Understanding the ecology and virulence of Legionella spp. populations in freshwater systems in Germany, Palestine and Israel - Phase 2
Microbial Ecology and Applied Microbiology
Final Report Abstract
The overall objective of this project was to advance current knowledge on the ecology of Legionella populations in man-made freshwater systems, the environmental factors affecting their occurrence, virulence potential and infectivity and to understand their transmission to humans. The overall research strategy to achieve this objective was to sample bacteria from Drinking Water Supply Systems (DWSS) in the three countries from relevant habitats, i. e. bulk water and biofilm of the surfaces of the DWSS. In parallel, clinical material (sputum and BAL) was obtained from the same geographical areas. Both types of samples were subjected to molecular and conventional microbiological analyses to determine the abundances of Legionella species with emphasis on L. pneumophila and its clonal structure. The DWSS of the city of Braunschweig, Germany, was analysed for its Legionella species composition. These analyses comprised the whole supply chain including raw water, treatment process and large-scale storage, and a seasonal study of finished drinking water sampled monthly from cold and hot tap water. Treatment of raw water had a major impact on Legionella species by reducing their diversity and abundances. During the seasonal cycle L. pneumophila was only observed at low relative abundances (<1%) in cold tap water. The first surprise was that in hot drinking water (50°C to 58°C) L. pneumophila was present during all seasons at high relative abundances (8% to 40%). This thermophilic Legionella community, including L. pneumophila, was able to grow in the hot water above 50°C. Such thermophilic Legionella populations are of general relevance for drinking water management and public health. For an understanding of the environmental factors, other than temperature, the abundances of L. pneumophila were compared with a set of chemical parameters determined in parallel for the related bulk water. Correlation analyses with these parameters indicated a decrease of L. pneumophila abundance for water and biofilm with increasing magnesium concentrations above 30 mg/l, most relevant in Palestine. From studies across the three countries, it could be concluded that different environmental factors influenced the abundances of Legionella species and genotypes of L. pneumophila due to the specific conditions and the differences of the DWSS. Detailed molecular analyses of the occurrence of specific genotypes of L. pneumophila indicated that a specific genotype cooccurred with high Legionella counts determined by conventional plate counts on agar medium. This finding pointed towards the need for high-resolution molecular analyses able to identify individual clones of L. pneumophila in DWSS. To this end, advanced molecular methods, such as genus(Legionella)-specific Next Generation Sequencing (NGS) and Nested PCR Sequence-Based Typing (NPSBT), were developed and applied to DNA obtained from environmental and clinical samples. These advanced molecular approaches enabled us to understand the relationship between Legionella infections and the environment by the analysis of clinical samples from pneumonia patients in comparison with related samples or isolates from the environment. Here, sputum from patients in Israel and Palestine were compared with the respective environmental material. The sputum samples that were Legionella-positive were inhabited by aquatic bacteria that have been observed in association with amoeba, indicating that amoeba might have transferred Legionella from the drinking water together with its bacterial microbiome. This was the second surprise in our project to demonstrate the co-occurrence of major bacterial commensals and pathogens in sputum with respect to the presence of Legionella. These observations lead to the hypothesis that Legionella infections are supported by co-infections with other potentially pathogenic bacteria co-transferred by the amoeba. The molecular findings were complemented by experimental studies of the virulence of relevant isolates indicating that the pathogenicity is clone dependent. Our findings and the developed high-resolution molecular diagnostic methodologies provide new surveillance tools for Legionella infections and can help improve the management of DWSS in the three countries. The International publications and press releases by the HZI solicited a substantial response in the German media and guaranteed the dissemination of the project’s results to the informed and general public.
Publications
- (2018) Virulence Traits of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes. Applied and environmental microbiology 84 (10)
Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Pecellin, Marina; Sela, Rotem; Peretz, Avi; Höfle, Manfred G.; Halpern, Malka; Brettar, Ingrid
(See online at https://doi.org/10.1128/AEM.00429-18) - (2015) Spatial distribution of Legionella pneumophila MLVA-genotypes in a drinking water system. Water Res. 77: 119-132
Rodríguez-Martínez, S., Y. Sharaby, M. Pecellin, I. Brettar, M. G. Höfle and M. Halpern
(See online at https://doi.org/10.1016/j.watres.2015.03.010) - (2016) Legionella species diversity and dynamics from surface reservoir to tap water: from cold adaptation to thermophily. ISME J 10: 1064- 1080
Lesnik, R., Brettar, I and M. G. Höfle
(See online at https://doi.org/10.1038/ismej.2015.199) - (2017) Bacterial community dynamics in a cooling tower with emphasis on pathogenic bacteria and Legionella species using universal and genus-specific deep sequencing. Water Res. 122:363-376
Pereira, R. P. A., R., Peplies, J., M. G. Höfle and I. Brettar, I
(See online at https://doi.org/10.1016/j.watres.2017.06.011) - (2017) Comparison of the microbiome from sputum samples of legionellosis associated patients with other pneumonia patients. Scientific Rep. 7: 40114
Mizrahi, H., Peretz, A., Rodriguez-Martinez, S., Aizenberg-Gershtein, Y., Sharaby, Y., Pastukh, N., Lesnik, R., Brettar, I., Höfle, M. G., and Halpern, M.
(See online at https://doi.org/10.1038/srep40114) - (2017) Development of a genusspecific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems. BMC Microbiol. 17:79
Pereira, R. P. A., Peplies, J., Brettar, I and M. G. Höfle
(See online at https://doi.org/10.1186/s12866-017-0987-5) - (2017) Temperature-dependent growth modeling of environmental and clinical Legionella pneumophila multilocus variable-number tandemrepeat analysis (MLVA) genotypes. Appl. Environ. Microbiol. e03295-16
Sharaby,Y, Rodríguez-Martínez, S., Oks, O., Pecellin, M., Mizrahi, H., Peretz, A., Brettar, I., Höfle, M., and Halpern, M.
(See online at https://doi.org/10.1128/AEM.03295-16) - (2018) Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific nextgeneration sequencing approach. bioRxiv
Pereira, R. P. A., J. Peplies, I. Brettar, and M. G. Höfle
(See online at https://dx.doi.org/10.1101/247445)
