Climate influence on Vibrio and associated human diseases during the past half-century in the coastal North Atlantic.

Climate change is having a dramatic impact on marine animal and plant communities but little is known of its influence on marine prokaryotes, which represent the largest living biomass in the world oceans and play a fundamental role in maintaining life on our planet. In this study, for the first time to our knowledge, experimental evidence is provided on the link between multidecadal climatic variability in the temperate North Atlantic and the presence and spread of an important group of marine prokaryotes, the vibrios, which are responsible for several infections in both humans and animals. Using archived formalin-preserved plankton samples collected by the Continuous Plankton Recorder survey over the past half-century (1958-2011), we assessed retrospectively the relative abundance of vibrios, including human pathogens, in nine areas of the North Atlantic and North Sea and showed correlation with climate and plankton changes. Generalized additive models revealed that long-term increase in Vibrio abundance is promoted by increasing sea surface temperatures (up to ∼1.5 °C over the past 54 y) and is positively correlated with the Northern Hemisphere Temperature (NHT) and Atlantic Multidecadal Oscillation (AMO) climatic indices (P < 0.001). Such increases are associated with an unprecedented occurrence of environmentally acquired Vibrio infections in the human population of Northern Europe and the Atlantic coast of the United States in recent years.

Virulence Traits of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different MLVA-8 (multilocus variable-number tandem-repeat analysis using 8 loci) genotypes dominated different sites of a drinking-water distribution system. Each genotype displayed a unique temperature-dependent growth behavior. Here we compared the pathogenicity potentials of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity toward amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity toward amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity toward macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strains showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective toward Acanthamoeba castellanii Moreover, Gt4 strains exhibited increased cytotoxicity toward macrophages and demonstrated a broader temperature range of amoebal lysis than Gt6 and Gt15 strains. Understanding the virulence traits of Legionella genotypes may improve the assessment of public health risks of Legionella in drinking water.IMPORTANCELegionella pneumophila is the causative agent of a severe form of pneumonia. Here we demonstrated that clinical strains were significantly more cytotoxic toward red blood cells than environmental strains, while their cytotoxicity toward macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic toward amoebae and macrophages and lysed amoebae in a broader temperature range than to the other studied genotypes. The results can explain the relatively high success of Gt4 in the environment and in clinical samples; thus, Gt4 strains should be considered a main factor for the assessment of public health risks of Legionella in drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of different L. pneumophila genotypes, which can be a valuable parameter for future modeling and quantitative microbial risk assessment of Legionella in drinking-water systems.

Divergence in Gene Regulation Contributes to Sympatric Speciation of Shewanella baltica Strains.

Niche partitioning and sequence evolution drive genomic and phenotypic divergence, which ultimately leads to bacterial diversification. This study investigated the genomic composition of two Shewanella baltica clades previously identified through multilocus sequencing typing and recovered from the redox transition zone in the central Baltic Sea. Comparative genomic analysis revealed significantly higher interclade than intraclade genomic dissimilarity and that a subset of genes present in clade A were associated with potential adaptation to respiration of sulfur compounds present in the redox transition zone. The transcriptomic divergence between two representative strains of clades A and D, OS185 and OS195, was also characterized and revealed marked regulatory differences. We found that both the transcriptional divergence of shared genes and expression of strain-specific genes led to differences in regulatory patterns between strains that correlate with environmental redox niches. For instance, under anoxic conditions of respiratory nitrate ammonification, OS185-the strain isolated from a nitrate-rich environment-upregulated nearly twice the number of shared genes upregulated by OS195-the strain isolated from an H2S-containing anoxic environment. Conversely, OS195 showed stronger induction of strain-specific genes, especially those associated with sulfur compound respiration, under thiosulfate-reducing conditions. A positive association between the level of transcriptional divergence and the level of sequence divergence for shared genes was also noted. Our results provide further support for the hypothesis that genomic changes impacting transcriptional regulation play an important role in the diversification of ecologically distinct populations.IMPORTANCE This study examined potential mechanisms through which co-occurring Shewanella baltica strains diversified to form ecologically distinct populations. At the time of isolation, the strains studied composed the major fraction of culturable nitrate-reducing communities in the Baltica Sea. Analysis of genomic content of 13 S. baltica strains from two clades representing different ecotypes demonstrated that one clade specifically possesses a number of genes that could favor successful adaptation to respire sulfur compounds in the portion of the water column from which these strains were isolated. In addition, transcriptional profiling of fully sequenced strains representative of these two clades, OS185 and OS195, under oxygen-, nitrate-, and thiosulfate-respiring conditions demonstrated that the strains exhibit relatively similar transcriptional responses during aerobic growth but more-distinct transcriptional responses under nitrate- and thiosulfate-respiring conditions. Results from this study provide insights into how genomic and gene regulatory diversification together impacted the redox specialization of the S. baltica strains.

