Morandi S, Cremonesi P, Capra E, Silvetti T, Decimo M, Bianchini V, Alves A C, Vargas A C, Costa G M, Ribeiro M G, Brasca M
Institute of Sciences of Food Production, Italian National Research Council, 20133 Milan, Italy.
Institute of Agricultural Biology and Biotechnology, National Research Council, 26900 Lodi, Italy.
J Dairy Sci. 2016 Aug;99(8):6436-6445. doi: 10.3168/jds.2016-10900. Epub 2016 May 26.
Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd.
由原壁菌引起的牛乳腺炎是一个严重且复杂的问题，给乳制品行业造成了巨大的经济损失。本研究的主要目的是在基因水平上鉴定和表征不同的原壁菌菌株，并提供关于原壁菌抗生素耐药性的最完整数据。该研究涉及从意大利（13株）和巴西（33株）患乳腺炎牛奶中分离出的46株菌株。这些菌株通过多重PCR和单链构象多态性分析进行鉴定，并通过随机扩增多态性DNA（RAPD）-PCR进行表征。此外，还评估了生物膜的产生和抗生素敏感性。42株菌株鉴定为魏氏原壁菌基因型2，而4株分离株可能属于一个潜在的新原壁菌物种。用3种引物（M13、OPA-4和OPA-18）进行的RAPD-PCR显示，分离株之间存在显著的异质性，并根据物种和地理来源对菌株进行了分组。生物膜的产生具有物种依赖性，魏氏原壁菌基因型2菌株被归类为强生物膜生产者。体外抗生素敏感性试验表明，原壁菌菌株对氨基糖苷类抗菌药物敏感；在硫酸多粘菌素、庆大霉素和奈替米星的情况下，观察到对原壁菌菌株的活性最高（100%的敏感菌株）。有趣的是，所有意大利魏氏原壁菌基因型2菌株的最低抑菌浓度值均低于巴西菌株。乳酸链球菌素比溶菌酶和山梨酸钾更有效，可抑制31%的菌株。本研究获得的结果证实，RAPD-PCR是一种快速、廉价且具有高度区分性的工具，可用于原壁菌菌株的表征，并可为更好地了解奶牛群中原壁菌乳腺炎提供良好的科学贡献。
