Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (∼14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells

Kinetics of CO<inf>2</inf>-fluid-rock reactions in a basalt aquifer, Soda Springs, Idaho

The dissolution of silicate minerals by CO2–rich fluids and the subsequent precipitation of CO2 as carbonate minerals represent a means of permanently storing anthropogenic CO2 waste products in a solid and secure form. Modelling the progression of these reactions is hindered by our poor understanding of the rates of mineral dissolution-precipitation reactions and mineral surface properties in natural systems. This study evaluates the chemical evolution of groundwater flowing through a basalt aquifer, which forms part of the leaking CO2-charged system of the Blackfoot Volcanic Field in south-eastern Idaho, USA. Reaction progress is modelled using changes in groundwater chemistry by inverse mass balance techniques. The CO2-promoted fluid-mineral reactions include the dissolution of primary plagioclase, orthoclase, pyroxene and gypsum which is balanced by the precipitation of secondary albite, calcite, zeolite, kaolinite and silica. Mineral mole transfers and groundwater flow rates estimated from hydraulic head data are used to determine the kinetics of plagioclase and orthoclase feldspar dissolution. Plagioclase surface area measurements were determined using the evolution of the U-series isotope ratios in the groundwater and are compared to published surface area measurements. Calculated rates of dissolution for plagioclase range from 2.4 x 10-12 to 4.6 x 10-16 mol/m2/s and orthoclase from 2.0 x 10-13 to 6.8 x 10-16 mol/m2/s respectively. These feldspar reaction rates, correlate with the degree of mineral-fluid disequilibrium and are similar to the dissolution rates for these mineral measured in other natural CO2-charged groundwater systems.Carbon research at Cambridge is supported by Natural Environment Research Council grant NE/F004699/1, part of the UK CRIUS (Carbon Research Into Underground Storage) consortium and DECC through the ‘£20 million’ competition. Niko Kampman acknowledges financial support from Shell Global Solutions International.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.apgeochem.2015.06.01

Mechanistic Studies of Liquid Metal Anode SOFCs: I. Oxidation of Hydrogen in Chemical - Electrochemical Mode

Liquid metal anode (LMA) solid oxide fuel cells (SOFCs) are a promising type of high temperature fuel cell suitable for the direct oxidation of gaseous or solid fuel. Depending upon the operating conditions they can be run in four different modes. In this first of a series of studies concerning the mechanism of reaction and species transport in LMA SOFCs, the oxidation of hydrogen fuel in a liquid tin anode has been investigated. An electrochemical model is developed based upon fast dissolution of hydrogen in a molten tin anode, slow, rate-determining homogeneous reaction of hydrogen with oxygen dissolved in the liquid tin, followed by anodic oxygen injection under diffusion control to replace the oxygen removed by reaction (so-called Chemical - Electrochemical mode or CE mode). Experimentally-generated data are used to validate the model. The model has introduced a new key parameter, z¯, which takes a value between zero and unity; its value is determined by geometric and convective factors in the cell as well as the partial pressure of the supplied hydrogen fuel. Current output of the cell is proportional to the value of z¯

Within-host spatiotemporal dynamics of systemic Salmonella infection during and after antimicrobial treatment

$\textbf{Objectives:}$ We determined the interactions between efficacy of antibiotic treatment, pathogen growth rates and between-organ spread during systemic $\textit{Salmonella}$ infections. $\textbf{Methods:}$ We infected mice with isogenic molecularly tagged subpopulations of either a fast-growing WT or a slow-growing $\Delta$$\textit{aroC Salmonella}$ strain. We monitored viable bacterial numbers and fluctuations in the proportions of each bacterial subpopulation in spleen, liver, blood and mesenteric lymph nodes (MLNs) before, during and after the cessation of treatment with ampicillin and ciprofloxacin. $\textbf{Results:}$ Both antimicrobials induced a reduction in viable bacterial numbers in the spleen, liver and blood. This reduction was biphasic in infections with fast-growing bacteria, with a rapid initial reduction followed by a phase of lower effect. Conversely, a slow and gradual reduction of the bacterial load was seen in infections with the slow-growing strain, indicating a positive correlation between bacterial net growth rates and the efficacy of ampicillin and ciprofloxacin. The viable numbers of either bacterial strain remained constant in MLNs throughout the treatment with a relapse of the infection with WT bacteria occurring after cessation of the treatment. The frequency of each tagged bacterial subpopulation was similar in the spleen and liver, but different from that of the MLNs before, during and after treatment. $\textbf{Conclusions:}$ In $\textit{Salmonella}$ infections, bacterial growth rates correlate with treatment efficacy. MLNs are a site with a bacterial population structure different to those of the spleen and liver and where the total viable bacterial load remains largely unaffected by antimicrobials, but can resume growth after cessation of treatment.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grant number BB/M000982/1 (http://www.bbsrc.ac.uk/research/grants/grants/AwardDetails.aspx?FundingReference=BB/M000982/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Transmission and dose–response experiments for social animals: a reappraisal of the colonization biology of Campylobacter jejuni in chickens

