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）分离株的毒力特征，最常从牛乳房分离的细菌，尚未得到很好的记录。我们目前已经鉴定出9株CNS分离物，它们能抑制革兰氏阳性细菌的体外生长，特别是金黄色葡萄球菌，并期望鉴定出更多的CNS分离物。这种抑制作用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的菌株中，每种变体都具有相应的免疫基因，以保护产生菌株免受其自身杀菌/抑菌产物的影响。本研究计划的目标有三个方面。***1)利用9株体外抑制CNS分离株的全基因组序列与非抑制CNS分离株进行比较，在计算机上鉴定具有已知细菌素或主要病原体免疫活性的候选基因。发现的细菌素的流行将在所有500个测序的中枢神经系统基因组中得到证实。此外，在这些分离株中存在杆菌素基因将得到表型证实。接下来，靶向诱变将用于确认已鉴定基因在观察到的主要病原体抑制中的作用。最后，通过随机转座子诱变筛选筛选出新的抑制基因和机制。***2)将采用qPCR技术研究CNS分离株和金黄色葡萄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，以评价该菌株作为益生菌的潜力。由于细菌基因表达在体内可能受到差异调节，我们将把体外细菌素表达试验扩展到小鼠乳腺炎模型中。此外，在细菌素启动子和荧光报告基因构建的帮助下，活体显微镜（IVM）将用于成像感染乳腺组织中的基因表达。***3) CNS菌株或蛋白质的体内抑制作用将通过小鼠和牛乳腺炎模型进行评估，以表征在软组织感染背景下，抑制性CNS菌株和主要病原体如何相互作用。将使用全身成像和IVM检测感染CNS菌株的小鼠乳内感染的持久性，实时观察和表征活体动物的宿主免疫反应、组织炎症和细菌动力学。接下来，CNS和金黄色葡萄球菌分离株的联合感染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菌株与缺乏候选细菌素的特定突变菌株的抑制效果，证实鉴定出的细菌素在CNS对主要病原体的抑制作用。奶牛乳房中枢神经系统感染的持续性将被研究，保护性细菌素的表达和产生将在牛乳腺炎模型中被量化

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.**