An acid-oxidizing solution containing hypochlorous acid reduces Staphylococcus aureus and improves bacterial diversity in epidermolysis bullosa wounds

Epidermolysis bullosa (EB) is a group of rare inherited skin disorders characterized by mechanobullous skin fragility and painful wounds that are readily infected by bacteria, particularly Staphylococcus (S.) aureus. More effective approaches for decreasing wound colonization and infection by pathogenic bacteria are necessary to reduce EB morbidity and mortality. To assess the ability of a low pH, hypochlorous acid (HOCl)-based spray to reduce S. aureus burden, promote wound healing, and restore a healthier skin microbiome. Pilot 12-week open-label study of APR-TD011, a low pH (2.5-3) acid-oxidizing spray containing HOCl (0.0038%), on chronic wounds of EB patients. Patients used the spray daily for 8 weeks on designated wounds. Wounds were assessed clinically and swabbed for microbiome analysis using full-length 16S rRNA sequencing before, at Weeks 4 and 8 of treatment, and at 4 weeks post-treatment. Thirteen of 15 enrolled patients, most of whom had dystrophic EB, were culture-positive for S. aureus at baseline. Sequencing data showed S. aureus had the highest relative abundance (34%), followed by Acinetobacter guillouiae and Pseudomonas poae. S. aureus relative abundance was reduced by Weeks 4 (to 11%), 8 (primary endpoint; to 10%, p<0.01), and still at Week 12 (to 9.7%), including for methicillin-resistant S. aureus. Overall, bacterial diversity increased with treatment and wound sizes decreased, correlating with reduction in S. aureus (p=0.64). These findings suggest that acid-oxidizing HOCl sprays may be an effective adjunctive therapy to reduce bacterial dysbiosis and infection risk in EB wounds, while promoting wound healing. Overall design: Genomic DNA was extracted using a Maxwell 16 LEV Blood DNA Kit (Promega, Madison, WI) implemented on a Maxwell 16 Instrument, following the manufacturer's instructions with minor modifications: a lysozyme incubation (10 ng/µl lysozyme; Thermo Fisher Scientific, Waltham, MA) for 30 minutes at 37°C and bead beating (5 min at 30 Hz twice, 2 min rest in between) using a TissueLyser III (Qiagen, Germantown, MD). Homogenized samples were transferred to the Maxwell cartridges for final DNA purification. Genomic DNA was prepared for sequencing on a PacBio Revio sequencer using a two-stage PCR protocol, similar to that described for Illumina amplicon library preparation.15 In the first stage of PCR, DNA was PCR amplified with primers 27F and 1492R using domain-level bacterial primers as described by PacBio. The underlined regions, however, represent Oxford Nanopore Universal sequences for tailing PCR primers' linker sequences. In the second stage of PCR, amplicons from the first stage were used as a template and amplified with primers that contained PacBio Kinnex adapter sequences, combinatorial dual indices, and the ONT linker sequences at the 3' ends of the primers). Kinnex adapters are bolded, combinatorial indices are indicated with “NNNNNNNNNN”, and ONT linkers are underlined. PCR products from the 2nd stage of amplification were pooled, purified, and subjected to Kinnex library preparation and loading on the Revio instrument. Two-stage library preparation was performed at the Genomics and Microbiome Core Facility (GMCF) at Rush University. Kinnex library preparation and PacBio sequencing was performed at the DNA Services Facility at the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Library preparation was performed using a two-stage PCR amplification protocol but with primers targeting the full 16S rRNA gene.16,17 Genomic DNA was initially amplified with an annealing temperature of 50°C for 24 cycles. Barcoding was performed during the 2nd stage, as described previously.