Comparison of in situ sequence type analysis of Legionella pneumophila in respiratory tract secretions and environmental samples of a hospital in East Jerusalem.

Legionella pneumophila genotyping is important for epidemiological investigation of nosocomial and community-acquired outbreaks of legionellosis. The prevalence of legionellosis in pneumonia patients in the West Bank was monitored for the first time, and the sequence types (STs) from respiratory samples were compared with STs of environmental samples from different wards of the hospital. Sputum (n = 121) and bronchoalveolar lavage (BAL) (n = 74) specimens were cultured for L. pneumophila; genomic DNA was tested by 16S rRNA polymerase chain reaction (PCR) amplification. Nested PCR sequence-based typing (NPSBT) was implemented on DNA of the respiratory and environmental PCR-positive samples. Only one respiratory specimen was positive for L. pneumophila by culture. BAL gave a higher percentage of L. pneumophila-positive samples, 35% (26/74) than sputum, 15% (18/121) by PCR. NPSBT revealed the following STs: ST 1 (29%, 7/24), ST 461 (21%, 5/24), ST 1037 (4%, 1/24) from respiratory samples, STs from environmental samples: ST 1 (28.5%, 4/14), ST 187 (21.4%, 3/14) and ST 2070, ST 461, ST 1482 (7.1%, 1/14) each. This study emphasises the advantage of PCR over culture for the detection of L. pneumophila in countries where antibiotics are indiscriminately used prior to hospital admission. ST 1 was the predominant ST in both respiratory and environmental samples.

Temperature-Dependent Growth Modeling of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

Legionella pneumophila causes waterborne infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA). Recently, we found that different MLVA genotypes of L. pneumophila dominated different sites in a small drinking-water network, with a genotype-related temperature and abundance regime. The present study focuses on understanding the temperature-dependent growth kinetics of the genotypes that dominated the water network. Our aim was to model mathematically the influence of temperature on the growth kinetics of different environmental and clinical L. pneumophila genotypes and to compare it with the influence of their ecological niches. Environmental strains showed a distinct temperature preference, with significant differences among the growth kinetics of the three studied genotypes (Gt4, Gt6, and Gt15). Gt4 strains exhibited superior growth at lower temperatures (25 and 30°C), while Gt15 strains appeared to be best adapted to relatively higher temperatures (42 and 45°C). The temperature-dependent growth traits of the environmental genotypes were consistent with their distribution and temperature preferences in the water network. Clinical isolates exhibited significantly higher growth rates and reached higher maximal cell densities at 37°C and 42°C than the environmental strains. Further research on the growth preferences of L. pneumophila clinical and environmental genotypes will result in a better understanding of their ecological niches in drinking-water systems as well as in the human body.IMPORTANCELegionella pneumophila is a waterborne pathogen that threatens humans in developed countries. The bacteria inhabit natural and man-made freshwater environments. Here we demonstrate that different environmental L. pneumophila genotypes have different temperature-dependent growth kinetics. Moreover, Legionella strains that belong to the same species but were isolated from environmental and clinical sources possess adaptations for growth at different temperatures. These growth preferences may influence the bacterial colonization at specific ecological niches within the drinking-water network. Adaptations for growth at human body temperatures may facilitate the abilities of some L. pneumophila strains to infect and cause illness in humans. Our findings may be used as a tool to improve Legionella monitoring in drinking-water networks. Risk assessment models for predicting the risk of legionellosis should take into account not only Legionella concentrations but also the temperature-dependent growth kinetics of the isolates.

