Equine Methicillin-Resistant Sequence Type 398 Staphylococcus aureus (MRSA) Harbor Mobile Genetic Elements Promoting Host Adaptation

Abstract

Continuing introduction of multi-drug resistant, zoonotic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) in horse clinics challenges the biosafety of employees and animal patients. This study was aimed to determine the occurrence of mobile genetic elements facilitating survival in the early stages of invasive infection in different host species, including humans and horses, in MRSA carried by equine patients admitted to a large horse clinic. A total of 341 equine patients were investigated for carriage of MRSA by hygiene screening directly at hospital admission. MRSA were further investigated by antimicrobial susceptibility testing, whole-genome sequencing and genomic composition, including virulence factors involved in immune evasion and host adaption. From a total of 340 validated specimens from equine nostrils, 3.5% yielded positive results for MRSA. All MRSA were found to be closely related belonging to sequence type (ST) 398_t011 with up to four additional antimicrobial resistances. All MRSA harbored a specific Staphylococcal Pathogenicity Island (SaPIbov5) involved in facilitating survival in ruminant and equine plasma. Moreover, a β-hemolysin (hlb) converting ΦSa3 phage encoding the human-specific Immune Evasion Cluster (IEC) was present in 72% of the isolates. An equid-specific leukotoxin encoded by a further temperate phage (Saeq1) was only rarely detected (22%). Despite the absence of β-hemolysin production for all IEC-positive ST398, a prominent hemolysis zone was demonstrable on sheep blood agar. Thus, IEC might remain undetected among the ST398 lineage, since the presence of IEC is commonly associated with reduction of hemolysis in S. aureus belonging to other genetic backgrounds. Here we describe MRSA-ST398 harboring different mobile genetic elements encoding variants of immune evasion factors and toxins previously shown to contribute to S. aureus invasive diseases in specific host species or ecologic niches. We suggest these combinations contribute to the adaptation of MRSA belonging to ST398 with respect to epidemic spread across different habitats and hosts, and may therefore confer a host "generalist" phenotype.

Keywords: MRSA; ST398; horses; host range; multi-drug resistance; one health.

Figure 1

Close relationship of 18 equine MRSA-ST398-t011 isolated from horses at hospital admission. Maximum Likelihood Phylogeny: Phylogenetic tree generated with RAxML, displayed with CLC Genomics Workbench on the basis of 2,346 orthologous genes from 19 ST398 S. aureus WGS, including livestock-associated methicillin-resistant Staphylococcus aureus (ST398) strain 08S00974 (CP020019) as an outgroup. Presence of mobile genetic elements harboring host adaptation factors is marked blue. phiNM3-var., ΦSa3 phage (40 kb) harboring an immune evasion cluster (IEC); Saeq1, 45 kb prophage Φ Saeq1; SaPIbov5, Staphylococcus pathogenicity island SaPIbov5. Two subgroups could be distinguished, cluster (A) and (B).

Figure 2

IEC-positive phiNM3-like phage integrated in hlb gene of equine MRSA-ST398. Gene map of the hlb-encoding phospholipase C (ß-hemolysin) region in equine MRSA-ST398. The colored bar at the top indicates the mean pairwise nucleotide sequence identity in the column: bright green = 100% identity; green-brown = <100% but >30% base pair identity. Prophage genes are colored based on putative or known function. Genomic attachment sites were shown in gray. The integration site (5′-GTATCCGAATTGG-3′) of 40,699 bp or 40,703 bp phiNM3-like phages (coverage: 96%, similarity 98%; DQ530361.1) harboring genes of the Immune Evasion Cluster (scn, sak) within the first 13 genomes is indicated by an orange arrow, resulting in a disrupted hlb gene (red) of 301 bp (left) and 825 bp (right). So far, ΦSa3 integration in hlb of ST-398 was rarely reported (Kraushaar et al., 2017).

Figure 3

Hemolysis of equine MRSA-ST398 on sheep blood agar plate. Hemolysis zones of closely related MRSAST398-t011 isolated from horses on sheep blood agar plates after 18 h at 37°C incubation followed by 4 h at 4°C. The β-hemolysin activity induced “double zone” hemolysis (halo) was noticed only for isolate 9, 12, 15, 16, and 18. Isolates 1–8, 10, 11, 13, 14, and 17 harbor a β-hemolysin disrupting phage carrying IEC (numbers as indicated in Table 2).

Figure 4

Lack of β-hemolysin production in MRSA-ST398 harboring IEC. (A–C) CAMP test results for Streptococcus agalactiae (grown as central diameter on each plate) together with 18 MRSA-ST398-t011 isolates of equine origin. grown as central diameter on each plate) together with 18 MRSA-ST398-t011 isolates of equine origin. (A) White arrow: large hemolytic zone irrespectively of CAMP factor presence in 1–8, 10, 11, 13, 14, and 17. These isolates harbor a β-hemolysin disrupting phage carrying IEC (numbers as indicated in Table 2). (B) White arrow: enlarged hemolysis zone induced by synergistic activity of CAMP factor- and β-hemolysin production in 9, 12, 15, 16, and 18. (C) White arrow: β-hemolysin production inhibits lysis by α-hemolysin (Traber et al., 2008), resulting in weaker total hemolysis zones for the IEC-negative isolates 9, 12, 15, 16, and 18. +, positive control S. aureus ATCC 25923.