RESUMEN
BACKGROUND: Chronic, non-healing wounds are often characterized by the persistence of bacteria within biofilms - aggregations of cells encased within a self-produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from planktonic, or culture-grown, bacterial phenotype, including diminished responses to antimicrobial therapy and persistence against host immune responses. Mesenchymal stromal cells (MSCs) are host cells characterized by their multifunctional ability to undergo differentiation into multiple cell types and modulation of host-immune responses by secreting factors that promote wound healing. While these characteristics make MSCs an attractive therapeutic for wounds, these pro-healing activities may be differentially influenced in the context of an infection (i.e., biofilm related infections) within chronic wounds. Herein, we evaluated the effect of soluble factors derived from biofilms of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on the viability, differentiation, and paracrine activity of human MSCs to evaluate the influence of biofilms on MSC activity in vitro. RESULTS: Exposure of MSCs to biofilm-conditioned medias of S. aureus and P. aeruginosa resulted in reductions in cell viability, in part due to activation of apoptosis. Similarly, exposure to soluble factors from biofilms was also observed to diminish the migration ability of cells and to hinder multi-lineage differentiation of MSCs. In contrast to these findings, exposure of MSCs to soluble factors from biofilms resulted in significant increases in the release of paracrine factors involved in inflammation and wound healing. CONCLUSIONS: Collectively, these findings demonstrate that factors produced by biofilms can negatively impact the intrinsic properties of MSCs, in particular limiting the migratory and differentiation capacity of MSCs. Consequently, these studies suggest use/application of stem-cell therapies in the context of infection may have a limited therapeutic effect.
Asunto(s)
Células Madre Mesenquimatosas/efectos de los fármacos , Compuestos Orgánicos/toxicidad , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Infección de Heridas/microbiología , Biopelículas/crecimiento & desarrollo , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Citocinas/metabolismo , Humanos , Células Madre Mesenquimatosas/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Compuestos Orgánicos/aislamiento & purificación , Compuestos Orgánicos/metabolismo , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/fisiologíaRESUMEN
Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of D-amino acids (D-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of D-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. D-Met, D-Phe, and D-Trp at concentrations of ≥ 5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (D-Met/D-Phe/D-Trp). When combined with D-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 µg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of D-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 µg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of D-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.
Asunto(s)
Aminoácidos/farmacología , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Tensoactivos/farmacología , Aminoglicósidos/farmacología , Biopelículas/crecimiento & desarrollo , Clindamicina/farmacología , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/fisiología , Vancomicina/farmacología , Heridas y Lesiones/tratamiento farmacológico , Heridas y Lesiones/microbiología , beta-Lactamas/farmacologíaRESUMEN
BACKGROUND: Macrophages are important in wound defense and healing. Dakin's solution (DS), buffered sodium hypochlorite, has been used since World War I as a topical antimicrobial for wound care. DS has been shown to be toxic to host cells, but effects on immune cells are not well documented. MATERIALS AND METHODS: DS at 0.5%, 0.125%, and ten-fold serial dilutions from 0.25%-0.00025% were evaluated for cellular toxicity on murine macrophages (J774A.1). The effect of DS on macrophage adhesion, phagocytosis, and generation of reactive oxygen species was examined. Macrophage polarization following DS exposure was determined by gene expression using quantitative real-time polymerase chain reaction. RESULTS: Concentrations of DS >0.0025% reduced macrophage viability to <5% in exposure times as short as 30 s. Similarly, phagocytosis of Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus flavus were significantly reduced at all tested concentrations by macrophages pretreated with DS. H2O2 production was reduced by 8%-38% following treatment with 0.00025%-0.125% DS. Macrophage adherence was significantly increased with >0.0025% DS after 15 min of exposure compared with controls. Quantitative real-time polymerase chain reaction demonstrated that DS exposure resulted in classical macrophage activation, with increased expression of inducible nitric oxide synthase 2, interferon-γ, and interleukin (IL)-1ß. CONCLUSIONS: DS at clinically used concentrations (0.025%-0.25%) was detrimental to macrophage survival and function. For optimal clinical use, understanding the impact of DS on macrophages is important as depletion may result in impaired pathogen clearance and delayed healing. These findings indicate that 0.00025% DS is a safe starting dose; however, optimal use of DS requires further validation with in vivo models.
