Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates

Abstract

Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus.

FIG. 1

Example of PFGE gel of SmaI macrorestriction fragments of bovine S. aureus isolates, showing isolates 17 to 40. Molecular sizes are indicated on the right.

FIG. 2

Dendrogram showing the level of similarity between SmaI macrorestriction patterns of 38 bovine S. aureus isolates as determined by PFGE and subsequent GelCompar analysis of digitized photographs. Scale indicates level of genetic relatedness within this set of strains. Capital letters indicate pulsotypes based on visual interpretation of PFGE results.

FIG. 3

Dendrogram showing the grouping of 38 bovine S. aureus strains on the basis of hybridization scores after binary typing with probes AW-1 to AW-15 (a) and after omission of probe AW-14, which is associated with hypervariable regions on the bovine staphylococcal genome (b). Isolate number, visual pulsotype, and binary code are given for all isolates. Scale indicates level of genetic relatedness within this set of strains.

FIG. 4

Dendrogram showing the grouping of 55 unrelated human S. aureus strains described previously (42) and 38 bovine S. aureus strains on the basis of hybridization scores after binary typing with 15 DNA probes. Isolate numbers and binary codes are shown for all isolates. Scale indicates level of genetic relatedness within this collection of strains.