Repurposing sunscreen as an antibiotic: zinc-activated avobenzone inhibits methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major healthcare concern with associated healthcare costs reaching over ${\$}$1 billion in a single year in the USA. Antibiotic resistance in S. aureus is now observed against last line of defense antibiotics, such as vancomycin, linezolid, and daptomycin. Unfortunately, high throughput drug discovery approaches to identify new antibiotics effective against MRSA have not resulted in much tangible success over the last decades. Previously, we demonstrated the feasibility of an alternative drug discovery approach, the identification of metallo-antibiotics, compounds that gain antibacterial activity only after binding to a transition metal ion and as such are unlikely to be detected in standard drug screens. We now report that avobenzone, the primary active ingredient of most sunscreens, can be activated by zinc to become a potent antibacterial compound against MRSA. Zinc-activated avobenzone (AVB-Zn) potently inhibited a series of clinical MRSA isolates [minimal inhibitory concentration (MIC): 0.62-2.5 µM], without pre-existing resistance and activity without zinc (MIC: >10 µM). AVB-Zn was also active against clinical MRSA isolates that were resistant against the commonly used zinc-salt antibiotic bacitracin. We found AVB-Zn exerted no cytotoxicity on human cell lines and primary cells. Last, we demonstrate AVB-Zn can be deployed therapeutically as lotion preparations, which showed efficacy in a mouse wound model of MRSA infection. AVB-Zn thus demonstrates Zn-activated metallo-antibiotics are a promising avenue for future drug discovery.

Acute Infection with a Tobramycin-Induced Small Colony Variant of Staphylococcus aureus Causes Increased Inflammation in the Cystic Fibrosis Rat Lung.

Cystic fibrosis (CF) disease is characterized by lifelong infections with pathogens such as Staphylococcus aureus, leading to eventual respiratory failure. Small colony variants (SCVs) of S. aureus have been linked to worse clinical outcomes for people with CF. Current studies of SCV pathology in vivo are limited, and it remains unclear whether SCVs directly impact patient outcomes or are a result of late-stage CF disease. To investigate this, we generated a stable menadione-auxotrophic SCV strain by serially passaging a CF isolate of S. aureus with tobramycin, an aminoglycoside antibiotic commonly administered for coinfecting Pseudomonas aeruginosa. This SCV was tobramycin resistant and showed increased tolerance to the anti-staphylococcal combination therapy sulfamethoxazole-trimethoprim. To better understand the dynamics of SCV infections in vivo, we infected CF rats with this strain compared with its normal colony variant (NCV). Analysis of bacterial burden at 3 days postinfection indicated that NCVs and SCVs persisted equally well in the lungs, but SCV infections ultimately led to increased weight loss and neutrophilic inflammation. Additionally, cellular and histopathological analyses showed that in CF rats, SCV infections yielded a lower macrophage response. Overall, these findings indicate that SCV infections may directly contribute to lung disease progression in people with CF.

Muc5b Contributes to Mucus Abnormality in Rat Models of Cystic Fibrosis.

Cystic fibrosis (CF) airway disease is characterized by excessive and accumulative mucus in the airways. Mucociliary clearance becomes defective as mucus secretions become hyperconcentrated and viscosity increases. The CFTR-knockout (KO) rat has been previously shown to progressively develop delayed mucociliary transport, secondary to increased viscoelasticity of airway secretions. The humanized-G551D CFTR rat model has demonstrated that abnormal mucociliary clearance and hyperviscosity is reversed by ivacaftor treatment. In this study, we sought to identify the components of mucus that changes as the rat ages to contribute to these abnormalities. We found that Muc5b concentrations, and to a lesser extent Muc5ac, in the airway were increased in the KO rat compared to WT, and that Muc5b concentration was directly related to the viscosity of the mucus. Additionally, we found that methacholine administration to the airway exacerbates these characteristics of disease in the KO, but not WT rat trachea. Lastly we determined that at 6 months of age, CF rats had mucus that was adherent to the airway epithelium, a process that is reversed by ivacaftor therapy in the hG551D rat. Overall, these data indicate that accumulation of Muc5b initiates the muco-obstructive process in the CF lung prior to infection.

Static mucus impairs bacterial clearance and allows chronic infection with Pseudomonas aeruginosa in the cystic fibrosis rat.