Asunto(s)
Desinfectantes/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Hipoclorito de Sodio/farmacología , Infección de la Herida Quirúrgica/tratamiento farmacológico , Cicatrización de Heridas/efectos de los fármacos , Adulto , Animales , Aspergilosis/tratamiento farmacológico , Aspergilosis/inmunología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Línea Celular , Polaridad Celular/efectos de los fármacos , Polaridad Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Femenino , Humanos , Ratones , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/inmunología , Soluciones/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/inmunología , Infección de la Herida Quirúrgica/inmunología , Cicatrización de Heridas/inmunologíaRESUMEN
BACKGROUND: Biofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR) species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics. METHODS: The biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC) from 2004-2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA) (n=23), Acinetobacter baumannii (n=53), Pseudomonas aeruginosa (n=36), Klebsiella pneumoniae (n=54), and Escherichia coli (n=39), were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM). Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro. RESULTS: Of the 205 clinical isolates tested, 126 strains (61.4%) were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro. CONCLUSIONS: This study is the first to evaluate biofilm formation in a large collection of infecting clinical isolates representing diverse types of infections. Our results demonstrate: (1) biofilm formation is a heterogeneous property amongst clinical strains which is associated with certain clonal types, (2) biofilm forming strains are more frequently isolated from non-fluid tissues, in particular bone and soft tissues, (3) MDR pathogens are more often biofilm formers, and (4) strains from patients with persistent infections are positive for biofilm formation.
Asunto(s)
Infecciones Bacterianas/microbiología , Biopelículas , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/fisiología , Antibacterianos/farmacología , Enfermedad Crónica , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/aislamiento & purificación , Humanos , RecurrenciaRESUMEN
BACKGROUND: Osteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L). METHODS: Osteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR. RESULTS: BCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls. CONCLUSIONS: Together these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Resorción Ósea/microbiología , Osteoblastos/microbiología , Osteoclastos/microbiología , Osteogénesis/fisiología , Staphylococcus aureus/fisiología , Fosfatasa Alcalina/metabolismo , Apoptosis , Biomarcadores/metabolismo , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Interacciones Huésped-Patógeno , Humanos , Microscopía Electrónica de Rastreo , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patología , Staphylococcus aureus/ultraestructuraRESUMEN
The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.
Asunto(s)
Candida albicans/genética , Candida albicans/patogenicidad , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Esteroles/metabolismo , Animales , Antifúngicos , Resistencia a Múltiples Medicamentos/genética , Regulación Fúngica de la Expresión Génica , Metabolismo de los Lípidos/genética , Ratones , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Microbial biofilms have been associated with the development of chronic human infections and represent a clinical challenge given their increased antimicrobial tolerance. Staphylococcus aureus is a major human pathogen causing a diverse range of diseases, of which biofilms are often involved. Staphylococcal attachment and the formation of biofilms have been shown to be facilitated by host factors that accumulate on surfaces. To better understand how host factors enhance staphylococcal biofilm formation, we evaluated the effect of whole human plasma on biofilm formation in clinical isolates of S. aureus and the expression of seven microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) known to be involved in biofilm formation by quantitative real-time PCR. We also evaluated whether plasma augmented changes in S. aureus biofilm morphology and antimicrobial resistance. RESULTS: Exposure of clinical isolates of S. aureus to human plasma (10%) within media, and to a lesser extent when coated onto plates, significantly enhanced biofilm formation in all of the clinical isolates tested. Compared to biofilms grown under non-supplemented conditions, plasma-augmented biofilms displayed significant changes in both the biofilm phenotype and cell morphology as determined by confocal scanning laser microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Exposure of bacteria to plasma resulted in a significant fold-increase in MSCRAMM expression in both a time and isolate-dependent manner. Additionally, plasma-augmented biofilms displayed an increased tolerance to vancomycin compared to biofilms grown in non-supplemented media. CONCLUSIONS: Collectively, these studies support previous findings demonstrating a role for host factors in biofilm formation and provide further insight into how plasma, a preferred growth medium for staphylococcal biofilm formation enhances as well as augments other intrinsic properties of S. aureus biofilms. Consequently, these findings indicate that incorporation of host factors may be necessary to better replicate in vivo conditions and for the best utility of a clinical biofilm assay to evaluate the process of biofilm formation and treatments.
