Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci 噬菌体耐药性改变了抗生素介导的肠球菌肠道扩张
Anushila Chatterjee, ^("a "){ }^{\text {a }} Cydney N. Johnson, ^("a "){ }^{\text {a }} Phat Luong, ^("a "){ }^{\text {a }} Karthik Hullahalli, ^("b "){ }^{\text {b }} Sara W. McBride, ^("a "){ }^{\text {a }} Alyxandria M. Schubert, ^("c "){ }^{\text {c }} Kelli L. Palmer, ^("b "){ }^{\text {b }} Paul E. Carlson, Jr., ^("c "){ }^{\text {c }} (D) Breck A. Duerkop ^("a "){ }^{\text {a }} Anushila Chatterjee， ^("a "){ }^{\text {a }} Cydney N. Johnson， ^("a "){ }^{\text {a }} Phat Luong， ^("a "){ }^{\text {a }} Karthik Hullahalli， ^("b "){ }^{\text {b }} Sara W. McBride， ^("a "){ }^{\text {a }} Alyxandria M. Schubert， ^("c "){ }^{\text {c }} Kelli L. Palmer， ^("b "){ }^{\text {b }} Paul E. Carlson， Jr.， ^("c "){ }^{\text {c }} （D） Breck A. Duerkop ^("a "){ }^{\text {a }}^("a "){ }^{\text {a }} Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA ^("a "){ }^{\text {a }} 美国科罗拉多州奥罗拉市科罗拉多大学医学院免疫学和微生物学系^("b "){ }^{\text {b }} Department of Biological Sciences, University of Texas at Dallas, Richardson, Texas, USA ^("b "){ }^{\text {b }} 德克萨斯大学达拉斯分校生物科学系，美国德克萨斯州理查森^("c "){ }^{\text {c }} Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluations and Research, Food and Drug Administration, Silver Spring, Maryland, USA ^("c "){ }^{\text {c }} 美国马里兰州银泉市食品药品管理局生物制品评估和研究中心疫苗研究与审查办公室细菌、寄生虫和过敏原产品司
Abstract 抽象
Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against EE. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalis epa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment. 粪肠球菌是一种人类肠道致病菌，对许多抗生素（包括万古霉素）具有内在和获得性耐药性。大自然提供了多种多样且几乎未开发的细菌病毒或噬菌体（噬菌体），可用于对抗耐多药肠球菌感染。细菌噬菌体耐药性代表了实施噬菌体疗法的潜在障碍，强调了研究噬菌体耐药性出现的分子机制的重要性。使用一组 19 个具有嗜性 EE 的环境裂解噬菌体。粪便中，我们发现这些噬菌体需要肠球菌多糖抗原 （Epa） 才能进行生产性感染。Epa 是一种表面暴露的异聚糖，由保守基因和菌株特异性基因编码的酶合成。我们发现，暴露于噬菌体选择压力有利于培养物和肠道定植小鼠模型中非保守 EPA 基因的突变。尽管获得了噬菌体抗性，但 EPA 突变菌株表现出对细胞壁靶向抗生素的耐药性丧失。最后，我们表明粪肠球菌 epa 突变菌株缺乏肠道定植，在抗生素驱动的肠道菌群失调时无法扩大其种群，并且无法在出生后有效地传播给幼年小鼠。这项研究表明，噬菌体疗法可以与抗生素联合使用，以靶向菌群失调中的肠球菌。通过产生耐药性来逃避噬菌体治疗的肠球菌可能不太适合在肠道定植并对万古霉素敏感，从而防止它们在抗生素治疗期间过度生长。
Enterococci are Gram-positive commensal bacteria native to the intestinal tracts of animals, including humans (1). Under healthy conditions, enterococci exist as minority members of the microbiota in asymptomatic association with their host. However, upon antibiotic disruption of the intestinal bacterial community, enterococcal populations can flourish, resulting in elevated intestinal colonization (2, 3). As dominant members of the intestinal microbiota, enterococci can breach the intestinal barrier, leading to bloodstream infections (3). The pathogenic success of the enterococci is largely attributed to the development of multidrug resistance (MDR) traits, including the emergence of vancomycin-resistant enterococci (VRE). Enterococcus faecalis and Enterococcus faecium represent the species most commonly associated with vancomycin resistance. In the hospital, MDR enterococcal strains can be transmitted rapidly, leading to dangerous outbreaks that put immunocompromised patients at risk (4,5)(4,5). This is especially troubling as clinical VRE isolates that are resistant to recently intro- 肠球菌是原生于动物（包括人类）肠道的革兰氏阳性共生菌 （1）。