Cystic fibrosis (CF) airway disease is characterised by chronic Pseudomonas aeruginosa infection. Successful eradication strategies have been hampered by a poor understanding of the mechanisms underlying conversion to chronicity. The CFTR-knockout (KO) rat harbors a progressive defect in mucociliary transport and viscosity. KO rats were infected before and after the appearance of the mucus defect, using a clinical, mucoid-isolate of P. aeruginosa embedded in agarose beads. Young KO rats that were exposed to bacteria before the development of mucociliary transport defects resolved the infection and subsequent tissue damage. However, older KO rats that were infected in the presence of hyperviscous and static mucus were unable to eradicate bacteria, but instead had bacterial persistence through 28 days post-infection that was accompanied by airway mucus occlusion and lingering inflammation. Normal rats responded to infection with increased mucociliary transport to supernormal rates, which reduced the severity of a second bacterial exposure. We therefore conclude that the aberrant mucus present in the CF airway permits persistence of P. aeruginosa in the lung.

Ivacaftor partially corrects airway inflammation in a humanized G551D rat.

Animal models have been highly informative for understanding the pathogenesis and progression of cystic fibrosis (CF) lung disease. In particular, the CF rat models recently developed have addressed mechanistic causes of the airway mucus defect characteristic of CF, and how these may change when cystic fibrosis transmembrane conductance regulator (CFTR) activity is restored using new modulator therapies. We hypothesized that inflammatory changes to the airway would develop spontaneously and progressively, and that these changes would be resolved with modulator therapy. To test this, we used a humanized-CFTR rat expressing the G551D variant that responds to the CFTR modulator ivacaftor. Markers typically found in the CF lung were assessed, including neutrophil influx, small airway histopathology, and inflammatory cytokine concentration. Young hG551D rats did not express inflammatory cytokines at baseline but did upregulate these in response to inflammatory trigger. As the hG551D rats aged, histopathology worsened, accompanied by neutrophil influx into the airway and increasing concentrations of TNF-α, IL-1α, and IL-6 in the airways. Ivacaftor administration reduced concentrations of these cytokines when administered to the rats at baseline but was less effective in the rats that had also received inflammatory stimulus. Therefore, we conclude that administration of ivacaftor resulted in an incomplete resolution of inflammation when rats received an external trigger, suggesting that CFTR activation may not be enough to resolve inflammation in the lungs of patients with CF.

Development of a Broadly Protective, Self-Adjuvanting Subunit Vaccine to Prevent Infections by Pseudomonas aeruginosa.

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa can be difficult to treat due to innate and acquired antibiotic resistance and this is exacerbated by the emergence of multi-drug resistant strains. Unfortunately, no licensed vaccine yet exists to prevent Pseudomonas infections. Here we describe a novel subunit vaccine that targets the P. aeruginosa type III secretion system (T3SS). This vaccine is based on the novel antigen PaF (Pa Fusion), a fusion of the T3SS needle tip protein, PcrV, and the first of two translocator proteins, PopB. Additionally, PaF is made self-adjuvanting by the N-terminal fusion of the A1 subunit of the mucosal adjuvant double-mutant heat-labile enterotoxin (dmLT). Here we show that this triple fusion, designated L-PaF, can activate dendritic cells in vitro and elicits strong IgG and IgA titers in mice when administered intranasally. This self-adjuvanting vaccine expedites the clearance of P. aeruginosa from the lungs of challenged mice while stimulating host expression of IL-17A, which may be important for generating a protective immune response in humans. L-PaF's protective capacity was recapitulated in a rat pneumonia model, further supporting the efficacy of this novel fusion vaccine.

The Potential of Cr3 [Triaqua-µ3 -Oxo-Hexa-µ-Propionatotrichromium(III) Chloride] to Reduce Birth Defects in the Offspring of Diabetic CD-1 Mice.

Diabetes mellitus is a growing concern worldwide and leads to multiple complications during pregnancy. Pharmacologic doses of chromium (Cr) have been linked with improving insulin sensitivity and other positive benefits in the treatment of diabetes in animal models. By using streptozotocin induced hyperglycemia in female CD-1 mice, reproductive outcomes of diabetic and chromium-dosed diabetic females were examined. After dosing 10 mg/kg Cr in the form of triaqua-µ3 -oxo-hexa-µ-propionatotrichromium(III) chloride or Cr3 during gestation days 8-16 (GD8-GD16), all females were sacrificed on gestation day 17 (GD17) and examined for maternal weight gain. The fetuses were examined for gross malformations and for skeletal malformations. The offspring of Cr3-dosed females tended to have a reduction in the incidence of supernumerary ribs. While hyperglycemia still had negative impacts on the health of dams and their offspring, administration of Cr led to an apparent trend in the reduction in the number of malformations and incidence of supernumerary ribs compared to those of untreated diabetic mothers.