Dataset Information

Comparative genomics of European avian pathogenic E. Coli (APEC).

ABSTRACT: Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which results in significant economic losses to the poultry industry worldwide. However, the diversity between isolates remains poorly understood. Here, a total of 272 APEC isolates collected from the United Kingdom (UK), Italy and Germany were characterised using multiplex polymerase chain reactions (PCRs) targeting 22 equally weighted factors covering virulence genes, R-type and phylogroup. Following these analysis, 95 of the selected strains were further analysed using Whole Genome Sequencing (WGS).The most prevalent phylogroups were B2 (47%) and A1 (22%), although there were national differences with Germany presenting group B2 (35.3%), Italy presenting group A1 (53.3%) and UK presenting group B2 (56.1%) as the most prevalent. R-type R1 was the most frequent type (55%) among APEC, but multiple R-types were also frequent (26.8%). Following compilation of all the PCR data which covered a total of 15 virulence genes, it was possible to build a similarity tree using each PCR result unweighted to produce 9 distinct groups. The average number of virulence genes was 6-8 per isolate, but no positive association was found between phylogroup and number or type of virulence genes. A total of 95 isolates representing each of these 9 groupings were genome sequenced and analysed for in silico serotype, Multilocus Sequence Typing (MLST), and antimicrobial resistance (AMR). The UK isolates showed the greatest variability in terms of serotype and MLST compared with German and Italian isolates, whereas the lowest prevalence of AMR was found for German isolates. Similarity trees were compiled using sequencing data and notably single nucleotide polymorphism data generated ten distinct geno-groups. The frequency of geno-groups across Europe comprised 26.3% belonging to Group 8 representing serogroups O2, O4, O18 and MLST types ST95, ST140, ST141, ST428, ST1618 and others, 18.9% belonging to Group 1 (serogroups O78 and MLST types ST23, ST2230), 15.8% belonging to Group 10 (serogroups O8, O45, O91, O125ab and variable MLST types), 14.7% belonging to Group 7 (serogroups O4, O24, O35, O53, O161 and MLST type ST117) and 13.7% belonging to Group 9 (serogroups O1, O16, O181 and others and MLST types ST10, ST48 and others). The other groups (2, 3, 4, 5 and 6) each contained relatively few strains. However, for some of the genogroups (e.g. groups 6 and 7) partial overlap with SNPs grouping and PCR grouping (matching PCR groups 8 (13 isolates on 22) and 1 (14 isolates on 16) were observable). However, it was not possible to obtain a clear correlation between genogroups and unweighted PCR groupings. This may be due to the genome plasticity of E. coli that enables strains to carry the same virulence factors even if the overall genotype is substantially different.The conclusion to be drawn from the lack of correlations is that firstly, APEC are very diverse and secondly, it is not possible to rely on any one or more basic molecular or phenotypic tests to define APEC with clarity, reaffirming the need for whole genome analysis approaches which we describe here. This study highlights the presence of previously unreported serotypes and MLSTs for APEC in Europe. Moreover, it is a first step on a cautious reconsideration of the merits of classical identification criteria such as R typing, phylogrouping and serotyping.

SUBMITTER: Cordoni G

PROVIDER: S-EPMC5120500 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Comparative genomics of European avian pathogenic E. Coli (APEC).

Cordoni Guido G Woodward Martin J MJ Wu Huihai H Alanazi Mishaal M Wallis Tim T La Ragione Roberto M RM

BMC genomics 20161122 1

<h4>Background</h4>Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which results in significant economic losses to the poultry industry worldwide. However, the diversity between isolates remains poorly understood. Here, a total of 272 APEC isolates collected from the United Kingdom (UK), Italy and Germany were characterised using multiplex polymerase chain reactions (PCRs) targeting 22 equally weighted factors covering virulence genes, R-type and phylogroup. Following these analy ...[more]

PMID: 27875980

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Project description:Many reports show an association between the Pst system, the Pho regulon related genes and bacterial virulence. Our previous results showed that a functional Pst system is required for full virulence, resistance to serum, polymyxin B and acid shock However, the interplay between the Pst system and virulence has an unknown molecular basis. To understand global APEC virulent strain responses to Pho regulon activation, we conducted transcriptome profiling experiments comparing the isogenic Pst mutant with its wild type strain chi7122 using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. To understand the global responses to Pho regulon activation of APEC, we use the Affymetrix technology. We conducted transcriptome profiling experiments comparing the APEC chi7122 strain and its isogenic Pst mutant grown in rich phosphate medium using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. The Affymetrix GeneChipÂ® E. coli Genome 2.0 Array contains the genome of the E. coli MG1655 and three pathogenic E. coli strain (EDL933, Sakai and CFT073) representing 20,366 genes. While comparing genes expression between Pst mutant and the wild type chi7122 strain, 471 genes are either up- (254) or down-regulated (217) of at least 1.5-fold, with a p-value inferior or equal to 0.05 and a false discovery rate of 2.71%. Experiment Overall Design: RNA extraction was performed onto the chi7122 and K3 strains on four biological replicates. An overnight culture grown at 37Â°C was diluted 100-fold into 5 ml of LB broth and was allowed to grow to mid-log phase (OD600 0,6). Cultures were centrifuged and RNAs were isolated by using the RiboPureâ¢-Bacteria Kit (Ambion, Austin, TX), according to the manufacturerâs recommendations, with the exception that the DNAse 1 treatment was performed twice. RNA extractions were performed on four different days (the chi7122 and K3 RNA were extracted at the same time) and a total of eight hybridizations were performed. Hybridization was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4). GeneChip scan was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4), and data were processed using the FlexArray software (Michal Blazejczyk, Mathieu Miron, Robert Nadon (2007). FlexArray: A statistical data analysis software for gene expression microarrays. Genome Quebec, Montreal, Canada). Raw data were normalized using the RMA algorithm and log2 were generated. The expression value was generated by subtracting the mean value of each replicate of the mutant strain by wild-type strain.

