Characterisation of Staphylococcus aureus Strains and Their Prophages That Carry Horse-Specific Leukocidin Genes lukP/Q

Abstract

Leukocidins of Staphylococcus (S.) aureus are bicomponent toxins that form polymeric pores in host leukocyte membranes, leading to cell death and/or triggering apoptosis. Some of these toxin genes are located on prophages and are associated with specific hosts. The genes lukP/Q have been described from equine S. aureus isolates. We examined the genomes, including the lukP/Q prophages, of S. aureus strains belonging to clonal complexes CC1, CC350, CC816, and CC8115. In addition to sequencing, phages were characterised by mitomycin C induction and transmission electron microscopy (TEM). All lukP/Q prophages integrated into the lip2=geh gene, and all included also the gene scn-eq encoding an equine staphylococcal complement inhibitor. The lukP/Q prophages clustered, based on gene content and allelic variants, into three groups. One was found in CC1 and CC97 sequences; one was present mainly in CC350 but also in other lineages (CC1, CC97, CC133, CC398); and a third one was exclusively observed in CC816 and CC8115. Prophages of the latter group additionally included a rare enterotoxin A allele (sea_320E). Moreover, a prophage from a CC522 goat isolate was found to harbour lukP. Its lukF component could be regarded as chimaera comprising parts of lukQ and of lukF-P83. A putative kinase gene of 1095 basepairs was found to be associated with equine strains of S. aureus. It was also localised on prophages. However, these prophages were different from the ones that carried lukP/Q, and three different integration sites of kinase-carrying phages were identified. These observations confirmed the presence of prophage-located important virulence-associated genes in equine S. aureus and that certain prophages might determine the host specificity of the staphylococcal strains they reside in.

Keywords: DNA-microarrays; Staphylococcus aureus; bacteriophages; electron microscopy; horse (Equus caballus); leukocidins; lukP/Q; prophages; whole-genome sequencing.

Figure 1

(a) Alignment of lukQ and lukF-P83, showing that sequences from CP138360, pubMLST ID 24359, and JXHY01000064 match the sequence of lukQ until position 702 and the one of lukF-PV83 henceforth. (b) Alignment of lukP/lukM sequences, showing that the sequences from CP138360, pubMLST ID 24359, and JXHY01000064 match the sequence of equine lukP rather than the one of bovine lukM.

Figure 1

(a) Alignment of lukQ and lukF-P83, showing that sequences from CP138360, pubMLST ID 24359, and JXHY01000064 match the sequence of lukQ until position 702 and the one of lukF-PV83 henceforth. (b) Alignment of lukP/lukM sequences, showing that the sequences from CP138360, pubMLST ID 24359, and JXHY01000064 match the sequence of equine lukP rather than the one of bovine lukM.

Figure 2

Transmission electron micrographs of phage particles from the preparation of strain IMT39173. (A), icosahedral phage. (B), prolate phage with large head diameter and oval shape. (C), prolate phage with small head diameter and angular shape. Negative contrast preparation with uranyl acetate. Size bars = 100 nm.

Figure 3

Transmission electron micrographs of phage particles from the preparation of strain IMT37083. (A), icosahedral phage. (B), phage with mildly elongated head. Negative contrast preparation with uranyl acetate. Size bars = 100 nm.

Figure 4

Transmission electron micrographs of phage particles from the preparation of strain V353. (A), prolate phage with large head diameter and oval shape. (B), prolate phage with small head diameter and angular shape. Negative contrast preparation with uranyl acetate. Size bars = 100 nm.

Figure 5

Transmission electron micrograph of incomplete phage particles from the preparation of strain V641. (A), two prolate phage particles, one with small head diameter and angular shape (1) and one with large head diameter and oval shape (2). (B), head of an icosahedral phage particle. Negative contrast preparation with uranyl acetate. Size bars = 100 nm.

Figure 6

Transmission electron micrographs of phage particles from the preparation of strain 17CS1042. (A), large icosahedral phage. (B), small icosahedral phage. Negative contrast preparation with uranyl acetate. Size bars = 100 nm.