Affiliation:
1. Laboratories of Food Microbiology
2. Microbiology, Wageningen University and Research Center, Wageningen
3. Kiwa N.V. Water Research, Nieuwegein, The Netherlands
Abstract
ABSTRACT
The need for protozoa for the proliferation of
Legionella pneumophila
in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of
L. pneumophila
in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous
L. pneumophila
originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-μm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable
L. pneumophila
, and for their concentrations of protozoa.
L. pneumophila
grew to high numbers (6.3 log CFU/cm
2
) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of
Hartmannella vermiformis
in all flasks in which
L. pneumophila
multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of
L. pneumophila
in trophozoites of
H. vermiformis
, with 25.9% ± 10.5% of the trophozoites containing
L. pneumophila
on day 10 and >90% containing
L. pneumophila
on day 14. Calculations confirmed that intracellular growth was most likely the only way for
L. pneumophila
to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher
L. pneumophila
concentrations, and therefore the growth of
L. pneumophila
within engineered water systems can be limited by controlling biofilm formation.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
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