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

      Pereira, Rui P A; Peplies, Jörg; Mushi, Douglas; Brettar, Ingrid; Höfle, Manfred G; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-01-01)
      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
    • Shewanella baltica Ecotypes Have Wide Transcriptional Variation under the Same Growth Conditions.

      Hambright, W S; Deng, Jie; Tiedje, James M; Brettar, Ingrid; Rodrigues, Jorge L M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-09)
      In bacterial populations, subtle expressional differences may promote ecological specialization through the formation of distinct ecotypes. In a barrier-free habitat, this process most likely precedes population divergence and may predict speciation events. To examine this, we used four sequenced strains of the bacterium Shewanella baltica, OS155, OS185, OS195, and OS223, as models to assess transcriptional variation and ecotype formation within a prokaryotic population. All strains were isolated from different depths throughout a water column of the Baltic Sea, occupying different ecological niches characterized by various abiotic parameters. Although the genome sequences are nearly 100% conserved, when grown in the laboratory under standardized conditions, all strains exhibited different growth rates, suggesting significant expressional variation. Using the Ecotype Simulation algorithm, all strains were considered to be discrete ecotypes when compared to 32 other S. baltica strains isolated from the same water column, suggesting ecological divergence. Next, we employed custom microarray slides containing oligonucleotide probes representing the core genome of OS155, OS185, OS195, and OS223 to detect natural transcriptional variation among strains grown under identical conditions. Significant transcriptional variation was noticed among all four strains. Differentially expressed gene profiles seemed to coincide with the metabolic signatures of the environment at the original isolation depth. Transcriptional pattern variations such as the ones highlighted here may be used as indicators of short-term evolution emerging from the formation of bacterial ecotypes. IMPORTANCE Eukaryotic studies have shown considerable transcriptional variation among individuals from the same population. It has been suggested that natural variation in eukaryotic gene expression may have significant evolutionary consequences and may explain large-scale phenotypic divergence of closely related species, such as humans and chimpanzees (M.-C. King and A. C. Wilson, Science 188:107-116, 1975, http://dx.doi.org/10.1126/science.1090005; M. F. Oleksiak, G. A. Churchill, and D. L. Crawford, Nat Genet 32:261-266, 2002, http://dx.doi.org/10.1038/ng983). However, natural variation in gene expression is much less well understood in prokaryotic organisms. In this study, we used four sequenced strains of the marine bacterium Shewanella baltica to better understand the natural transcriptional divergence of a stratified prokaryotic population. We found substantial low-magnitude expressional variation among the four S. baltica strains cultivated under identical laboratory conditions. Collectively, our results indicate that transcriptional variation is an important factor for ecological speciation.
    • Spatial distribution of Legionella pneumophila MLVA-genotypes in a drinking water system.

      Rodríguez-Martínez, Sarah; Sharaby, Yehonatan; Pecellín, Marina; Brettar, Ingrid; Höfle, Manfred; Halpern, Malka; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015-06-15)
      Bacteria of the genus Legionella cause water-based infections, resulting in severe pneumonia. To improve our knowledge about Legionella spp. ecology, its prevalence and its relationships with environmental factors were studied. Seasonal samples were taken from both water and biofilm at seven sampling points of a small drinking water distribution system in Israel. Representative isolates were obtained from each sample and identified to the species level. Legionella pneumophila was further determined to the serotype and genotype level. High resolution genotyping of L. pneumophila isolates was achieved by Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Within the studied water system, Legionella plate counts were higher in summer and highly variable even between adjacent sampling points. Legionella was present in six out of the seven selected sampling points, with counts ranging from 1.0 × 10(1) to 5.8 × 10(3) cfu/l. Water counts were significantly higher in points where Legionella was present in biofilms. The main fraction of the isolated Legionella was L. pneumophila serogroup 1. Serogroup 3 and Legionella sainthelensis were also isolated. Legionella counts were positively correlated with heterotrophic plate counts at 37 °C and negatively correlated with chlorine. Five MLVA-genotypes of L. pneumophila were identified at different buildings of the sampled area. The presence of a specific genotype, "MLVA-genotype 4", consistently co-occurred with high Legionella counts and seemed to "trigger" high Legionella counts in cold water. Our hypothesis is that both the presence of L. pneumophila in biofilm and the presence of specific genotypes, may indicate and/or even lead to high Legionella concentration in water. This observation deserves further studies in a broad range of drinking water systems to assess its potential for general use in drinking water monitoring and management.
    • Temperature-dependent growth modeling of environmental and clinical Legionella pneumophila MLVA-genotypes.

      Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Oks, Olga; Pecellin, Marina; Mizrahi, Hila; Peretz, Avi; Brettar, Ingrid; Höfle, Manfred; Halpern, Malka; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-03)
      Legionella pneumophila cause waterborne infections resulting in severe pneumonia. High resolution genotyping of L. pneumophila isolates can be achieved by Multiple-Locus Variable number of 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 current 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 compare it with 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 (25-30 °C) while Gt15 strains appeared to be best adapted to relatively higher temperatures (42-45 °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 better understanding of their ecological niches in drinking water systems as well as in the human body.
    • Virulence traits of environmental and clinicalLegionella pneumophilaMLVA genotypes.

      Sharaby, Yehonatan; Rodríguez-Martínez, Sarah; Pecellin, Marina; Sela, Rotem; Peretz, Avi; Höfle, Manfred; Halpern, Malka; Brettar, Ingrid; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-03-09)
      Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different variable numbers of tandem repeat analysis (MLVA-8) genotypes dominated different sites of a drinking-water distribution system, with a genotype-related temperature and abundance regime. Here we compare the pathogenicity potential of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity towards amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity towards amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity towards macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strain showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective towardsAcanthamoeba castellaniiMoreover, Gt4 strain exhibited increased cytotoxicity towards macrophages and demonstrated a broader temperature range of amoebal lysis compared to Gt6 and Gt15. Understanding the virulence traits ofLegionellagenotypes may improve the assessment of public health risks ofLegionellain drinking water.ImportanceLegionella pneumophilais the causative agent of a severe form of pneumonia. Here we demonstrate that clinical strains were significantly more cytotoxic towards red blood cells compared to environmental strains, while their cytotoxicity towards macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic towards amoebae, macrophages, and lysed amoebae in a broader temperature range, compared to the other studied genotypes. The results can explain the Gt4 relatively high success in the environment and in clinical samples; thus Gt4 strains should be considered as a main factor for the assessment of public health risks ofLegionellain drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of differentL. pneumophilagenotypes that can be a valuable parameter for future modelling and Quantitative Microbial Risk Assessment ofLegionellain drinking water systems.