Experimental Toxocara cati infection in gerbils and rats

Mongolian gerbils and Wistar rats were inoculated orally with 240 and 2,500 Toxocara cati embryonated eggs, respectively, to evaluate the larval recovery in different tissues and organs, such as the liver, lungs, heart, kidney, and skeletal muscles after 5, 30, 49, 70, and 92 days post-infection (PI). Larval recovery rates were 1.7-30.0% in Mongolian gerbils on days 5-92 PI and 0.2-3.8% in rats on the same days. These results indicate that Mongolian gerbils and Wistar rats are suitable experimental paratenic hosts for the study of neurological toxocariasis as well as visceral toxocariasis.     

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.     

The response regulator PhoP negatively regulates Yersinia pseudotuberculosis and Yersinia pestis biofilms

A few Yersinia pseudotuberculosis strains form biofilms on the head of the nematode Caenorhabditis elegans , but numerous others do not. We show that a widely used Y. pseudotuberculosis strain, YPIII, is biofilm positive because of a mutation in phoP , which encodes the response regulator of a two-component system. For two wild-type Y. pseudotuberculosis that do not make biofilms on C. elegans , deletion of phoP was sufficient to produce robust biofilms. In Yersinia pestis , a phoP mutant made more extensive biofilms in vitro than did the wild type. Expression of HmsT, a diguanylate cyclase that positively regulates biofilms, is diminished in Y. pseudotuberculosis strains with functional PhoP.     

Response and resilience of soil biocrust bacterial communities to chronic physical disturbance in arid shrublands

The impact of 10 years of annual foot trampling on soil biocrusts was examined in replicated field experiments at three cold desert sites of the Colorado Plateau, USA. Trampling detrimentally impacted lichens and mosses, and the keystone cyanobacterium, Microcoleus vaginatus, resulting in increased soil erosion and reduced C and N concentrations in surface soils. Trampled biocrusts contained approximately half as much extractable DNA and 20–52% less chlorophyll a when compared with intact biocrusts at each site. Two of the three sites also showed a decline in scytonemin-containing, diazotrophic cyanobacteria in trampled biocrusts. 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses of soil bacteria from untrampled and trampled biocrusts demonstrated a reduced proportion (23–65% reduction) of M. vaginatus and other Cyanobacteria in trampled plots. In parallel, other soil bacterial species that are natural residents of biocrusts, specifically members of the Actinobacteria, Chloroflexi and Bacteroidetes, became more readily detected in trampled than in untrampled biocrusts. Replicate 16S rRNA T-RFLP profiles from trampled biocrusts at all three sites contained significantly more fragments (n=17) than those of untrampled biocrusts (n⩽6) and exhibited much higher variability among field replicates, indicating transition to an unstable disturbed state. Despite the dramatic negative impacts of trampling on biocrust physical structure and composition, M. vaginatus could still be detected in surface soils after 10 years of annual trampling, suggesting the potential for biocrust re-formation over time. Physical damage of biocrusts, in concert with changing temperature and precipitation patterns, has potential to alter performance of dryland ecosystems for decades.     

The dynamic bacterial communities of a melting High Arctic glacier snowpack

Snow environments can occupy over a third of land surface area, but little is known about the dynamics of snowpack bacteria. The effect of snow melt on bacterial community structure and diversity of surface environments of a Svalbard glacier was examined using analyses of 16S rRNA genes via T-RFLP, qPCR and 454 pyrosequencing. Distinct community structures were found in different habitat types, with changes over 1 week apparent, in particular for the dominant bacterial class present, Betaproteobacteria. The differences observed were consistent with influences from depositional mode (snowfall vs aeolian dusts), contrasting snow with dust-rich snow layers and near-surface ice. Contrary to that, slush as the decompositional product of snow harboured distinct lineages of bacteria, further implying post-depositional changes in community structure. Taxa affiliated to the betaproteobacterial genus Polaromonas were particularly dynamic, and evidence for the presence of betaproteobacterial ammonia-oxidizing bacteria was uncovered, inviting the prospect that the dynamic bacterial communities associated with snowpacks may be active in supraglacial nitrogen cycling and capable of rapid responses to changes induced by snowmelt. Furthermore the potential of supraglacial snowpack ecosystems to respond to transient yet spatially extensive melting episodes such as that observed across most of Greenland''s ice sheet in 2012 merits further investigation.     

