Growth conditions and environmental factors impact aerosolization but not virulence of Francisella tularensis infection in mice

Abstract

In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller–Hinton broth (MHb) or Chamberlain's chemically defined medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 min for BHI compared to <50% for MHb and CCDM). Removal of the capsule by hypertonic treatment impacted the spray factor for CCDM-grown LVS or MHb-grown LVS but not BHI-grown LVS, suggesting the choice of culture media altered the adherence of the capsule to the cell membrane. The choice of growth media did not impact the LD50 of LVS but the LD99 of BHI-grown LVS was 1 log lower than that for MHb-grown LVS or CCDM-grown LVS. Splenomegaly was prominent in mice that succumbed to MHb- and BHI-grown LVS but not CCDM-grown LVS. Environmental factors and growth conditions should be evaluated when developing new animal models for aerosol infection, particularly for vegetative bacterial pathogens.

Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

“…For aerosol exposures, virulent F. tularensis strain SCHU S4 was grown as previously described using brain heart infusion (BHI) broth (28,29). After the exposures were completed, nebulizer and all-glass impinger (AGI) contents were quantified on cysteine heart agar (CHA).…”

Discordant Results Obtained with Francisella tularensis during In Vitro and In Vivo Immunological Studies Are Attributable to Compromised Bacterial Structural Integrity

“…, TNF, IL-1β, IL-6, IL-12, and IFN-γ) from a variety of cell-types within 24 h of in vitro infection, such T H 1-oriented molecules are absent during the first 72 h of in vivo infection [5], [14], [15]. Hazlett, et al [16] and others [17]–[20] have reported that in vitro growth conditions have a profound qualitative and quantitative effect on the in vitro as well as in vivo host response to Ft LVS and SchuS4. Ft cultivated under conditions that preclude host-adaptation ( e.g.…”

Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei

“…The strains of B. pseudomallei and choice of culture medium were also different, which may contribute to the different results obtained in the two studies. It has been noted with F. tularensis and B. pseudomallei that culture conditions alter gene and protein expression, which can impact virulence and aerosolization (Chantratita et al, 2007;Hazlett et al, 2008;Faith et al, 2012).…”

“…Bacteria were grown first on solid media prior to an overnight culture in 25 ml liquid broth media using a baffled, vented 125 ml polycarbonate Erlenmeyer flask incubated in a bacterial shaker set at 200 r.p.m. Francisella tularensis SchuS4 strain was grown on cysteine heart agar (CHA Becton Dickinson, La Jolla, CA) for 2 days at 37 uC prior to culture in brain heart infusion broth (BHI; Becton Dickinson, La Jolla, CA) for 17-19 h at 37 uC (Faith et al, 2012). Burkholderia pseudomallei strain 1026b was grown on Luria-Bertani (LB; Becton Dickinson, La Jolla, CA) Agar for 1 day at 37 uC and then cultured overnight in BHI for 11 h at 37 uC.…”