Differential development of Legionella sub-populations during short- and long-term starvation

Abstract

Legionellae are among the most important waterborne pathogens in industrialized countries. Monitoring and surveillance of Legionella in engineered water systems is usually performed with culture-based methods. Since the advent of culture-independent techniques, it has become clear that Legionella concentrations are often several orders of magnitude higher than those measured by culture-based techniques and that a variable proportion of these non-culturable cells are viable. In engineered water systems, the formation of these viable but non-culturable (VBNC) cells can be caused by different kinds of stress, such as, and most importantly, nutrient starvation, oxidative stress and heat. In this study, the formation of VBNC cells of six Legionella strains under conditions of starvation was monitored in mono-species microcosms for up to one year using a combination of different viability indicators. Depending on the strain, complete loss of culturability was observed from 11 days to 8 weeks. During the starvation process, three distinct phases and different sub-populations of VBNC cells were identified. Until complete loss of culturability, the number of membrane-intact cells decreased rapidly to 5.5-69% of the initial cell concentration. The concentration of the sub-population with low esterase activity dropped to 0.03-55%, and the concentration of the highly esterase-active sub-population dropped to 0.01-1.2% of the initial concentration; these sub-populations remained stable for several weeks to months. Only after approximately 200 days of starvation, the number of VBNC cells started to decrease below detection limits. The most abundant VBNC sub-populations were characterized by partially damaged membranes and low esterase-activity. With this study, we showed that upon starvation, a stable VBNC Legionella community may be present over several months in a strain-dependent manner even under harsh conditions. Even after one year of starvation, a small proportion of L. pneumophila cells with high esterase-activity was detected. We speculate that this highly active VBNC subpopulation is able to infect amoebae and human macrophages.

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

“…All microcosms used in ELISA and FCM were prepared in a standardized procedure described in detail elsewhere [ 18 ]. In brief, Legionella strains were grown in liquid buffered yeast extract (liBYE, modified [ 24 ]) and sub-cultured once, until the population reached the stationary phase, assessed by optical density (OD) measurement (OD-values of stationary phase: 3–4 depending on the strain, OD analysis was conducted after eight-fold dilution in liBYE).…”

“…Approximately 10 8 cells mL − 1 (final concentration, using a conversion factor comparing OD values with total cell counts determined by SYBR green 1/propidium iodide staining [ 26 ] and epifluorescence microscopy (EFM) [ 27 ]) were inoculated to sterile glass bottles prefilled with 350 mL of autoclaved ultrapure water (starvation microcosms). After an initial sampling, all microcosm bottles were incubated at 45 °C without shaking and periodically sampled by pouring out 15 mL of each bottle and aliquoting the sample for further analysis [ 18 , 21 ]. 500 μl were used for immunofluorescence (IF)-FCM.…”

“…Encountering such conditions, legionellae change gene expression to optimize stress management and consecutively enter a dormant and viable but nonculturable (VBNC) state [ 17 ]. Those cells cannot be cultured on routine media anymore, but may retain viability and infectivity for long periods of time [ 18 – 21 ].…”

“…To close this gap in knowledge we analysed the impact of starvation on the reactivity of different mAbs targeting outer membrane structures and thus, possible changes on the corresponding epitopes which would in turn influence the potential of VBNC L. pneumophila to infect host cells [ 6 ]. Moreover, the results are compared to information on culturability, viability and infectivity of the cells from parallel investigations [ 18 , 21 ]. The outcome of the study is of high importance for the development of mAb-based detection techniques for culture-independent enumeration of Legionella in man-made water systems.…”

Population analysis ofLegionella pneumophilareveals the basis of resistance to complement-mediated killing

“…The trend of lag-1 gene loss or deactivation was not observed among longitudinal ST1 healthcare facility-associated isolates from a cluster sequenced in a recent study by David and colleagues (David et al, 2017). However, a study on starvation of L. pneumophila in ultrapure water showed that in a short-term period, the viable cell numbers of all mAb 3/1-positive strains decreased strongly compared to the other strains suggesting a negative selection for lag-1 function in some water environments (Schrammel et al, 2018). Overall, these data indicate that the presence of a functional lag-1 correlates with enhanced resistance to serum killing.…”