Identification and Further Characterization of Streptococcus uberis and Streptococcus parauberis Isolated from Bovine Milk Samples

Abstract

In the present study 130 S. uberis strains and one S. parauberis strain isolated from bovine milk samples of 59 different farms of various locations in Hesse, Germany, were comparatively investigated together with four reference strains of both species for cultural, biochemical and serological properties. The S. uberis and S. parauberis strains generally did not grow on media specific for enterococci, whereas after cultivation on blood agar both species were alpha -or non-hemolytic. All S. uberis strains produced the enzyme beta-D-glucuronidase, the S. parauberis were negative for this enzyme. Serogrouping revealed a positive reaction for 42 S. uberis strains and one S. parauberis reference strain with group E specific antiserum and for some strains with specific antisera of the serological groups P, U and A. However, most of the S. uberis and two S. parauberis strains were non-groupable. Some of the S. uberis strains agglutinated with the lectin of Helix pomatia, and showed a CAMP-like reaction of complete hemolysis in the zone of staphylococcal beta-toxin. The S. uberis and S. parauberis strains displayed a generally hydrophilic surface. This could be demonstrated by determination of growth patterns in fluid media and soft agar and by salt aggregation tests. Antibiotic resistances could be observed for most of the strains for colistin, sulphamethoxazole/trimethoprim, tetracycline, clindamycin, erythromycin and gentamicin. All strains were sensitive to penicillin-G.

The S. uberis and S. parauberis isolates were further analyzed by molecular methods. This was performed by amplification of the gene encoding the S. uberis and S. parauberis 16S rRNA gene by polymerase chain reaction and subsequent digestion of the respective gene with the restriction enzymes RsaI and AvaII. The species identity could additionally be confirmed by amplifying species-specific parts of the genes encoding the 16S rRNA, the 23S rRNA and of the 16S-23S rDNA intergenic spacer region. For CAMP positive S. uberis strains the gene cfu could be amplified using two different primer pairs. In addition the gene for the potential virulence factor streptokinase, the skc/pauA gene, could be amplified for 128 of the 132 S. uberis but not for S. parauberis.