Asunto(s)
Biopelículas/efectos de los fármacos , Medios de Cultivo/farmacología , Plasma/química , Staphylococcus aureus/efectos de los fármacos , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Medios de Cultivo/química , Expresión Génica , Humanos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/ultraestructura , Vancomicina/farmacología , Resistencia a la VancomicinaRESUMEN
Infectious complications of open fractures continue to be a significant factor contributing to non-osseous union and extremity amputation. The persistence of bacteria within biofilms despite meticulous debridement and antibiotic therapy is believed to be a major cause of chronic infection. Considering the difficulties in treating biofilm-associated infections, the use of biofilm dispersal agents as a therapeutic strategy for the prevention of biofilm-associated infections has gained considerable interest. In this study, we investigated whether local delivery of D-Amino Acids (D-AAs), a biofilm dispersal agent, protects scaffolds from contamination and reduces microbial burden within contaminated rat segmental defects in vivo. In vitro testing on biofilms of clinical isolates of Staphylococcus aureus demonstrated that D-Met, D-Phe, D-Pro, and D-Trp were highly effective at dispersing and preventing biofilm formation individually, and the effect was enhanced for an equimolar mixture of D-AAs. Incorporation of D-AAs into polyurethane scaffolds as a mixture (1:1:1 D-Met:D-Pro:D-Trp) significantly reduced bacterial contamination on the scaffold surface in vitro and within bone when implanted into contaminated femoral segmental defects. Our results underscore the potential of local delivery of d-AAs for reducing bacterial contamination by targeting bacteria within biofilms, which may represent a treatment strategy for improving healing outcomes associated with open fractures.
Asunto(s)
Aminoácidos/farmacología , Biopelículas/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Fémur/patología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/fisiología , Andamios del Tejido/microbiología , Animales , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Fémur/efectos de los fármacos , Fémur/microbiología , Humanos , Poliuretanos/farmacología , Ratas , Infecciones Estafilocócicas/patología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/ultraestructuraRESUMEN
Implant associated osteomyelitis (OM) is difficult to treat with antibiotics, and outcomes remain poor. Some reports suggest that hyperbaric oxygen treatment is a safe and effective means of treating OM. We tested this hypothesis in a murine model. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Klebsiella pneumoniae were used. The mice were infected with each of the three pathogens, treated with 100% oxygen at high pressure, hyperbaric oxygen (HBO), and monitored for the ability of HBO to prevent and/or clear the OM infection. Assessments included bacterial burden of the tibias and lesion scores, as well as receptor activator of NF-κB ligand (RANKL) and myeloperoxidase (MPO) concentrations. HBO resulted in more severe lesion scores and higher RANKL and MPO concentrations for MRSA. A significant positive correlation was found between RANKL concentration and lesion score. No significant difference was found with HBO in P. aeruginosa infections and K. pneumoniae seems to either not infect bone well or get cleared before establishing an infection. The model is useful for studying OM infections caused by MRSA and P. aeruginosa, but HBO does not appear to be an efficacious treatment of an implant-associated OM infection.