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems.

BACKGROUND: Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. METHODS: A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples. RESULTS: Our results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers. CONCLUSIONS: The developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.

Stability, genotypic and phenotypic diversity of Shewanella baltica in the redox transition zone of the Baltic Sea.

Studying how bacterial strains diverge over space and time and how divergence leads to ecotype formation is important for understanding structure and dynamics of environmental communities. Here we assess the ecological speciation and temporal dynamics of a collection of Shewanella baltica strains from the redox transition zone of the central Baltic Sea, sampled at three time points over a course of 12 years, with a subcollection containing 46 strains subjected to detailed genetic and physiological characterization. Nine clades were consistently recovered by three different genotyping approaches: gyrB gene sequencing, multilocus sequence typing (MLST) and whole genome clustering of data from comparative genomic hybridization, and indicated specialization according to nutrient availability, particle association and temporal distribution. Genomic analysis suggested higher intra- than inter-clade recombination that might result from niche partitioning. Substantial heterogeneity in carbon utilization and respiratory capabilities suggested rapid diversification within the same 'named' species and physical habitat and showed consistency with genetic relatedness. At least two major ecotypes, represented by MLST clades A and E, were proposed based on genetic, ecological and physiological distinctiveness. This study suggests that genetic analysis in conjunction with phenotypic evaluation can provide better understanding of the ecological framework and evolutionary trajectories of microbial species.

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.

Genome sequencing of five Shewanella baltica strains recovered from the oxic-anoxic interface of the Baltic Sea.

Here we describe five Shewanella baltica genomes recovered from the same sample, as well as 12 years apart from the same sampling station. These genomes expand the collection of previously sequenced S. baltica strains and represent a valuable resource for assessing the role of environmental settings on genome adaptation.

Analysis of structure and composition of bacterial core communities in mature drinking water biofilms and bulk water of a citywide network in Germany.

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity.

Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.

The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water.

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.

High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.

Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.

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.

Molecular assessment of bacterial pathogens - a contribution to drinking water safety.

Human bacterial pathogens are considered as an increasing threat to drinking water supplies worldwide because of the growing demand of high-quality drinking water and the decreasing quality and quantity of available raw water. Moreover, a negative impact of climate change on freshwater resources is expected. Recent advances in molecular detection technologies for bacterial pathogens in drinking water bear the promise in improving the safety of drinking water supplies by precise detection and identification of the pathogens. More importantly, the array of molecular approaches allows understanding details of infection routes of waterborne diseases, the effects of changes in drinking water treatment, and management of freshwater resources.

Antimicrobial agent susceptibilities of Legionella pneumophila MLVA-8 genotypes.

Legionella pneumophila causes human lung infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA-8). Legionella infections in humans occur as a result of inhalation of bacteria-containing aerosols, thus, our aim was to study the antimicrobial susceptibilities of different MLVA-8 genotypes to ten commonly used antimicrobial agents in legionellosis therapy. Epidemiological cut-off values were determined for all antibiotics. Significant differences were found between the antimicrobial agents' susceptibilities of the three studied environmental genotypes (Gt4, Gt6, and Gt15). Each genotype exhibited a significantly different susceptibility profile, with Gt4 strains (Sequence Type 1) significantly more resistant towards most studied antimicrobial agents. In contrast, Gt6 strains (also Sequence Type 1) were more susceptible to six of the ten studied antimicrobial agents compared to the other genotypes. Our findings show that environmental strains isolated from adjacent points of the same water system, exhibit distinct antimicrobial resistance profiles. These differences highlight the importance of susceptibility testing of Legionella strains. In Israel, the most extensively used macrolide for pneumonia is azithromycin. Our results point at the fact that clarithromycin (another macrolide) and trimethoprim with sulfamethoxazole (SXT) were the most effective antimicrobial agents towards L. pneumophila strains. Moreover, legionellosis can be caused by multiple L. pneumophila genotypes, thus, the treatment approach should be the use of combined antibiotic therapy. Further studies are needed to evaluate specific antimicrobial combinations for legionellosis therapy.

