Daniel G. Meeker

Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs

Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as “ESKAPE pathogens” on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections.

Comparative impact of diverse regulatory loci on Staphylococcus aureus biofilm formation.

The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin‐resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur‐ and mgrA‐enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin‐sensitive strain UAMS‐1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS‐1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin‐resistant versus methicillin‐sensitive strains.

Impact of sarA and Phenol-Soluble Modulins on the Pathogenesis of Osteomyelitis in Diverse Clinical Isolates of Staphylococcus aureus

ABSTRACT We used a murine model of acute, posttraumatic osteomyelitis to evaluate the virulence of two divergent Staphylococcus aureus clinical isolates (the USA300 strain LAC and the USA200 strain UAMS-1) and their isogenic sarA mutants. The results confirmed that both strains caused comparable degrees of osteolysis and reactive new bone formation in the acute phase of osteomyelitis. Conditioned medium (CM) from stationary-phase cultures of both strains was cytotoxic to cells of established cell lines (MC3TC-E1 and RAW 264.7 cells), primary murine calvarial osteoblasts, and bone marrow-derived osteoclasts. Both the cytotoxicity of CM and the reactive changes in bone were significantly reduced in the isogenic sarA mutants. These results confirm that sarA is required for the production and/or accumulation of extracellular virulence factors that limit osteoblast and osteoclast viability and that thereby promote bone destruction and reactive bone formation during the acute phase of S. aureus osteomyelitis. Proteomic analysis confirmed the reduced accumulation of multiple extracellular proteins in the LAC and UAMS-1 sarA mutants. Included among these were the alpha class of phenol-soluble modulins (PSMs), which were previously implicated as important determinants of osteoblast cytotoxicity and bone destruction and repair processes in osteomyelitis. Mutation of the corresponding operon reduced the cytotoxicity of CM from both UAMS-1 and LAC cultures for osteoblasts and osteoclasts. It also significantly reduced both reactive bone formation and cortical bone destruction by CM from LAC cultures. However, this was not true for CM from cultures of a UAMS-1 psm α mutant, thereby suggesting the involvement of additional virulence factors in such strains that remain to be identified.

Evaluation of Antibiotics Active against Methicillin-Resistant Staphylococcus aureus Based on Activity in an Established Biofilm.

ABSTRACT We used in vitro and in vivo models of catheter-associated biofilm formation to compare the relative activity of antibiotics effective against methicillin-resistant Staphylococcus aureus (MRSA) in the specific context of an established biofilm. The results demonstrated that, under in vitro conditions, daptomycin and ceftaroline exhibited comparable activity relative to each other and greater activity than vancomycin, telavancin, oritavancin, dalbavancin, or tigecycline. This was true when assessed using established biofilms formed by the USA300 methicillin-resistant strain LAC and the USA200 methicillin-sensitive strain UAMS-1. Oxacillin exhibited greater activity against UAMS-1 than LAC, as would be expected, since LAC is an MRSA strain. However, the activity of oxacillin was less than that of daptomycin and ceftaroline even against UAMS-1. Among the lipoglycopeptides, telavancin exhibited the greatest overall activity. Specifically, telavancin exhibited greater activity than oritavancin or dalbavancin when tested against biofilms formed by LAC and was the only lipoglycopeptide capable of reducing the number of viable bacteria below the limit of detection. With biofilms formed by UAMS-1, telavancin and dalbavancin exhibited comparable activity relative to each other and greater activity than oritavancin. Importantly, ceftaroline was the only antibiotic that exhibited greater activity than vancomycin when tested in vivo in a murine model of catheter-associated biofilm formation. These results emphasize the need to consider antibiotics other than vancomycin, most notably, ceftaroline, for the treatment of biofilm-associated S. aureus infections, including by the matrix-based antibiotic delivery methods often employed for local antibiotic delivery in the treatment of these infections.

