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Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections

Andreas Raemy1, Mireille Meylan1*, Simona Casati2, Valeria Gaia2, Beat Berchtold1, Renate Boss3, Anja Wyder1 and Hans U Graber3

Author Affiliations

1 Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse-Faculty, University of Berne, Bremgartenstrasse 109a, PO Box 8466, 3001 Berne, Switzerland

2 Istituto Cantonale di Microbiologia, Via Mirasole 22a, 6500 Bellinzona, Switzerland

3 Agroscope Liebefeld-Posieux Research Station ALP, Schwarzenburgstrasse 161, 3003 Berne, Switzerland

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Acta Veterinaria Scandinavica 2013, 55:53  doi:10.1186/1751-0147-55-53

Published: 18 July 2013

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.

Keywords:
Mastitis; Cattle; Streptococci; Identification; Mass spectroscopy