<sec>
<title>Objectives</title>
<p>The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant <italic>Staphylococcus aureus</italic> (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed.</p></sec>
<sec>
<title>Methods</title>
<p>Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually.</p></sec>
<sec>
<title>Results</title>
<p>Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the <italic>Siphoviridae</italic> family and the <italic>Caudovirales</italic> order.</p></sec>
<sec>
<title>Conclusion</title>
<p>Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.</p></sec>
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Objectives
Bacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). The aim of this study was to clone and express LysK-CHAP domain in <i>Escherichia coli BL21 (DE3)</i> using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter. Methods
The <i>E. coli</i> cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the <i>E. coli</i> strain BL21. Isopropyl β-<sc>d</sc>-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein. Results
In this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to <i>E. coli DH5α</i>. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to <i>E. coli BL21</i> and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression. Conclusion
In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and <i>E. coli BL21 (DE3)</i>.
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