Does ampicillin resistance or clade type determine GI colonization by E. faecium?

描述(申请人提供)：粪肠球菌(EFM)在过去引起的感染很少，但近年来显著增加，包括心内膜炎和其他严重感染，其中一些是医学上无法治疗的(例如，医院内EFM的80%是万古霉素耐药(VRE)，几乎所有的都是氨苄西林耐药，传统药物的选择)。这导致了EFM被归入似乎没有ESKAPE的“坏虫子”一类。EFM从“共生”转变为“致病”的原因尚不清楚，但已被归咎于抗生素耐药性、可移动的遗传元件和/或增加致病性或适合性的基因，因为这些基因更常见于EFM克隆复合体，几乎所有暴发/医院相关(HA)分离株都是由这些基因引起的。我们最近的基因组比较表明，EFM由两个不同的分支组成，它们的核心基因序列相差&gt；4%。几乎所有的HA EFM都属于一个(HA)分支，而几乎所有来自健康志愿者组的社区相关(CA)分离株都属于另一个(CA)分支；这种核心基因差异在粪肠球菌中没有观察到，尽管它也有HA克隆复合体。我们还发现，pbp5是编码耐氨苄西林的Ha-Clade菌株的核心基因，与CA-clade分离株的pBP5-S(氨苄西林最低抑菌浓度)相差约5%。我们的分子时钟估计表明，这两个分支在现代抗生素时代(30万~1100万年)之前很久就出现了分歧。因此，最近院内EFM的增加是由于CA被HA菌株所取代，而不是像人们所说的那样，是社区优势(CA)菌株中最近的进化。然而，目前尚不清楚是什么原因导致了这种替换，也不清楚为什么在健康志愿者中很少看到HA菌株，也不清楚HA分离株是否更具致病性。也许CA菌株实际上定植得更好，这解释了它们在社区粪便中的巨大优势，也可能仅仅是HA分支的抗生素耐药性使其能够战胜CA分离株，生长到高密度，然后在医疗保健环境中引起感染。这两个分支之间的内在区别是对氨苄西林的耐药性；值得注意的是，在全球医院VRE出现之前，HA分支氨苄西林耐药菌株(也是高度耐头孢菌素的菌株)。这一提议的假设是，核心基因组，甚至可能完全是HA分支的pbp5-R，已经允许该分支在接受β-内酰胺类药物的患者中取代CA菌株。在目标1中，我们将研究哪一种定植效果更好：HA-clade EFM菌株和CA-Clade EFM菌株，然后在小鼠胃肠道定植模型中确定β-内酰胺类药物对HA-Clade分离株(PBP5-R)对CA分离株(PBP5-S)定植的促进作用。在目标2中，我们将通过在CA和HA菌株之间交换pbp5-S和-R来研究pbp5等位基因在β-内酰胺类药物使用中EFM定植中的特定作用。这一结果可能为预防感染开辟新的途径，或者，如果HA菌株即使在没有β-内酰胺类抗生素的情况下也比CA菌株更好地定植，那么未来可能会寻找新的途径来揭示其他核心和/或获得性基因的贡献。

DESCRIPTION (provided by applicant): Enterococcus faecium (Efm) caused very few infections in the past but these have increased markedly in recent years, including endocarditis and other serious infections, some of which are medically untreatable (e.g., >80% of nosocomial Efm are vancomycin resistant (VRE), almost all of which are ampicillin resistant, the traditional drugs of choice). This led to Efm's inclusion in the group of "bad bugs" from which we seem to have "no ESKAPE". The factors behind the shift of Efm from "commensal" to "pathogenic" are not well understood but antibiotic resistance, mobile genetic elements and/or genes conferring increased pathogenicity or fitness have been blamed, since such genes are more commonly found in the Efm clonal complexes that account for almost all outbreak/nosocomial (hospital-associated (HA)) isolates. Our recent genomic comparisons showed that the species Efm consists of 2 distinct clades whose core gene sequences differ by >4%. Almost all HA Efm belong to one (HA) clade whereas almost all community-associated (CA) isolates from healthy volunteers group in the other (CA) clade; such core gene differences are not observed in E. faecalis although it too has HA clonal complexes. We also found that pbp5 is a core gene that encodes, in HA-clade strains, PBP5-R (ampicillin resistance) and varies from PBP5-S (ampicillin MICs <4) of CA-clade isolates by ~5%. Our molecular clock estimates indicate that the 2 clades diverged long before (300,000 ~ e1 million years) the modern antibiotic era. Thus, the recent increase of nosocomial Efm is due to replacement of CA by HA strains, rather than, as had been suggested, recent evolution within community-predominant (CA) strains. However, it is not clear what explains this replacement, why HA strains are seldom seen in healthy volunteers nor if HA isolates are more pathogenic. Perhaps CA strains actually colonize better, explaining their vast predominance in community feces, and perhaps it is simply the antibiotic resistance of the HA clade that allows its isolates to overcome CA isolates, grow to high density and then cause infections in the healthcare setting. An inherent difference between these 2 clades is resistance to ampicillin; of note, HA-clade ampicillin-resistant strains (which are also highly cephalosporin resistant) preceded nosocomial VRE emergence worldwide. The hypothesis of this proposal is that the core genome, and perhaps entirely pbp5-R of the HA clade, has allowed this clade to replace CA strains in patients receiving ?-lactams. In Aim 1, we will investigate which colonize better: HA-clade Efm strains or CA-clade Efm strains, and then determine the impact of ?-lactam use on promoting colonization by HA-clade isolates (PBP5-R) over CA isolates (PBP5-S) in the mouse GI colonization model. In Aim 2, we will examine the specific role of pbp5 alleles in Efm colonization during ?-lactam use by swapping pbp5-S and -R between CA and HA strains. The results could open new avenues for preventing infection or, if HA strains colonize better than CA ones even without ?-lactam antibiotics, new avenues to pursue in the future to unravel the contributions of other core and/or acquired genes.