Novel recombinant endolysin ointment with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus isolated from wounds and burns.

The major problem in the management of burn wounds is infections. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of infection in burn wounds. Antibiotic-resistant bacteria around the world have become a major therapeutic challenge. Bacteriophages and their lysin are suggested as an antimicrobial alternative agent. The approach of this study was to evaluate the potential of recombinant phage lysin ointment efficacy in MRSA burn wound infection in vitro. Whole genome sequencing was performed to the three isolated bacteriophages by ABM, USA using Illumina next-generation sequencing (NGS) technology. De novo assembly and genetic analysis carried out. Expression of lysin genes was performed by cloning using Escherichia coli JM109. Lysin protein extraction and purification was performed before and after cloning using precipitation by ammonium sulfate, dialysis, and gel filtration chromatography. Dose-dependent assay and time-kill curve experiment was performed for 2 lysins showed that recombinant lysin 2 functions more than its non-recombinant lysins 2 with the same concentration of 0.5 µg/mL. Both lysins' ointment was prepared and compared with commercial ointments. 62 (78.4%) out of 79 wounds a burns swabs were detected as S. aureus and S methicillin-resistant S. aureus (MRSA) rate was determined to be 29 (46.8%) in total, while 33 isolates (53.2%) determined as methicillin-sensitive S. aureus (MSSA). According to the antibiotic susceptibility test results, all S. aureus isolates were identified as sensitive against vancomycin, ceftaroline, and linezolid. Results shows one lysogenic bacteriophage and three distinct lytic specific S. aureus bacteriophage were isolated from sewage. For each of the three samples, a single contig was possible to be obtained. Sample BP-SA2 had the best coverage, and the contig was slightly longer than the other bacteriophages. In addition, BLAST search identified Staphylococcus bacteriophage vB-SscM-1 (accession KX171212.1) as the closest match to the public database. Finally, the gene annotation was checked, and two potential lysin genes were identified. Besides the two ends, there are only 4 SNPs between the three genomes. It should be noted that the two lysin genes from the genomes have no SNPs, and are identical across the three genomes. It can be seen that the three bacteriophages (BP-SA1), (BP-SA 2), and (BP-SA3) form their own tight cluster. It can be seen that (BP-SA 2) is more closely related to Staphylococcus bacteriophage vB-SscM-1 genome and most noticeable 5' region of S5 and vB-SscM-1 are now located at 3' end of vB-Sau-Clo6. The investigation of the two lysin genes in (BP-SA 2) by whole genome sequencing showed that there is some homology with vB-SscM-1; although the first gene is annotated as hypothetical protein, the second gene is annotated as amidase. The same two lysin genes are identified in all three bacteriophage genomes by the RAST. The putative protein sequences of the discovered phage lysin was analyzed using protein search with UniProt/Swiss-Prot database, and all matches suggest that the putative protein of the discovered phage lysin is a real endolysin. The three samples of bacteriophage were harboring both (Lysin 1 and lysin 2) genes were amplified. Afterward, 2-lysin genes were cloned successfully; for the dose-dependent assay, the same incubation time of recombinant lysins and its two non-recombinant lysins with the bacteria for 30 min. It is found that the bactericidal activity of these groups increased in correlation with their concentrations. For the time-kill curve experiment, it showed that Recombinant lysin 2 functions more than its non-recombinant lysins 2 with the same concentration of 0.5 µg/mL. Both lysins' ointments have potential activity against S. aureus isolates more than mupirocin and have a similar activity with Fusidic acid through applying 10 µL from lysin 1 ointment, lysin 2 ointment, mupirocin ointment 2%, and Fusidic acid cream 2%. In vitro lytic spectrum analysis revealed that 100% (29/29) tested S. aureus were sensitive. One dose of lysin ointment resulted in a reduction of 3.3 log units in the number of bacteria (from an initial count of 2 × 10