Genomic Variations Underlying Speciation and Niche Specialization of Shewanella baltica.

Shewanella baltica was the dominant culturable nitrate-reducing bacterium in the eutrophic and strongly stratified Baltic Sea in the 1980s, where it primarily inhabited the oxic-anoxic transition zone. The genomic structures of 46 of these isolates were investigated through comparative genomic hybridization (CGH), which revealed a gradient of genomic similarity, ranging from 65% to as high as 99%. The core genome of the S. baltica species was enriched in anaerobic respiration-associated genes. Auxiliary genes, most of which locate within a few genomic islands (GIs), were nonuniformly distributed among the isolates. Specifically, hypothetical and mobile genetic element (MGE)-associated genes dominated intraclade gene content differences, whereas gain/loss of functional genes drove gene content differences among less related strains. Among the major S. baltica clades, gene signatures related to specific redox-driven and spatial niches within the water column were identified. For instance, genes involved in anaerobic respiration of sulfur compounds may provide key adaptive advantages for clade A strains in anoxic waters where sulfur-containing electron acceptors are present. Genes involved in cell motility, in particular, a secondary flagellar biosynthesis system, may be associated with the free-living lifestyle by clade E strains. Collectively, this study revealed characteristics of genome variations present in the water column and active speciation of S. baltica strains, driven by niche partitioning and horizontal gene transfer (HGT).IMPORTANCE Speciation in nature is a fundamental process driving the formation of the vast microbial diversity on Earth. In the central Baltic Sea, the long-term stratification of water led to formation of a large-scale vertical redoxcline that provided a gradient of environmental niches with respect to the availability of electron acceptors and donors. The region was home to Shewanella baltica populations, which composed the dominant culturable nitrate-reducing bacteria, particularly in the oxic-anoxic transition zone. Using the collection of S. baltica isolates as a model system, genomic variations showed contrasting gene-sharing patterns within versus among S. baltica clades and revealed genomic signatures of S. baltica clades related to redox niche specialization as well as particle association. This study provides important insights into genomic mechanisms underlying bacterial speciation within this unique natural redoxcline.

Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower.

Pseudomonas species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. P. aeruginosa is the species most commonly associated with human disease, causing a wide variety of infections with links to its presence in freshwater systems. Though several other Pseudomonas species are of ecological and public health importance, little knowledge exists regarding environmental abundances of these species. In the present study, an Illumina-based next-generation sequencing (NGS) approach using Pseudomonas-specific primers targeting the 16S rRNA gene was evaluated and applied to a set of freshwater samples from different environments including a cooling tower sampled monthly during 2 years. Our approach showed high in situ specificity and accuracy. NGS read counts revealed a precise quantification of P. aeruginosa and a good correlation with the absolute number of Pseudomonas genome copies in a validated genus-specific qPCR assay, demonstrating the ability of the NGS approach to determine both relative and absolute abundances of Pseudomonas species and P. aeruginosa. The characterization of Pseudomonas communities in cooling tower water allowed us to identify 43 phylotypes, with P. aeruginosa being the most abundant. A shift existed within each year from a community dominated by phylotypes belonging to P. fluorescens and P. oleovorans phylogenetic groups to a community where P. aeruginosa was highly abundant. Co-occurrence was observed between P. aeruginosa and other phylotypes of P. aeruginosa group as well as the potentially pathogenic species P. stutzeri, but not with phylotypes of the P. fluorescens group, indicating the need to further investigate the metabolic networks and ecological traits of Pseudomonas species. This study demonstrates the potential of deep sequencing as a valuable tool in environmental diagnostics and surveillance of health-related pathogens in freshwater environments.