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Lytic Activity of Polyvalent Staphylococcal Bacteriophage PhiSA012 and Its Endolysin Lys-PhiSA012 Against Antibiotic-Resistant Staphylococcal Clinical Isolates From Canine Skin Infection Sites.

ABSTRACT: The spread of antibiotic-resistant bacteria (ARB) in human and veterinary medicine is of global concern. Notably, the emergence of methicillin-resistant Staphylococcus pseudintermedius has become a serious problem. In this context, bacteriophages and their lytic enzymes, endolysins, have received considerable attention as therapeutics for infectious diseases in place of antibiotics. The aim of the present study was to investigate the antibiotic-resistance patterns of staphylococcal species isolated from canine skin at a primary care animal hospital in Tokyo, Japan and evaluate the lytic activity of the staphylococcal bacteriophage phiSA012 and its endolysin Lys-phiSA012 against isolated antibiotic-resistant staphylococcal strains. Forty clinical staphylococcal samples were isolated from infection sites of dogs (20 from skin and 20 from the external ear canal). Susceptibility to antimicrobial agents was determined by a disk diffusion method. The host range of phiSA012 was determined by using a spot test against staphylococcal isolates. Against staphylococcal isolates that showed resistance toward five classes or more of antimicrobials, the lytic activity of phiSA012 and Lys-phiSA012 was evaluated using a turbidity reduction assay. Twenty-three S. pseudintermedius, 16 Staphylococcus schleiferi, and 1 Staphylococcus intermedius were detected from canine skin and ear infections, and results revealed 43.5% methicillin resistance in S. pseudintermedius and 31.3% in S. schleiferi. In addition, the prevalence multidrug resistance (MDR) S. pseudintermedius was 65.2%. PhiSA012 could infect all staphylococcal isolates by spot testing, but showed little lytic activity by turbidity reduction assay against MDR S. pseudintermedius isolates. On the other hand, Lys-phiSA012 showed lytic activity and reduced significantly the number of staphylococcal colony-forming units. These results demonstrated that ARB issues underlying in small animal hospital and proposed substitutes for antibiotics. Lys-phiSA012 has broader lytic activity than phiSA012 against staphylococcal isolates; therefore, Lys-phiSA012 is a more potential candidate therapeutic agent for several staphylococcal infections including that of canine skin.

SUBMITTER: Nakamura T

PROVIDER: S-EPMC7298730 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Lytic Activity of Polyvalent Staphylococcal Bacteriophage PhiSA012 and Its Endolysin Lys-PhiSA012 Against Antibiotic-Resistant Staphylococcal Clinical Isolates From Canine Skin Infection Sites.

Nakamura Tomohiro T Kitana Junya J Fujiki Jumpei J Takase Masayuki M Iyori Keita K Simoike Kenta K Iwano Hidetomo H

Frontiers in medicine 20200610

The spread of antibiotic-resistant bacteria (ARB) in human and veterinary medicine is of global concern. Notably, the emergence of methicillin-resistant <i>Staphylococcus pseudintermedius</i> has become a serious problem. In this context, bacteriophages and their lytic enzymes, endolysins, have received considerable attention as therapeutics for infectious diseases in place of antibiotics. The aim of the present study was to investigate the antibiotic-resistance patterns of staphylococcal specie ...[more]

PMID: 32587860

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Project description:BACKGROUND: Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAP(K)), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAP(K) cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge. METHODS: The CHAP(K) domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants. RESULTS: The structure of CHAP(K) revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity. CONCLUSIONS: The high-resolution structure of the CHAP(K) domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAP(K) with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.

Project description:ϕEf11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for an N-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathione S-transferase tag. It produced rapid, profound lysis in E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+ Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages.IMPORTANCE The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains of Enterococcus faecalis The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

Dataset Information

Lytic Activity of Polyvalent Staphylococcal Bacteriophage PhiSA012 and Its Endolysin Lys-PhiSA012 Against Antibiotic-Resistant Staphylococcal Clinical Isolates From Canine Skin Infection Sites.

Publications

Lytic Activity of Polyvalent Staphylococcal Bacteriophage PhiSA012 and Its Endolysin Lys-PhiSA012 Against Antibiotic-Resistant Staphylococcal Clinical Isolates From Canine Skin Infection Sites.

Similar Datasets

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