Cold temperature-induced modifications to the composition and structure of the lipopolysaccharide of Yersinia pestis

Following a report of variations in the lipopolysaccharide (LPS) structure of Yersinia pestis at mammalian (37 degrees C) and flea (25 degrees C) temperatures, a number of changes to the LPS structure were observed when the bacterium was cultivated at a temperature of winter-hibernating rodents (6 degrees C). In addition to one of the known Y. pestis LPS types, LPS of a new type was isolated from Y. pestis KM218 grown at 6 degrees C. The core of the latter differs in: (i) replacement of terminal galactose with terminal d-glycero-d-manno-heptose; (ii) phosphorylation of terminal oct-2-ulosonic acid with phosphoethanolamine; (iii) a lower content of GlcNAc, and; (iv) the absence of glycine; lipid A differs in the lack of any 4-amino-4-deoxyarabinose and presumably partial (di)oxygenation of a fatty acid(s). The data obtained suggest that cold temperature switches on an alternative mechanism of control of the synthesis of Y. pestis LPS.     

The incidence of bacterial endosymbionts in terrestrial arthropods

Intracellular endosymbiotic bacteria are found in many terrestrial arthropods and have a profound influence on host biology. A basic question about these symbionts is why they infect the hosts that they do, but estimating symbiont incidence (the proportion of potential host species that are actually infected) is complicated by dynamic or low prevalence infections. We develop a maximum-likelihood approach to estimating incidence, and testing hypotheses about its variation. We apply our method to a database of screens for bacterial symbionts, containing more than 3600 distinct arthropod species and more than 150 000 individual arthropods. After accounting for sampling bias, we estimate that 52% (CIs: 48–57) of arthropod species are infected with Wolbachia, 24% (CIs: 20–42) with Rickettsia and 13% (CIs: 13–55) with Cardinium. We then show that these differences stem from the significantly reduced incidence of Rickettsia and Cardinium in most hexapod orders, which might be explained by evolutionary differences in the arthropod immune response. Finally, we test the prediction that symbiont incidence should be higher in speciose host clades. But while some groups do show a trend for more infection in species-rich families, the correlations are generally weak and inconsistent. These results argue against a major role for parasitic symbionts in driving arthropod diversification.     

Proteomic characterization of host response to Yersinia pestis and near neighbors

Host-pathogen interactions result in protein expression changes within both the host and the pathogen. Here, results from proteomic characterization of host response following exposure to Yersinia pestis, the causative agent of plague, and to two near neighbors, Yersinia pseudotuberculosis and Yersinia enterocolitica, are reported. Human monocyte-like cells were chosen as a model for macrophage immune response to pathogen exposure. Two-dimensional electrophoresis followed by mass spectrometry was used to identify host proteins with differential expression following exposure to these three closely related Yersinia species. This comparative proteomic characterization of host response clearly shows that host protein expression patterns are distinct for the different pathogen exposures, and contributes to further understanding of Y. pestis virulence and host defense mechanisms. This work also lays the foundation for future studies aimed at defining biomarkers for presymptomatic detection of plague.     

Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus)

Bacterial gut symbiont communities are critical for the health of many insect species. However, little is known about how microbial communities vary among host species or how they respond to anthropogenic disturbances. Bacterial communities that differ in richness or composition may vary in their ability to provide nutrients or defenses. We used deep sequencing to investigate gut microbiota of three species in the genus Bombus (bumble bees). Bombus are among the most economically and ecologically important non-managed pollinators. Some species have experienced dramatic declines, probably due to pathogens and land-use change. We examined variation within and across bee species and between semi-natural and conventional agricultural habitats. We categorized as ‘core bacteria'' any operational taxonomic units (OTUs) with closest hits to sequences previously found exclusively or primarily in the guts of honey bees and bumble bees (genera Apis and Bombus). Microbial community composition differed among bee species. Richness, defined as number of bacterial OTUs, was highest for B. bimaculatus and B. impatiens. For B. bimaculatus, this was due to high richness of non-core bacteria. We found little effect of habitat on microbial communities. Richness of non-core bacteria was negatively associated with bacterial abundance in individual bees, possibly due to deeper sampling of non-core bacteria in bees with low populations of core bacteria. Infection by the gut parasite Crithidia was negatively associated with abundance of the core bacterium Gilliamella and positively associated with richness of non-core bacteria. Our results indicate that Bombus species have distinctive gut communities, and community-level variation is associated with pathogen infection.     

Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.     

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected ¹³C- and ¹⁵N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate ¹³C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.     

Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides

In this study, we examine microbial communities of early developmental stages of the coral Porites astreoides by sequence analysis of cloned 16S rRNA genes, terminal restriction fragment length polymorphism (TRFLP), and fluorescence in situ hybridization (FISH) imaging. Bacteria are associated with the ectoderm layer in newly released planula larvae, in 4-day-old planulae, and on the newly forming mesenteries surrounding developing septa in juvenile polyps after settlement. Roseobacter clade-associated (RCA) bacteria and Marinobacter sp. are consistently detected in specimens of P. astreoides spanning three early developmental stages, two locations in the Caribbean and 3 years of collection. Multi-response permutation procedures analysis on the TRFLP results do not support significant variation in the bacterial communities associated with P. astreoides larvae across collection location, collection year or developmental stage. The results are the first evidence of vertical transmission (from parent to offspring) of bacteria in corals. The results also show that at least two groups of bacterial taxa, the RCA bacteria and Marinobacter, are consistently associated with juvenile P. astreoides against a complex background of microbial associations, indicating that some components of the microbial community are long-term associates of the corals and may impact host health and survival.     

Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil

Priming effect (PE) is defined as a stimulation of the mineralization of soil organic matter (SOM) following a supply of fresh organic matter. This process can have important consequences on the fate of SOM and on the management of residues in agricultural soils, especially in tropical regions where soil fertility is essentially based on the management of organic matter. Earthworms are ecosystem engineers known to affect the dynamics of SOM. Endogeic earthworms ingest large amounts of soil and assimilate a part of organic matter it contains. During gut transit, microorganisms are transported to new substrates and their activity is stimulated by (i) the production of readily assimilable organic matter (mucus) and (ii) the possible presence of fresh organic residues in the ingested soil. The objective of our study was to see (i) whether earthworms impact the PE intensity when a fresh residue is added to a tropical soil and (ii) whether this impact is linked to a stimulation/inhibition of bacterial taxa, and which taxa are affected. A tropical soil from Madagascar was incubated in the laboratory, with a ¹³C wheat straw residue, in the presence or absence of a peregrine endogeic tropical earthworm, Pontoscolex corethrurus. Emissions of ¹²CO₂ and ¹³CO₂ were followed during 16 days. The coupling between DNA-SIP (stable isotope probing) and pyrosequencing showed that stimulation of both the mineralization of wheat residues and the PE can be linked to the stimulation of several groups especially belonging to the Bacteroidetes phylum.     

A link between ectoparasite infection and susceptibility to bacterial disease in rainbow trout

Rainbow trout, Oncorhynchus mykiss, were infected concomitantly with Argulus coregoni and Flavobacterium columnare and their survival was compared with that of fish infected with either the parasite or the bacterium alone. The mortality of fish challenged with A. coregoni was negligible while infection with F. columnare alone led to significantly lower survival. However, compared with single infections, the mortality was significantly higher and the onset of disease condition was earlier among fish, which were concomitantly infected by A. coregoni and F. columnare. This data presents, for the first time, experimental support for the hypothesis that an ectoparasite infection increases susceptibility of fish to a bacterial pathogen.     

Interaction between host genotype and environmental conditions affects bacterial density in Wolbachia symbiosis

Regulation of microbial population density is a necessity in stable symbiotic interactions. In Wolbachia symbiosis, both bacterial and host genotypes are involved in density regulation, but environmental factors may also affect bacterial population density. Here, we studied the interaction between three strains of Wolbachia in two divergent homozygous lines of the wasp Leptopilina heterotoma at two different temperatures. Wolbachia density varied between the two host genotypes at only one temperature. Moreover, at this temperature, reciprocal-cross F1 insects displayed identical Wolbachia densities, which were intermediate between the densities in the two parental lines. While these findings confirm that the host genotype plays an important role in Wolbachia density, they also highlight its interaction with environmental conditions, making possible the evolution of local adaptations for the regulation of Wolbachia density.     

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.