A practical method for preparation of pneumococcal and nontypeable Haemophilus influenzae inocula that preserves viability and immunostimulatory activity.

Background: Convenience is a major reason for using killed preparations of bacteria to investigate host-pathogen interactions, however, host responses to such preparations can result in different outcomes when compared to live bacterial stimulation. We investigated whether cryopreservation of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) permitted investigation of host responses to infection without the complications of working with freshly prepared live bacteria on the day of experimental challenge.

Findings: S. pneumoniae and NTHi retained >90% viability following cryopreservation in fetal calf serum for at least 8 weeks. Host responses to live, cryopreserved (1 week and 4 weeks), heat-killed or ethanol-killed S. pneumoniae and NTHi were assessed by measuring cytokine release from stimulated peripheral blood mononuclear cells (PBMCs). We found that cryopreserved bacteria, in contrast to heat-killed and ethanol-killed preparations, resulted in comparable levels of inflammatory cytokine release from PBMCs when compared with fresh live bacterial cultures.

Conclusion: Cryopreservation of S. pneumoniae and NTHi does not alter the immunostimulatory properties of these species thereby enabling reproducible and biologically relevant analysis of host responses to infection. This method also facilitates the analysis of multiple strains on the same day and allows predetermination of culture purity and challenge dose.