Detection of DNA sequences distinguishing two closely related genomes of Staphylococcus aureus from subclinical versus gangrenous ewe mastitis strains
Abstract number: p1701
Chevalier N., Huard C., Thiery R., Vautor E.
Staphylococcus aureus is a common cause of mastitis in dairy sheep. The severity of mastitis ranges from subclinical to gangrenous forms. Subclinical mastitis is an inflammation that is not readily detected clinically whereas gangrenous form is an acute necrotizing mastitis. With the ain to find genetic markers or virulence factors that are only present in gangrenous strains a suppression substractive hybridisation (SSH) method was used in the present study to compare two strains of S. aureus respectively recovered from subclinical or gangrenous mastitis in the same dairy sheep herd.
42 ewes were held in the investigated farm. The subclinical strain was recovered in January 2002 from the milk of 6 ewes. The gangrenous strain was recovered in December 2002 from an primipare dairy sheep that subsequently died from this acute mastitis. DNA extracted from the strains were first compared by pulsed field gel electrophoresis (PFGE). Then, SSH was performed by using DNA from the subclinical strain (driver), as described in a commercial kit (Clontech PCR-Select Bacterial Genome Substraction Kit).
Using PFGE, four band differences were found between the two strains. Two DNA fragments, presumably specific from the gangrenous strain were detected by SSH and sequenced: (i) a 262 bp (98% of homology with the sulfide quinone reductase contained in Orf 11 pathogenicity island of the MRSA 252 strain) (ii) 280 bp (98% homology with a gene coding a bacteriophage holine contained in the S. aureus N315 genome). Control PCR tests using primers designed from these specific gene candidates confirmed that they were only present in the S. aureus gangrenous strain.
According to Tenover et al. (1995), a 4 band difference using PFGE indicates that the strains may possibly be related genetically. Although genes classically involved in the virulence of S. aureus were not detected in the present study, two putative virulence factors were detected. The sulfide quinone reductase allows S. aureus to growth on sulfide (found in animal manure). The holine protein breaks the internal membrane of S. aureus to release daughter phages suggesting that a mechanism of horizontal gene transfer could have been mediated by bacteriophages and could explain the acquisition of virulence factors.
|Session name:||XXIst ISTH Congress|
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