A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia

Abstract

Francisella tularensis, a gram–negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

Francisella tularensis : FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation

“…In agreement with the latter hypothesis, we observed that a highly FQ-resistant mutant of F. novicida (U112_Fno3) bearing a functionally-inert DNA gyrase mutation (GyrA_ΔE524, ΔS525) also had a nonsense mutation in FTN_0444 ( fupA ) [7]. Moreover, fupA was identified as one of seven mutated genes in a ciprofloxacin-resistant mutant of F. tularensis SCHU S4, alongside gyrA and parE [14].…”

Development, Phenotypic Characterization and Genomic Analysis of a Francisella tularensis Panel for Tularemia Vaccine Testing

“…Therefore, LD 50 analysis was performed with all 10 strains by intranasal challenge of BALB/c mice ( Figure 7 and Table 4 ). As expected for both versions of Schu S4 (FRAN244 and FRAN249), and in agreement with previous results from our laboratory and others ( Chance et al, 2017 ), the LD 50 measurements for both versions were found to be <1 CFU.…”

A Francisella novicida Mutant, Lacking the Soluble Lytic Transglycosylase Slt, Exhibits Defects in Both Growth and Virulence

“…To assess the contribution of the slt gene to Francisella virulence, BALB/c mice were challenged via the intranasal route with various doses of either the WT strain or the slt mutant. As previously demonstrated, BALB/c mice were highly susceptible to challenge with the parent strain, and the LD 50 was calculated to be less than 1 CFU (Figure 7 and Table 2) (Chance et al, 2017). In contrast, the slt mutant was shown to be highly attenuated in the murine model, and a LD 50 value was not able to be measured as only 4/10 mice succumbed to infection at the highest challenge dose (620 CFU).…”

“…28 days post-vaccination, the three groups of Fn slt vaccinated mice and naïve control mice ( n = 20) were split into two groups of 10 and challenged intranasally with either a high (37 CFU) or low (6 CFU) dose of Ft Schu S4. Based upon our previous published data, the IN LD 50 for BALB/c mice is 1 CFU of Ft Schu S4 (Chance et al, 2017). However, despite the promising results with protection against Fn challenge, no protection was afforded to the mice receiving any dose of the slt mutant strain when challenged with the Ft Schu S4 strain (Figure 12).…”