Genomic epidemiology and characterization of Staphylococcus aureus isolates from raw milk in Jiangsu, China: emerging broader host tropism strain clones ST59 and ST398

Abstract

Staphylococcus aureus is highly pathogenic and can cause disease in both humans and domestic animals. The aim of this study was to investigate the genomic epidemiology of S. aureus isolates from raw milk in Jiangsu Province, China, to identify predominant lineages and their associated genomic and phenotypic characteristics. In this study, we identified 117 S. aureus isolates collected from 1,062 samples in Jiangsu Province between 2021 and 2022. Based on whole-genome sequencing (WGS) data from 117 S. aureus isolates, molecular analyses indicated CC1-ST1 (26.50%, 31/117), CC97-ST97 (18.80%, 22/117), CC398-ST398 (10.26%, 12/117), CC8-ST630 (7.69%, 9/117) and CC59-ST59 (2.56%, 3/117) were the major lineages. The prevalence of mecA-positive strains was 11.11%. Four methicillin-resistant S. aureus (MRSA) lineages were found, including MRSA-ST59-t172 (n = 3), OS-MRSA-ST398-t011 (n = 1), MRSA-ST630-t2196 (n = 2) and OS-MRSA-ST630-t2196 (n = 7). Phenotypic resistance to penicillin (30.77%, 36/117), ciprofloxacin (17.09%, 20/117) and erythromycin (15.38%, 18/117) was observed which corresponded with resistance genotypes. All of the isolates could produce biofilms, and 38.46% (45/117) of isolates had invasion rates in mammary epithelial cells (MAC-T) of greater than 1%. Interestingly, most biofilm-producing and invading isolates harbored ebp-icaA-icaB-icaC-icaR-clfA-clfB-fnbA-fnbB-sdrC-sdrD-sdrE-map-can (27.35%, 32/117) and ebp-icaA-icaB-icaC-icaD-icaR-clfA-clfB-fnbA-fnbB-sdrC-sdrD-sdrE-map (33.33%, 39/117) adherence-associated gene patterns and belonged to lineages CC1 and CC97, respectively. Virulence factor assays showed that 47.01% of the isolates contained at least enterotoxin genes. Isolates harboring the immune evasion cluster (IEC) genes (sea, sak, chp, and scn) were predominantly categorized as STs 464, 398, and 59. IEC-positive ST398 and ST59 isolates contained a very high proportion of virulence genes located on prophages, whereas most IEC-negative ST398 clade isolates carried broad-spectrum drug resistance genes. Meanwhile, the IEC-positive ST398 clade showed a close genetic relationship with isolates from the pork supply chain and hospital surgical site infections. MRSA-ST59 strains showed the closest genetic relationship with an isolate from quick-frozen products. High-risk livestock-associated strains ST398 and MRSA-ST59 were detected in raw milk, indicating a potential public health risk of S. aureus transmission between livestock and humans. Our study highlights the necessity for S. aureus surveillance in the dairy industry.

Keywords: Staphylococcus aureus; biofilm formation; invasion; phylogenetic analysis; virulence factors; whole-genome sequencing.

Figure 1

Phylogenetic analysis of Staphylococcus aureus isolates. Showing distribution of clonal complexes (CCs), sequence types (STs), polymorphism of protein A gene (spa typing), accessory gene regulator gene (agr typing) and carrying of mecA. Mash phylogenetic trees were constructed based on the global mutation distance of the whole genome using Mash (v2.1) and overlayed with information regarding CCs, STs, spa typing, and agr typing using iTOL.

Figure 2

Heatmap of antibiotic resistance phenotypes and genotypes, biofilm formation, invasion rate of mammary epithelial cells (MAC-T) and carriage of virulence genes for 117 S. aureus isolates. The distributions of antimicrobial genes, antibiotic susceptibility profiles and carriage of virulence genes are plotted against the core genome phylogeny of the S. aureus isolates. BF, biofilm formation; IR, invation rate.

Figure 3

Antimicrobial phenotype and genotype results of S. aureus isolated from raw milk samples. (A) Antimicrobial susceptibility tests of 117 S. aureus strains isolated from raw milk samples. AMI, amikacin; AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; KAN, kanamycin; MOX, moxifloxacin; OXA, oxacillin; PEN, penicillin; RIF, rifampicin; SRT, streptomycin; SUL, sulfamethoxazole; TET, tetracycline; VAN, vancomycin. (B) Detection of antimicrobial resistance genes in 117 S. aureus strains isolated from raw milk samples.

Figure 4

Distributions of virulence and antimicrobial genes, and genetic structure of prophages carrying immune evasion cluster (IEC) genes in ST398 and ST59 isolates. (A) The main virulence and antimicrobial genes in ST398 and ST59 isolates. The Mash phylogenetic tree was constructed based on global mutation distances of the whole genome using Mash (v2.1). (B) Genetic structure of prophages carrying immune evasion cluster genes identified from ST59 and ST398 isolates in this study.

Figure 5

Phylogenetic analysis of ST398 isolates. Classification of location, source, polymorphism of protein A gene (spa typing) and presence of mecA for 79 domestic ST398 isolates. Strains from this study and publicly-available reference genomes are indicated by leaf color. The Mash phylogenetic tree was constructed based on global mutation distances of the whole genome using Mash (v2.1) and cleaned and overlayed using iTOL.

Figure 6

Phylogenetic analysis of ST59 isolates. Classification of location, source, polymorphism of protein A gene (spa typing) and presence of mecA for 81 domestic ST59 isolates. Isolates from this study and publicly-available reference genomes are indicated by leaf color. The Mash phylogenetic tree was constructed based on the global mutation distance of the whole genome using Mash (v2.1) and cleaned and overlayed using iTOL.