed of glucose and palmitic acid. The glycolipid prevented adhesion of C. albicans BH, P. aeruginosa PAO1 and B. pumilus TiO1. The glycolipid also disrupted preformed biofilms of these cultures in microtitre plates. Confocal laser scanning microscopy and electron microscopy confirmed the effective removal of biofilms from glass surfaces. The glycolipid derived from S. marcescens could thus serve as a potential anti-biofilm agent.
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Organismos Acuáticos/metabolismo , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Glucolípidos/farmacología , Serratia marcescens/metabolismo , Tensoactivos/farmacología , Organismos Acuáticos/aislamiento & purificación , Bacillus/efectos de los fármacos , Secuencia de Bases , Candida albicans/efectos de los fármacos , Glucolípidos/química , Glucolípidos/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Serratia marcescens/aislamiento & purificación , Tensoactivos/química , Tensoactivos/aislamiento & purificaciónRESUMEN
Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.
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Antiinfecciosos/metabolismo , Bacterias/crecimiento & desarrollo , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Hongos/crecimiento & desarrollo , Percepción de Quorum/efectos de los fármacos , Agua de Mar/microbiología , Tensoactivos/metabolismo , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Incrustaciones Biológicas , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Hongos/efectos de los fármacos , Interacciones Microbianas , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Clima TropicalRESUMEN
This study investigated the efficacy of a biphasic synthetic ß-tricalcium phosphate/calcium sulfate (ß-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded ß-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. ß-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded ß-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of ß-TCP/CS in the management of orthopaedic infections.
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Antibacterianos , Biopelículas/efectos de los fármacos , Fosfatos de Calcio , Sulfato de Calcio , Portadores de Fármacos , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/fisiología , Antibacterianos/química , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Fosfatos de Calcio/química , Fosfatos de Calcio/farmacología , Sulfato de Calcio/química , Sulfato de Calcio/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacologíaRESUMEN
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 3â¯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).
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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.
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Quorum sensing (QS) has received significant attention in the past few decades. QS describes population density dependent cell to cell communication in bacteria using diffusible signal molecules. These signal molecules produced by bacterial cells, regulate various physiological processes important for social behavior and pathogenesis. One such process regulated by quorum sensing molecules is the production of a biosurfactant, rhamnolipid. Rhamnolipids are important microbially derived surface active agents produced by Pseudomonas spp. under the control of two interrelated quorum sensing systems; namely las and rhl. Rhamnolipids possess antibacterial, antifungal and antiviral properties. They are important in motility, cell to cell interactions, cellular differentiation and formation of water channels that are characteristics of Pseudomonas biofilms. Rhamnolipids have biotechnological applications in the uptake of hydrophobic substrates, bioremediation of contaminated soils and polluted waters. Rhamnolipid biosurfactants are biodegradable as compared to chemical surfactants and hence are more preferred in environmental applications. In this review, we examine the biochemical and genetic mechanism of rhamnolipid production by P. aeruginosa and propose the application of QS signal molecules in enhancing the rhamnolipid production.
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Biopelículas , Glucolípidos/biosíntesis , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum , Biodegradación Ambiental , Comunicación Celular , Pseudomonas aeruginosa/genética , Transducción de Señal , Tensoactivos/metabolismoRESUMEN
Electrochemically generated bactericidal compounds have been shown to eradicate bacterial lawn biofilms through electroceutical treatment. However, the ultrastructure of biofilms exposed to these species has not been studied. Moreover, it is unknown if the efficacy of electroceutical treatment extends to antibiotic-resistant variants that emerge in lawn biofilms after antibiotic treatment. In this report, the efficacy of the in vitro electroceutical treatment of Pseudomonas aeruginosa biofilms is demonstrated both at room temperature and in an incubator, with a ~4 log decrease (p < 0.01) in the biofilm viability observed over the anode at both conditions. The ultrastructure changes in the lawn biofilms imaged using transmission electron microscopy demonstrate significant bacterial cell damage over the anode after 24 h of electroceutical treatment. A mix of both damaged and undamaged cells was observed over the cathode. Finally, both eradication and prevention of the emergence of tobramycin-resistant variants were demonstrated by combining antibiotic treatment with electroceutical treatment on the lawn biofilms.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Técnicas Electroquímicas/métodos , Pseudomonas aeruginosa/ultraestructura , Tobramicina/farmacología , Farmacorresistencia Bacteriana , Electrodos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Transmisión , Pseudomonas aeruginosa/fisiologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0231791.].
