Genotypes And Phenotypes Of Staphylococci On Selected Dairy Farms In Vermont

The genus Staphylococcus contains at least 47 species and 23 subspecies. Bacteria in this genus are ubiquitous; many are commensals on human and animal skin and can be opportunistic pathogens. In dairy cattle, staphylococci are the leading cause of intramammary infections (IMI) and mastitis. Mastitis is the inflammation of the mammary gland, and is one of the leading infectious diseases causing production losses in the dairy industry. Based on the ability to clot blood plasma in vitro, members of the genus can be divided into two groups: coagulase positive staphylococci (CPS) and coagulase negative staphylococci (CNS). In the dairy industry, Staphylococcus aureus is the most common CPS causing mastitis and is considered a major mastitis pathogen compared to the CNS, which as a group have been described as minor mastitis pathogens. The CNS species are increasingly recognized as an important cause of bovine mastitis, although the relative role of some species is still uncertain. Our understanding of the local and global epidemiology of CNS mastitis is improving with application of more accurate DNA sequence-based species identification methods and techniques to discriminate between strains within species. These factors have led to a shift in perspective, with the CNS being recognized as a heterogeneous group where some species are more important than others in bovine mastitis. The major goals of this thesis were to describe Staphylococcus mastitis epidemiology, and to identify phenotypes that may contribute to persistence in various niches on selected dairy farms in Vermont. We conducted 2 field studies on 2 groups of farms in Vermont. In the first study, we collected S. aureus isolates from bulk tank milk of 44 certified organic dairy farms. In the second field study, we completed quarter milk, cow skin, and environmental sampling of 5 herds that make farmstead cheeses. In both studies, we used non-selective and selective agar medium to isolate staphylococci from the farm sources. From these studies, we collected 1,853 Staphylococcus spp. isolates. We used PCR-amplicon sequence-based species identification to describe Staphylococcus species diversity on these selected Vermont dairy farms. S. aureus isolates were straintyped using an established Multilocus Sequence Typing (MLST) scheme. A novel MLST scheme was developed to investigate the molecular epidemiology of S. chromogenes, one of the leading CNS species causing bovine mastitis in this and other studies. We also evaluated antibiotic resistance and biofilm formation phenotypes and genotypes of staphylococci to test the hypothesis that these phenotypes may be associated with strain types. In the study of organic dairy farms, 20 S. aureus strain types (STs) were identified, including ten novel STs. The majority of STs belonged to lineages or clonal complexes (CCs) previously identified as cattle adapted (e.g. CC97 and CC151). Associations between ST and carriage of beta-lactam resistance and biofilm forming capacity were identified among the S. aureus isolates from these farms. In the 5-herd study, a total of 27 different staphylococci species were identified from various niches including humans, but only five species; S. chromogenes, S. aureus, S. haemolyticus, S. simulans, and S. xylosus were commonly identified to cause IMI. S. aureus and S. chromogenes strain types were niche specific. ii ACKNOWLEDGEMENTS This dissertation wouldn’t have been finished without the help from a lot of people over the past five years. First and foremost, I would like to acknowledge God for perfect health and wisdom. Secondly, I thank my advisor John Barlow for believing and accepting me to work in his laboratory even when he didn’t know me so much. I acknowledge his mentorship and desire to make me a better scientist who I am today, evidenced by discussions we have heard about data interpretation, statistics, reviewing manuscripts, grant writing, and availing resources for me to go to scientific conferences. I appreciate all the past and present undergraduate and graduate students, postdocs who have helped me in one way or another. I can’t forget our former laboratory technician Amanda Ochoa who helped me get acclimatized to the laboratory in my initial years, as well as field sample collection. I also extend my sincere gratitude to my thesis committee members for their immense advice. I acknowledge Dr. George Sensabaugh who was influential in the development of the novel S. chromogenes MLST scheme. Last but not least, I extend my appreciation to my family, UVM office of international education, and friends for their immeasurable academic, emotional, social, and spiritual support during my five-year tenure in graduate school.