BIBF 1120 cell line anthracis spores, most in vitro infection models have been conducted using culture media

containing FBS and/or specific L-amino acids or nucleotides at concentrations previously demonstrated to promote germination of spores in vitro [20, 28–34]. Under such conditions, it is likely that, in these previous studies, host cells were infected with heterogeneous populations of germinated and dormant spores. The objective of this BLZ945 study was to experimentally address existing gaps in knowledge as to how the germination state of B. anthracis spores, as dictated by the presence or absence of serum during in vitro infections, influences the uptake of spores into mammalian cells, as well as the subsequent fate of both intracellular B. anthracis and infected cells. Germinating and non-germinating culture conditions were used to compare the interaction of see more spores prepared from B. anthracis Sterne 7702 with RAW264.7 macrophage-like cells, as well as several other cell lines. These studies revealed that the uptake of B. anthracis into cells was largely unaffected by the germination state of spores. In contrast, the number of viable, intracellular B. anthracis recovered from infected cells, as well as the viability of the infected cells, was dependent on the germination state of spores during uptake. These results support the idea that

the germination state of spores is an important consideration when interpreting results from in vitro infections with B. anthracis spores. Results and Discussion The composition of cell culture medium influences the germination and outgrowth of B. anthracis spores Several commonly used mammalian cell culture media, in the presence or absence of fetal bovine serum (FBS), were first evaluated for the capacity to induce germination initiation, which is the earliest set of changes in dormant spores triggered by the presence of germinants. Spore outgrowth, which is the transition of germinated spores into vegetative bacilli [35–37], was also evaluated. These studies revealed

that, regardless Edoxaban of the medium tested, dormant spores prepared from B. anthracis Sterne 7702 (1.0 × 108 spores/mL) underwent germination initiation when incubated at 37°C and under 5% CO2 in the presence of FBS, as indicated by increased sensitivity of the spores to heat treatment [38] and a time-dependent decrease in spore refractility, which indicates rehydration of the spore core following germination initiation [39] (Table 1, Figure 1A, B). When incubated in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% FBS, or, Roswell Park Memorial Institute (RPMI) 1640 medium plus 10% FBS, 86.0 ± 5.2% and 83.4 ± 2.6% of total spores, respectively, converted from heat-resistant to heat sensitive forms within 10 min, while 97.6 ± 0.2% and 96.6 ± 2.