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Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.
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Linfócitos B , Células Dendríticas , Endodesoxirribonucleases , Microbioma Gastrointestinal , Animais , Feminino , Humanos , Masculino , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Microbioma Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismo , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos Endogâmicos C57BLRESUMO
The sudden mortality of African elephants (Loxodonta africana) in Botswana and Zimbabwe in 2020 provoked considerable public interest and speculation. Poaching and malicious poisoning were excluded early on in the investigation. Other potential causes included environmental intoxication, infectious diseases, and increased habitat stress due to ongoing drought. Here we show evidence of the mortalities in Zimbabwe as fatal septicaemia associated with Bisgaard taxon 45, an unnamed close relative of Pasteurella multocida. We analyse elephant carcasses and environmental samples, and fail to find evidence of cyanobacterial or other intoxication. Post-mortem and histological findings suggest a bacterial septicaemia similar to haemorrhagic septicaemia caused by P. multocida. Biochemical tests and 16S rDNA analysis of six samples and genomic analysis of one sample confirm the presence of Bisgaard taxon 45. The genome sequence contains many of the canonical P. multocida virulence factors associated with a range of human and animal diseases, including the pmHAS gene for hyaluronidase associated with bovine haemorrhagic septicaemia. Our results demonstrate that Bisgaard taxon 45 is associated with a generalised, lethal infection and that African elephants are susceptible to opportunistically pathogenic Pasteurella species. This represents an important conservation concern for elephants in the largest remaining metapopulation of this endangered species.
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Elefantes , Septicemia Hemorrágica , Pasteurella multocida , Humanos , Animais , Bovinos , Septicemia Hemorrágica/veterinária , Septicemia Hemorrágica/microbiologia , Pasteurella , Pasteurella multocida/genética , EcossistemaRESUMO
Most biological rates depend on the rate of respiration. Temperature variation is typically considered the main driver of daily plant respiration rates, assuming a constant daily respiration rate at a set temperature. Here, we show empirical data from 31 species from temperate and tropical biomes to demonstrate that the rate of plant respiration at a constant temperature decreases monotonically with time through the night, on average by 25% after 8 h of darkness. Temperature controls less than half of the total nocturnal variation in respiration. A new universal formulation is developed to model and understand nocturnal plant respiration, combining the nocturnal decrease in the rate of plant respiration at constant temperature with the decrease in plant respiration according to the temperature sensitivity. Application of the new formulation shows a global reduction of 4.5 -6 % in plant respiration and an increase of 7-10% in net primary production for the present-day.
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Folhas de Planta , Plantas , Dióxido de Carbono , Ecossistema , Respiração , Temperatura , ÁrvoresRESUMO
Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause is Mycobacterium bovis, which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole-genome sequencing to examine the population structure of M. bovis in Ethiopia. A total of 134 M. bovis isolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure of M. bovis, based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.
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Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Tuberculose Bovina/transmissão , Animais , Técnicas de Tipagem Bacteriana , Bovinos , Etiópia/epidemiologia , Europa (Continente) , Genótipo , Gado , Repetições Minissatélites , Mycobacterium bovis/isolamento & purificação , Análise de Sequência , Tuberculose Bovina/epidemiologia , Sequenciamento Completo do GenomaRESUMO
An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.
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Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants' reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary.
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Outbreaks of highly pathogenic avian influenza virus (HPAIV) often result in the infection of millions of poultry, causing up to 100% mortality. HPAIV has been shown to emerge from low pathogenicity avian influenza virus (LPAIV) in field outbreaks. Direct evidence for the emergence of H7N7 HPAIV from a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified in the UK in 2008. The DBCS contained an additional basic amino acid compared to commonly circulating LPAIVs that harbor a single-basic amino acid at the cleavage site (SBCS). Using reverse genetics, outbreak HPAIVs were rescued with a DBCS (H7N7DB), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7SB). Passage of H7N7DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7SB was serially passaged showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS appears naturally unstable, enabling rapid evolution to HPAIV. Evaluation in embryo tissue presents a useful approach to study AIV evolution and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.
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Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H7N7/patogenicidade , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Sequência de Aminoácidos , Animais , Embrião de Galinha , Galinhas , Evolução Molecular , Genoma Viral/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H7N7/metabolismo , Influenza Aviária/patologia , Mutação , Fenótipo , Doenças das Aves Domésticas/patologia , Taxa de Sobrevida , Tripsina/metabolismo , Virulência/genéticaRESUMO
We previously reported that costimulation blockade by abatacept limits the decline of ß-cell function and the frequency of circulating CD4+ central memory T cells (TCM) (CD45RO+CD62L+) in new-onset type 1 diabetes. In human subjects receiving placebo, we found a significant association between an increase in CD4+ TCM cells and the decline of ß-cell function. To extend and refine these findings, we examined changes in human CD4+ and CD8+ naive and memory T cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group, we successfully reproduced the original finding of a significant association between TCM and ß-cell function and extended this to other T cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4+ conventional and regulatory T cell subsets; in general, Ag-naive subsets increase and Ag-experienced subsets decrease, whereas CD8+ T cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated costimulation. Importantly, abatacept uncouples the relationship between changes in T cell subsets and ß-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in ß-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers nonpredictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.
