Host antimicrobial peptide S100A12 disrupts the fungal membrane by direct binding and inhibits growth and biofilm formation of Fusarium species.

Fungal keratitis is the foremost cause of corneal infections worldwide, of which Fusariumspp. is the common etiological agent that causes loss of vision and warrants surgical intervention. An increase in resistance to the available drugs along with severe side effects of the existing antifungals demands for new effective antimycotics. Here, we demonstrate that antimicrobial peptide S100A12 directly binds to the phospholipids of the fungal membrane, disrupts the structural integrity, and induces generation of reactive oxygen species in fungus. In addition, it inhibits biofilm formation by Fusariumspp. and exhibits antifungal property against Fusariumspp. both in vitro and in vivo. Taken together, our results delve into specific effect of S100A12 against Fusariumspp. with an aim to investigate new antifungal compounds to combat fungal keratitis.

Safety and efficacy of McCarey-Kaufman medium supplemented with colistin (polymyxin E) and amphotericin B in inhibiting the multidrug-resistant Pseudomonas aeruginosa using an ex vivo donor corneal infection model.

PURPOSE: This study aimed to evaluate the efficacy and safety of McCarey-Kaufman (MK) medium supplemented with colistin and amphotericin B in inhibiting the growth of multidrug-resistant Pseudomonas (P.) aeruginosa , using an ex vivo experimental model with human donor corneas. METHODS: Cadaveric human corneas deemed unsuitable for corneal transplantation were obtained, and MK media were supplemented with colistin and amphotericin B. Multidrug-resistant P. aeruginosa was cultured and used to infect the human donor corneas ex vivo . Infected corneas were placed in the MK media with additional antibiotics (colistin and amphotericin B) and the standard MK media, which served as the control arm for comparison. Corneal opacity due to infiltration and quantitative analysis of colony-forming units (CFUs) were assessed. The viability of the corneal endothelium was assessed using trypan blue staining. RESULTS: Corneas incubated in MK media supplemented with additional antibiotics showed less corneal opacification compared with those in standard MK media at both 48- and 96-hour (hr) time points. Quantitative analysis revealed a lower bacterial load and a significant reduction in CFU in the corneas incubated in MK media with additional antibiotics compared with the control group. At 48 hrs, there was 84% ( P value = 0.024) reduction in bacterial load, and at 96 hr, a 53% ( P value = 0.016) reduction was observed in comparison with those placed in standard MK media. The trypan blue staining tests revealed that the extent of endothelial cell loss in corneas incubated in supplemented MK media was comparable to the ones in standard MK media. CONCLUSION: The addition of colistin and amphotericin B to MK media demonstrated efficacy in inhibiting the growth of multidrug-resistant P. aeruginosa in an ex vivo cornea infection model. The supplemented media had no detrimental effect on the corneal endothelium. The findings suggest that supplementing the MK media with these broad-spectrum antimicrobial agents may help mitigate the risk of postoperative donor-related infection in the recipients by reducing and containing the load of microbial contamination in donor corneas.

3D-Printed Inherently Antibacterial Contact Lens-Like Patches Carrying Antimicrobial Peptide Payload for Treating Bacterial Keratitis.

Delivery of therapeutic agents through contact lenses-like patches is a promising strategy to achieve significant bioavailability with negligible eye drainage. The present study investigates the preparation and 3D printing of mucoadhesive gelatin methacryloyl (GelMA)/chitosan methacryloyl (ChiMA) hydrogels to fabricate them as contact lens-like patches (CLP) loaded with antimicrobial peptide, S100A12 (AMP) for treating bacterial keratitis (BK). Extrusion technology is used to print the patches layer by layer to form a hemispherical scaffold suitable for eyewear, and 3D-printed CLP is crosslinked using Irgacure 2959 under UV light. The results from the in vivo experiment conducted on Pseudomonas aeruginosa-infected BK rabbit model after the treatment with AMP-loaded CLP have shown a significant decrease in bacterial load when plated for CFU. The newly developed delivery system containing AMP has great potential to overcome the treatment challenges of multidrug resistance (MDR) in bacteria and eliminate the frequent dosing associated with eye drops. The presence of chitosan in the formulation provides a synergetic effect on the AMP in disrupting bacterial biofilms. The ease of using 3D printing will open new avenues for optimizing the dosage depending on the severity of the BK in the patients, which can be used as personalized medicine.

