Gene flow in environmental Legionella pneumophila leads to genetic and pathogenic heterogeneity within a Legionnaires’ disease outbreak

Background

Legionnaires’ disease is a severe form of pneumonia caused by the environmental bacterium Legionella pneumophila. Outbreaks commonly affect people with known risk factors, but the genetic and pathogenic complexity of L. pneumophila within an outbreak is not well understood. Here, we investigate the etiology of the major Legionnaires’ disease outbreak that occurred in Edinburgh, UK, in 2012, by examining the evolutionary history, genome content, and virulence of L. pneumophila clinical isolates.

Results

Our high resolution genomic approach reveals that the outbreak was caused by multiple genetic subtypes of L. pneumophila, the majority of which had diversified from a single progenitor through mutation, recombination, and horizontal gene transfer within an environmental reservoir prior to release. In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution. Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.

Conclusions

Taken together, our study indicates previously cryptic levels of pathogen heterogeneity within a Legionnaires’ disease outbreak, a discovery that impacts on source attribution for future outbreak investigations. Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0504-1) contains supplementary material, which is available to authorized users.     

Diagnostic testing for Legionnaires’ disease

Legionnaires’ disease is commonly diagnosed clinically using a urinary antigen test. The urinary antigen test is highly accurate for L. pneumophila serogroup 1, however other diagnostic tests should also be utilized in conjunction with the urinary antigen as many other Legionella species and serogroups are pathogenic. Culturing of patient specimens remains the gold standard for diagnosis of Legionnaires’ disease. Selective media, BYCE with the addition of antibiotics, allows for a high sensitivity and specificity. Culturing can identify all species and serogroups of Legionella. A major benefit of culturing is that it provides the recovery of a patient isolate, which can be used to find an environmental match. Other diagnostic tests, including DFA and molecular tests such as PCR and LAMP, are useful tests to supplement culturing. Molecular tests provide much more rapid results in comparison to culture, however these tests should not be a primary diagnostic tool given their lower sensitivity and specificity in comparison to culturing. It is recommended that all laboratories develop the ability to culture patient specimens in-house with the selective media.     

Alpine crossroads or origin of genetic diversity? Comparative phylogeography of two sympatric microgastropod species

The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa--Carychium minimum O.F. Müller, 1774 and Carychium tridentatum (Risso, 1826) (Gastropoda, Eupulmonata, Carychiidae)--by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant 'hot-spot' for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general.     

Molecular genetic studies of familial Meniere’s disease

<正>Dear Editor.Meniere's disease (MD,MIM 156000),a chronic clinical illness affecting the inner ear,presents as episodes of spontaneous vertigo,fluctuating sensorineural hearing loss,tinnitus,and aural fullness.Endolymphatic hydrops in the cochlear duct and vestibular organs is considered the underlying histopathologic characteristic of MD.Most MD cases are sporadic (sporadic Meniere's disease,SMD),and approximately 4%-20%of patients with MD have a familial history.Familial Meniere's disease (FMD) is defined as the clinical symptoms of at least one patient's relative (first-or     

Can novel genetic analyses help to identify low-dispersal marine invasive species?

Genetic methods can be a powerful tool to resolve the native versus introduced status of populations whose taxonomy and biogeography are poorly understood. The genetic study of introduced species is presently dominated by analyses that identify signatures of recent colonization by means of summary statistics. Unfortunately, such approaches cannot be used in low‐dispersal species, in which recently established populations originating from elsewhere in the species' native range also experience periods of low population size because they are founded by few individuals. We tested whether coalescent‐based molecular analyses that provide detailed information about demographic history supported the hypothesis that a sea squirt whose distribution is centered on Tasmania was recently introduced to mainland Australia and New Zealand through human activities. Methods comparing trends in population size (Bayesian Skyline Plots and Approximate Bayesian Computation) were no more informative than summary statistics, likely because of recent intra‐Tasmanian dispersal. However, IMa2 estimates of divergence between putatively native and introduced populations provided information at a temporal scale suitable to differentiate between recent (potentially anthropogenic) introductions and ancient divergence, and indicated that all three non‐Tasmanian populations were founded during the period of European settlement. While this approach can be affected by inaccurate molecular dating, it has considerable (albeit largely unexplored) potential to corroborate nongenetic information in species with limited dispersal capabilities.     

