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Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime.

ABSTRACT: Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin's host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.

SUBMITTER: Oh HK

PROVIDER: S-EPMC7999683 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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

Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime.

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