Carlos J. Sanchez

Biofilm formation by clinical isolates and the implications in chronic infections

BackgroundBiofilm 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.MethodsThe 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.ResultsOf 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.ConclusionsThis 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.

Effects of local delivery of D-amino acids from biofilm-dispersive scaffolds on infection in contaminated rat segmental defects

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.

d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

ABSTRACT 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.

Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro

BackgroundOsteomyelitis 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).MethodsOsteoblasts 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.ResultsBCM 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.ConclusionsTogether 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.

Human plasma enhances the expression of Staphylococcal microbial surface components recognizing adhesive matrix molecules promoting biofilm formation and increases antimicrobial tolerance In Vitro.

BackgroundMicrobial 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.ResultsExposure 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.ConclusionsCollectively, 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.

Soluble Factors from Biofilms of Wound Pathogens Modulate Human Bone Marrow-derived Stromal Cell Differentiation, Migration, Angiogenesis, and Cytokine Secretion

BackgroundChronic, 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.ResultsExposure 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.ConclusionsCollectively, 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.

In Vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

Time-Dependent Effectiveness of Locally Applied Vancomycin Powder in a Contaminated Traumatic Orthopaedic Wound Model.

Objectives: To evaluate the effectiveness of locally applied vancomycin powder at different times postinfection in a contaminated traumatic animal model. Methods: This study used an established segmental defect rat femur model contaminated with Staphylococcus aureus UAMS-1 followed by treatment at 6 or 24 hours postinfection. Three treatments were evaluated: debridement and irrigation alone (control group) or in combination with either vancomycin powder or vancomycin-impregnated poly(methyl methacrylate) beads. Serum vancomycin levels were determined at scheduled time points over 14 days; bone, surrounding muscle, and implants were harvested for bacterial and inflammatory analyses. Results: Locally applied vancomycin powder and impregnated beads significantly reduced bacteria both within the bone and implant when treatment was performed at 6 hours. Delaying treatment to 24 hours significantly reduced the therapeutic efficacy of locally applied vancomycin of both groups. Serum vancomycin levels were detectable in all animals treated with vancomycin powder at 24 hours, but absorption was negligible from beads. At 14 days, vancomycin was detectable in the surrounding musculature of all animals and in serum of 20% of animals treated with vancomycin powder. Conclusions: This study suggests that vancomycin powder is a promising adjunctive therapy for preventing infection in traumatic wounds when treatment is performed early. This time-dependent effectiveness of vancomycin powder is similar to that observed with systemic and other local delivery adjuncts, which is likely attributable to biofilm formation after contamination, conferring intrinsic recalcitrance to antimicrobials.

Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

ABSTRACT Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity.

Activity of Gallium Meso- and Protoporphyrin IX against Biofilms of Multidrug-Resistant Acinetobacter baumannii Isolates

Acinetobacter baumannii is a challenging pathogen due to antimicrobial resistance and biofilm development. The role of iron in bacterial physiology has prompted the evaluation of iron-modulation as an antimicrobial strategy. The non-reducible iron analog gallium(III) nitrate, Ga(NO3)3, has been shown to inhibit A. baumannii planktonic growth; however, utilization of heme-iron by clinical isolates has been associated with development of tolerance. These observations prompted the evaluation of iron-heme sources on planktonic and biofilm growth, as well as antimicrobial activities of gallium meso- and protoporphyrin IX (Ga-MPIX and Ga-PPIX), metal heme derivatives against planktonic and biofilm bacteria of multidrug-resistant (MDR) clinical isolates of A. baumannii in vitro. Ga(NO3)3 was moderately effective at reducing planktonic bacteria (64 to 128 µM) with little activity against biofilms (≥512 µM). In contrast, Ga-MPIX and Ga-PPIX were highly active against planktonic bacteria (0.25 to 8 µM). Cytotoxic effects in human fibroblasts were observed following exposure to concentrations exceeding 128 µM of Ga-MPIX and Ga-PPIX. We observed that the gallium metal heme conjugates were more active against planktonic and biofilm bacteria, possibly due to utilization of heme-iron as demonstrated by the enhanced effects on bacterial growth and biofilm formation.