Genomic Analysis of Bovine Staphylococcus aureus Isolates from Milk To Elucidate Diversity and Determine the Distributions of Antimicrobial and Virulence Genes and Their Association with Mastitis

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p><jats:named-content content-type="genus-species">Staphylococcus aureus</jats:named-content> causes persistent clinical and subclinical bovine intramammary infections (IMI) worldwide. However, there is a lack of comprehensive information regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for <jats:named-content content-type="genus-species">S. aureus</jats:named-content> in bovine milk in Canada. Here, we performed whole-genome sequencing (WGS) of 119 Canadian bovine milk <jats:named-content content-type="genus-species">S. aureus</jats:named-content> isolates and determined they belonged to 8 sequence types (ST151, ST352, ST351, ST2187, ST2270, ST126, ST133, and ST8), 5 clonal complexes (CC151, CC97, CC126, CC133, and CC8), and 18 distinct Spa types. Pan-, core, and accessory genomes were composed of 6,340, 1,279, and 2,431 genes, respectively. Based on phenotypic screening for AMR, resistance was common against beta-lactams (19% of isolates) and sulfonamides (7% of isolates), whereas resistance against pirlimycin, tetracycline, ceftiofur, and erythromycin and to the combination of penicillin and novobiocin was uncommon (3, 3, 3, 2, and 2% of all isolates, respectively). We also determined distributions of 191 virulence factors (VFs) in 119 <jats:named-content content-type="genus-species">S. aureus</jats:named-content> isolates after classifying them into 5 functional categories (adherence [<jats:italic>n</jats:italic> = 28], exoenzymes [<jats:italic>n</jats:italic> = 21], immune evasion [<jats:italic>n</jats:italic> = 20], iron metabolism [<jats:italic>n</jats:italic> = 29], and toxins [<jats:italic>n</jats:italic> = 93]). Additionally, we calculated the pathogenic potential of distinct CCs and STs and determined that CC151 (ST151 and ST351) had the highest pathogenic potential (calculated by subtracting core-VFs from total VFs), followed by CC97 (ST352 and ST2187) and CC126 (ST126 and ST2270), potentially linked to their higher prevalence in bovine IMI worldwide. However, there was no statistically significant link between the presence of VF genes and mastitis.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> <jats:named-content content-type="genus-species">Staphylococcus aureus</jats:named-content> is a major cause of bovine intramammary infections, leading to significant economic losses to dairy industry in Canada and worldwide. There is a lack of knowledge regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for <jats:named-content content-type="genus-species">S. aureus</jats:named-content> isolated from bovine milk in Canada. Based on whole-genome sequencing and genomic analysis, we have determined the phylogeny and diversity of <jats:named-content content-type="genus-species">S. aureus</jats:named-content> in bovine milk and concluded that it had a large accessory genome, limited distribution of AMR genes, variable VF gene profiles and sequence types (ST), and clonal complex (CC)-specific pathogenic potentials. Comprehensive information on the population structure, as well as the virulence and resistance characteristics of <jats:named-content content-type="genus-species">S. aureus</jats:named-content> from bovine milk, will allow for source attribution, risk assessment, and improved therapeutic approaches in cattle.</jats:p>

Genomic Analysis of Staphylococcus aureus Isolates Associated With Peracute Non-gangrenous or Gangrenous Mastitis and Comparison With Other Mastitis-Associated Staphylococcus aureus Isolates

“…To our knowledge, CC522 has not been previously reported in bovine mastitis. The other four CCs detected in our study have also been among the predominant CCs in earlier bovine S. aureus mastitis studies ( Hoekstra et al, 2020 ; Naushad et al, 2020 ). Three CCs, namely CC133, CC479, and CC522, were significantly associated with peracute gangrenous S. aureus mastitis.…”

“…The majority, 17 of 20 (85%) of our isolates belonged to three CCs: CC151, CC133, and CC97, which have been reported as common bovine-associated complexes ( Hoekstra et al, 2020 ; Naushad et al, 2020 ). Two of our isolates in the peracute group belonged to CC522, which is commonly found in isolates from mastitis in small ruminants ( Kahila Bar-Gal et al, 2015 ; Romano et al, 2020 ).…”

“…The single CC479 isolate in our study originated from peracute gangrenous mastitis and clustered in ribotyping with the two isolates belonging to CC522, also from peracute gangrenous mastitis. CC522 was not detected by Hoekstra et al (2020), nor by Naushad et al (2020). Both CC522 and CC133 are commonly found in small ruminants (Kahila Bar-Gal et al, 2015;Romano et al, 2020).…”

“…Several studies have focused on the possible differences in virulence gene profiles between S. aureus isolates from clinical and subclinical mastitis, but have failed to detect any differences ( Åvall-Jääskeläinen et al, 2018 ; Rocha et al, 2019 ; Naushad et al, 2020 ). Mastitis is a dynamic phenomenon; when the microbe enters the mammary gland, the leukocytes in the milk and mammary-gland epithelial cells react and initiate the immunologic defense process, leading to large numbers of neutrophils migrating to the mammary gland and attempting to kill the microbial cells ( Wellnitz and Bruckmaier, 2012 ).…”

“…The 3rd most common isolate in that study, CC479, represented 11.6% of the isolates, while only one isolate (5%) in our study belonged to CC479. In another recent whole-genome sequencing study analyzing 119 S. aureus isolates from clinical and subclinical bovine mastitis from Canada ( Naushad et al, 2020 ), CC151 and CC97 were the most common, covering 92% of the isolates. Only one isolate in that study belonged to CC133, while CC479 and CC522 were not detected at all.…”