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

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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).

Results: 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.

Results: 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.

Conclusions: 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.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3289-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Cladogram based on the 22 virulence factors analysed using multiplex PCRs. Based on this analysis nine main groups were identified and are highlighted in different colours. The branches of the tree are proportional to the distance between the strains. Legend: group 1 = dark green, group 2 = yellow, group3 = blue, group 4 = light green, group 5 = red, group 6 = dark yellow, group 7 = turquoise group 8 = dark blue, group 9 = dark red
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Fig1: Cladogram based on the 22 virulence factors analysed using multiplex PCRs. Based on this analysis nine main groups were identified and are highlighted in different colours. The branches of the tree are proportional to the distance between the strains. Legend: group 1 = dark green, group 2 = yellow, group3 = blue, group 4 = light green, group 5 = red, group 6 = dark yellow, group 7 = turquoise group 8 = dark blue, group 9 = dark red

Mentions: In order to explore the genetic diversity between the 95 strains analysed, a whole genome Single-Nucleotide Polymorphisms (SNPs) analysis was performed using the online software CSI Phylogeny 1.0a [32]. Here, the analysis focuses upon all the genes shared in common by the strains and the number and location of the single nucleotide differences within those common genes. This provided a detailed overview of deeper phylogenetic relatedness. To facilitate this the strains were investigated for SNPs and compared to the reference APEC O78 strain deposited in the NCBI database (NC_020163). This analysis facilitated the grouping of strains according to their overall similarity with the O78 reference strain, as reported in Fig. 1, where the branches of the 10 groups found are highlighted in different colours.Fig. 1


Comparative genomics of European avian pathogenic E. Coli (APEC)
Cladogram based on the 22 virulence factors analysed using multiplex PCRs. Based on this analysis nine main groups were identified and are highlighted in different colours. The branches of the tree are proportional to the distance between the strains. Legend: group 1 = dark green, group 2 = yellow, group3 = blue, group 4 = light green, group 5 = red, group 6 = dark yellow, group 7 = turquoise group 8 = dark blue, group 9 = dark red
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120500&req=5

Fig1: Cladogram based on the 22 virulence factors analysed using multiplex PCRs. Based on this analysis nine main groups were identified and are highlighted in different colours. The branches of the tree are proportional to the distance between the strains. Legend: group 1 = dark green, group 2 = yellow, group3 = blue, group 4 = light green, group 5 = red, group 6 = dark yellow, group 7 = turquoise group 8 = dark blue, group 9 = dark red
Mentions: In order to explore the genetic diversity between the 95 strains analysed, a whole genome Single-Nucleotide Polymorphisms (SNPs) analysis was performed using the online software CSI Phylogeny 1.0a [32]. Here, the analysis focuses upon all the genes shared in common by the strains and the number and location of the single nucleotide differences within those common genes. This provided a detailed overview of deeper phylogenetic relatedness. To facilitate this the strains were investigated for SNPs and compared to the reference APEC O78 strain deposited in the NCBI database (NC_020163). This analysis facilitated the grouping of strains according to their overall similarity with the O78 reference strain, as reported in Fig. 1, where the branches of the 10 groups found are highlighted in different colours.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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).

Results: 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.

Results: 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.

Conclusions: 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.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3289-7) contains supplementary material, which is available to authorized users.

No MeSH data available.