Salmonella TraDIS in immunodeficient mice

Salmonella enterica causes systemic diseases (typhoid and paratyphoid fever), nontyphoidal septicemia (NTS), and gastroenteritis in humans and other animals worldwide. An important but underrecognized emerging infectious disease problem in sub-Saharan Africa is NTS in children and immunocompromised adults. A current goal is to identify Salmonella mutants that are not pathogenic in the absence of key components of the immune system such as might be found in immunocompromised hosts. Such attenuated strains have the potential to be used as live vaccines. We have used transposon-directed insertion site sequencing (TraDIS) to screen mutants of Salmonella enterica serovar Typhimurium for their ability to infect and grow in the tissues of wild-type and immunodeficient mice. This was to identify bacterial genes that might be deleted for the development of live attenuated vaccines that would be safer to use in situations and/or geographical areas where immunodeficiencies are prevalent. The relative fitness of each of 9,356 transposon mutants, representing mutations in 3,139 different genes, was determined in gp91(-/-) phox mice. Mutations in certain genes led to reduced fitness in both wild-type and mutant mice. To validate these results, these genes were mutated by allelic replacement, and resultant mutants were retested for fitness in the mice. A defined deletion mutant of cysE was attenuated in C57BL/6 wild-type mice and immunodeficient gp91(-/-) phox mice and was effective as a live vaccine in wild-type mice.This work was supported by a Medical Research Council (MRC) grant G1100102. Tn libraries were previously constructed with funding from the Biotechnology and Biological Sciences Research Council grant APG19115. O.O. was supported by a Newton Trust grant awarded to A.J.G. M.M. was supported by the Wellcome Trust grant number WT098051. The authors have no conflicting financial interests. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/IAI.01423-1

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

OBJECTIVES: The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. METHODS: Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. RESULTS: A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. CONCLUSIONS: This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial

Further developments towards a genome-scale metabolic model of yeast

BACKGROUND: To date, several genome-scale network reconstructions have been used to describe the metabolism of the yeast Saccharomyces cerevisiae, each differing in scope and content. The recent community-driven reconstruction, while rigorously evidenced and well annotated, under-represented metabolite transport, lipid metabolism and other pathways, and was not amenable to constraint-based analyses because of lack of pathway connectivity. RESULTS: We have expanded the yeast network reconstruction to incorporate many new reactions from the literature and represented these in a well-annotated and standards-compliant manner. The new reconstruction comprises 1102 unique metabolic reactions involving 924 unique metabolites - significantly larger in scope than any previous reconstruction. The representation of lipid metabolism in particular has improved, with 234 out of 268 enzymes linked to lipid metabolism now present in at least one reaction. Connectivity is emphatically improved, with more than 90% of metabolites now reachable from the growth medium constituents. The present updates allow constraint-based analyses to be performed; viability predictions of single knockouts are comparable to results from in vivo experiments and to those of previous reconstructions. CONCLUSIONS: We report the development of the most complete reconstruction of yeast metabolism to date that is based upon reliable literature evidence and richly annotated according to MIRIAM standards. The reconstruction is available in the Systems Biology Markup Language (SBML) and via a publicly accessible database http://www.comp-sys-bio.org/yeastnet/

Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405 : comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, BB/G018553/1, BB/S002103/1, and BB/S005897/1), the UK Department for Environment, Food and Rural Affairs, and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. Funding for LL provided by the ‘National Natural Science Foundation of China’ (No.31520103917). MTGH and DH were supported by the Wellcome Trust (grant number 098051).We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.Publisher PDFPeer reviewe

Effects of Environmental and Management-Associated Factors on Prevalence and Diversity of Streptococcus suis in Clinically Healthy Pig Herds in China and the United Kingdom.

Streptococcus suis, a global zoonosis of pigs, shows regional differences in the prevalence of human-associated disease for Asian and non-Asian countries. The isolation rates and diversities of S. suis on tonsils of healthy slaughter pigs in China and the United Kingdom were studied for effects of geography, temperature, pig age, and farm type. Isolates underwent analysis of molecular serotype and multilocus sequence type and virulence-associated genotyping. Although we found no significant difference in positive isolation rates between Chinese and UK farms, the prevalences of serotypes previously associated with human disease were significantly greater in the Chinese collection (P = 0.003). A significant effect of temperature was found on the positive isolation rate of the Chinese samples and the prevalence of human disease-associated serotypes in the UK S. suis population (China, P = 0.004; United Kingdom, P = 0.024) and on the prevalence of isolates carrying key virulence genes in China (P = 0.044). Finally, we found marked diversity among S. suis isolates, with statistically significant temperature effects on detection of multiple strain types within individual pigs. This study highlighted the high carriage prevalence and diversity of S. suis among clinically healthy pig herds of China and the United Kingdom. The significant effect of temperature on prevalence of isolation, human disease-associated serotypes, and diversity carried by individual pigs may shed new light on geographic variations in human S. suis-associated disease.IMPORTANCEStreptococcus suis is a global zoonotic pathogen and also a normal colonizer mainly carried on the tonsil of pigs. Thus, it is important to study the effect of environmental and management-associated factors on the S. suis populations in clinically healthy pigs. In this research, we investigated the similarities and differences between the S. suis populations obtained from different pig ages, seasons, and farm management systems and discovered the relationship between high climatic temperature and the prevalence of S. suis

Normalized rates for a number of reactions, selected for the high inter-group variance in their rates.

<p>Reaction abbreviations are used in place of reaction names, but a full list can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181365#pone.0181365.s011" target="_blank">S11 Table</a>. Note that the x-axis scale has been subjected to cubic root scaling to highlight smaller changes. Normalization is against the <i>in vitro</i> group (termed Input), hence this value is always at 100%.</p

Figure to show the different experimental conditions.

<p>The figure shows the three different <i>in vivo</i> Groups and <i>in vitro</i> grown Input, as well as indicating the time points for the transcriptome (RNA-Seq) and proteome analysis for the bacteria, and transcriptome (microarray) analysis of the host.</p