Integrative computational evaluation of genetic markers for Alzheimer’s disease

Recent studies have reported hundreds of genes linked to Alzheimer’s Disease (AD). However, many of these candidate genes may be not identified in different studies when analyses were replicated. Moreover, results could be controversial. Here, we proposed a computational workflow to curate and evaluate AD related genes. The method integrates large scale literature knowledge data and gene expression data that were acquired from postmortem human brain regions (AD case/control: 31/32 and 22/8). Pathway Enrichment, Sub-Network Enrichment, and Gene-Gene Interaction analysis were conducted to study the pathogenic profile of the candidate genes, with 4 metrics proposed and validated for each gene. By using our approach, a scalable AD genetic database was developed, including AD related genes, pathways, diseases and info of supporting references. The AD case/control classification supported the effectiveness of the 4 proposed metrics, which successfully identified 21 well-studied AD genes (i.g. TGFB1, CTNNB1, APP, IL1B, PSEN1, PTGS2, IL6, VEGFA, SOD1, AKT1, CDK5, TNF, GSK3B, TP53, CCL2, BDNF, NGF, IGF1, SIRT1, AGER and TLR) and highlighted one recently reported AD gene (i.g. ITGB1). The computational biology approach and the AD database developed in this study provide a valuable resource which may facilitate the understanding of the AD genetic profile.     

Sequence analyses of presenilin mutations linked to familial Alzheimer’s disease

Familial Alzheimer’s disease (FAD)-linked presenilin (PS) mutations show gain-of-toxic-function characteristics. These FAD PS mutations are scattered throughout the PS molecule, reminiscent of the distribution of cystic fibrosis transmembrane conductance regulator and p53 mutations. Because of the scattered distribution of PS mutations, it is difficult to infer mechanistic insights about how these mutations cause the disease similarly. Recent careful reexamination of γ-secretase activity indicates that some PS mutations decrease the proteolytic activity of γ-secretase, suggesting a loss-of-function nature of PS mutations. To extend this observation to all known PS mutations, a large number of PS mutations were evaluated using bioinformatic tools. The analyses reveal that as many as one third of PS1 residues are highly conserved, that about 75% of FAD mutations are located to the highly conserved residues, and that most PS mutations likely damage the activity of PS. These results are consistent with the idea that the majority of PS mutations lower the activity of PS/γ-secretase.     

GSEA-SNP identifies genes associated with Johne’s disease in cattle

SNP-based gene-set enrichment analysis from single nucleotide polymorphisms, or GSEA-SNP, is a tool to identify candidate genes based on enrichment analysis of sets of genes rather than single SNP associations. The objective of this study was to identify modest-effect genes associated with Mycobacterium avium subsp. paratuberculosis (Map) tissue infection or fecal shedding using GSEA-SNP applied to KEGG pathways or Gene Ontology (GO) gene sets. The Illumina Bovine SNP50 BeadChip was used to genotype 209 Holstein cows for the GSEA-SNP analyses. For each of 13,744 annotated genes genome-wide located within 50 kb of a Bovine SNP50 SNP, the single SNP with the highest Cochran-Armitage Max statistic was used as a proxy statistic for that gene’s strength of affiliation with Map. Gene-set enrichment was tested using a weighted Kolmogorov-Smirnov-like running sum statistic with data permutation to adjust for multiple testing. For tissue infection and fecal shedding, no gene sets in KEGG pathways or in GO sets for molecular function or cellular component were enriched for signal. The GO biological process gene set for positive regulation of cell motion (GO:0051272, q = 0.039, 5/11 genes contributing to the core enrichment) was enriched for Map tissue infection, while no GO biological process gene sets were enriched for fecal shedding. GSEA-SNP complements traditional SNP association approaches to identify genes of modest effects as well as genes with larger effects as demonstrated by the identification of one locus that we previously found to be associated with Map tissue infection using a SNP-by-SNP genome-wide association study.     

Characterization and resuscitation of ‘non-culturable’ cells of Legionella pneumophila

Background

Legionella pneumophila is a waterborne pathogen responsible for Legionnaires’ disease, an infection which can lead to potentially fatal pneumonia. After disinfection, L. pneumophila has been detected, like many other bacteria, in a “viable but non culturable” state (VBNC). The physiological significance of the VBNC state is unclear and controversial: it could be an adaptive response favoring long-term survival; or the consequence of cellular deterioration which, despite maintenance of certain features of viable cells, leads to death; or an injured state leading to an artificial loss of culturability during the plating procedure. VBNC cells have been found to be resuscitated by contact with amoebae.