Dose-response experiments characterize the relationship between infectious agents and their hosts. These experiments are routinely used to estimate the minimum effective infectious dose for an infectious agent, which is most commonly characterized by the dose at which 50 per cent of challenged hosts become infected-the ID(50). In turn, the ID(50) is often used to compare between different agents and quantify the effect of treatment regimes. The statistical analysis of dose-response data typically makes the assumption that hosts within a given dose group are independent. For social animals, in particular avian species, hosts are routinely housed together in groups during experimental studies. For experiments with non-infectious agents, this poses no practical or theoretical problems. However, transmission of infectious agents between co-housed animals will modify the observed dose-response relationship with implications for the estimation of the ID(50) and the comparison between different agents and treatments. We derive a simple correction to the likelihood for standard dose-response models that allows us to estimate dose-response and transmission parameters simultaneously. We use this model to show that: transmission between co-housed animals reduces the apparent value of the ID(50) and increases the variability between replicates leading to a distinctive all-or-nothing response; in terms of the total number of animals used, individual housing is always the most efficient experimental design for ascertaining dose-response relationships; estimates of transmission from previously published experimental data for Campylobacter spp. in chickens suggest that considerable transmission occurred, greatly increasing the uncertainty in the estimates of dose-response parameters reported in the literature. Furthermore, we demonstrate that accounting for transmission in the analysis of dose-response data for Campylobacter spp. challenges our current understanding of the differing response of chickens with respect to host-age and in vivo passage of bacteria. Our findings suggest that the age-dependence of transmissibility between hosts-rather than their susceptibility to colonization-is the mechanism behind the 'lag-phase' reported in commercial flocks, which are typically found to be Campylobacter free for the first 14-21 days of life.A.J.K.C. is funded by DEFRA grant PU/T/WL/07/46 - SE3230, sponsored by the Veterinary Laboratories Agency. This research was developed during an earlier project funded by the Biotechnology and Biological Sciences Research Council/Defra Government Partnership Award, grants BB/500852/1 and BB/500936/1

Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs.

Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis, the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population

The essential genome of Streptococcus agalactiae.

BACKGROUND: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis. METHODS: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries-with at least 135,000 mutants per library-at serial 24 h time points after outgrowth in rich media. RESULTS: After statistical analysis of transposon insertion density and distribution, we identified 13.5 % of genes as essential and 1.2 % as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93 % concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS. CONCLUSIONS: We believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.This work was supported by NIH/NIAID R01 AI092743, R33 AI098654, and R21 AI11020 to A.J.R.; NIH/NICHD K23 HD065844 to T.M.R.; John M. Driscoll, Jr., M.D. Children’s Fund (Columbia University Department of Pediatrics) and the Pediatric Scientist Development Program (NIH/NICHD K12 HD000850) to T.A.H

Overexpression of antibiotic resistance genes in hospital effluents over time

$\textbf{Objectives}$: Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance gene-containing effluents originating from sites of varying antibiotic usage to the receiving environment. $\textbf{Methods}$: Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. $\textbf{Results}$: We found that mean antibiotic resistance gene and transcript abundances were correlated for both hospital (ρ=0.9, two-tailed $\textit{P}$<0.0001) and farm (ρ=0.5, two-tailed $\textit{P}$<0.0001) effluents and that two β-lactam resistance genes ($\textit{bla}$GES and $\textit{bla}$OXA) were overexpressed in all hospital effluent samples. High β-lactam resistance gene transcript abundance was related to hospital antibiotic usage over time and hospital effluents contained antibiotic residues. $\textbf{Conclusions}$: We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source.This research was funded by the Biotechnology and Biological Sciences Research Council, GlaxoSmithKline and the Centre for Environment, Fisheries and Aquaculture Science

Pathotyping the Zoonotic Pathogen Streptococcus suis: Novel Genetic Markers To Differentiate Invasive Disease-Associated Isolates from Non-Disease-Associated Isolates from England and Wales.

Streptococcus suis is one of the most important zoonotic bacterial pathogens of pigs, causing significant economic losses to the global swine industry. S. suis is also a very successful colonizer of mucosal surfaces, and commensal strains can be found in almost all pig populations worldwide, making detection of the S. suis species in asymptomatic carrier herds of little practical value in predicting the likelihood of future clinical relevance. The value of future molecular tools for surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. Here we describe the use of genome-wide association studies to identify genetic markers associated with the observed clinical phenotypes (i) invasive disease and (ii) asymptomatic carriage on the palatine tonsils of pigs on UK farms. Subsequently, we designed a multiplex PCR to target three genetic markers that differentiated 115 S. suis isolates into disease-associated and non-disease-associated groups, that performed with a sensitivity of 0.91, a specificity of 0.79, a negative predictive value of 0.91, and a positive predictive value of 0.79 in comparison to observed clinical phenotypes. We describe evaluation of our pathotyping tool, using an out-of-sample collection of 50 previously uncharacterized S. suis isolates, in comparison to existing methods used to characterize and subtype S. suis isolates. In doing so, we show our pathotyping approach to be a competitive method to characterize S. suis isolates recovered from pigs on UK farms and one that can easily be updated to incorporate global strain collections.This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) Knowledge Transfer Network CASE studentship co-funded by Zoetis (previously Pfizer Animal Health UK) and with significant contribution from BQP Ltd (Award Reference: BB/L502479/1). Funding bodies provided scholarship support but had no part in study design, data collection, analysis and interpretation of data or in writing the manuscript. AWT is supported by a BBSRC Longer and Larger (LoLa) grant (Award Reference: BB/G019274/1). LAW is supported by a Dorothy Hodgkin Fellowship funded by the Royal Society (Grant Number: DH140195) and a Sir Henry Dale Fellowship co-funded by the Royal Society and Wellcome Trust (Grant Number: 109385/Z/15/Z)