Characteristics of coagulase-negative staphylococci influencing virulence, antimicrobial resistance and inhibition of other mastitis pathogens

凝固酶阴性葡萄球菌（CNS）分离株是最常从牛乳房中分离的细菌，其毒力特征尚未得到很好的记录。我们目前已经确定了9个CNS分离株，抑制体外生长的革兰氏阳性菌，特别是金黄色葡萄球菌，并期望确定更多。这种抑制可能是由细菌素赋予的。这些细菌素的多种变体存在于给定菌株中，每种变体都具有相应的免疫基因，这些免疫基因保护生产菌株免受其自身的杀菌/抑菌产物的影响。该研究项目的目标有三个方面。 1)与非抑制性分离株相比，将使用9种体外抑制性CNS分离株生成的全基因组序列通过计算机模拟鉴定具有已知细菌素或主要病原体免疫活性的候选基因。将在所有500个测序的CNS基因组中证实发现的细菌素的流行。此外，将在表型上证实这些分离株中存在杆菌素基因。接下来，将使用靶向诱变来确认鉴定的基因在观察到的主要病原体抑制中的作用。最后，新的抑制基因和机制将确定随机转座子诱变筛选一些选择的菌株。 2)将通过qPCR研究CNS分离株和链球菌的纯培养物和共培养物中细菌素和相应免疫基因的表达。金黄色葡萄球菌，以评估菌株作为益生菌的潜力。由于细菌基因表达可能在体内受到差异调节，我们将我们的体外细菌素表达测定扩展到乳腺炎的小鼠模型中。此外，活体显微镜（IVM）将用于感染乳腺组织中的基因表达成像，借助细菌素启动子和荧光报告基因构建体。 3)将使用鼠和牛乳腺炎模型评估CNS菌株或蛋白质的体内抑制作用，以表征抑制性CNS菌株和主要病原体在软组织感染背景下如何相互作用。将使用全身成像和IVM分析乳房内感染CNS菌株的小鼠的感染持续性，以实时可视化和表征活体动物中的宿主免疫应答、组织炎症和细菌动力学。其次是CNS和S.金黄色葡萄球菌分离株将使我们了解两种细菌菌株如何在体内相互作用、定位和分离。最后，通过直接比较野生型CNS菌株与候选细菌素缺陷的特定突变菌株的抑制作用，将证实所鉴定的细菌素在CNS抑制主要病原体中的作用。将研究奶牛乳房中CNS感染的持续性，并在奶牛乳房炎模型中体内定量保护性细菌素的表达和产生。

Virulence characteristics of coagulase-negative staphylococci (CNS) isolates, the most frequently isolated bacteria from bovine udders, have not been well documented. We have currently identified 9 CNS isolates that inhibit in vitro growth of Gram-positive bacteria, particularly Staphylococcus aureus, and expect to identify more. This inhibition is likely conferred by bacteriocins. Multiple variants of these bacteriocins exist within a given strain, each with corresponding immunity genes that protect the producing strains from their own bacteriocidal/bacteriostatic products. Objectives of this research program are threefold. 1) Candidate genes with known bacteriocin or immunity activity of major pathogens will be identified in silico using whole-genome sequences generated from the 9 in vitro inhibitory CNS isolates in comparison with non-inhibitory isolates. Prevalence of discovered bacteriocins will be confirmed in all 500 sequenced CNS genomes. Furthermore, the presence of bacterocin genes in these isolates will be confirmed phenotypically. Next, targeted mutagenesis will be used to confirm the role of identified genes in the observed inhibition of major pathogens. Finally, novel inhibitory genes and mechanisms will be identified by random transposon mutagenesis screens on some select isolates. 2) Expression of bacteriocins and corresponding immunity genes will be studied by qPCR in pure and co-cultures of CNS isolates and S. aureus to evaluate the potential of the strain as a probiotic. As bacterial gene expression may be differentially regulated in vivo, we will extend our in vitro bacteriocin expression assays into a murine model of mastitis. Additionally, intravital microscopy (IVM) will be used to image gene expression in infected mammary tissue, with the aid of bacteriocin promotor and fluorescent reporter gene constructs. 3) The in vivo inhibitory effect of CNS strains or proteins will be assessed using both murine and bovine models of mastitis to characterize how inhibitory CNS strains and major pathogens interact within the context of a soft-tissue infection. Mice infected intramammarily with CNS strains will be assayed for persistence of infection using whole-body imaging and IVM to visualize and characterize, in real-time, host immune response, tissue inflammation, and bacterial dynamics in live animals. Next, coinfection with CNS and S. aureus isolates will allow us to understand how 2 bacterial strains interact, localize and segregate in vivo. Finally, the role of identified bacteriocins in inhibition of major pathogens by CNS will be confirmed by directly comparing the inhibitory effect of wildtype CNS strains to specific mutant strains deficient for candidate bacteriocins. Persistence of CNS infections in cow udders will be studied and expression and production of protective bacteriocins will be quantified in vivo in a bovine mastitis model.