J Vet Sci.  2018 Nov;19(6):771-781. 10.4142/jvs.2018.19.6.771.

Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis

Affiliations
  • 1Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 for Veterinary Science, Seoul National University, Seoul 08826, Korea. kwonhj01@snu.ac.kr
  • 2Laboratory of Avian Diseases, College of Veterinary Medicine and BK21 for Veterinary Science, Seoul National University, Seoul 08826, Korea.
  • 3Research Institute for Veterinary Science, College of Veterinary Medicine and BK21 for Veterinary Science, Seoul National University, Seoul 08826, Korea.

Abstract

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

Keyword

Staphylococcus aureus; bovine mastitis; molecular epidemiology; molecular prophage typing; rpoB sequence typing

MeSH Terms

Agriculture
Animals
Cattle
Computer Simulation
Dairy Products
Databases, Nucleic Acid
Drug Resistance, Microbial
Female
Foodborne Diseases
Humans
Mastitis, Bovine*
Methods
Molecular Epidemiology
Prophages*
Staphylococcus aureus*
Staphylococcus*

Figure

  • Fig. 1 Phylogenetic analysis of the complete rpoB sequences of 90 representative rpoB sequence types and consensus sequence of Staphylococcus aureus. The phylogenetic tree was constructed by using the neighbor-joining method (p-distance and 1,000 bootstrapping replicates) with MEGA software (ver. 7) [23]. The bovine isolates analyzed in this study are marked with closed circles.

  • Fig. 2 Amplification of terminase large subunit genes of prophage groups 1, 2, 3, 6, 7, 8, 9, and 10. Amplicons of prophage groups 5 and 12 are not shown because positive bovine isolates and human strains were not available. Lane M, 1000 bp size marker; Lane 1, prophage group 1 (1585 bp); Lane 2, prophage group 2 (1212 bp); Lane 3, prophage group 3 (813 bp); Lane 4, prophage group 6 (575 bp); Lane 5, prophage group 7 (895 bp); Lane 6, prophage group 8 (1116 bp); Lane 7, prophage group 9 (1223 bp); Lane 8, prophage group 10 (1769 bp).

  • Fig. 3 Evolution of Staphylococcus aureus on dairy farms through the acquisition of temperate phages. The first, second, and third numbers of each isolate's name are identifiers of the farm, individual cow, and udder, respectively. mPPT, molecular prophage type; + pp, acquisition of prophage.

  • Fig. 4 Virulence genes of prophage groups 3 and 7. The staphylokinase (A) and chemotaxis inhibiting (B) genes were detected by polymerase chain reaction. (A) Lane M, 100 bp size marker; Lane 1, PMB 81-1; Lane 2, PMB 232-1; Lane 3, PMB 67-1; Lane 4, PMB 177-1; Lane 5, PMB 188-1; Lane 6, PMB 196-1; Lane 7, PMB 208-1. (B) Lane M, 100 bp size marker; Lane 1, PMB 67-1; Lane 2, PMB 177-1; Lane 3, PMB 188-1; Lane 4, PMB 196-1; Lane 5, PMB 208-1.


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