Antibiotic-loaded phosphatidylcholine inhibits staphylococcal bone infection

AIM To test antibiotic-loaded coating for efficacy in reducing bacterial biofilm and development of osteomyelitis in an orthopaedic model of implant infection. METHODS Phosphatidylcholine coatings loaded with 25% vancomycin were applied to washed and sterilized titanium wires 20 mm in length. A 10 mm segment was removed from rabbit radius (total = 9; 5 coated, 4 uncoated), and the segment was injected with 1 × 10(6) colony forming units (CFUs) of Staphylococcus aureus (UAMS-1 strain). Titanium wires were inserted through the intramedullary canal of the removed segment and into the proximal radial segment and the segment was placed back into the defect. After 7 d, limbs were removed, X-rayed, swabbed for tissue contamination. Wires were removed and processed to determine attached CFUs. Tissue was swabbed and streaked on agar plates to determine bacteriological score. RESULTS Antibiotic-loaded coatings resulted in significantly reduced biofilm formation (4.7 fold reduction in CFUs; P < 0.001) on titanium wires and reduced bacteriological score in surrounding tissue (4.0 ± 0 for uncoated, 1.25 ± 0.5 for coated; P = 0.01). Swelling and pus formation was evident in uncoated controls at the 7 d time point both visually and radiographically, but not in antibiotic-loaded coatings. CONCLUSION Active antibiotic was released from coated implants and significantly reduced signs of osteomyelitic symptoms. Implant coatings were well tolerated in bone. Further studies with additional control groups and longer time periods are warranted. Antibiotic-loaded phosphatidylcholine coatings applied at the point of care could prevent implant-associated infection in orthopaedic defects.

Regulatory Mutations Impacting Antibiotic Susceptibility in an Established Staphylococcus aureus Biofilm

ABSTRACT We previously determined the extent to which mutations of different Staphylococcus aureus regulatory loci impact biofilm formation as assessed under in vitro conditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitro and in vivo conditions using two clinical isolates of S. aureus (LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigB was found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarA was comparable to that of a mutation in sigB and greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarA and/or sigB have the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureus biofilm-associated infections.

XerC contributes to diverse forms of Staphylococcus aureus infection via agr-dependent and agr-independent pathways

ABSTRACT We demonstrate that mutation of xerC, which reportedly encodes a homologue of an Escherichia coli recombinase, limits biofilm formation in the methicillin-resistant Staphylococcus aureus strain LAC and the methicillin-sensitive strain UAMS-1. This was not due to the decreased production of the polysaccharide intracellular adhesin (PIA) in either strain because the amount of PIA was increased in a UAMS-1 xerC mutant and undetectable in both LAC and its isogenic xerC mutant. Mutation of xerC also resulted in the increased production of extracellular proteases and nucleases in both LAC and UAMS-1, and limiting the production of either class of enzymes increased biofilm formation in the isogenic xerC mutants. More importantly, the limited capacity to form a biofilm was correlated with increased antibiotic susceptibility in both strains in the context of an established biofilm in vivo. Mutation of xerC also attenuated virulence in a murine bacteremia model, as assessed on the basis of the bacterial loads in internal organs and overall lethality. It also resulted in the decreased accumulation of alpha toxin and the increased accumulation of protein A. These findings suggest that xerC may impact the functional status of agr. This was confirmed by demonstrating the reduced accumulation of RNAIII and AgrA in LAC and UAMS-1 xerC mutants. However, this cannot account for the biofilm-deficient phenotype of xerC mutants because mutation of agr did not limit biofilm formation in either strain. These results demonstrate that xerC contributes to biofilm-associated infections and acute bacteremia and that this is likely due to agr-independent and -dependent pathways, respectively.

