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Identification of Staphylococcal Superantigens in Coagulase Negative Staphylococci from Bovine Intramammary Gland Infection for Submission of an USDA Afri Proposal

Institutions
Mississippi State University
Start date
2010
End date
2012
Objective
Staphylococcal enterotoxins (SEs), SE-like toxins (SEls) and toxic shock syndrome toxin-1 (TSST-1) belong to the family of superantigens (SAgs) and are important virulence factors of S. aureus. Currently, at least 20 different SAg genes have been reported. Some SAg genes in S. aureus are located on various kinds of mobile genetic element (MGE) including temperate bacteriophages, plasmids, or pathogenicity islands (SaPIs). Studies have shown that several SaPIs (SaPI1, SaPI2, and SaPIbov harboring SAg genes) can be excised and replicated by certain staphylococcal prophages including ?11 and 80?, and are transferred at high frequencies to several strains of S. aureus as well as to related species including coagulase-negative staphylococci (CNS) and Listeria monocytogenes in vitro. Our preliminary data demonstrated that SAg genes are also present in certain species of CNS isolates from bovine IMI suggesting certain species of CNS could be as nearly virulent as Staphylococcus aureus or potentially spread SAg genes to other pathogens. However, direct evidence supporting that CNS could be a possible source of SAgs is limited. Obtaining such knowledge is critical to developing more efficacious methods for treatment and prevention. To build upon these previous investigations, the objectives of the proposed study are: 1. Investigate the prevalence and distribution of CNS isolates from clinical samples that possess the genes for classical and/or newly described SAgs 2. Elucidate the mechanism by which SAgs are transferred.
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NON-TECHNICAL SUMMARY:
Coagulase-negative staphylococci (CNS) are a group of more than 30 species that cause intramammary infections (IMI) in bovine and endocarditis and urinary infections in humans. As a group, they are the most frequent isolates associated with bovine IMI and some clinical samples. However, the CNS are considered minor pathogens as they are not normally associated with clinical bovine mastitis. Compared to S. aureus, a relatively limited numbers of studies have been reported regarding the detection of SAg genes in CNS clinical isolates. Early study described the identification of CNS that produce TSST-1 or SEA or both from patients with toxic shock syndrome. However, several larger subsequent surveys in the 1980s and 1990s failed to detect genes encoding SAgs in CNS from human and veterinary specimens and concluded that SAg genes are generally not associated with CNS. However, our preliminary data demonstrated that SAg genes are also present in certain species of CNS isolates from bovine IMI suggesting certain species of CNS could be as nearly virulent as Staphylococcus aureus or potentially spread SAg genes to other pathogens. Survey studies on the relationship of CNS pathogens and genetic constructs for SAg production heretofore have been limited in scope, with either a limited number of samples collected or a limited number of SAg genes probed.

APPROACH:
All CNS isolates will be presumptively identified as CNS by primary phenotypic identification methods based on colony morphology, catalase, coagulase tube test, and Gram staining. Genomic DNA will be purified using Phenol extraction methods. A multiplex PCR method described by Omoe et al. (2005) will be used to amplify 19 different SAg genes (18 SE and TSST-1 genes). Phage will be induced from SAg positive CNS isolates using mitomycin C. Phage will be transduced to a recipient strains (SAg negative CNS) to identify transducibility.

PROGRESS: 2010/07 TO 2012/06
OUTPUTS: Staphylococci are a worldwide cause of human and animal infections including life-threatening cases of bacteraemia, wound infections, pyogenic lesions and mastitis. Enterotoxins produced by some staphylococcal species were recognized as causative agents of staphylococcal food poisoning (SFP), being also able to interrupt human and animal immune responses. Only enterotoxins produced by Staphylococcus aureus were as yet well characterized. Much less is known about enterotoxigenic potential of coagulase-negative species of genus Staphylococcus (CNS). The pathogenic role of CNS and their enterotoxigenicity in developing SFP has not been well established. Although it has been reported that enterotoxigenic CNS strains have been associated with human infections and food poisoning, most of research lacked a deeper insight into structure of elements encoding CNS enterotoxins. Recent studies provided us with strong evidence for the presence and localization of enterotoxin-coding elements in CNS genomes and production of enterotoxins. Thus, the importance of pathogenic potential of CNS has been highlighted in human and animal infections as well as food poisoning. PARTICIPANTS: Keun Seok Seo, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University TARGET AUDIENCES: Cattle farmers and Korean government are interested in developing a vaccine. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

PROGRESS: 2011/01/01 TO 2011/12/31
OUTPUTS: Coagulase-negative staphylococci (CNS) are a heterogeneous group that is composed of more than 30 Staphylococcus spp. CNS have been considered minor pathogens because they are not usually as virulent as the major pathogens such as S. aureus; yet as a group, the CNS are the most prevalent agents associated with bovine mastitis and some clinical samples. CNS may have a potential impact on the public health that CNS could cause food poisoning by producing staphylococcal superantigen (SAg) or spread SAg to other mastitis and food pathogens. However, direct evidence supporting that CNS could be a possible source of SAg is limited. Our study provides evidence that CNS does possess potential virulence factors such as SAg and could transfer these virulence factors to Staphylococcus aureus and other CNS. This is an important finding that we need to pay more attention to CNS and there is a need to investigate the role of CNS in several important staphylococcal diseases such as bovine mastitis. We submitted a research grant to Korean government and awarded a research fund for three years and are currently writing a scientific report on our findings. PARTICIPANTS: Keun Seok Seo, Department of Basic Sciences, Mississippi State University, College of Veterinary Medicine TARGET AUDIENCES: Cattle Farmers and Korean governement is interested in developing a vaccine. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
MISV-401110
Accession number
223945
Categories
Bacterial Pathogens