You are here:
Hartmannella vermiformis inhibition of Legionella pneumophila cultivability
Citation:
Buse, H., M. Donohue, AND N. ASHBOLT. Hartmannella vermiformis inhibition of Legionella pneumophila cultivability. MICROBIAL ECOLOGY. Springer, New York, NY, 66(3):715-726, (2013).
Impact/Purpose:
Original research to report novel finding of amoebae inhibition of Legionella growth.
Description:
Hartmannella vermiformis is frequently isolated from drinking water (DW) and is permissive to Legionella pneumophila intracellular replication. Thus, H. vermiformis may play an important role in the growth and survival potential of such environmental pathogens. In this study, Page’s amoeba saline (PAS) was used as an encystment medium for H. vermiformis monolayers, and the culture supernatants (HvS) were collected to determine if any extracellular factor(s) may modulate growth or cultivability of L. pneumophila. Compared to PAS only controls, HvS significantly inhibited cultivability of L. pneumophila strain Philadelphia-1 (Ph-1) by 3 log10 CFU mL-1 after 3 d of exposure compared to < 0.5 log10 CFU mL-1 reduction of strain Lp02 (P < 0.001). To better characterize this inhibition, flow cytometric analysis was utilized and changes in the percentage and cultivability of strain Ph-1 sub-populations were assessed based on differences in bacterial membrane permeability, which identified three sub-population types: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). The MT sub-population decreased significantly after 3 d of exposure to HvS compared to the control PAS exposed Ph-1 cells (31.6 % vs 67.2 %, respectively, P < 0.001). Conversely, ISM and UD sub-populations of HvS exposed L. pneumophila increased after 3 d of exposure (+26.7 % and +6.9 %, respectively) with the ISM sub-population appearing as viable but non-culturable cells that were absent in PAS exposed controls. Furthermore, the HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the < 5 kDa fraction (P < 0.001). Reverse phase liquid chromatography analysis revealed two different profiles for the PAS and HvS < 5 kDa fractions, which indicated that the inhibitory molecule(s) are probably polar and is eluted from a Novapak C18 column between 6-11 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila growth/cultivability and probably promote the VBNC state for this bacterium.
