Comparative genomics of European Avian Pathogenic E. coli (APEC)

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. 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. 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

Understanding the fitness burden of ESBL plasmids in Avian Pathogenic Escherichia Coli (APEC).

Extended spectrum β-lactamases (ESBLs) are enzymes produced by bacteria that confer resistance to cephalosporins, antibiotics that are widely used to treat infections in animals and humans. The emergence of plasmid-borne ESBL resistance in humans and animals, especially poultry, is of particular concern. Avian pathogenic E. coli (APEC) cause colibacillosis, an economically important disease of poultry, but have also been linked to human disease. Of growing concern is the potential transfer of ESBL-producing plasmids between E. coli isolates and their impact on the host bacterium. Thus, the aim of this study was to characterise ESBL plasmids in APEC isolated from UK poultry and examine their impact on host bacterial fitness and virulence. Plasmid profiling and genome sequencing revealed that all three APEC isolates harboured at least two plasmids, belonging to the IncF and IncI replicon types. The ESBL plasmid was identical in each isolate, being of 105,610 Kb in size, belonging to the IncI1-Iy family and carrying the blaCTX-M-1 allele. Upstream of blaCTX-M-1 gene was an ISEcp1 insertion element, which likely contributes to the transmissibility of the element. The plasmid also carried genes coding for tetracycline resistance that were co-transferred with the β-lactamase genes. The APEC isolates also harboured a large virulence plasmid of similar size, but belonging to the IncF replicon type. Competitive growth studies in rich or minimal media found no significant differences in the growth of CTX-M-1 plasmid-harbouring and plasmid-cured derivatives. However, the presence of the CTX-M-1 plasmid in APEC O78 was beneficial to their growth in iron-limited media, but caused a greater fitness burden at low pH. When present as the only plasmid in APEC, the ESBL-containing derivatives produced more biofilm at human and chicken body temperatures and were better able to invade and survive in human (THP-1) and chicken (HD11) macrophage cell lines than the cured isolates. Finally, the substrate utilisation profiles of the plasmid-containing derivatives differed to that of the parent strains, indicating that the plasmid may influence the metabolic capability of the organism. The studies presented here indicate that ESBL plasmids of the IncI1-1Y type spread readily to unrelated APEC isolates and can influence the fitness of APEC under certain conditions

Additional file 2: of Comparative genomics of European avian pathogenic E. Coli (APEC)

Cumulative table. (XLSX 87 kb

Additional file 3: of Comparative genomics of European avian pathogenic E. Coli (APEC)

Hits. (XLSX 2118 kb

Additional file 1: of Comparative genomics of European avian pathogenic E. Coli (APEC)

Assembly statistics. (XLSX 51 kb

Multiplex PCR results.xls

<p>276 APEC were investigated for R type, phylogroup and virulence factors using multiplex pcr as described in </p><p> </p><p>Comparative genomics of European Avian Pathogenic E. coli (APEC) (in press)</p