Clinic for Ruminants, Department of Clinical Veterinary Medicine, Vetsuisse-Faculty, University of Berne, Berne, Switzerland.
Acta Vet Scand. 2013 Jul 18;55(1):53. doi: 10.1186/1751-0147-55-53.
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.
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.
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.
链球菌属和其他革兰氏阳性、触酶阴性球菌（PNC）形成了一个庞大的微生物群体，可在患有乳腺炎的奶牛的牛奶中发现。最常见的 PNC 乳腺炎病原体（主要病原体）是无乳链球菌、停乳链球菌和乳房链球菌。其余的 PNC 包括少数次要病原体和一大组非病原体。需要改进方法来准确识别和区分 PNC。从患有乳腺炎的奶牛中收集了 151 株 PNC，并通过 16S rRNA 测序作为参考方法进行了明确鉴定。九项表型微生物学测试（α-溶血、CAMP 反应、七叶灵水解、在卡那霉素七叶灵叠氮琼脂和氯化钠琼脂上生长、菊糖发酵、马尿酸水解、亮氨酸氨基肽酶和吡咯烷酮肽酶活性）、用于三种主要病原体的多重 PCR（无乳链球菌、停乳链球菌和乳房链球菌的靶基因：pauA、16S rRNA 和 sklA3）和基质辅助激光解吸电离飞行时间（ MALDI-TOF MS）使用质谱法评估了三种临床上最相关的 PNC 的诊断和鉴别。
将 9 项表型测试结果结合起来，正确鉴定一株无乳链球菌、停乳链球菌和乳房链球菌的概率分别为 92%、90%和 100%。应用多重 PCR，三种主要病原体的所有菌株均被正确鉴定，未出现假阳性结果。MALDI-TOF MS 可正确鉴定所有无乳链球菌和乳房链球菌菌株。在停乳链球菌的情况下，亚种水平观察到一些可变性，但所有菌株均分配到一个单一的聚类中。
本研究结果表明，通过使用菌落形态、溶血类型和触酶反应以及针对这 3 种病原体的特异性引物的多重 PCR，可以获得对临床上最相关的 PNC（无乳链球菌、乳房链球菌和停乳链球菌）的可靠鉴定。MALDI-TOF MS 是一种快速的方法，显示出有希望的结果，尽管在亚种水平上鉴定停乳链球菌尚不完全令人满意。
