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.     

Artificial Sweeteners Stimulate Adipogenesis and Suppress Lipolysis Independently of Sweet Taste Receptors

G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3.     

Repeated bouts of eccentrically biased endurance exercise stimulate salivary IgA secretion rate

To determine the salivary secretory immunoglobulin A (sIgA) response to repeated bouts of unaccustomed, downhill running (eccentrically biased) and examine potential protective immunological adaption from a repeated bout effect. Eleven active but untrained males (age: 19.7±0.4 years; VO_2peak: 47.8± 3.6 ml · kg⁻¹ · min ⁻¹) performed two 60 min bouts (Run 1 and Run 2) of downhill running (−13.5% gradient), separated by 14 days, at a speed eliciting 75% of their VO_2peak on a level grade. Saliva samples were collected before (baseline), immediately post exercise (IPE), and every hour for 12 h and every 24 h for 6 days after each run. Salivary sIgA concentration was measured and sIgA secretion rate was calculated. Results were analysed using repeated measures ANOVA (12 h period: 2x14; 24 h intervals: 2x7; p ≤ 0.05) with Tukey post-hoc tests where appropriate. Results are reported as means ± SE. There was a significant (p < 0.0001) interaction effect for sIgA secretion rate, IPE, with higher values after Run 2, as well as a significant (p < 0.01) time effect with elevated levels IPE and between 24 h and 144 h. There was a run effect (p < 0.0001), with the sIgA secretion rate significantly higher after Run 2. Repeated bouts of unaccustomed, eccentrically biased exercise induced alterations in the salivary sIgA secretion rate. This may serve as a protective mucosal adaptation to exercise-induced tissue damage.     

Genetic characterization of the lobster pathogen Aerococcus viridans var. homari by 16S rRNA gene sequence and RAPD

A combination of 16S rRNA sequencing and random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity within Aerococcus viridans var. homari, the causative agent of gaffkemia in lobsters. A collection of 7 A. viridans var. homari strains and 2 avirulent A. viridans-like cocci isolated from homarid lobsters harvested from different regions on the Atlantic Coast of North America were analyzed. The isolates are separated geographically and temporally between the years 1947 and 2000. Sequencing of 16S rRNA genes confirmed the inclusion of all 9 isolates in the monophyletic A. viridans clade (99.8 to 100% similarity). RAPD analysis revealed that the 9 A. viridans var. homari isolates could be separated into 2 distinct subtypes. Subtype 1 included the 7 pathogenic lobster isolates and constituted a homogeneous group regardless of their geographical, temporal or virulence differences. Subtype 2 contained the 2 avirulent A. viridans-like cocci that had distinct RAPD patterns and clustered separately with the non-marine A. viridans. RAPD analysis represented a useful method for determining molecular subtyping for the intraspecific classification and epidemiological investigations of A. viridans var. homari.     

Effects of the protozoan parasite Sarcocystis rauschorum on open-field behaviour of its intermediate vertebrate host, Dicrostonyx richardsoni

Behaviour and activity levels were measured in varying lemmings experimentally infected with the heteroxenous parasite, Sarcocystis rauschorum to test the hypothesis that the parasite alters behaviour of this intermediate host and thereby increases probability of transmission to the definitive host, the snowy owl (Nyctea scandiaca). Measures of short-term activity levels on a running wheel indicated no effect of the parasite, either directly, or indirectly as a result of illness. We observed behaviour of infected lemmings placed in an "open field" (arena). Lemmings would increase their susceptibility to predators if they spent more time away from cover, used crypsis (stationary postures) less, spent more time exploring (especially in unfamiliar areas), or responded inappropriately to threats from predators. We found that only exploratory activity showed significant change after infection. The frequency of exploratory activity increased and became disassociated from the usual fear response. This may increase the lemmings' susceptibility to aerial predation. The mechanism for this effect is unknown, but neurological lesions have been observed. The examination of the modes of transmission of the S. rauschorum parasite within lemming populations and of a possible fecundity compensation strategy adopted by the lemmings, and their relevance to population control, are suggested as areas for future study.     

In vitro excystation of Sarcocystis capracanis, Sarcocystis cruzi and Sarcocystis tenella (Apicomplexa)

Improved rates of in vitro excystation of sporozoites from sporocysts of Sarcocystis capracanis, Sarcocystis cruzi, and Sarcocystis tenella were obtained by pretreating sporocysts with an aqueous sodium hypochlorite (NaOCl) solution followed by incubation in excysting fluid (EF). After pretreatment with NaOCl, sporocysts were washed 4 times in Hanks' balanced salt solution and then incubated in various EF (pH 7.4) at 38.5 C in 5% CO2-95% air. Maximum rates of excystation (free sporozoites/(sporozoites in sporocysts + free sporozoites) X 100) for all 3 species of Sarcocystis occurred at 4 hr after incubation in EF. These rates were 17% for S. capracanis after incubation in EF containing 2% trypsin + 10% caprine bile; 90% for S. cruzi in 2% trypsin + 10% bovine bile; and 20% for S. tenella in 2% trypsin + 10% caprine bile. Only a 40% excystation rate occurred in sporocysts of S. cruzi that had been stored previously for 14 days in aqueous potassium dichromate. Excysted sporozoites of S. capracanis, S. cruzi, and S. tenella penetrated and developed to mature meronts in bovine pulmonary artery endothelial cells or bovine monocytes.     

An inexpensive tag for short-term studies in milkfish (Cbanos chanos Forsskal) and in seabass (Lates calcarifer Bloch)

An opercular tag for marking adult milkfish ( Chanos chanos Forsskal) and seabass ( Lates calcarifer Bloch) is described. High tag retention and relatively low mortality rates were observed in adult fish handled two to ten times during 14-to 60-day tests. The features and advantages of the tag for marking large-sized fish in short-term studies are discussed.

Zusammenfassung

Eine preiswerte Markierung für Kurzzeitstudien des Milchfisches (Chanos chanos Forsskal) und der Centropomidae (Lates calcarifer Bloch)
Eine Kiemendeckel-Markierung für adulte Milchfische (Chanos chanos Forsskal) und Centropomidae (Lates calcacifer Bloch) wird beschrieben. Sie zeichnet sich durch gute Haltbarkeit aus und verursacht relativ geringe Mortalität bei adulten Fischen, die in Versuchen von 14 bis 60 Tage Dauer 2-bis 10mal untersucht wurden. Die Eigenschaften und Vorteile dieser Markierung für große Fische in Kurzzeitstudien werden diskutiert.

Résumé

Un marquage économique pour des études de courte durée du chanidé (Chanos chanos Forsskal) et du centropomidé (Lates calcarifer Bloch)
Un marquage d'opercule pour les chanidés (Chanos chanos Forsskal) et les centropomidés (Lates calcarifer Bloch) adultes est décrit. Une bonne conservation et une mortalité relativement basse ont été observées chez des poissons adultes examinés 2 à 10 fois pendant des expériences d'une durée de 14 à 60 jours. Les caractéristiques et les avantages du marquage de poissons de grande taille pendant des expériences d'une courte durée sont discutés.