Francisella novicida and F. philomiragia biofilm features conditionning fitness in spring water and in presence of antibiotics

Abstract

Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.

Long-Term Survival of Virulent Tularemia Pathogens outside a Host in Conditions That Mimic Natural Aquatic Environments

“…The only strain that produced significant levels of biofilm under the conditions utilized in this study (i.e., cold and low-nutrient water) was strain U112, which corroborates previous studies that suggest that biofilm formation is an environmental survival mechanism for F. novicida ( 32 ). Originally, F. novicida was isolated from a turbid water sample ( 1 ), and it has since been detected in brackish and saltwater sources, but no association with arthropod vectors or animals has been demonstrated in nature ( 1 , 23 , 40 – 42 ).…”

“…Compared with F. tularensis , the genome of F. novicida is larger and more complex; it shows a higher degree of metabolic competence, higher levels of recombination, and dN / dS ratios (ratios of nonsynonymous to synonymous substitutions) consistent with a free-living environmental niche ( 43 , 44 ). Several studies have contributed to the characterization of F. novicida biofilm formation and composition, revealing that the biofilm matrix consist of nucleic acids, proteins, exopolysaccharides (including cellulose), and polymeric β-1,4-linked N -acetylglucosamine ( 26 , 27 , 29 , 31 , 32 ). A gene cluster present in F. novicida but absent in F. tularensis is involved in the synthesis and degradation of the secondary messenger molecule c-di-GMP, which regulates the switch from a free-living planktonic lifestyle to a biofilm-associated lifestyle ( 45 ).…”

“…holarctica and F. tularensis subsp. tularensis, all have been induced to form biofilm on the wells of plastic culturing plates (26)(27)(28)(29)(30)(31)(32).…”

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

“…The metabolic activity of F. novicida wild-type and Δ ldcF was estimated through their capacity to reduce resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide; λ max = 600 nm ; Sigma-Aldrich) into the pink fluorescent compound resorufin (λ max = 570 nm). This oxidation–reduction indicator of mitochondrial function was conveniently used to evaluate cell viability of several bacterial species 88 including F. tularensis, F. novicida and F. philomiragia 89 , 90 . Exponential growth phase bacteria grown in MMH at 37 °C under shaking were transferred (200 µL) into a 96-well plate.…”

“…Assessment of the biofilm formation was carried out using crystal violet assay and following optimized protocol for F. novicida starting from a 200 µL bacterial inoculum (1.10 7 cells/mL) in flat-bottom 96-well plates. The biofilm biomass was quantified following 24 h incubation at 37 °C in a 5% CO 2 incubator without shaking 90 .…”