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

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

Genetic Determinants of Antibiotic Resistance in Francisella

“…Francisella has been shown to form biofilms ( Dean et al, 2009 ; Durham-Colleran et al, 2010 ; Margolis et al, 2010 ; Chung et al, 2014 ; Champion et al, 2019 ). Francisella embedded in these biofilms were shown to have increase AMR compared to planktonic cells ( Biot et al, 2020 ; Siebert et al, 2020 ), similar to what has been observed with other bacteria ( Singh et al, 2017 ). This is thought to be mainly due to poor diffusion of the antibiotics through the extracellular polysaccharide matrix that makes up the biofilm.…”

“…Generally, doxycycline is very effective against tularemia with an MIC reported between 0.064 and 4 mg/L and no doxycycline-resistant strains have been reported in nature ( Caspar and Maurin, 2017 ). Interestingly, ciprofloxacin-resistant Francisella strains have an approximately 2–10-fold increase in the MIC for doxycycline in two studies, suggesting that some cross-resistance may be happening ( Biot et al, 2020 ; Sutera et al, 2014b ). There is overall very little research on the mechanism and frequency of doxycycline resistance in Francisella .…”

“…A recently study of spontaneous ciprofloxacin- and streptomycin-resistant strains of Francisella had the interesting finding that all the F. novicida ciprofloxacin-resistant mutants had significantly less biofilm formation than the F. novicida wild-type parent strain, while the F. novicida streptomycin-resistant strains did not show this effect ( Biot et al, 2020 ). Molecular analysis of the off-target mutations in the ciprofloxacin resistant strains did not reveal any obvious regulators of biofilm production or degradation, thus this biofilm effect may be due to a general increase in stress for these mutants, indicated by their reduced fitness ( Biot et al, 2020 ).…”

“…Central to this concern is the fitness of organisms carrying such AMR characteristics and their ability to infect and replicate in the host. Recently, a study was undertaken by Biot et al (2020) to assess the fitness of spontaneous ciprofloxacin- and streptomycin-resistant F. novicida and F. holarctica LVS. Fitness was assessed through intracellular replication in host macrophages as well as through in vivo infection in mice.…”

Beating the Bio-Terror Threat with Rapid Antimicrobial Susceptibility Testing

“…Moreover, as F. tularensis is a facultative intracellular bacterium, any offered antibiotic should have intracellular pharmacokinetic and pharmacodynamic activity [ 30 , 31 ]. To note, resistant strains can be easily selected in vitro [ 32 , 33 , 34 , 35 , 36 ], thus ASTs should be applied prior to treatment selection, especially following a bioterror event.…”