Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

Abstract

Staphylococcus hyicus and Staphylococcus agnetis are two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus, S. agnetis, and Staphylococcus aureus Isolates (n = 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus, S. agnetis, and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis (n = 43) or S. hyicus (n = 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections.

Keywords: Staphylococcus; cattle; milk.

FIG 1

Multiplex PCR amplification profiles. The M lanes contain a 100-bp molecular weight ladder marker. Lane 1, S. chromogenes ATCC 43764^T; lane 2, S. chromogenes MU 970; lane 3, coagulase-negative S. agnetis DSM 23656^T; lane 4, coagulase-positive S. agnetis 43OE; lane 5, S. hyicus ATCC 11249^T; lane 6, S. aureus ATCC 12600^T, lane 7, S. aureus ATCC 29740. The N lane contains a negative control.

FIG 2

Comparison and alignment of aroD gene sequences from all S. agnetis isolates. The dendrogram shows that all isolates were at least 99.25% homologous. The dendrogram includes the S. agnetis type strain (DSM 23656 [6VT]), the reference strain DSM 23658 (55 OE), and two strains (100 VT and 43 OE) originally isolated from cows in Finland (10). A consensus sequence is at the top of the figure, with all other isolates below. All nucleotide bases in agreement with the consensus sequence are noted by a vertical bar (|), and any single-nucleotide changes are noted by the presence of the variant nucleotide base present within that column.

FIG 3

Comparison of all S. agnetis isolates (n = 39) from 8 commercial dairy herds in which more than one S. agnetis isolate was identified from bovine milk samples. A total of 6 different strains were identified (strains A to F). Strains B, C, and D include isolates from more than one herd. ID, identification.