Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage.

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: • vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. • vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. • vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.

Characterization of novel of temperate phages of Staphylococcus aureus isolated from bovine milk.

Bovine mastitis is an important disease in dairy cows, and Staphylococcus aureus is the most prevalent microorganism. Bacteriophages are considered an alternative to treat bacterial infections due to antimicrobial resistance crisis. In this study, we isolated and characterized novel S. aureus temperate phages, namely B_UFSM4 and B_UFSM5, from bovine milk. The complete genomes of B_UFSM4 and B_UFSM5 have 41.396 bp and 41.829 bp, respectively. The viruses have double-stranded DNA and linear architecture. Phylogenic similarity was observed by proteome with Staphylococcus phage phiPV83, CN125 and JS01. Therefore, the phages were classified into the family Siphoviridae, genus Biseptimavirus and order Caudovirales. In the host range, the B_UFSM4 and B_UFSM5 had lytic activity of 45.8% and 54.16%, respectively, inclusive on isolates from Staphylococcus sciuri and Rothia terrae. Thus, in this study, species novel of S. aureus temperate phages was isolated and characterized, these phages reveal similarities to each other; however, they are distinct from other species of S. aureus phages of the family Siphoviridae.

A plasmid vector for isolation of strong promoters in Escherichia coli.

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

Phage-mediated transfer of tetracycline resistance in Staphylococcus aureus isolated from cattle in Brazil.

Tetracycline-resistant strains of Staphylococcus aureus isolated from cattle in Brazil, were used as prospective donors for the transfer of resistance to laboratory strains, using mixed-culture and filter-mating protocols. Three lysogenic donors transferred tetracycline resistance in both mixed culture and during filter mating. In contrast, when a non-lysogenic strain was used as prospective donor, transfer was not detected using either mating protocol. In order to evaluate the involvement of phage, successful transfer experiments were repeated with the addition of sodium citrate, which sequestered calcium ions. Mixed-culture and filter-mating protocols did not result in the transfer of resistance. These results support the notion that transfer of the resistance determinant under both sets of conditions described here involve the same bacteriophage-mediated mechanism. Although transfer of tetracycline resistance was detected, without any attempt to create specialized transduction agents or to extract phages, the co-transfer of additional resistance markers indicated that it could not be conventional transduction.