在健康条件下，肠球菌作为微生物群的少数成员存在，与宿主无症状关联。然而，在抗生素破坏肠道细菌群落后，肠球菌种群可以大量繁殖，导致肠道定植增加 （2， 3）。作为肠道菌群的优势成员，肠球菌可以突破肠道屏障，导致血流感染 （3）。肠球菌的致病成功主要归因于多药耐药 （MDR） 性状的发展，包括万古霉素耐药肠球菌 （VRE） 的出现。粪肠球菌 和 屎肠球菌 是最常与万古霉素耐药相关的物种。在医院，耐多药肠球菌菌株可以迅速传播，导致危险的爆发，使免疫功能低下的患者处于危险 (4,5)(4,5) 之中。这尤其令人不安，因为临床 VRE 分离株对最近引入的
Received 30 January 2019 收稿日期 2019 年 1 月 30 日
Returned for modification 18 February 2019 2019 年 2 月 18 日返回修改
Accepted 25 March 2019 2019 年 3 月 25 日接受
Accepted manuscript posted online 1 April 2019 录用手稿于 2019 年 4 月 1 日在线发布
Published 21 May 2019 发布时间 21 May 2019
duced “last-line-of-defense” antibiotics have been discovered (6-10). With limited treatment options to combat the continuing rise of MDR enterococci, it is imperative to develop alternative therapeutic approaches in addition to conventional antibiotic therapy. 已经发现了所谓的“最后一道防线”抗生素 （6-10）。由于对抗耐多药肠球菌持续上升的治疗选择有限，因此除了常规抗生素治疗外，还必须开发替代治疗方法。
Bacteriophages (phages), viruses that infect bacteria, could be used for the eradication of difficult-to-treat E. faecalis and E. faecium infections. Many of these are obligate lytic phages belonging to the Siphoviridae and Myoviridae families of tailed double-stranded DNA phages (11). Current efforts in the development of phages as antienterococcal agents have focused on the treatment of systemic infections or surface-associated biofilms (12-14), although they may also be effective in decolonizing the intestines of individuals in a hospital setting. 噬菌体（噬菌体）是感染细菌的病毒，可用于根除难以治疗的粪肠球菌和屎肠球菌感染。其中许多是专性裂解噬菌体，属于有尾双链 DNA 噬菌体的 Siphoviridae 和 Myoviridae 家族 （11）。目前开发噬菌体作为抗肠球菌剂的努力集中在治疗全身感染或表面相关生物膜 （12-14），尽管它们也可能有效地在医院环境中对个体的肠道进行去定植。
The utility of phages as effective antienterococcal therapeutics relies on having a detailed understanding of phage infection mechanisms to understand how enterococci subvert phage infection through the development of resistance. To date, only a single membrane protein, PIP _("EF ")_{\text {EF }} (phage infection protein of E. faecalis), has been definitively identified as a phage receptor for EE. faecalis (15). The enterococcal polysaccharide antigen (Epa) is involved in phage adsorption to E. faecalis cells and may act as a phage receptor (16-18). Considering that at least a dozen well-characterized lytic enterococcal phages have the potential to be used for phage therapy (11), identification of receptors used by phages could allow for the generation of more-efficient phage cocktails to be used for the treatment of enterococcal infections. This is particularly important since phage therapies can employ the use of multivalent phage cocktails to limit the emergence of bacterial resistance (19,20)(19,20), and knowledge of phage receptors can lead