Results

We used quantitative microscopic analysis, to investigate this “resuscitation” phenomenon in L. pneumophila in a model involving amending solid plating media with ROS scavengers (pyruvate or glutamate), and co-culture with amoebae. Our results suggest that the restoration observed in the presence of pyruvate and glutamate may be mostly due to the capacity of these molecules to help the injured cells to recover after a stress. We report evidence that this extracellular signal leads to a transition from a not-culturable form to a culturable form of L. pneumophila, providing a technique for recovering virulent and previously uncultivated forms of L. pneumophila.

Conclusion

These new media could be used to reduce the risk of underestimation of counts of virulent of L. pneumophila cells in environmental samples.     

Cohen’s h for detection of disease association with rare genetic variants

Background

The power of the genome wide association studies starts to go down when the minor allele frequency (MAF) is below 0.05. Here, we proposed the use of Cohen’s h in detecting disease associated rare variants. The variance stabilizing effect based on the arcsine square root transformation of MAFs to generate Cohen’s h contributed to the statistical power for rare variants analysis. We re-analyzed published datasets, one microarray and one sequencing based, and used simulation to compare the performance of Cohen’s h with the risk difference (RD) and odds ratio (OR).

Results

The analysis showed that the type 1 error rate of Cohen’s h was as expected and Cohen’s h and RD were both less biased and had higher power than OR. The advantage of Cohen’s h was more obvious when MAF was less than 0.01.

Conclusions

Cohen’s h can increase the power to find genetic association of rare variants and diseases, especially when MAF is less than 0.01.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-875) contains supplementary material, which is available to authorized users.     

Can dominance genetic variance be ignored in evolutionary quantitative genetic analyses of wild populations?

Accurately estimating genetic variance components is important for studying evolution in the wild. Empirical work on domesticated and wild outbred populations suggests that dominance genetic variance represents a substantial part of genetic variance, and theoretical work predicts that ignoring dominance can inflate estimates of additive genetic variance. Whether this issue is pervasive in natural systems is unknown, because we lack estimates of dominance variance in wild populations obtained in situ. Here, we estimate dominance and additive genetic variance, maternal variance, and other sources of nongenetic variance in eight traits measured in over 9000 wild nestlings linked through a genetically resolved pedigree. We find that dominance variance, when estimable, does not statistically differ from zero and represents a modest amount (2-36%) of genetic variance. Simulations show that (1) inferences of all variance components for an average trait are unbiased; (2) the power to detect dominance variance is low; (3) ignoring dominance can mildly inflate additive genetic variance and heritability estimates but such inflation becomes substantial when maternal effects are also ignored. These findings hence suggest that dominance is a small source of phenotypic variance in the wild and highlight the importance of proper model construction for accurately estimating evolutionary potential.     

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

The polyphyly of Neotragus – Results from genetic and morphometric analyses

Dwarf antelope species were commonly united in the tribe “Neotragini” (Bovidae, Mammalia) due to their general morphological appearance. However, phylogenetic analyses have shown that not all dwarf antelopes are closely related, so it was suggested to restrict the name Neotragini to the type genus Neotragus. In our study we use mitochondrial cytochrome b sequences and linear skull measurements to further investigate the similarity of all three Neotragus species. Our analyses support the close relationship of N. moschatus and N. batesi. However, N. pygmaeus – the type species, which was never before included in phylogenetic analyses – is not closely related. It might share a most recent common ancestor with another “dwarf antelope”, the Klipspringer Oreotragus oreotragus, and the duikers in the taxon Cephalophini. Hence, we suggest resurrecting the genus Nesotragus von Dueben, 1846 for Nesotragus moschatus and N. batesi.     

