Isolation of a Novel Lytic Bacteriophage against a Nosocomial Methicillin-Resistant Staphylococcus aureus Belonging to ST45

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide range of infections from mild to life-threatening conditions. Its enhanced antibiotic resistance often leads to therapeutic failures and therefore alternative eradication methods must be considered. Potential candidates to control MRSA infections are bacteriophages and their lytic enzymes, lysins. In this study, we isolated a bacteriophage against a nosocomial MRSA strain belonging to the ST45 epidemiologic group. The phage belonging to Caudovirales, Siphoviridae, showed a narrow host range and stable lytic activity without the emergence of resistant MRSA clones. Phylogenetic analysis showed that the newly isolated Staphylococcus phage R4 belongs to the Triavirus genus in Siphoviridae family. Genetic analysis of the 45 kb sequence of R4 revealed 69 ORFs. No remnants of mobile genetic elements and traces of truncated genes were observed. We have localized the lysin (N-acetylmuramoyl-L-alanine amidase) gene of the new phage that was amplified, cloned, expressed, and purified. Its activity was verified by zymogram analysis. Our findings could potentially be used to develop specific anti-MRSA bacteriophage- and phage lysin-based therapeutic strategies against major clonal lineages and serotypes.

Figure 1

Transmission electron microscopic picture of phage R4 showed the typical characteristic of the Siphoviridae family, with an elongated head.

Figure 2

Phylogeny tree of R4 phage. Genome-BLAST Distance Phylogeny (GBDP) tree inferred using the formula D0 and yielding average support of 2%. The numbers above branches are GBDP pseudobootstrap support values from 100 replications. The branch lengths of the resulting VICTOR trees are scaled in terms of the respective distance formula used. The OPTSIL clustering yielded twenty species clusters, one cluster at the genus level, and one at the family level. Accession numbers are indicated next to the phage names. R4 phage is marked with a black asterisk.

Figure 3

Genome organization of the R4 phage. The 45 kb double-stranded DNA (black parallel lines) contains 69 coding genes, each represented by arrows, annotated as indicated. The modular genome organization, characteristic for Staphylococcal siphophages is highlighted by colors, indicating the coding regions belonging to one of the six functional modules. The 1455 bp long gene orf27, coding for the endolysin (amidase), marked by a black asterisk, was cloned into the 2.9 kb pRSET A vector. The rightmost black image represents the result of XhoI-HindIII probe digestion of orf27-pRSET A construct (lane “A”) and pRSET A control (lane “B”) on 1% agarose gel. Excised 1.5 kb orf27 band is marked by a white asterisk. (Lane “L”: DNA ladder.)

Figure 4

Visualization of the expressed recombinant R4lys endolysin. Comparison of sonicated and boiled crude expression cell lysates and purified amidase by SDS-PAGE (left) and by zymogram (right) analysis. The letters are indicating that the same samples were added to both gels in the corresponding lanes. Expressing BL21 cells were harboring the orf27-pRSET A plasmid. (a) Sonicated crude BL21 cells, boiled, with 5x sample buffer. (b) Column purified endolysin, boiled, with 5x sample buffer. (c) Sonicated crude BL21 cells, not boiled, with 5x sample buffer. (d) Column purified endolysin, not boiled, with 5x sample buffer. (e) Column purified endolysin, not boiled, without 5x sample buffer. SDS-PAGE revealed the presence of a protein, slightly below 60 kDa (black arrow is showing 60 kDa on the protein ladder), regardless of the sample preparation method. In spite of the R4lys protein's presence, zymography only showed its cell wall destroying amidase activity when the sample was not boiled (white bands in lanes (c, d, e)).