Project description:BackgroundAvian pathogenic Escherichia coli (APEC) is a major cause of disease impacting animal health. The bone marrow is the reservoir of immature immune cells; however, it has not been examined to date for gene expression related to developmental changes (cell differentiation, maturation, programming) after APEC infection. Here, we study gene expression in the bone marrow between infected and non-infected animals, and between infected animals with mild (resistant) versus severe (susceptible) pathology, at two times post-infection.ResultsWe sequenced 24 bone marrow RNA libraries generated from the six different treatment groups with four replicates each, and obtained an average of 22 million single-end, 100-bp reads per library. Genes were detected as differentially expressed (DE) between APEC treatments (mild pathology, severe pathology, and mock-challenged) at a given time point, or DE between 1 and 5 days post-infection (dpi) within the same treatment group. Results demonstrate that many immune cells, genes and related pathways are key contributors to the different responses to APEC infection between susceptible and resistant birds and between susceptible and non-challenged birds, at both times post-infection. In susceptible birds, lymphocyte differentiation, proliferation, and maturation were greatly impaired, while the innate and adaptive immune responses, including dendritic cells, monocytes and killer cell activity, TLR- and NOD-like receptor signaling, as well as T helper cells and many cytokine activities, were markedly enhanced. The resistant birds' immune system, however, was similar to that of non-challenged birds.ConclusionThe DE genes in the immune cells and identified signaling models are representative of activation and resolution of infection in susceptible birds at both post-infection days. These novel results characterizing transcriptomic response to APEC infection reveal that there is combinatorial activity of multiple genes controlling myeloid cells, and B and T cell lymphopoiesis, as well as immune responses occurring in the bone marrow in these early stages of response to infection.

Project description:Purpose: This study is to more comprehensively understand the genome-wide host response to avian pathogen E. coli (APEC). Methods: Male broiler chicks were challenged with APEC (or mock-challenged as controls), and bone marrow was harvested at 1 and 5 days post-infection (dpi). Based upon necropsy-scored lesions on liver, pericardium, and air sacs, the challenged birds were assigned to mild or severe pathology categories, representing resistant and susceptible phenotypes, respectively. RNA sequence data were first analyzed using the R package EdgeR to identify differentially expressed genes.GO and pathway analysis using the R package GOseq identified many immune-related pathways. Results: RNA sequencing resulted in 11 to 40 million single-end raw reads of 100 bp per sample. After alignment, an average of 80 % of the reads, with 5 % representing multiple mapping, were mapped to the chicken reference transcriptome. Among these detected unique transcripts, there were 2,404 novel genes mainly distributed on chromosomes 1, 2, 4, 3, Z, 5 in decreasing number. Genes were detected as differentially expressed (DE) between treatments (susceptible, resistant, and mock-challenged) at a given time point, or DE between 1 and 5 dpi within the same treatment group. Compared to mock-challenged birds on 5 dpi, susceptible birds exhibited extensive enhancement of their T cell and B cell development, as well as innate and adaptive immune response. However, for the 1 dpi group, susceptible birds exhibited a decreased gene expression of T and B cell development. The differences between the two phenotypes, susceptible and resistant birds, at 5 dpi showed that susceptible birds had increased gene expression of B-cell development and adaptive immune response. Conclusions: This is the first report to our knowledge examining the role of bone marrow cells in responding to APEC infection in broilers. This transcriptome study provides insight and global overview into the response of genes involved in the earliest phases of the immune response to APEC infection. Our data indicate a dynamic interaction between the innate and adaptive immune responses to APEC infection in susceptible birds, providing flexibility and redundancy in the host’s induction of cytokines and chemokines. Additionally, B cell and T cell development are also extensively affected by APEC infection for susceptible birds, resulting in drastic host impairment in early response to infection. The findings of present study shed light on the underlying chicken immune modulation against APEC infection. Also, this study will build a solid foundation for identifying host genetic variation that may be manipulated to enhance resistance to infection and may be useful as colibacillosis control targets.

Project description:BACKGROUND: Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain chi7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. RESULTS: Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain chi7122. CONCLUSION: Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence.

Dataset Information

Comparative genomics of European avian pathogenic E. Coli (APEC).

Publications

Comparative genomics of European avian pathogenic E. Coli (APEC).

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