Prevalence, Genetic Diversity, and Virulence Potential of Legionella spp. Summary of the Doctoral Thesis

Abstract

Legionella spp. is one of the most important water-borne pathogens that can cause both outbreaks and sporadic cases. Legionella is spread by water aerosols containing the bacteria. In the form of aerosols, bacteria can spread several kilometres from the primary source of infection while remaining viable. Legionellosis is a preventable disease and a form of atypical pneumonia caused by Legionella pneumophila and related bacteria. Clinical manifestations of the disease range from a mild, self-limiting flu-like illness (Pontiac fever) to potentially fatal pneumonia (Legionnaires' disease). The aim of the study was to investigate Legionella spp. distribution trends, genetic diversity and virulence potential for diagnosis and prevention of legionellosis in Latvia. Materials and methods. To assess the seroprevalence of L. pneumophila in the general healthy population and to identify associated risk factors, 2007 blood samples were collected from healthy blood donors. To evaluate Legionella spp. distribution in water supply systems, 1467water samples were taken in different types of buildings. 137 L. pneumophila isolates were selected for the study of genetic diversity and virulence genes were determined for 58 isolates. Immunoenzymatic reaction was performed for all blood samples to detect IgG antibodies against L. pneumophila SG 1-6. Determination of Legionella spp. in water and biofilm samples was performed according to a standardized method. Free-living protozoa were determined morphologically and by three different PCR protocols. Whole genome sequencing of L. pneumophila isolates was performed with Illumina MiSeq. Sequence-based typing was performed according to the scheme developed by ESCMID. cgMLST typing was performed using the previously described protocol. The database of virulence factors was used to determine virulence determinants. Results. The seroprevalence of L. pneumophila SG 1-6 in blood donors was 4.8 % and 0.2 % of donors were seropositive for L. pneumophila SG1. Seroprevalence was higher in women (5.9 %) than men (3.3 %), and in regions with a larger population, ranging from 3.5 % in rural regions to 6.8 % in the capital Riga. Legionella spp. prevalence in water supply systems varied from an average of 25 % in cold water samples to 35 % in hot water samples. The predominant L. pneumophila serogroup (SG) was SG 3, which was identified in 208 of 482 cases (43.2 %). At least one free-living amoeba genus was detected in at least 77.2 % of the samples. The average hot water temperature was 47.8 ± 0.7 °C. Temperature measurements showed that only 249 out of 1275 hot water samples exceeded 55 °C. Overall, 137 L. pneumophila strains included in the study represented 46 sequence types, including new 10 sequence types, previously unrecorded in the world. Using cgMLST typing 116 genotypes were obtained. 420 virulence genes were identified of which 260 genes were found in all sequenced L. pneumophila isolates. Virulence factors encoding enhC, htpB, omp28 and mip genes were detected in all isolates, suggesting that adhesion, attachment and entry into the host cell are enabled for all isolates. Relative frequency of virulence genes among L. pneumophila isolates was high. Conclusions. Residents of apartment buildings are exposed to a higher risk of legionellosis than those living in private houses (OR = 2.23; p = 0.011). The highest risk of seropositivity was for residents of buildings with centralized hot water supply (OR = 3.16; p = 0.001). High Legionella spp. prevalence in water supply systems is related to low hot water temperature (average 47.8 ± 0.7 °C) and high presence of free-living amoebae (84.2 %). Environmental isolates of L. pneumophila exhibited a high diversity of SBT and cgMLST sequence types and showed signs of regional clonal expansion, with ST-338, ST-366 and ST-1104 as the predominant sequence type group characteristic of our region. The relative frequency of virulence genes suggests that L. pneumophila isolates have a high virulence potential and the presence of a wide range of virulence factors.