Background. Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against ...experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection. Methods. In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined. Results. In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation). Conclusions. Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.
The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with ...experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.
Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached ...adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties--as well as elastin binding--and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by >=10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.
The HtrA surface protease is involved in the virulence of many pathogens, mainly by its role in stress resistance and bacterial survival. Staphylococcus aureus encodes two putative HtrA-like ...proteases, referred to as HtrA₁ and HtrA₂. To investigate the roles of HtrA proteins in S. aureus, we constructed htrA₁, htrA₂, and htrA₁ htrA₂ insertion mutants in two genetically different virulent strains, RN6390 and COL. In the RN6390 context, htrA₁ inactivation resulted in sensitivity to puromycin-induced stress. The RN6390 htrA₁ htrA₂ mutant was affected in the expression of several secreted virulence factors comprising the agr regulon. This observation was correlated with the disappearance of the agr RNA III transcript in the RN6390 htrA₁ htrA₂ mutant. The virulence of this mutant was diminished in a rat model of endocarditis. In the COL context, both HtrA₁ and HtrA₂ were essential for thermal stress survival. However, only HtrA₁ had a slight effect on exoprotein expression. The htrA mutations did not diminish the virulence of the COL strain in the rat model of endocarditis. Our results indicate that HtrA proteins have different roles in S. aureus according to the strain, probably depending on specific differences in the regulation of virulence factor and stress protein expression. We propose that HtrA₁ and HtrA₂ contribute to pathogenicity by controlling the production of certain extracellular factors that are crucial for bacterial dissemination, as revealed in the RN6390 background. We speculate that HtrA proteins act in the agr-dependent regulation pathway by assuring folding and/or maturation of some surface components of the agr system.
Candida spp. are able to survive on hospital surfaces and causes healthcare-associated infections (HCAIs). Since surface cleaning and disinfecting interventions are not totally effective to eliminate ...Candida spp., new approaches should be devised. Copper (Cu) has widely recognized antifungal activity and the use of Cu-sputtered surfaces has recently been proposed to curb the spread of HCAIs. Moreover, the activity of Cu under the action of actinic light remains underexplored. We investigated the antifungal activity of Cu-sputtered polyester surfaces (Cu-PES) against azole-resistant Candida albicans and Candida glabrata under dark and low intensity visible light irradiation (4.65mW/cm2). The surface properties of Cu-PES photocatalysts were characterized by diffuse reflectance spectroscopy (DRS) and X-ray fluorescence (XRF). Under dark, Cu-PES showed a fungicidal activity (≥3log10CFU reduction of the initial inoculum) against both C. albicans DSY296 and C. glabrata DSY565 leading to a reduction of the starting inoculum of 3.1 and 3.0log10CFU, respectively, within 60min of exposure. Under low intensity visible light irradiation, Cu-PES exhibited an accelerated fungicidal activity against both strains with a reduction of 3.0 and 3.4log10CFU, respectively, within 30min of exposure. This effect was likely due to the semiconductor Cu2O/CuO charge separation. The decrease in cell viability of the two Candida strains under dark and light conditions correlated with the progressive loss of membrane integrity. These results indicate that Cu-PES represent a promising strategy for decreasing the colonization of surfaces by yeasts and that actinic light can improve its self-disinfecting activity.
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•Candida spp. can survive on hospital surfaces and are important causes of infections.•Copper-sputtered polyesters (Cu-PES) showed a rapid fungicidal activity under dark.•The antifungal activity of Cu-PES was faster upon visible light irradiation.•The yeast envelop was damaged after Cu-Pes exposure.•Self-disinfecting Cu-sputtered surfaces could be implemented in healthcare facilities.
Background. Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pill, pil2, ...and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pill locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C(Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. Methods. S. gallolyticus UCN34, Pill⁺ and Pill ⁻, expressing various levels of pill, and recombinant Lactococcus lactis strains, constitutively expressing pill, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pill was tested in a rat model of endocarditis. Results. We showed that the pill locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-exp ressing pill. We demonstrated that Pill pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. Conclusions. This study identifies Pill as the first virulence factor characterized in S. gallolyticus.
In this study, silver/copper (Ag/Cu)-coated catheters were investigated for their efficacy in preventing methicillin-resistant Staphylococcus aureus (MRSA) infection in vitro and in vivo Ag and Cu ...were sputtered (67/33% atomic ratio) on polyurethane catheters by direct-current magnetron sputtering. In vitro, Ag/Cu-coated and uncoated catheters were immersed in phosphate-buffered saline (PBS) or rat plasma and exposed to MRSA ATCC 43300 at 10(4) to 10(8) CFU/ml. In vivo, Ag/Cu-coated and uncoated catheters were placed in the jugular vein of rats. Directly after, MRSA (10(7) CFU/ml) was inoculated in the tail vein. Catheters were removed 48 h later and cultured. In vitro, Ag/Cu-coated catheters preincubated in PBS and exposed to 10(4) to 10(7) CFU/ml prevented the adherence of MRSA (0 to 12% colonization) compared to uncoated catheters (50 to 100% colonization; P < 0.005) and Ag/Cu-coated catheters retained their activity (0 to 20% colonization) when preincubated in rat plasma, whereas colonization of uncoated catheters increased (83 to 100%; P < 0.005). Ag/Cu-coating protection diminished with 10(8) CFU/ml in both PBS and plasma (50 to 100% colonization). In vivo, Ag/Cu-coated catheters reduced the incidence of catheter infection compared to uncoated catheters (57% versus 79%, respectively; P = 0.16) and bacteremia (31% versus 68%, respectively; P < 0.05). Scanning electron microscopy of explanted catheters suggests that the suboptimal activity of Ag/Cu catheters in vivo was due to the formation of a dense fibrin sheath over their surface. Ag/Cu-coated catheters thus may be able to prevent MRSA infections. Their activity might be improved by limiting plasma protein adsorption on their surfaces.
Background The potential of phage therapy for the treatment of endovascular
infections remains to be evaluated. Methods and Results The efficacy of a phage cocktail combining
phage vB_SauH_2002 and
...phage 66 was evaluated against a methicillin-sensitive
strain in vitro and in vivo in a rodent model of experimental endocarditis. Six hours after bacterial challenge, animals were treated with (1) the phage cocktail. (2) subtherapeutic flucloxacillin dosage, (3) combination of the phage cocktail and flucloxacillin, or (4) saline. Bacterial loads in cardiac vegetations at 30 hours were the primary outcome. Secondary outcomes were phage loads at 30 hours in cardiac vegetations, blood, spleen, liver, and kidneys. We evaluated phage resistance 30 hours post infection in vegetations of rats under combination treatment. In vitro, phages synergized against
planktonic cells and the cocktail synergized with flucloxacillin to eradicated biofilms. In infected animals, the phage cocktail achieved bacteriostatic effect. The addition of low-dose flucloxacillin elevated bacterial suppression (∆ of -5.25 log
colony forming unit/g CFU/g versus treatment onset,
<0.0001) and synergism was confirmed (∆ of -2.15 log
CFU/g versus low-dose flucloxacillin alone,
<0.01). Importantly, 9/12 rats given the combination treatment had sterile vegetations at 30 hours. In vivo phage replication was partially suppressed by the antibiotic and selection of resistance to the
component of the phage cocktail occurred. Plasma-mediated inhibition of phage killing activity was observed in vitro. Conclusions Combining phages with a low-dose standard of care antibiotic represents a promising strategy for the treatment of
infective endocarditis.
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