Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.
Hot water recirculation systems (HWRS) in hotels and nursing homes, which are common in countries such as Spain, have been related to outbreaks of legionellosis. To establish the relationships of microbial and physicochemical parameters, especially protozoa, with the occurrence of Legionella in HWRS, 231 samples from hotels and nursing homes were analysed for Legionella, protozoa, heterotrophic plate counts (HPC) at 22 and 37 °C, Pseudomonas, metals, temperature and others. Legionella pneumophila was the dominant species isolated, and 22 % were sg. 1. The sampling method became particularly important in order to define which factors were involved on the occurrence of Legionella. Results showed that the bacteria and the accompanying microbiota were more abundant in the first flush water whose temperature was lower. The bacteria occurred in those samples with high HPC and were inversely correlated with high temperatures. Multivariate regression showed that a concentration above 1 × 10(5) CFU/100 mL of HPC at 37 °C, Fe above 0.095 ppm and the presence of protozoa increased significantly the risk of Legionella colonization, while univariant regression showed that the presence of Cu above 0.76 ppm and temperature above 55 °C diminished it. Therefore, to reduce the risk associated with Legionella occurrence in HWRS these parameters should be taken into consideration.
Legionella infections are among the most important waterborne infections with constantly increasing numbers of cases in industrialized countries, as a result of aging populations, rising numbers of immunocompromised individuals and increased need for conditioned water due to climate change. Surveillance of water systems is based on microbiological culture-based techniques; however, it has been shown that high percentages of the Legionella populations in water systems are not culturable. In the past two decades, the relevance of such viable but non-culturable (VBNC) legionellae has been controversially discussed, and whether VBNC legionellae can directly infect human macrophages, the primary targets of Legionella infections, remains unclear. In this study, it was demonstrated for the first time that several starved VBNC Legionella strains (four L. pneumophila serogroup 1 strains, a serogroup 6 strain and a L. micdadei strain) can directly infect different types of human macrophages and amoebae even after one year of starvation in ultrapure water. However, under these conditions, the strains caused infection with reduced efficacy, as represented by the lower percentages of infected cells, prolonged time in co-culture and higher multiplicities of infection required. Interestingly, the VBNC cells remained mostly non-culturable even after multiplication within the host cells. Amoebal infection by starved VBNC Legionella, which likely occurs in oligotrophic biofilms, would result in an increase in the bacterial concentration in drinking-water systems. If cells remain in the VBNC state, the real number of active legionellae will be underestimated by the use of culture-based standard techniques. Thus, further quantitative research is needed in order to determine, whether and how many starved VBNC Legionella cells are able to cause disease in humans.
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.
The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17-4.59) and 1.65 (0.15-3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24-2.93), 0.64 (0.12-1.97), 0.45 (0.04-2.54) and 0.72 (0.22-2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.