Table_1_Evolution of Antibiotic Resistance in Surrogates of Francisella tularensis (LVS and Francisella novicida): Effects on Biofilm Formation and Fitness.docx

Francisella tularensis, the causative agent of tularemia, is capable of causing disease in a multitude of mammals and remains a formidable human pathogen due to a high morbidity, low infectious dose, lack of a FDA approved vaccine, and ease of aerosolization. For these reasons, there is concern over the use of F. tularensis as a biological weapon, and, therefore, it has been classified as a Tier 1 select agent. Fluoroquinolones and aminoglycosides often serve as the first line of defense for treatment of tularemia. However, high levels of resistance to these antibiotics has been observed in gram-negative bacteria in recent years, and naturally derived resistant Francisella strains have been described in the literature. The acquisition of antibiotic resistance, either natural or engineered, presents a challenge for the development of medical countermeasures. In this study, we generated a surrogate panel of antibiotic resistant F. novicida and Live Vaccine Strain (LVS) by selection in the presence of antibiotics and characterized their growth, biofilm capacity, and fitness. These experiments were carried out in an effort to (1) assess the fitness of resistant strains; and (2) identify new targets to investigate for the development of vaccines or therapeutics. All strains exhibited a high level of resistance to either ciprofloxacin or streptomycin, a fluoroquinolone and aminoglycoside, respectively. Whole genome sequencing of this panel revealed both on-pathway and off-pathway mutations, with more mutations arising in LVS. For F. novicida, we observed decreased biofilm formation for all ciprofloxacin resistant strains compared to wild-type, while streptomycin resistant isolates were unaffected in biofilm capacity. The fitness of representative antibiotic resistant strains was assessed in vitro in murine macrophage-like cell lines, and also in vivo in a murine model of pneumonic infection. These experiments revealed that mutations obtained by these methods led to nearly all ciprofloxacin resistant Francisella strains tested being completely attenuated while mild attenuation was observed in streptomycin resistant strains. This study is one of the few to examine the link between acquired antibiotic resistance and fitness in Francisella spp., as well as enable the discovery of new targets for medical countermeasure development.

土拉弗朗西斯菌（Francisella tularensis）是土拉菌病（tularemia）的致病菌，可感染多种哺乳动物，且因高发病率、低感染剂量、缺乏FDA批准的疫苗以及易于气溶胶传播等特性，仍是极具威胁的人类病原菌。基于上述原因，土拉弗朗西斯菌被用作生物武器的风险备受关注，因此被列为一级选控生物制剂（Tier 1 select agent）。氟喹诺酮类（fluoroquinolones）与氨基糖苷类（aminoglycosides）药物通常是土拉菌病治疗的一线用药。然而近年来，革兰氏阴性菌对这类抗生素的耐药性水平居高不下，且已有文献报道了天然耐药的弗朗西斯菌菌株。无论是天然还是人工诱导的抗生素耐药性，均为医疗应对措施的研发带来了挑战。本研究通过抗生素压力筛选，构建了抗生素耐药性新凶手弗朗西斯菌（F. novicida）与土拉弗朗西斯菌活疫苗株（Live Vaccine Strain, LVS）的替代菌株集，并对这些菌株的生长特性、生物膜形成能力与适应度进行了表征。本实验旨在达成两大目标：（1）评估耐药菌株的适应度；（2）筛选可用于疫苗或治疗药物研发的新型靶点。所有受试菌株均对环丙沙星（ciprofloxacin）或链霉素（streptomycin）表现出高水平耐药性，二者分别属于氟喹诺酮类与氨基糖苷类药物。对该菌株集的全基因组测序结果显示，其突变分为通路内突变与通路外突变两类，且活疫苗株的突变数量更多。针对新凶手弗朗西斯菌，我们发现相较于野生型菌株，所有环丙沙星耐药菌株的生物膜形成能力均有所下降，而链霉素耐药菌株的生物膜形成能力则无明显变化。我们通过体外小鼠巨噬细胞系实验与体内肺炎型感染小鼠模型，对代表性抗生素耐药菌株的适应度进行了评估。实验结果表明，经上述方法获得的突变可使几乎所有受试环丙沙星耐药弗朗西斯菌菌株完全减毒，而链霉素耐药菌株仅表现出轻度减毒。本研究是少数几项探究弗朗西斯菌属（Francisella spp.）获得性抗生素耐药性与适应度之间关联的研究之一，同时也为医疗应对措施的研发提供了新型靶点发掘路径。