Modeling CRISPR to Preserve Antibiotics

描述(申请人提供)：肠球菌是70‘S和80’S医院获得性多药耐药感染的主要原因，目前仅次于葡萄球菌，占医院获得性感染的12.1%。具有抗生素耐药性的HAI每年增加超过200亿美元的额外医疗费用。因此，每年24.2亿美元以及约12,000人的死亡可归因于由肠球菌引起的HAI，使其成为主要的公共卫生问题。此外，他们现在正在将对最后一种药物万古霉素的耐药性传递给葡萄球菌。在初步数据中，我们发现肠球菌作为医院主要病原菌的出现与70‘S和80’S菌株的生长相吻合，这些菌株缺乏对噬菌体、质粒和其他可移动元件进入基因组的防御。我们的比较基因组学研究表明，在强大的抗生素压力下，没有CRISPR的肠球菌在获得抗生素耐药决定因素方面具有优势。它们对多种抗生素产生了抗药性，并在医院中增殖。恰逢失去了 CRISPR、噬菌体溶原化医院菌株，致病岛、转座子等质粒和可移动元件进入。因此，医院地方性肠球菌的基因组比共生菌株大25%以上，毒力特征和抗生素耐药性的协同趋同。在自然界中，大多数证据表明，噬菌体压力已经选择了在亿万年的进化中发生和维持CRISPR防御。然而，在过去的50年里，突然大量使用高水平的抗生素，已经选择了缺乏这种防御的菌株，据推测，获得新的抗生素耐药性的能力增强了。在这一探索性的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紧张关系，以导致该基因座的丢失/维持。这将为一个更大的项目提供初步数据，以确定在肠球菌种群中保存CRISPR的最佳抗生素使用参数，从而抑制抗生素耐药成分的转移，并保护日益稀缺的有效抗生素的货架期。

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.