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Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Memória Imunológica/imunologia , Abatacepte/farmacologia , Linfócitos B/efeitos dos fármacos , Biomarcadores/sangue , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Diabetes Mellitus Tipo 1/sangue , Humanos , Memória Imunológica/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study.
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Surtos de Doenças/veterinária , Patos/virologia , Vírus da Influenza A Subtipo H5N8/classificação , Vírus da Influenza A Subtipo H5N8/genética , Infecções por Orthomyxoviridae/veterinária , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodos , Animais , Genoma Viral , Infecções por Orthomyxoviridae/virologia , RNA Viral/análise , RNA Viral/genética , Análise de Sequência de DNARESUMO
Remote sensing of vegetation provides important information for ecological applications and environmental assessments. The association between vegetation composition and structure with its spectral signal can most fully be assessed with hyperspectral data. Particularly field spectroscopy data can improve such understanding as the spectral data can be linked with the vegetation under consideration without the geographic registration uncertainties of aerial or satellite imagery. The data provided in this article contain field spectroscopy measurements from non-arable, grass-dominated objects on four farms in an intensively used agricultural landscape in the South-East of the UK. Detailed data on the plant species composition of the objects are also supplied with this article to support further analysis. Reuse potential includes linking the vegetation data with the spectral response using spectral unmixing techniques to map certain plant species or including the field spectroscopy data in a larger study with data from a wider area. This data article is related to the paper 'Classifying grass-dominated habitats from remotely sensed data: the influence of spectral resolution, acquisition time and the vegetation classification system on accuracy and thematic resolution' (Bradter et al., 2019) in which the ability to classify the recorded vegetation from the field spectroscopy data was analysed.
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Detailed maps of vegetation facilitate spatial conservation planning. Such information can be difficult to map from remotely sensed data with the detail (thematic resolution) required for ecological applications. For grass-dominated habitats in the South-East of the UK, it was evaluated which of the following choices improved classification accuracies at various thematic resolutions: 1) Hyperspectral data versus data with a reduced spectral resolution of eight and 13 bands, which were simulated from the hyperspectral data. 2) A vegetation classification system using a detailed description of vegetation (sub)-communities (the British National Vegetation Classification, NVC) versus clustering based on the dominant plant species (Dom-Species). 3) The month of imagery acquisition. Hyperspectral data produced the highest accuracies for vegetation away from edges using the NVC (84-87%). Simulated 13-band data performed also well (83-86% accuracy). Simulated 8-band data performed poorer at finer thematic resolutions (77-78% accuracy), but produced accuracies similar to those from simulated 13-band or hyperspectral data for coarser thematic resolutions (82-86%). Grouping vegetation by NVC (84-87% accuracy for hyperspectral data) usually achieved higher accuracies compared to Dom-Species (81-84% for hyperspectral data). Highest discrimination rates were achieved around the time vegetation was fully developed. The results suggest that using a detailed description of vegetation (sub)-communities instead of one based on the dominating species can result in more accurate mapping. The NVC may reflect differences in site conditions in addition to differences in the composition of dominant species, which may benefit vegetation classification. The results also suggest that using hyperspectral data or the 13-band multispectral data can help to achieve the fine thematic resolutions that are often required in ecological applications. Accurate vegetation maps with a high thematic resolution can benefit a range of applications, such as species and habitat conservation.
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Poaceae , Ecossistema , Monitoramento Ambiental , PlantasRESUMO
Plasmids may maintain antibiotic resistance genes in bacterial populations through conjugation, in the absence of direct selection pressure. However, the costs and benefits of conjugation for plasmid and bacterial fitness are not well understood. Using invasion and competition experiments with plasmid mutants we explicitly tested how conjugation contributes to the maintenance of a plasmid bearing a single extended-spectrum ß-lactamase (ESBL) gene (blaCTX-M-14). Surprisingly, conjugation had little impact on overall frequencies, although it imposed a substantial fitness cost. Instead, stability resulted from the plasmid conferring fitness benefits when rare. Frequency dependent fitness did not require a functional blaCTX-M-14 gene, and was independent of culture media. Fitness benefits when rare are associated with the core plasmid backbone but are able to drive up frequencies of antibiotic resistance because fitness burden of the blaCTX-M-14 gene is very low. Negative frequency dependent fitness can contribute to maintaining a stable frequency of resistance genes in the absence of selection pressure from antimicrobials. In addition, persistent, low cost resistance has broad implications for antimicrobial stewardship.
