Genome sequencing and comparative genomic analysis of bovine mastitis-associated Staphylococcus aureus strains from India

Abstract

Background: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India.

Results: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes.

Conclusion: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

Keywords: Bovine mastitis; MLST; Resistome; Staphylococcus aureus; Virulome; cgMLST.

Fig. 1

Pan-genome of 18 clinical and 23 subclinical mastitis-associated S. aureus isolates obtained from India. A- Pie chart depicting the numbers of core-, soft core-, shell- and cloud genomes. B- Gene family accumulation curves of the pan-genome and the core-genome

Fig. 2

The genome-wide SNP-based maximum likelihood phylogenetic tree of mastitis-associated S. aureus isolates obtained from India. The S. aureus strain K5 (NCBI accession No: NZ_CP020656.1) was used as the reference genome to map and screen the SNPs. The phylogenetic tree was constructed using The CSI Phylogeny v1.4 and visualized using iTOL v6. The scale bar indicates 0.1 substitutions per nucleotide position. The strain names, sequence types (STs), clonal complexes (CCs), spa types, clinical (CL) or subclinical (SCL) mastitis, and five different states in India (GJ-Gujarat, KA-Karnataka, ML-Meghalaya, TG-Telangana, UP-Uttar Pradesh) from where the strains were isolated are indicated, respectively

Fig. 3

The minimum spanning tree based on the cgMLST profiles of 198 S. aureus genome sequences reported from India. The clade numbers, along with the names of the clonal complexes (CCs) or the sequence types (STs), if CC could not be assigned, are shown. The red nodes indicate the mastitis-associated S. aureus isolates. The blue node in the middle highlights the strain MUF256

Fig. 4

Isolation sources and distribution of clonal complexes (CCs) and sequence types (STs) among 198 S. aureus genome sequences reported from India

Fig. 5

The resistome of 41 mastitis-associated S. aureus isolates hierarchically clustered based on the presence (green) or absence (red) of 17 antimicrobial resistance (AMR) genes

Fig. 6

The virulome of 41 mastitis-associated S. aureus isolates hierarchically clustered based on the presence (green) or absence (red) of 108 virulence genes