title: Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.
abstract: BACKGROUND
  Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC.
  
  
  RESULTS
  The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster.
  
  
  CONCLUSIONS
  The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.
authors.affiliation: Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse-Faculty, University of Berne, Berne, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Raemy
authors.forename: Mireille
authors.initials: M
authors.lastname: Meylan
authors.forename: Simona
authors.initials: S
authors.lastname: Casati
authors.forename: Valeria
authors.initials: V
authors.lastname: Gaia
authors.forename: Beat
authors.initials: B
authors.lastname: Berchtold
authors.forename: Renate
authors.initials: R
authors.lastname: Boss
authors.forename: Anja
authors.initials: A
authors.lastname: Wyder
authors.forename: Hans U
authors.initials: HU
authors.lastname: Graber
mesh_terms: D000818:Animals; D002417:Cattle; D005260:Female; D008414:Mastitis, Bovine / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013290:Streptococcal Infections / Q000382:microbiology / Q000662:veterinary*; D013291:Streptococcus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification