Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains

Abstract

BackgroundLegionella pneumophila, the causative agent of Legionnaire’s disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water.ResultsWith ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb- stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population.ConclusionsThe results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1220-x) contains supplementary material, which is available to authorized users.

Viability and infectivity of viable but nonculturable Legionella pneumophila strains induced at high temperatures

“…A L. pneumophila SG1 subtype mAb Benidorm strain isolated from a clinical source (LpClin) and a L. pneumophila SG1 mAb subtype Philadelphia strain (LpParis) were used for all experiments (Schrammel et al, 2018a, 2018b). The two strains belong to the most virulent mAb 3/1 subtype according to the Dresden panel (Helbig et al, 1997).…”

“…For each temperature, different sampling time points were chosen. Bottles were shaken 20 times and from each microcosm, samples of 15-20 mL were taken and cooled by placing the tube in cold water (approximately 18°C) for 2 min and vortexed for 20 s. Samples were then prepared for further analyses: culturability, analysis of cellular integrity and DNA content, viability indicators, presence of OMEs and infection assays (Dietersdorfer et al, 2018; Schrammel et al, 2018a, 2018b).…”

“…The details of all staining procedures, the FCM protocol analysis, the gating strategy and the FCM results calculation are described extensively in our previous publications (Schrammel et al, 2018a, 2018b) and can be found in the Supplemental Information (SI). Legionella microcosms were analysed using an Attune NxT Flow Cytometer (Life Technologies, Darmstadt, Germany) equipped with a 488 nm flat-top laser at 50 mW.…”

“…Legionella microcosms were analysed using an Attune NxT Flow Cytometer (Life Technologies, Darmstadt, Germany) equipped with a 488 nm flat-top laser at 50 mW. The flow cytometric assays had a limit of quantification (LOQ) of 1.3 × 10 4 cells/mL (Schrammel et al, 2018a, 2018b).…”

“…For example, the diversity on the lipopolysaccharide (LPS) and specifically the outer-membrane epitopes (OMEs) of L. pneumophila have been used in the past years for serotyping L. pneumophila strains for diagnostic purposes (Helbig et al, 1997). However, little is known about the persistence of such OMEs under different environmental conditions and their relation to Legionella viability and infectivity (Schrammel et al, 2018b).…”