Modeling CRISPR to Preserve Antibiotics

描述（由申请人提供）：肠球菌在70年代和80年代成为多重耐药医院获得性感染（HAI）的主要原因。它们目前仅次于葡萄球菌，占所有HAI的12.1%。抗生素耐药HAI每年增加超过200亿美元的额外医疗保健费用。因此，每年有24.2亿美元，沿着约12，000例死亡可归因于肠球菌引起的HAI，使其成为主要的公共卫生问题。此外，它们现在正在将对最后一线药物万古霉素的耐药性传递给葡萄球菌。在初步数据中，我们表明肠球菌作为主要医院病原体的出现与70年代和80年代缺乏针对噬菌体、质粒和其他移动的元件进入基因组的CRISPR防御的菌株的生长相一致。我们的比较基因组学研究表明，在强烈的抗生素压力下，没有CRISPR的肠球菌在获得抗生素耐药决定簇方面具有优势。它们对多种抗生素产生了耐药性，并在医院中扩散。与失去的 CRISPR使医院菌株溶原化;致病岛、转座子和其他质粒以及移动的元件进入。因此，医院流行性肠球菌的基因组比医院菌株大25%以上，具有毒力性状和抗生素耐药性的协同收敛。在自然界中，大多数证据表明，噬菌体压力选择了CRISPR防御的发生和维持。然而，在过去的50年里，高水平抗生素的突然和大规模应用选择了缺乏这种防御的菌株，可能增强了获得新抗生素耐药性的能力。在这个探索性的R21应用中，我们建议量化CRISPR基因座对质粒和噬菌体进入粪肠球菌的防御水平，并使用不同的参数来模拟抗生素选择和噬菌体选择之间的张力，以丢失/维持该基因座。这将为一个更大的项目提供初步数据，以确定在肠球菌群体中保留CRISPR的最佳抗生素使用参数，从而抑制抗生素耐药元件的转移，并保留日益稀缺的有效抗生素的保质期。

DESCRIPTION (provided by applicant): The enterococci emerged as leading causes of multidrug resistant hospital acquired infection (HAI) in the 70's and 80's. They currently rank second only to the staphylococci, causing 12.1% of all HAI. Antibiotic resistant HAI add over $ 20 Billion annually in excess health care costs. Therefore $ 2.42 Billion annually, along with ~ 12,000 deaths are ascribable to HAI caused by enterococci, making it a leading public health concern. Moreover, they are now transmitting resistance to the last line drug, vancomycin, to the staphylococci. In preliminary data, we showed that the emergence of enterococci as leading hospital pathogens coincided with the outgrowth of strains in the 70's and 80's that lacked CRISPR defense against phage, plasmid and other mobile element entry into the genome. Our comparative genomics study indicated that under intense antibiotic pressure, enterococci without CRISPR were at an advantage for acquiring antibiotic resistance determinants. They became resistant to multiple antibiotics and proliferated in hospitals. Coinciding with the loss of CRISPR, phages lysogenized the hospital strains; and pathogenicity islands, transposons and other plasmids and mobile elements entered. As a result, hospital endemic enterococci have genomes over 25% larger than commensal strains, with a synergistic convergence of virulence traits and antibiotic resistances. In nature, most evidence indicates that phage pressure has selected for the occurrence and maintenance of the CRISPR defense over eons of evolution. However, the sudden and massive application of high levels of antibiotics in the last 50 years has selected for strains lacking that defense, with presumably enhanced ability to acquire new antibiotic resistances. In this exploratory R21 application, we propose to quantify the level of defense against plasmid and phage entry into Enterococcus faecalis accorded by the CRISPR locus, and to model using varying parameters, the tension between antibiotic selection and phage selection for the loss/maintenance of this locus. This will provide the preliminary data for a larger program to identify the optimum antibiotic use parameters for preserving CRISPR in the enterococcal population, thereby inhibiting transfer of antibiotic resistant elements, and preserving the shelf life of increasingly scarce, effective antibiotics.