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Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Plasmídeos/genética , beta-Lactamases/genética , Conjugação Genética , Escherichia coli/enzimologiaRESUMO
Helicobacter pylori has been implicated in the pathogenesis of Parkinson's disease (PD). Its eradication, in a randomized placebo-controlled trial, improved PD hypokinesia. Helicobacter species zoonosis might explain excess mortality from PD and non-Hodgkin lymphoma in livestock, but not arable, farmers. Indeed, Helicobacter is causally-associated with gastric lymphoma. We have previously shown that the relative-frequency, H. suis to H. pylori, was 10-times greater in 60 PD-patients than in 256 controls. We now go on to evaluate the pathological significance of H. suis, detected in gastric-biopsy DNA-extracts by ureA-based species-specific qPCR, validated by amplicon sequencing. The methodology had been cross-validated by a carR-based PCR. The pathological significance is put in context of H. pylori detection [urea-breath-test (UBT) with biopsy-culture, and, if negative, PCR], and the potential reservoir in pigs. Here, we explore, in these 60 PD-patients, associations of H. suis status with all-cause-mortality, and with orthostatic cardiovascular and blood profiling. H. suis had been detected in 19 of the 60 PD-patients on one or more occasion, only two (with co-existent H. pylori) being UBT positive. We found that the hazard-of-death (age-at-diagnosis- and gender-adjusted) was 12 (95% CI 1,103) times greater (likelihood-ratio test, P = 0.005) with H. suis-positivity (6/19) than with negativity (2/40: one lost to follow-up). UBT-values did not influence the hazard. H. suis-positivity was associated with lower standing mean-arterial-pressure [6 (1, 11) mmHg], H. pylori-positivity having no effect. The lower total lymphocyte count with H. pylori-positivity [-8 (-1, -14) %] was not seen with H. suis, where T-cell counts were higher [24 (2, 52) %]. Regarding the potential zoonotic reservoir in the UK, Helicobacter-like-organism frequency was determined in freshly-slaughtered pigs, nature ascertained by sequencing. Organisms immunostaining for Helicobacter, with corkscrew morphology typical of non-H. pylori Helicobacter, were seen in 47% of 111 pig-antra. We conclude that H. suis is associated with all-cause-mortality in PD and has a potential zoonotic reservoir.
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Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.
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Biologia Computacional/métodos , Simulação por Computador , Sequenciamento de Nucleotídeos em Larga Escala/normas , Ensaio de Proficiência Laboratorial/estatística & dados numéricos , Análise de Sequência de DNA/normas , Vírus/genética , Análise de Dados , Europa (Continente) , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Colaboração Intersetorial , Ensaio de Proficiência Laboratorial/organização & administração , Reprodutibilidade dos Testes , Análise de Sequência de DNA/estatística & dados numéricos , Vírus/patogenicidadeRESUMO
Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.
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Brachyspira hyodysenteriae/genética , Infecções por Bactérias Gram-Negativas/veterinária , Doenças dos Suínos/microbiologia , Animais , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Genoma Bacteriano/genética , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas Hemolisinas/genética , Hemólise/genética , Tipagem de Sequências Multilocus/veterinária , Fenótipo , Filogenia , Análise de Sequência de DNA/veterinária , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.
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One of the major transmission routes for the foodborne bacterial pathogen Campylobacter is undercooked poultry meat, contaminated from intestinal contents during processing. In broilers, Campylobacter can grow to very high densities in the caeca, and is often considered to be a commensal or an opportunistic pathogen in poultry. Reduction of caecal loads of Campylobacter may assist in lowering incidence rates of Campylobacter food poisoning. To achieve this, there needs to be a better understanding of the dynamics of Campylobacter colonization in its natural niche, and the effect of the local microbiome on colonization. Previous studies have shown that the microbiome differed between Campylobacter colonized and non-colonized chicken intestinal samples. To characterize the microbiome of Campylobacter-colonized broilers, caecal samples of 100 randomly selected birds from four farms were analyzed using amplified 16S rRNA gene sequences. Bacterial taxonomic analysis indicated that inter-farm variation was greater than intra-farm variation. The two most common bacterial groups were Bacteroidetes and Firmicutes which were present in all samples and constituted 29.7-63.5 and 30.2-59.8% of the bacteria present, respectively. Campylobacter was cultured from all samples, ranging from 2 to 9 log10 CFU g-1. There was no clear link between Campylobacter counts and Firmicutes, Bacteroidetes, or Tenericutes levels in the 16S rRNA operational taxonomic unit (OTU)-based analysis of the caecal microbiome, but samples with high Campylobacter counts (>9 log CFU g-1) contained increased levels of Enterobacteriaceae. A decrease in Lactobacillus abundance in chicken caeca was also associated with high Campylobacter loads. The reported associations with Lactobacillus and Enterobacteriaceae match changes in the intestinal microbiome of chickens and mice previously reported for Campylobacter infection, and raises the question about temporality and causation; as to whether increases in Campylobacter loads create conditions adverse to Lactobacilli and/or beneficial to Enterobacteriaceae, or that changes in Lactobacilli and Enterobacteriaceae levels created conditions beneficial for Campylobacter colonization. If these changes can be controlled, this may open opportunities for modulation of chicken microbiota to reduce Campylobacter levels for improved food safety.
