Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

链球菌性乳汁过少症亚种。停乳链球菌（SDSD），一个兰斯菲尔德C组链球菌（GCS），是牛乳腺炎的常见原因。这种高度流行的疾病是乳业最昂贵的疾病。粘附和生物膜产生是链球菌的重要因素发病。我们之前已经描述了SDSD分离物在人细胞中的粘附和内在化，现在我们描述了这种细菌的生物膜生产能力。在这项工作中，我们结合了微生物学，成像和计算方法来评估SDSD分离物的生物膜生产能力。评估生物膜生产者中生物膜调节蛋白BrpA同源物的存在；并预测BrpA样蛋白的结构模型及其与假定抑制剂的结合。我们的结果表明，SDSD分离物在非生物表面上形成生物膜，例如玻璃（亲水）和聚苯乙烯（疏水），在玻璃中观察到最强的生物膜形成。蛋白酶K和胰蛋白酶处理后，生物膜的分散证实了这种能力主要与蛋白质细胞外基质有关。还通过共聚焦激光扫描显微镜（CLSM）和扫描电子显微镜（SEM）确认了SDSD分离物中的生物膜形成。在SEM观察下，VSD16分离物在生物膜生长期间形成细胞聚集体，而VSD9和VSD10形成光滑且膜状的层。我们证明brpA在SDSD生物膜生产菌株中存在并表达了类似的基因，其表达水平与生物膜生产能力相关，与生物膜生长相比，在浮游生物生长的指数后期，其表达水平更高。Fisetin是已知的生物膜抑制剂和推定的BrpA结合分子，在测试浓度下可显着抑制SDSD分离物形成的生物膜，但不影响浮游生物的生长。同源性建模用于预测BrpA样蛋白的3D结构。使用高通量虚拟筛选和分子对接，我们选择了五个对蛋白质的疏水性裂口具有强结合亲和力的配体分子，使其成为SDSD BrpA样蛋白质的潜在抑制剂候选物。