to rational design of such cocktails. In addition, phages often target conserved components of the bacterial cell surface, which bacteria can mutate to subvert phage infection. If a phage receptor is essential to bacterial physiology, mutation often imposes a fitness cost (21-23). Therefore, therapeutic phages could be selected that force bacterial targets to trade a fitness benefit in return for phage resistance, making them less pathogenic and possibly more susceptible to current antimicrobials (21,24)(21,24). 噬菌体作为有效的抗肠球菌治疗剂的效用取决于对噬菌体感染机制的详细了解，以了解肠球菌如何通过产生耐药性来破坏噬菌体感染。迄今为止，只有一种膜蛋白 PIP _("EF ")_{\text {EF }} （粪肠球菌噬菌体感染蛋白）被明确鉴定为 EE .粪 （15）。肠球菌多糖抗原 （Epa） 参与噬菌体对粪肠球菌细胞的吸附，并可能充当噬菌体受体 （16-18）。考虑到至少有十几种特征明确的裂解肠球菌噬菌体有可能用于噬菌体治疗 （11），鉴定噬菌体使用的受体可以产生更有效的噬菌体混合物，用于治疗肠球菌感染。这一点尤其重要，因为噬菌体疗法可以利用多价噬菌体混合物来限制细菌耐药 (19,20)(19,20) 性的出现，并且对噬菌体受体的了解可以导致此类混合物的合理设计。此外，噬菌体通常以细菌细胞表面的保守成分为目标，细菌可以突变这些成分以破坏噬菌体感染。如果噬菌体受体对细菌生理学至关重要，那么突变通常会带来适应成本 （21-23）。因此，可以选择治疗性噬菌体，迫使细菌靶标以牺牲健身益处换取噬菌体抗性，使它们的致病性降低，并且可能更容易受到当前抗菌剂 (21,24)(21,24) 的影响。
The E. faecalis genome contains both broadly conserved and strain-variable epa genes (25). Using EE. faecalis and a collection of uncharacterized virulent phages, we identify genes located in the variable region of the epa locus to be critical for phage infection. Epa is directly involved in phage attachment to the bacterial surface. Exposure of EE. faecalis to certain phages, both in vitro and in the mouse intestine, selects for mutations in the epa locus, primarily in epa variable genes. Loss-of-function mutations in two epa variable genes, epaS and epaAC, resulted in cell surface alterations that increase the sensitivity of EE. faecalis to cell wall-targeting antibiotics. During colonization of the mouse intestine, an EE. faecalis epas mutant had a colonization defect in both adult mice and juvenile mice shortly following birth. The epaS mutant also failed to outgrow in the intestine efficiently upon antibiotic-mediated perturbation of the native commensal bacteria. Together, these data suggest that during enterococcal intestinal dysbiosis, phages could be harnessed to selectively modify the enterococcal population in favor of epa mutants that could be targeted more efficiently with concurrent antibiotic therapies. 粪肠球菌基因组包含广泛保守和菌株可变的 epa 基因 （25）。使用 EE .粪和一组未表征的毒力噬菌体，我们确定了位于 EPA 基因座可变区的基因对噬菌体感染至关重要。EPA 直接参与噬菌体附着到细菌表面。曝光 EE .粪对某些噬菌体，无论是在体外还是在小鼠肠道中，都会选择 EPA 基因座的突变，主要是在 EPA 可变基因中。两个 epa 可变基因 epaS 和 epaAC 的功能丧失突变导致细胞表面改变，从而增加 EE .粪到细胞壁靶向抗生素。在小鼠肠道定植过程中，. EE Faecalis epas 突变体在成年小鼠和幼年小鼠出生后不久均存在定植缺陷。在抗生素介导的天然共生菌扰动下，epaS 突变体也未能在肠道中有效地生长。总之，这些数据表明，在肠球菌肠道菌群失调期间，可以利用噬菌体选择性地改变肠球菌种群，以支持 EPA 突变体，这些突变体可以通过同步抗生素治疗更有效地靶向。
RESULTS 结果
