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

Abstract

Thermal disinfection is commonly used to prevent the proliferation of culturable Legionella in engineered water systems (EWS). In response to such stress, culturable Legionella populations can switch into a viable but nonculturable (VBNC) state. The importance of such VBNC Legionella cells is currently hotly debated. Here, we investigated the stress response patterns and transitions of the bacteria to the VBNC state at 55°C, 60°C and 70°C on two L. pneumophila strains for >80 days using a combination of cell-based viability indicators. Complete loss of culturability at 55°C, 60°C and 70°C occurred after 3-8 hours, 60 min and <2 min, respectively. In contrast, L. pneumophila strains required 9 days at 55°C, 8 hours at 60°C and 20 min at 70°C to achieve a 2 log reduction in cells with intact membranes and high esterase activity; a 4 log reduction was achieved only after 150, 8-15 and 1-4 days, respectively. In parallel, the presence of diagnostic outer-membrane epitopes (OMEs) and changes in the infectivity patterns of the two strains towards amoebae and THP-1 cells were assessed. OMEs were more persistent than viability indicators, showing their potential as targets for VBNC Legionella detection. L. pneumophila strains infected amoebae and THP-1 cells for at least 85 days at 55°C and 60°C and for up to 8 days at 70°C. However, they did so with reduced efficiency, requiring prolonged co-incubation times with the hosts and higher Legionella cell numbers in comparison to culturable cells. Consequently, infection of amoebae by thermally induced VBNC L. pneumophila with lowered virulence can be expected in EWS. Although the gold standard method cannot detect VBNC Legionella, it provides important information about the most virulent bacterial subpopulations. Our results indicate that a prolonged thermal regime ≥60°C at the central parts of warm water systems is not only effective against culturable L. pneumophila but in the long run even against VBNC cells.

Legionella and Biofilms—Integrated Surveillance to Bridge Science and Real-Field Demands

“…Warm temperatures, hydrodynamic patterns (specially stagnation) [35], water quality (pH and hardness) [44][45][46][47], corrosion and scaling (as nutrient sources) [48], and surface materials [49,50] have been linked to Legionella prevalence. Additionally, biocidal programs that are not properly set or managed may promote Legionella colonization in several installations [51][52][53][54]. Mimicking the diversity and dynamic conditions of engineered water systems is an unfeasible task in the laboratory.…”

Induction of Escherichia coli O157:H7 into a viable but non‐culturable state by high temperature and its resuscitation

“…Most bacteria live in a dynamic environment where the temperature varies, which requires rapid adaptation to stress. Responding to high temperature, many pathogens are known to enter into a viable but non-culturable (VBNC) state, including Escherichia coli (Bruhn-Olszewska et al, 2018), Legionella pneumophila (Cervero-Arago et al, 2019), Listeria monocytogenes (Zolfaghari et al, 2016) and Salmonella typhimurium (Liao et al, 2018). In the VBNC state, cells remain viable but they are no longer culturable with a culturing method but retain certain features of viable cells, such as cellular membrane integrity, metabolic activity and virulence (Li et al, 2014).…”

A valuable experimental setup to model exposure to Legionella’s aerosols generated by shower-like systems

“…In the context of this study, we demonstrated a new data that the Legionella physiological states were not modified by the nebulization process or by its transportation through the 12-stage DLPI or the anatomical model. Thermally or chlorine-induced VBNC Legionella pneumophila may still infect amoebae and pulmonary cells after resuscitation on amoebae (Cervero-Aragó et al, 2019;Epalle et al, 2015;Mustapha et al, 2015). VBNC cell concentrations in the water network must be included in the QMRA models.…”