Identification of rare genetic variants in novel loci associated with Paget’s disease of bone

In genome-wide association studies, single nucleotide polymorphisms located in five novel loci were associated with PDB. We aimed at identifying rare genetic variants of candidate genes located in these loci and search for genetic association with PDB in the French-Canadian population. Exons, promoter and exon–intron junctions from patients with familial PDB and healthy individuals were sequenced in candidate genes, located within novel loci associated with PDB in our population. Rare variant was defined by a minor allele frequency <0.05 or absent from dbSNP (NCBI). We sequenced seven genes in 1p13 locus, three genes in 7q33, three genes in 8q22, and five genes in 15q24 locus. We identified 126 rare variants in at least one patient with PDB of whom 55 were located in 1p13 locus, 32 in 7q33, 10 in 8q22 and 29 in 15q24 locus. We located 71 of these 126 rare variants in an intron, 30 in an exon and 9 in an untranslated region. 60 % of these variants were located in functionally relevant gene regions. Among the 12 missense rare variants in PDB, two (rs62620995 in TM7SF4; rs62641691 in CD276) were predicted to be damaging by in silico analysis tools. Rs62620995, which altered a conserved amino acid (p.Leu397Phe) in the TM7SF4 gene, encoding the DC-STAMP protein involved in osteoclastogenesis through RANK signaling pathway, was found to have a marginal association with PDB (p = 0.09). Rs35500845, located in the CTHRC1 gene, which encodes a regulator of collagen matrix deposition, was also associated with PDB in the French-Canadian population (p = 0.046).     

Precipitation Increases the Occurrence of Sporadic Legionnaires’ Disease in Taiwan

Legionnaires’ disease (LD) is an acute form of pneumonia, and changing weather is considered a plausible risk factor. Yet, the relationship between weather and LD has rarely been investigated, especially using long-term daily data. In this study, daily data was used to evaluate the impacts of precipitation, temperature, and relative humidity on LD occurrence in Taiwan from 1995–2011. A time-stratified 2:1 matched-period case-crossover design was used to compare each case with self-controlled data using a conditional logistic regression analysis, and odds ratios (ORs) for LD occurrence was estimated. The city, gender and age were defined as a stratum for each matched set to modify the effects. For lag day- 0 to 15, the precipitation at lag day-11 significantly affected LD occurrence (p<0.05), and a 2.5% (95% CIs = 0.3–4.7%) increased risk of LD occurrence was associated with every 5-mm increase in precipitation. In addition, stratified analyses further showed that positive associations of precipitation with LD incidence were only significant in male and elderly groups and during the warm season ORs = 1.023–1.029). However, such an effect was not completely linear. Only precipitations at 21–40 (OR = 1.643 (95% CIs = 1.074–2.513)) and 61–80 mm (OR = 2.572 (1.106–5.978)) significantly increased the risk of LD occurrence. Moreover, a negative correlation between mean temperature at an 11-day lag and LD occurrence was also found (OR = 0.975 (0.953–0.996)). No significant association between relative humidity and LD occurrence was identified (p>0.05). In conclusion, in warm, humid regions, an increase of daily precipitation is likely to be a critical weather factor triggering LD occurrence where the risk is found particularly significant at an 11-day lag. Additionally, precipitation at 21–40 and 61–80 mm might make LD occurrence more likely.     

A genetic mechanism of species replacement in European waterfrogs?

Introduced Rana ridibunda currentlyreplace the native waterfrogs R. lessonaeand R. esculenta in several areas ofcentral Europe. The unusual reproductive systemin waterfrogs of the Rana esculentacomplex suggests that this replacement may bedriven by a genetic mechanism: Ranaesculenta, a hybrid between R. ridibundaand R. lessonae, eliminates the lessonae genome from the germline and clonallytransmits the ridibunda genome(hybridogenesis). Hybrids form mixedpopulations with R. lessonae (L-E-system)in which they persist by backcrossing with theparental species. Matings between hybrids areunsuccessful, because their ridibundagenomes contain fixed recessive deleteriousmutations. When introduced into a L-E-system,R. ridibunda can mate with both nativetaxa, producing R. ridibunda offspringwith R. esculenta, and R. esculentaoffspring with R. lessonae (primaryhybridizations). If primary hybrids arehybridogenetic, they produce viable R.ridibunda offspring in matings with otherhybrids, because their clonal genomes areunlikely to share the deleterious allelespresent in the ancient clones. Thus, R.ridibunda will increase in the population atthe expense of both native taxa, eventuallyleaving a pure R. ridibunda population.We provide three lines of evidence for thisprocess from a currently invaded population inSwitzerland: (1) Primary hybridizations takeplace, as roughly 10% of hybrids in thepopulation possess ridibunda genomesderived from the introduced frogs. (2)Hybridogenesis occurs in primary hybrids,although at a low frequency. (3) Many hybrid ×hybrid matings in the population indeed produceviable offspring. Hence, the proposed geneticmechanism appears to contribute to the speciesreplacement, although its importance may belimited.