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Periprosthetic joint infections (PJIs) are a devastating complication that occurs in 2% of patients following joint replacement. These infections are costly and difficult to treat, often requiring multiple corrective surgeries and prolonged antimicrobial treatments. The Gram-positive bacterium Staphylococcus aureus is one of the most common causes of PJIs, and it is often resistant to a number of commonly used antimicrobials. This tolerance can be partially attributed to the ability of S. aureus to form biofilms. Biofilms associated with the surface of indwelling medical devices have been observed on components removed during chronic infection, however, the development and localization of biofilms during PJIs remains unclear. Prior studies have demonstrated that synovial fluid, in the joint cavity, promotes the development of bacterial aggregates with many biofilm-like properties, including antibiotic resistance. We anticipate these aggregates have an important role in biofilm formation and antibiotic tolerance during PJIs. Therefore, we sought to determine specifically how synovial fluid promotes aggregate formation and the impact of this process on surface attachment. Using flow cytometry and microscopy, we quantified the aggregation of various clinical S. aureus strains following exposure to purified synovial fluid components. We determined that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. To determine how synovial fluid mediated aggregation affects surface attachment, we utilized microscopy to measure bacterial attachment. Surprisingly, we found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. We conclude from this study that fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI. Collectively, we propose that synovial fluid may have conflicting protective roles for the host by preventing adhesion to surfaces, but by promoting bacterial aggregation is also contributing to the development of antibiotic tolerance.
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Adhesión Bacteriana/fisiología , Biopelículas/crecimiento & desarrollo , Staphylococcus aureus/fisiología , Líquido Sinovial/microbiología , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Fibrinógeno/farmacología , Fibronectinas/farmacología , Humanos , Staphylococcus aureus/efectos de los fármacos , Líquido Sinovial/efectos de los fármacos , Factores de TiempoRESUMEN
Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE) have emerged as multidrug-resistant (MDR) pathogens associated with periprosthetic joint infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ALCSB) in inhibiting bacterial growth, encouraging biofilm formation and killing preformed biofilms of CRE and VRE. Three strains of Klebsiella pneumoniae (KP) and a strain of Enterococcus faecalis (EF) were used. ALCSB of 4.8-mm diameter were loaded with vancomycin (V) and gentamicin (G), V and rifampicin (R), V and tobramycin (T) or R and meropenem (M), and placed onto tryptic soy agar (TSA), spread with one of the test strains and incubated for 24 h at 37 °C. Beads were transferred daily onto fresh TSA spread plates and the zone of inhibition (ZOI) was recorded until no inhibition was observed. ALCSB containing R + M or R + V produced the most extensive ZOI up to 5 weeks. Biofilm prevention efficacy was investigated by challenging ALCSB daily with 5 × 105 CFU/mL bacterial cells and analyzing for biofilm formation at challenges 1, 2 and 3. In the biofilm killing experiments, ALCSB were added to pre-grown 3-day biofilms of KP and EF strains, which were then analyzed at days 1 and 3 post-exposure. The CFU counts and confocal images of the attached cells showed that ALCSB treatment reduced colonization and biofilm formation significantly (5-7 logs) with combinations of R + M or R + V, compared to unloaded beads. This study provides evidence that the local release of antibiotics from ALCSB may be useful in treating the biofilms of multidrug-resistant strains of CRE and VRE.
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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.
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Bacterias/aislamiento & purificación , Fenómenos Fisiológicos Bacterianos , Biopelículas/crecimiento & desarrollo , Técnicas Microbiológicas/métodos , Ortopedia/métodos , Prótesis e Implantes/microbiología , Infecciones Relacionadas con Prótesis/microbiología , Adulto , Agar , Anciano , Anciano de 80 o más Años , Remoción de Dispositivos , Femenino , Humanos , Masculino , Infecciones Relacionadas con Prótesis/diagnóstico , Reproducibilidad de los ResultadosRESUMEN
Electroceutical wound dressings, especially those involving current flow with silver based electrodes, show promise for treating biofilm infections. However, their mechanism of action is poorly understood. We have developed an in vitro agar based model using a bioluminescent strain of Pseudomonas aeruginosa to measure loss of activity and killing when direct current was applied. Silver electrodes were overlaid with agar and lawn biofilms grown for 24 h. A 6 V battery with 1 kΩ ballast resistor was used to treat the biofilms for 1 h or 24 h. Loss of bioluminescence and a 4-log reduction in viable cells was achieved over the anode. Scanning electron microscopy showed damaged cells and disrupted biofilm architecture. The antimicrobial activity continued to spread from the anode for at least 2 days, even after turning off the current. Based on possible electrochemical ractions of silver electrodes in chlorine containing medium; pH measurements of the medium post treatment; the time delay between initiation of treatment and observed bactericidal effects; and the presence of chlorotyrosine in the cell lysates, hypochlorous acid is hypothesized to be the chemical agent responsible for the observed (destruction/killing/eradication) of these biofilm forming bacteria. Similar killing was obtained with gels containing only bovine synovial fluid or human serum. These results suggest that our in vitro model could serve as a platform for fundamental studies to explore the effects of electrochemical treatment on biofilms, complementing clinical studies with electroceutical dressings.
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Biopelículas/crecimiento & desarrollo , Electricidad , Pseudomonas aeruginosa/fisiología , Animales , Vendajes/microbiología , Bovinos , Electrodos , Concentración de Iones de Hidrógeno , Líquido Sinovial/microbiologíaRESUMEN
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.