Quantitative Microbial Ecology through Stable Isotope Probing

Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon''s density shift relative to that taxon''s density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in ¹⁸O and ¹³C composition after exposure to [¹⁸O]water or [¹³C]glucose. The addition of glucose increased the assimilation of ¹⁸O into DNA from [¹⁸O]water. However, the increase in ¹⁸O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.     

Impacts of environmental stress on resistance and resilience of algal‐associated bacterial communities

Algal‐associated bacteria are fundamental to the ecological success of marine green macroalgae such as Caulerpa. The resistance and resilience of algal‐associated microbiota to environmental stress can promote algal health and genetic adaptation to changing environments. The composition of bacterial communities has been shown to be unique to algal morphological niches. Therefore, the level of response to various environmental perturbations may in fact be different for each niche‐specific community. Factorial in situ experiments were set up to investigate the effect of nutrient enrichment and temperature stress on the bacterial communities associated with Caulerpa cylindracea. Bacteria were characterized using the 16S rRNA gene, and the community compositions were compared between different parts of the algal thallus (endo‐, epi‐, and rhizomicrobiome). Resistance and resilience were calculated to further understand the changes of microbial composition in response to perturbations. The results of this study provide evidence that nutrient enrichment has a significant influence on the taxonomic and functional structure of the epimicrobiota, with a low community resistance index observed for both. Temperature and nutrient stress had a significant effect on the rhizomicrobiota taxonomic composition, exhibiting the lowest overall resistance to change. The functional performance of the rhizomicrobiota had low resilience to the combination of stressors, indicating potential additive effects. Interestingly, the endomicrobiota had the highest overall resistance, yet the lowest overall resilience to environmental stress. This further contributes to our understanding of algal microbiome dynamics in response to environmental changes.     

Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two m embers of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probi otic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.     

Soil minerals affect taxon-specific bacterial growth

Secondary minerals (clays and metal oxides) are important components of the soil matrix. Clay minerals affect soil carbon persistence and cycling, and they also select for distinct microbial communities. Here we show that soil mineral assemblages—particularly short-range order minerals—affect both bacterial community composition and taxon-specific growth. Three soils with different parent material and presence of short-range order minerals were collected from ecosystems with similar vegetation and climate. These three soils were provided with ¹⁸O-labeled water and incubated with or without artificial root exudates or pine needle litter. Quantitative stable isotope probing was used to determine taxon-specific growth. We found that the growth of bacteria varied among soils of different mineral assemblages but found the trend of growth suppression in the presence of short-range order minerals. Relative growth of bacteria declined with increasing concentration of short-range order minerals between 25–36% of taxa present in all soils. Carbon addition in the form of plant litter or root exudates weakly affected relative growth of taxa (p = 0.09) compared to the soil type (p < 0.01). However, both exudate and litter carbon stimulated growth for at least 34% of families in the soils with the most and least short-range order minerals. In the intermediate short-range order soil, fresh carbon reduced growth for more bacterial families than were stimulated. These results highlight how bacterial-mineral-substrate interactions are critical to soil organic carbon processing, and how growth variation in bacterial taxa in these interactions may contribute to soil carbon persistence and loss.Subject terms: Biogeochemistry, Microbial ecology, Biogeochemistry     

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.     

Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.     

Facility-based disease surveillance and Bayesian hierarchical modeling to estimate endemic typhoid fever incidence,Kilimanjaro Region,Tanzania, 2007–2018

Growing evidence suggests considerable variation in endemic typhoid fever incidence at some locations over time, yet few settings have multi-year incidence estimates to inform typhoid control measures. We sought to describe a decade of typhoid fever incidence in the Kilimanjaro Region of Tanzania. Cases of blood culture confirmed typhoid were identified among febrile patients at two sentinel hospitals during three study periods: 2007–08, 2011–14, and 2016–18. To account for under-ascertainment at sentinel facilities, we derived adjustment multipliers from healthcare utilization surveys done in the hospital catchment area. Incidence estimates and credible intervals (CrI) were derived using a Bayesian hierarchical incidence model that incorporated uncertainty of our observed typhoid fever prevalence, of healthcare seeking adjustment multipliers, and of blood culture diagnostic sensitivity. Among 3,556 total participants, 50 typhoid fever cases were identified. Of typhoid cases, 26 (52%) were male and the median (range) age was 22 (<1–60) years; 4 (8%) were aged <5 years and 10 (20%) were aged 5 to 14 years. Annual typhoid fever incidence was estimated as 61.5 (95% CrI 14.9–181.9), 6.5 (95% CrI 1.4–20.4), and 4.0 (95% CrI 0.6–13.9) per 100,000 persons in 2007–08, 2011–14, and 2016–18, respectively. There were no deaths among typhoid cases. We estimated moderate typhoid incidence (≥10 per 100 000) in 2007–08 and low (<10 per 100 000) incidence during later surveillance periods, but with overlapping credible intervals across study periods. Although consistent with falling typhoid incidence, we interpret this as showing substantial variation over the study periods. Given potential variation, multi-year surveillance may be warranted in locations making decisions about typhoid conjugate vaccine introduction and other control measures.     

In vitro differentiation of mouse embryonic yolk sac cells

The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

SELECTION AND EVOLUTION OF CAUSALLY COVARYING TRAITS

When traits cause variation in fitness, the distribution of phenotype, weighted by fitness, necessarily changes. The degree to which traits cause fitness variation is therefore of central importance to evolutionary biology. Multivariate selection gradients are the main quantity used to describe components of trait‐fitness covariation, but they quantify the direct effects of traits on (relative) fitness, which are not necessarily the total effects of traits on fitness. Despite considerable use in evolutionary ecology, path analytic characterizations of the total effects of traits on fitness have not been formally incorporated into quantitative genetic theory. By formally defining “extended” selection gradients, which are the total effects of traits on fitness, as opposed to the existing definition of selection gradients, a more intuitive scheme for characterizing selection is obtained. Extended selection gradients are distinct quantities, differing from the standard definition of selection gradients not only in the statistical means by which they may be assessed and the assumptions required for their estimation from observational data, but also in their fundamental biological meaning. Like direct selection gradients, extended selection gradients can be combined with genetic inference of multivariate phenotypic variation to provide quantitative prediction of microevolutionary trajectories.