Legionella pneumophila Presence in Dental Unit Waterlines: A Cultural and Molecular Investigation in the West Bank, Palestine.

A Legionella pneumophila bacterium is ubiquitous in water distribution systems, including dental unit waterlines (DUWLs). Legionellosis is atypical pneumonia, including Legionnaires' disease (LD) and the less acute form of Pontiac fever. Legionellosis occurs as a result of inhalation/aspiration of aerosolized Legionella-contaminated water by susceptible patients, health workers, and dentists. In this study, we undertook to determine the prevalence of Legionella in water and biofilm samples from Tap and DUWLs collected from five sites of dental clinics and faculties across the West Bank. Water samples were tested for physical and chemical parameters. The study samples included 185 samples, 89 (48%) water samples, and 96 (52%) biofilm swabs, which were analyzed by cultivation-dependent analysis (CDA) and by the cultivation-independent technique (CIA). Also, partial sequencing of the 16S rRNA gene for fifteen L. pneumophila isolates was performed for quality assurance and identification. L. pneumophila was isolated from 28 (15%) of 185 samples using CDA and was detected in 142 (77%) of 185 samples using CIA. The abundance of culturable L. pneumophila was low in DUWL of the sampling sites (range: 27-115 CFU/Liter). PCR was 5× more sensitive than the culture technique. L. pneumophila Sg 1 was detected in (75%) of the isolates, while (25%) isolates were L. pneumophila Sg 2-14. All fifteen sequenced Legionella isolates were identified as L. pneumophila ≥ 94.5%. The analysis of phylogenetic tree showed that L. pneumophila branch clearly identified and distinguished from other branches. These results show that DUWLs of the examined dental clinics and faculties are contaminated with L. pneumophila. This finding reveals a serious potential health risk for infection of immunocompromised patients and dentists' post-exposure.

Comparative Genomics of Legionella pneumophila Isolates from the West Bank and Germany Support Molecular Epidemiology of Legionnaires' Disease.

Legionella pneumophila is an environmental bacterium and clinical pathogen that causes many life-threating outbreaks of an atypical pneumonia called Legionnaires' disease (LD). Studies of this pathogen have focused mainly on Europe and the United States. A shortage in L. pneumophila data is clearly observed for developing countries. To reduce this knowledge gap, L. pneumophila isolates were studied in two widely different geographical areas, i.e., the West Bank and Germany. For this study, we sequenced and compared the whole genome of 38 clinical and environmental isolates of L. pneumophila covering different MLVA-8(12) genotypes in the two areas. Sequencing was conducted using the Illumina HiSeq 2500 platform. In addition, two isolates (A194 and H3) were sequenced using a Pacific Biosciences (PacBio) RSII platform to generate complete reference genomes from each of the geographical areas. Genome sequences from 55 L. pneumophila strains, including 17 reference strains, were aligned with the genome sequence of the closest strain (L. pneumophila strain Alcoy). A whole genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using the ParSNP software v 1.0. The reference genomes obtained for isolates A194 and H3 consisted of circular chromosomes of 3,467,904 bp and 3,691,263 bp, respectively. An average of 36,418 SNPs (min. 8569, max. 70,708 SNPs) against our reference strain L. pneumophila str. Alcoy, and 2367 core-genes were identified among the fifty-five strains. An analysis of the genomic population structure by SNP comparison divided the fifty-five L. pneumophila strains into six branches. Individual isolates in sub-lineages in these branches differed by less than 120 SNPs if they had the same MLVA genotype and were isolated from the same location. A bioinformatics analysis identified the genomic islands (GIs) for horizontal gene transfer and mobile genetic elements, demonstrating that L. pneumophila showed high genome plasticity. Four L. pneumophila isolates (H3, A29, A129 and L10-091) contained well-defined plasmids. On average, only about half of the plasmid genes could be matched to proteins in databases. In silico phage findings suggested that 43 strains contained at least one phage. However, none of them were found to be complete. BLASTp analysis of proteins from the type IV secretion Dot/Icm system showed those proteins highly conserved, with less than 25% structural differences in the new L. pneumophila isolates. Overall, we demonstrated that whole genome sequencing provides a molecular surveillance tool for L. pneumophila at the highest conceivable discriminatory level, i.e., two to eight SNPs were observed for isolates from the same location but several years apart.

Whole-genome sequencing of the clinical isolate of Legionella pneumophila ALAW1 from the West Bank allows high-resolution typing and determination of pathogenicity mechanisms.

Background: Legionella pneumophila is water-based bacterium causing Legionnaires' disease (LD). We describe the first documented case of nosocomial LD caused by L. pneumophila sequence type (ST) 461 and serogroup 6. The etiology of LD was confirmed by culturing the bronchoalveolar lavage sample retrieving L. pneumophila strain ALAW1. A 7-days treatment of the LD patient with Azithromycin and Levofloxacin allowed complete recovery. Methods: In details, we sequenced the whole genome of the L. pneumophila ALAW1 using Illumina HiSeq platform. The sequence of ALAW1 was aligned with the genome sequence from the closely related reference strain Alcoy 2300/99 and a whole-genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using Parsnp software. Also, the TYGS web-server was used in order to compare the genome with type strain. Results: An analysis of the population structure by SNP and TYGS comparison clustered ALAW1 with the reference genome Alcoy 2300/99. Blastp analysis of the type IV secretion Dot/Icm system genes showed that these genes were highly conserved with (≤25%) structural differences at the protein level. Conclusions: Overall, this study provides insights into detailed genome structure and demonstrated the value of whole-genome sequencing as the ultimate typing tool for Legionella.

Cytotoxicity, Intracellular Replication, and Contact-Dependent Pore Formation of Genotyped Environmental Legionella pneumophila Isolates from Hospital Water Systems in the West Bank, Palestine.

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.

Biogeography and Environmental Drivers of Legionella pneumophila Abundance and Genotype Composition Across the West Bank: Relevance of a Genotype-Based Ecology for Understanding Legionella Occurrence.

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.

Characterization of Legionella pneumophila Populations by Multilocus Variable Number of Tandem Repeats (MLVA) Genotyping from Drinking Water and Biofilm in Hospitals from Different Regions of the West Bank.

The West Bank can be considered a high-risk area for Legionnaires' disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. Legionella, mostly L. pneumophila, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of Legionella spp and L. pneumophila using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of Legionella in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 L. pneumophila strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of L. pneumophila. Overall, our results provide important insights into the detailed population structure of L. pneumophila, allowing for better risk assessment for DWDS.