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Type 2 diabetes (T2D) has been linked with increased intestinal permeability, but the clinical significance of this phenomenon remains unknown. The objective of this study was to investigate the potential link between glucose control, intestinal permeability, diet and intestinal microbiota in patients with T2D. Thirty-two males with well-controlled T2D and 30 age-matched male controls without diabetes were enrolled in a case-control study. Metabolic parameters, inflammatory markers, endotoxemia, and intestinal microbiota in individuals subdivided into high (HP) and normal (LP) colonic permeability groups, were the main outcomes. In T2D, the HP group had significantly higher fasting glucose (P = 0.034) and plasma nonesterified fatty acid levels (P = 0.049) compared with the LP group. Increased colonic permeability was also linked with altered abundances of selected microbial taxa. The microbiota of both T2D and control HP groups was enriched with Enterobacteriales. In conclusion, high intestinal permeability was associated with poorer fasting glucose control in T2D patients and changes in some microbial taxa in both T2D patients and nondiabetic controls. Therefore, enrichment in the gram-negative order Enterobacteriales may characterize impaired colonic permeability prior to/independently from a disruption in glucose tolerance.
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Diabetes Mellitus Tipo 2/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal , Intestinos/patologia , Permeabilidade , Glicemia , Estudos de Casos e Controles , Enterobacteriaceae , Humanos , Resistência à Insulina/fisiologia , Intestinos/microbiologia , Masculino , Pessoa de Meia-IdadeRESUMO
Between 2011 and 2014 outbreaks of septicaemia due to Klebsiella pneumoniae subspecies pneumoniae (Kpp) were diagnosed on thirteen English pig farms. The most consistent features were rapid deaths of pigs from ten-days-old to weaning, seasonal occurrence (May to September), affected farms being outdoor breeding herds and the location of all but one of the outbreaks in the East Anglia region in Eastern England. Molecular characterisation of the outbreak Kpp isolates showed that by multilocus sequencing all were sequence type 25 (ST25) of K2 capsular type with a combination of a 4.3kb plasmid (pKPMC25), three phage sequences and the rmpA virulence gene. No archived Kpp isolates of porcine origin pre-dating 2011 were identified as ST25. In 2013 there was the first detection of an outbreak Kpp isolate showing antimicrobial resistance to six antibiotics. Human infection with Kpp ST25 has not been reported in the UK.
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Infecções por Klebsiella/veterinária , Sepse/veterinária , Doenças dos Suínos/microbiologia , Animais , Resistência Microbiana a Medicamentos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Sepse/microbiologia , Suínos , VirulênciaRESUMO
Host shift events play an important role in epizootics as adaptation to new hosts can profoundly affect the spread of the disease and the measures needed to control it. During the late 1990s, an epizootic in Turkey resulted in a sustained maintenance of rabies virus (RABV) within the fox population. We used Bayesian inferences to investigate whole genome sequences from fox and dog brain tissues from Turkey to demonstrate that the epizootic occurred in 1997 (±1 year). Furthermore, these data indicated that the epizootic was most likely due to a host shift from locally infected domestic dogs, rather than an incursion of a novel fox or dog RABV. No evidence was observed for genetic adaptation to foxes at consensus sequence level and dN/dS analysis suggested purifying selection. Therefore, the deep sequence data were analysed to investigate the sub-viral population during a host shift event. Viral heterogeneity was measured in all RABV samples; viruses from the early period after the host shift exhibited greater sequence variation in comparison to those from the later stage, and to those not involved in the host shift event, possibly indicating a role in establishing transmission within a new host. The transient increase in variation observed in the new host species may represent virus replication within a new environment, perhaps due to increased replication within the CNS, resulting in a larger population of viruses, or due to the lack of host constraints present in the new host reservoir.