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The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56-25 μg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract's lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies.

Widespread antibiotic resistance is on the rise and current therapies are becoming increasingly limited in both scope and efficacy. Methicillin-resistant Staphylococcus aureus (MRSA) represents a major contributor to this trend. Quorum sensing controlled virulence factors include secreted toxins responsible for extensive damage to host tissues and evasion of the immune system response; they are major contributors to morbidity and mortality. Investigation of botanical folk medicines for wounds and infections led us to study Schinus terebinthifolia (Brazilian Peppertree) as a potential source of virulence inhibitors. Here, we report the inhibitory activity of a flavone rich extract “430D-F5” against all S. aureus accessory gene regulator ( agr ) alleles in the absence of growth inhibition. Evidence for this activity is supported by its agr -quenching activity (IC 50 2–32 μg mL −1 ) in transcriptional reporters, direct protein outputs (α-hemolysin and δ-toxin), and an in vivo skin challenge model. Importantly, 430D-F5 was well tolerated by human keratinocytes in cell culture and mouse skin in vivo ; it also demonstrated significant reduction in dermonecrosis following skin challenge with a virulent strain of MRSA. This study provides an explanation for the anti-infective activity of peppertree remedies and yields insight into the potential utility of non-biocide virulence inhibitors in treating skin infections.

Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of single or coinfection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2-3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger than single infection throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis was not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1-7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1β, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1β has an important role in controlling bacterial replication. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses.

USA100 health care-associated MRSA isolates are highly antibiotic resistant and can cause invasive disease across all patient populations. Even though USA100 strains are some of the most frequently identified causes of infections, little is known about virulence regulation in these isolates. Our study demonstrates that the USA100 agr quorum-sensing system is important for the control of toxin and exoenzyme production and that the agr system has a key role in skin infection. In some USA100 isolates, the agr system is important for growth in the presence of low levels of antibiotics. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence and that it may make a contribution to the optimal survival of these MRSA strains in the presence of antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) infections impact all patient populations both in the community and in health care settings. Despite advances in our knowledge of MRSA virulence, little is known about the regulatory mechanisms of USA100 health care-associated MRSA isolates, which are the second most frequently identified MRSA isolates found in all infections. This work focused on the contribution of the USA100 agr type II quorum-sensing system to virulence and antibiotic resistance. From a MRSA strain collection, we selected 16 representative USA100 isolates, constructed mutants with Δ agr mutations, and characterized selected strain pairs for virulence factor expression, murine skin infection, and antibiotic resistance. For each strain pair, hemolysis and extracellular protease expression were significantly greater in the wild-type (WT) strains than in the Δ agr mutants. Similarly, mice challenged with the WT strains had larger areas of dermonecrosis and greater weight loss than those challenged with the Δ agr mutants, demonstrating that the USA100 agr system regulates virulence. Although USA100 isolates exhibit a high level of antibiotic resistance, the WT and Δ agr strain pairs showed no difference in MICs by MIC testing. However, in the presence of a sub-MIC of vancomycin, most of the USA100 Δ agr mutants exhibited slower growth than the WT isolates, and a couple of the Δ agr mutants also grew more slowly in the presence of a sub-MIC of cefoxitin. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence, and it may have a contribution to the optimal survival of these MRSA strains in the presence of antibiotics. IMPORTANCE USA100 health care-associated MRSA isolates are highly antibiotic resistant and can cause invasive disease across all patient populations. Even though USA100 strains are some of the most frequently identified causes of infections, little is known about virulence regulation in these isolates. Our study demonstrates that the USA100 agr quorum-sensing system is important for the control of toxin and exoenzyme production and that the agr system has a key role in skin infection. In some USA100 isolates, the agr system is important for growth in the presence of low levels of antibiotics. Altogether, our findings demonstrate that the USA100 agr system is a critical regulator of virulence and that it may make a contribution to the optimal survival of these MRSA strains in the presence of antibiotics.

The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56-25 Mg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract's lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies.