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Legionella pneumophila is the causative agent of Legionnaires' disease. Due to the hot climate and intermittent water supply, the West Bank, Palestine, can be considered a high-risk area for this often fatal atypical pneumonia. L. pneumophila occurs in biofilms of natural and man-made freshwater environments, where it infects and replicates intracellularly within protozoa. To correlate the genetic diversity of the bacteria in the environment with their virulence properties for protozoan and mammalian host cells, 60 genotyped isolates from hospital water systems in the West Bank were analyzed. The L. pneumophila isolates were previously genotyped by high resolution Multi Locus Variable Number of Tandem Repeat Analysis (MLVA-8(12)) and sorted according to their relationship in clonal complexes (VACC). Strains of relevant genotypes and VACCs were compared according to their capacity to infect Acanthamoeba castellanii and THP-1 macrophages, and to mediate pore-forming cytotoxicity in sheep red blood cells (sRBCs). Based on a previous detailed analysis of the biogeographic distribution and abundance of the MLVA-8(12)-genotypes, the focus of the study was on the most abundant L. pneumophila- genotypes Gt4(17), Gt6 (18) and Gt10(93) and the four relevant clonal complexes [VACC1, VACC2, VACC5 and VACC11]. The highly abundant genotypes Gt4(17) and Gt6(18) are affiliated with VACC1 and sequence type (ST)1 (comprising L. pneumophila str. Paris), and displayed seroroup (Sg)1. Isolates of these two genotypes exhibited significantly higher virulence potentials compared to other genotypes and clonal complexes in the West Bank. Endemic for the West Bank was the clonal complex VACC11 (affiliated with ST461) represented by three relevant genotypes that all displayed Sg6. These genotypes unique for the West Bank showed a lower infectivity and cytotoxicity compared to all other clonal complexes and their affiliated genotypes. Interestingly, the L. pneumophila serotypes ST1 and ST461 were previously identified by in situ-sequence based typing (SBT) as main causative agents of Legionnaires' disease (LD) in the West Bank at a comparable level. Overall, this study demonstrates the site-specific regional diversity of L. pneumophila genotypes in the West Bank and suggests that a combination of MLVA, cellular infection assays and hierarchical agglomerative cluster analysis allows an improved genotype-based risk assessment.

The West Bank can be considered as a high-risk area for Legionella prevalence in drinking water due to high ambient temperature, intermittent water supply, frequent pressure loss, and storage of drinking water in roof containers. To assess occurrence of Legionella species, especially L. pneumophila, in the drinking water of the West Bank, the drinking water distribution systems of eight hospitals were sampled over a period of 2.3 years covering the seasonal cycle and the major geographic regions. To gain insight into potential environmental drivers, a set of physico-chemical and microbiological parameters was recorded. Sampling included drinking water and biofilm analyzed by culture and PCR-based methods. Cultivation led to the isolation of 180 strains of L. pneumophila that were genotyped by Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). Surprisingly, the abundance of culturable L. pneumophila was low in drinking water of the sampling sites, with only three out of eight sites where Legionella was observed at all (range: 30-500 CFU/liter). By contrast, biofilm and PCR-based analyses showed a higher prevalence. Statistical analyses with physico-chemical parameters revealed a decrease of L. pneumophila abundance for water and biofilm with increasing magnesium concentrations (>30 mg/l). MLVA-genotype analysis of the L. pneumophila isolates and their spatial distribution indicated three niches characterized by distinct physico-chemical parameters and inhabited by specific consortia of genotypes. This study provides novel insights into mechanisms shaping L. pneumophila populations and triggering their abundance leading to an understanding of their genotype-specific niches and ecology in support of improved prevention measures.

Legionella pneumophila is an environmental bacterium and a human pathogen causing life-threating outbreaks of an atypical pneumonia called Legionnaires’ disease. This study aimed to understand the diversity of L. pneumophila isolates, their clonal populations and the environmental driver of their abundance and prevalence in the West Bank. For this, a multi-annual seasonal sampling of nine drinking water sites in the West Bank were performed with a detailed recording of physico-chemical parameters. A total of 180 L. pneumophila isolates were obtained and analysed by high resolution genotyping (Multi-Locus Variable Number of Tandem Repeats (MLVA-8(12)). Also, physiological and virulence traits were studied. Genotyping and the studied traits led to the selection of representative strains submitted to high through-put genome sequencing (Illumina HiSeq and Pacific Biosciences platforms). Analysis of genotype prevalence in correspondence to environmental factors was used to elucidate genotype consortia and their environmental niches. The 180 isolates represented twenty-six individual MLVA-8(12) genotypes (Gt). The most frequently genotype was Gt4(17) (41.1%). All MLVA genotypes were clustered into four MLVA clonal complexes. Analysis of the prevalence of genotype indicated genotype consortia that seemed to be triggered by a set of environmental drivers. The concentration of several ions (Mg, Ca, Cl, SO4 and TDS) and turbidity seemed to determine niches for three different sets of genotypes and may explain their regional variability. Also, the abundance of L. pneumophila was influenced by these environmental drivers with Mg having a negative effect. The virulence of a representative subset of sixty environmental strains was assessed by five different in-vitro assays. Virulence traits were shown to be genotype dependent. A carefully selected subset of thirty-eight L. pneumophila isolates were genome sequenced and compared to already published reference genomes. Genome sequences were aligned with the sequence of the respective reference genome and analysed with respect to core-single nucleotide polymorphisms (core-SNPs), genomic islands and genes related to virulence traits. Overall, this study provided important insights into detailed population structure, the ecology and pathogenicity of this pathogen in the West Bank., Legionella pneumophila ist ein Umweltbakterium in Süsswassersystemen und humanpathogen; es verursacht lebensbedrohliche Ausbrüche der Legionärskrankheit, einer atypischen Pneumonie. Das Ziel der Doktorarbeit ist es, in der West Bank die Diversität von L. pneumophila-Isolaten, ihre clonale Populationsstruktur und die wesentlichen Umweltfaktoren, die Abundanz und Prävalenz steuern, zu verstehen. Hierzu wurde ein mehrjähriges saisonales Probenahmeprogramm für 9 Trinkwasserprobe-nahmestellen repräsentatv für die West Bank durchgeführt, bei dem physico-chemische und mikrobiologische Parameter mitaufgezeichnet wurden. 180 Isolate wurden hierbei gewonnen, hochauflösend genotypisiert (Multi-Locus Variable Number of Tandem Repeats (MLVA-8(12)), und hinsichtlich relevanter physiologischer Eigenschaften und Virulenzfaktoren analysiert. Die umfassende Charakterisierung der Isolate bildete die Grundlage zur Auswahl repräsentativer Stämme für die Genomsequenzierung mittels Illumina HiSeq und der Pac-Biosciences Platform. Die statistische Analyse von regionaler Genotypprävalenz und Umweltfaktoren wurde dazu genutzt um Genotypkonsortien und ihre Umweltnischen zu charakterisieren. Die 180 Isolate aus der Westbank zeigten eine hohe Genotypdiversität von 26 MLVA-8(12)-Genotypen, zugehörig zu vier MLVA clonalen Komplexen. Analysen der Prävalenz von Genotypen im Hinblick auf Umweltfaktoren zeigte, daß Konsortien von Genotypen durch Umweltfaktoren getriggert wurden. Unterschiedliche Konzentrationen von Ionen im Trinkwasser (Mg, Ca, Cl, SO4, TDS) and Trübung schienen Nischen für drei unterschiedliche Konsortien von Genotypen zu bestimmen, und könnten die räumliche Variabilität der Genotypen in der West Bank erklären und die Abundanz von L. pneumophila zu beeinflussen. Dabei schien Mg eine relevante Rolle zu spielen. Die Virulenz einer repräsentativen Untergruppe von 60 Isolaten wurde mit 5 in-vitro Ansätzen getestet. Die Virulenzstärke variierte in Abhängigkeit vom Genotyp mit relativ hoher Virulenz der häufigen Genotypen Gt4(17) und Gt6(18). Eine repräsentative Auswahl von 38 L. pneumophila Isolaten wurden genomsequenziert und mit Referenzgenomen und generierten eigenen Referenzgenomen verglichen, und hinsichtlich core-SNPs, genomischen Inseln und Virulenz-assoziierten Genen analysiert. Insgesamt gesehen, liefert die vorliegende Studie wichtige Einblicke zum Verständnis von Populationsstruktur, Ökologie und Pathogenität dieses pathogenen Bakteriums in der West Bank.