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, 10 (4)

Characterization of a New Staphylococcus Aureus Kayvirus Harboring a Lysin Active Against Biofilms

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Characterization of a New Staphylococcus Aureus Kayvirus Harboring a Lysin Active Against Biofilms

Luís D R Melo et al. Viruses.

Abstract

Staphylococcus aureus is one of the most relevant opportunistic pathogens involved in many biofilm-associated diseases, and is a major cause of nosocomial infections, mainly due to the increasing prevalence of multidrug-resistant strains. Consequently, alternative methods to eradicate the pathogen are urgent. It has been previously shown that polyvalent staphylococcal kayviruses and their derived endolysins are excellent candidates for therapy. Here we present the characterization of a new bacteriophage: vB_SauM-LM12 (LM12). LM12 has a broad host range (>90%; 56 strains tested), and is active against several MRSA strains. The genome of LM12 is composed of a dsDNA molecule with 143,625 bp, with average GC content of 30.25% and codes for 227 Coding Sequences (CDSs). Bioinformatics analysis did not identify any gene encoding virulence factors, toxins, or antibiotic resistance determinants. Antibiofilm assays have shown that this phage significantly reduced the number of viable cells (less than one order of magnitude). Moreover, the encoded endolysin also showed activity against biofilms, with a consistent biomass reduction during prolonged periods of treatment (of about one order of magnitude). Interestingly, the endolysin was shown to be much more active against stationary-phase cells and suspended biofilm cells than against intact and scraped biofilms, suggesting that cellular aggregates protected by the biofilm matrix reduced protein activity. Both phage LM12 and its endolysin seem to have a strong antimicrobial effect and broad host range against S. aureus, suggesting their potential to treat S. aureus biofilm infections.

Keywords: Kayvirus; Staphylococcus aureus; bacteriophage; biofilms; endolysin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphology of LM12. Transmission electron microscopic image of Staphylococcus myovirus LM12 negatively stained with 2% (w/v) uranyl acetate. Scale bar represents 50 nm.
Figure 2
Figure 2
Genome overview of the Staphylococcus phage LM12. Genome map with predicted 227 coding sequences (CDSs) numbered and colored (yellow shows hypothetical proteins, blue shows DNA replication and transcription genes, green represents DNA packaging and phage morphogenesis genes, and red indicates cells lysis genes) according to their predicted function. Some important CDSs are highlighted. Above the genome, the nucleotide position in kb is given. The figure was performed using Geneious 9.1.4.
Figure 3
Figure 3
Phylogenetic tree from LM12 genome and its most closely related phage genomes. The tree was generated with a Maximum-Likelihood phylogenetic (PHYML) algorithm, with a bootstrapping of 100. Four groups are indicated (green, orange, purple, and blue), and arrows indicates phage LM12. The figure was performed using Geneious 9.1.4.
Figure 4
Figure 4
Endolysin E-LM12 activity against S. aureus biofilms, using 8 μM (a) intact biofilms; (b) scraped biofilms. Data was assessed by CFU counting, and the values represent the mean (plus or minus) of three independent experiments performed in duplicate. Statistical differences (p < 0.01) between the control biofilms and the LM12-treated biofilms (*) were determined by two-way repeated-measures analysis of variance (ANOVA) with a Sidak post-test.
Figure 5
Figure 5
Endolysin E-LM12 activity against 24 h S. aureus planktonic cells, using 8 μM (a) exponential phase cells, (b) stationary phase cells, and (c) biofilm suspended cells. Data was assessed by CFU counting, and the values represent the mean (plus or minus) of three independent experiments performed in duplicate. Statistical differences (p < 0.01) between control biofilms and LM12-treated biofilms (*) were determined by two-way repeated-measures analysis of variance (ANOVA) with a Sidak post-test.

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