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)是牛乳房中最常见的分离菌，其毒力特征尚未得到很好的报道。我们目前已经鉴定了9株抑制革兰氏阳性菌体外生长的CNS菌株，特别是金黄色葡萄球菌，并预计会鉴定出更多。这种抑制作用可能是由细菌素引起的。在一个给定的菌株中存在这些细菌素的多个变种，每个变种都有相应的免疫基因，以保护生产菌株免受其自己的杀菌/抑菌产品的影响。这项研究计划的目标有三个。 1)利用9株体外抑制性CNS分离株与非抑制性分离株的全基因组序列，在电子计算机中鉴定具有已知细菌素或主要病原体免疫活性的候选基因。已发现细菌素的流行率将在所有500个已测序的中枢神经系统基因组中得到确认。此外，细菌素基因在这些分离株中的存在将被确认为表型。下一步，将使用定向突变来确认已识别的基因在观察到的对主要病原体的抑制中的作用。最后，通过对部分菌株进行随机转座子诱变筛选，将发现新的抑制基因和机制。 2)通过定量聚合酶链式反应研究细菌素及其免疫相关基因在中枢神经系统分离株和金黄色葡萄球菌的纯培养和共培养中的表达，以评价该菌株作为益生菌的潜力。由于细菌基因的表达在体内可能受到不同的调节，我们将把我们的体外细菌素表达分析扩展到乳房炎的小鼠模型。此外，在细菌素启动子和荧光报告基因构建的辅助下，活体显微镜(IVM)将用于成像受感染的乳腺组织中的基因表达。 3)将使用小鼠和牛乳房炎模型来评估CNS菌株或蛋白质的体内抑制效果，以表征抑制性CNS菌株和主要病原体如何在软组织感染的背景下相互作用。通过乳房内感染CNS毒株的小鼠将使用全身成像和IVM来检测感染的持久性，以实时可视化和表征活体动物中的宿主免疫反应、组织炎症和细菌动力学。接下来，与CNS和金黄色葡萄球菌分离株的混合感染将使我们了解两个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菌株和缺乏候选细菌素的特定突变菌株的抑制效果，将证实已鉴定的细菌素在CNS对主要病原体的抑制中的作用。将研究中枢神经系统感染在奶牛乳房中的持久性，并将在牛乳房炎模型中对保护性细菌素的表达和生产进行体内量化。

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.