Influence of carbon concentration, shear stress and temperature on survival of Legionella pneumophila in drinking water biofilms

Abstract

Biofilms can form under stressful conditions and offer protection
against mechanical and chemical disruptions, hence providing
a refuge for pathogens. One of the possible waterborne
colonisers in drinking water distribution system biofilms is
Legionella pneumophila, which is still responsible for many outbreaks
of Legionellosis worldwide. The aim of this work was
to study the influence of important operational parameters,
including shear stress, carbon concentration and temperature,
on persistence of this pathogen in drinking water biofilms.
The biofilm studies were carried out using a two-stage chemostat
system. The outflow culture of the first vessel fed three
secondary chemostats in parallel and under different conditions
of shear stress and carbon concentration. After 10 days the
chemostats attained steady conditions and PVC coupons were
then immersed to allow biofilm formation. The coupons were
removed at different times (up to 32 days) and scraped with
sterile glass beads. Planktonic and sessile cells were quantified
by standard cultivation techniques (R2A and BCYE agar) and
SYTO9 staining. In addition, a specific 16S rRNA peptide
nucleic acid probe (PNA) was used to quantify the total numbers
of L. pneumophila in the biofilm. The conditions described
above were tested at two different temperatures, 15 and 20ºC.
Concerning sessile cells, the number of total fluorescently
labelled bacteria and L. pneumophila were similar for the three
conditions studied at both temperatures. However more cultivable
cells were seen when chemostats were operated with high
shear and high carbon concentrations, particularly the latter.
This might indicate the existence of Viable But Non Cultivable
L. pneumophila that can recover cultivability under certain
conditions. For example, the total of fluorescently labelled L.
pneumophila was higher at 15ºC. In conclusion, temperature was
the most important factor influencing the formation of biofilm
and persistence of L. pneumophila. Carbon concentration also
had a considerable influence on the number of total cells in
water and biofilms. Shear stress played only a minor role in the
numbers of planktonic and sessile cells present.

ASM Biofilm Conference, Quebec City, 25-29th March 2007. ISBN: 1-55581-428-X

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Identifiers

ORCID for Sandra Wilks: orcid.org/0000-0002-4134-9415

ORCID for Charles Keevil: orcid.org/0000-0003-1917-7706

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