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

Fabrice V Biot,Gustavo F Palacios,Christopher P Klimko,Joel A Bozue,Christopher K Cote,Ronald G Toothman,Beth A Bachert,Sean P Lovett,Kevin D Mlynek,Jason T Ladner,Galina I Koroleva

doi:10.3389/fmicb.2020.593542

Fabrice V Biot, Gustavo F Palacios + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2020.593542

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Abstract

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.

Highlights

The facultative intracellular gram-negative bacterium Francisella tularensis, found ubiquitously across the northern hemisphere, is responsible for the zoonotic disease known as tularemia or more commonly “rabbit fever.” The disease is typically spread between hosts by arthropod vectors, namely through the bite of an infected mosquito or tick (Ellis et al, 2002)
As fluoroquinolones and aminoglycosides often serve as the first line of defense for treatment of tularemia, we sought to characterize a surrogate panel of antibiotic resistant Francisella strains in an effort to (1) assess the fitness of such strains; and (2) identify new targets to investigate for the development of vaccines or therapeutics
Strains were designated as follows- F. novicida (Fn) or LVS, Cip or Strep, followed by the concentration of antibiotic used in the final step of passaging and the colony number

Summary

Introduction

The facultative intracellular gram-negative bacterium Francisella tularensis, found ubiquitously across the northern hemisphere, is responsible for the zoonotic disease known as tularemia or more commonly “rabbit fever.” The disease is typically spread between hosts by arthropod vectors, namely through the bite of an infected mosquito or tick (Ellis et al, 2002). The facultative intracellular gram-negative bacterium Francisella tularensis, found ubiquitously across the northern hemisphere, is responsible for the zoonotic disease known as tularemia or more commonly “rabbit fever.”. Holarctica (Type B), which are responsible for the majority of tularemia infections in humans. A third closely related subspecies, Francisella novicida, is only associated with brackish water and soil, and rarely causes disease in humans. F. novicida is commonly used as a laboratory surrogate since it has a high degree of genetic similarity to the virulent F. tularensis subspecies, is able to infect macrophages in vitro and cause disease in mice, and can be handled under BSL-2 conditions (Kingry and Petersen, 2014)

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