Antimicrobial peptide S100A12 (calgranulin C) inhibits growth, biofilm formation, pyoverdine secretion and suppresses type VI secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen and is the major cause of corneal infections in India and worldwide. The increase in antimicrobial resistance among Pseudomonas has prompted rise in significant research to develop alternative therapeutics. Antimicrobial peptides (AMPs) are considered as potent alternatives to combat bacterial infections. In this study, we investigated the role of S100A12, a host defense peptide, against PAO1 and an ocular clinical isolate. Increased expression of S100A12 was observed in corneal tissues obtained from Pseudomonas keratitis patients by immunohistochemistry. S100A12 significantly inhibited growth of Pseudomonas in vitro as determined from colony forming units. Furthermore, recombinant S100A12 reduced the corneal opacity and the bacterial load in a mouse model of Pseudomonas keratitis. Transcriptome changes in PAO1 in response to S100A12 was investigated using RNA sequencing. The pathway analysis of transcriptome data revealed that S100A12 inhibits expression of genes involved in pyoverdine synthesis and biofilm formation. It also impedes several important pathways like redox, pyocyanin synthesis and type 6 secretion system (T6SS). The transcriptome data was further validated by checking the expression of several affected genes by quantitative PCR. Our study sheds light on how S100A12 impacts Pseudomonas and that it might have the potential to be used as therapeutic intervention in addition to antibiotics to combat infection in future.

Hydroxypropyl methacrylamide-based copolymeric nanoparticles loaded with moxifloxacin as a mucoadhesive, cornea-penetrating nanomedicine eye drop with enhanced therapeutic benefits in bacterial keratitis.

Bacterial keratitis (BK) is a leading cause of visual impairment. The fluoroquinolone antibiotic moxifloxacin (Mox), being highly water-soluble, suffers from poor corneal penetration leading to unsatisfactory therapeutic outcomes in BK. Here, we prepared Mox-loaded co-polymeric nanoparticles (NPs) by entrapping the drug in co-polymeric NPs constituted by the self-assembly of a water-soluble copolymer, poly(ethylene glycol)-b-p(hydroxypropyl) methacrylamide (mPH). The polymer (mPH) was prepared using a radical polymerization technique at different mPEG: HPMA ratios of 1:70/100/150. The polymer/nanoparticles were characterized by GPC, CAC, DLS, SEM, XRD, DSC, FTIR, % DL, % EE, and release studies. The ex vivo muco-adhesiveness and corneal permeation ability were judged using a texture analyzer and Franz Diffusion Cells. In vitro cellular uptake, cytotoxicity, and safety assessment were performed using HCE cells in monolayers, spheroids, and multilayers in transwells. The DOE-optimized colloidal solution of Mox-mPH NPs (1:150) displayed a particle size of ~116 nm, superior drug loading (8.3%), entrapment (83.2%), robust mucoadhesion ex vivo, and ocular retention in vivo (~6 h) (judged by in vivo image analysis). The non-irritant formulation, Mox-mPH NPs (1:150) (proven by HET-CAM test) exhibited intense antimicrobial activity against P. aeruginosa, S. pneumoniae, and S. aureus in vitro analyzed by live-dead cells assay, zone of inhibition studies, and by determining the minimum inhibitory and bactericidal concentrations. The polymeric nanoparticles, mPH (1:150), decreased the opacity and the bacterial load compared to the other treatment groups. The studies warrant the safe and effective topical application of the Mox-mPH NPs solution in bacterial keratitis.

Plasmodium vivax liver stage assay platforms using Indian clinical isolates.

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Differential Expression of Antimicrobial Peptides in Streptococcus pneumoniae Keratitis and STAT3-Dependent Expression of LL-37 by Streptococcus pneumoniae in Human Corneal Epithelial Cells.

Streptococcus pneumoniae is the leading cause of bacterial keratitis in the developing world with a growing trend of acquiring resistance against various antibiotics. In the current study, we determined the expression of different antimicrobial peptides (AMPs) in response to S. pneumoniae in patients, as well as in primary and immortalized human corneal epithelial cells. We further focused on LL-37 and determined its expression in human cornea infected with S. pneumoniae and studied the killing ability of LL-37 against S. pneumoniae. The expression of AMPs was determined by quantitative PCR and the phosphorylation of signaling proteins was evaluated by immunoblot analysis. LL-37 expression was also determined by immunofluorescence and Western blot method and the killing ability of LL-37 against S. pneumoniae was determined by colony-forming units. Differential expression of antimicrobial peptides was observed in patients with S. pneumoniae keratitis. Although S. pneumoniae induced expression of the AMPs in human corneal epithelial cells (HCEC), it did not induce AMP expression in U937, a human monocyte cell line. S. pneumoniae also caused activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB)and mitogen activated protein kinase (MAPK) pathways in corneal epithelial cells. LL-37 was found to be effective against both laboratory and clinical strains of S. pneumoniae. LL-37 induction by S. pneumoniae in human corneal epithelial cells was mediated by signal transducer and activator of transcription 3 (STAT3) activation, and inhibition of STAT3 activation significantly reduced LL-37 expression. Our study determines an extensive profile of AMPs expressed in the human cornea during S. pneumoniae infection, and suggests the potential of LL-37 to be developed as an alternative therapeutic intervention to fight increasing antibiotic resistance among bacteria.