Novel linear lipopeptide paenipeptins with potential for eradicating biofilms and sensitizing Gram-negative bacteria to rifampicin and clarithromycin

We report the structure-activity relationship analyses of 17 linear lipopeptide paenipeptin analogues. Analogues 7, 12, and 17 were more potent than the lead compound. Analogue 17 was active against carbapenem-resistant and polymyxin-resistant pathogens. This compound at 40 μg/mL resulted in 3 log and 2.6 log reductions of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, respectively, in catheter-associated biofilms in vitro. Analogue 17 showed little hemolysis at 32 μg/mL and lysed 11% of red blood cells at 64 μg/mL. Analogues 9 and 16 were nonhemolytic and retained potent P. aeruginosa-specific antimicrobial activity. These two analogues when used alone lacked activity against Acinetobacter baumannii and Klebsiella pneumoniae; however, analogue 9 and 16 at 4 μg/mL decreased the MIC of rifampicin and clarithromycin against the same pathogens from 16 to 32 μg/mL to nanomolar levels (sensitization factor: 2048-8192). Therefore, paenipeptins, alone or in combination with rifampicin or clarithromycin, are promising candidates for treating bacterial infections.

Impact of Staphylococcus aureus regulatory mutations that modulate biofilm formation in the USA300 strain LAC on virulence in a murine bacteremia model

ABSTRACT Staphylococcus aureus causes acute and chronic forms of infection, the latter often associated with formation of a biofilm. It has previously been demonstrated that mutation of atl, codY, rot, sarA, and sigB limits biofilm formation in the USA300 strain LAC while mutation of agr, fur, and mgrA has the opposite effect. Here we used a murine sepsis model to assess the impact of these same loci in acute infection. Mutation of agr, atl, and fur had no impact on virulence, while mutation of mgrA and rot increased virulence. In contrast, mutation of codY, sarA, and sigB significantly attenuated virulence. Mutation of sigB resulted in reduced accumulation of AgrA and SarA, while mutation of sarA resulted in reduced accumulation of AgrA, but this cannot account for the reduced virulence of sarA or sigB mutants because the isogenic agr mutant was not attenuated. Indeed, as assessed by accumulation of alpha toxin and protein A, all of the mutants we examined exhibited unique phenotypes by comparison to an agr mutant and to each other. Attenuation of the sarA, sigB and codY mutants was correlated with increased production of extracellular proteases and global changes in extracellular protein profiles. These results suggest that the inability to repress the production of extracellular proteases plays a key role in attenuating the virulence of S. aureus in acute as well as chronic, biofilm-associated infections, thus opening up the possibility that strategies aimed at the de-repression of protease production could be used to broad therapeutic advantage. They also suggest that the impact of codY, sarA, and sigB on protease production occurs via an agr-independent mechanism.

Biodistribution and toxicity assessment of photoactivatable antibody-conjugated, antibiotic loaded gold nanocages for the treatment of bacterial infections (Conference Presentation)

We previously explored the use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of bacterial infections. Using Staphylococcus aureus as a proof-of-principle pathogen, we confirmed that nanocages coated with polydopamine and loaded with daptomycin could be effectively targeted to bacterial cells using an antibody targeting S. aureus surface-associated protein A. We also confirmed that laser irradiation could then be used to achieve a lethal photothermal effect and localized release of the antibiotic, the synergistic effect of which was capable of eradicating viable bacteria even from a therapeutically recalcitrant biofilm. To assess the possibility that this comes at the cost of adverse side effects, we used multispectral optoacoustic tomography (MSOT) to track the biodistribution of our nanocages following intravenous administered and determined whether their administration was associated with toxic side effects. The results of our MSOT analysis confirmed that our nanocages accumulate primarily in the liver, spleen and kidney irrespective of infection status. However, in an infected animal, they also confirmed that nanocages ultimately do reach the site of infection. MSOT results were consistent with studies involving the direct analysis of these tissues, which confirmed the correlation between MSOT signals and the presence of gold nanocages. More importantly, they also demonstrated that the presence of nanocages was not associated with appreciable histopathology in the spleen, liver, kidney, lung or heart. This suggests that our use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of infection offers significant promise that would not be compromised by systemic toxicity.