Host range and morphology of enterococcal bacteriophages. We obtained a library of 19 enterococcus-specific bacteriophages through the Biological Defense Research Directorate of the Naval Medical Research Center (NMRC). These phages were isolated from environmental sources as described previously (26). Phage spot agar assays were performed (27,28)(27,28) to assess phage infectivity against 19 E. faecalis strains whose susceptibility profiles for these phages were unknown. Phage lysates formed clear, opaque, or no spots against specific E. faecalis strains, indicating strong infection, weak infection, or no infection, respectively (Fig. 1A). With the exception of phi44 and phi49, each phage infected at least one E. faecalis strain, and there was host range variability among the phages. Phages phi4, phi17, and phi19 had the broadest host 肠球菌噬菌体的宿主范围和形态。我们通过海军医学研究中心 （NMRC） 的生物防御研究理事会获得了 19 个肠球菌特异性噬菌体的文库。如前所述，这些噬菌体是从环境来源中分离出来的 （26）。进行 (27,28)(27,28) 噬菌体斑点琼脂测定以评估噬菌体对 19 种粪肠球菌菌株的感染性，这些菌株对这些噬菌体的敏感性尚不清楚。噬菌体裂解物对特定的粪肠球菌菌株形成透明、不透明或无斑点，分别表明强感染、弱感染或无感染（图 1A）。除 phi44 和 phi49 外，每个噬菌体至少感染一种粪肠球菌菌株，并且噬菌体之间存在宿主范围差异。噬菌体 phi4、phi17 和 phi19 的宿主范围最广
We performed transmission electron microscopy to determine the structural features of three broad-host-range and two narrow-host-range NMRC phages. Phages phi4, phi47, and phi51 belong to the Siphoviridae family of long-noncontractile-tailed phages. phi4 has a cubic icosahedral capsid symmetry (Fig. 2A), whereas phi47 and phi51 have elongated prolate capsids (Fig. 2B and E) (29). Phages phi17 and phi19 belong to the Myoviridae family with icosahedral capsids and sheathed contractile tails (Fig. 2C and D) (29). 我们进行了透射电子显微镜检查，以确定 3 个宽宿主范围和 2 个窄宿主范围 NMRC 噬菌体的结构特征。噬菌体 phi4、phi47 和 phi51 属于长尾非收缩尾噬菌体的 Siphoviridae 家族。phi4 具有立方二十面体衣壳对称性（图 2A），而 phi47 和 phi51 具有细长的长状衣壳（图 2B 和 E）（29）。噬菌体 phi17 和 phi19 属于肌病毒科，具有二十面体衣壳和鞘状收缩尾巴（图 2C 和 D）（29）。
NMRC phages infect E\boldsymbol{E}. faecalis independent of PIP _("EF ")_{\text {EF }}. To determine how NMRC phages infect EE. faecalis, we tested their ability to infect EE. faecalis BDU50, a pip mutant strain of EE. faecalis V583 that is resistant to phage infection (15). Phages from the NMRC collection had identical tropisms for both wild-type E. faecalis V583 and BDU50, indicating that NMRC phages infect EE. faecalis in a PIP _(EF){ }_{E F}-independent manner (see Fig. S1A and S1B in the supplemental material). To determine the molecular mechanism underlying NMRC phage infection, we selected EE. faecalis phage-resistant isolates using both broad-host-range (phi4, phi17, and phi19) and narrow-host-range (phi47 and phi51) phages, using E. faecalis strains V583 (phi4, phi17, and phi19), SF28073 (phi47), and X98 (phi51) (Table S1). Phages were mixed with E. faecalis in top NMRC 噬菌体感染 E\boldsymbol{E} 。粪 独立于 PIP _("EF ")_{\text {EF }} 。确定 NMRC 噬菌体如何感染 EE .粪便，我们测试了它们的感染能力 EE 。粪 BDU50 是 EE .粪 V583 对噬菌体感染具有抵抗力 （15）。来自 NMRC 收藏的噬菌体对野生型粪肠球菌 V583 和 BDU50 具有相同的趋向性，表明 NMRC 噬菌体感染 EE .粪以 PIP _(EF){ }_{E F} 非依赖性方式（参见补充材料中的图 S1A 和 S1B）。为了确定 NMRC 噬菌体感染的分子机制，我们选择了 EE 。使用广泛宿主范围（PHI4、PHI17 和 PHI19）和窄宿主范围（PHI47 和 PHI51）噬菌体，使用粪肠球菌菌株 V583（PHI4、PHI17 和 PHI19）、SF28073 （PHI47） 和 X98 （PHI51） 的粪噬菌体抗性分离株（表 S1）。噬菌体与粪肠球菌混合在 Top
