Refining the definition of the avian pathogenic Escherichia coli (APEC) pathotype through inclusion of high-risk clonal groups

Abstract

Colibacillosis in poultry is a unique disease manifestation of Escherichia coli in the animal world, as one of the primary routes of entry is via the respiratory tract of birds. Because of this, a novel extraintestinal pathogenic E. coli (ExPEC) subpathotype coined avian pathogenic E. coli (or APEC) has been described. Like other ExPEC, this pathotype has been challenging to clearly define, and in the case of APEC, its role as an opportunistic pathogen has further complicated these challenges. Using 3,479 temporally matched genomes of poultry-source isolates, we show that the APEC plasmid, previously considered a defining trait of APEC, is highly prevalent in clinical isolates from diseased turkeys. However, the plasmid is also quite prevalent among cecal E. coli isolates from healthy birds, including both turkeys and broilers. In contrast, we identify distinct differences in clonal backgrounds of turkey clinical versus cecal strains, with a subset of sequence types (STs) dominating the clinical landscape (ST23, ST117, ST131, ST355, and ST428), which are rare within the cecal landscape. Because the same clinical STs have also dominated the broiler landscape, we performed lethality assays using strains from dominant STs from clinical or cecal landscapes in embryonated turkey and chicken eggs. We show that, irrespective of plasmid carriage, dominant clinical STs are significantly more virulent than dominant cecal STs. We present a revised APEC screening tool that incorporates APEC plasmid carriage plus markers for dominant clinical STs. This revised APEC pathotyping tool improves the ability to identify high-risk APEC clones within poultry production systems, and identifies STs of interest for mitigation targets.

Keywords: APEC; Escherichia coli; colibacillosis; pathotype; poultry.

Figure 1

Single nucleotide polymorphism-based phylogenetic tree depicting relationships between Turkey Clinical isolates sequenced in this study (N = 397). The inner ring depicts isolates colored by company (blinded) with different colors representing different companies submitting clinical isolates. The outer ring highlights the five dominant sequence types identified amongst this collection.

Figure 2

Patterns of prevalence of selected genes of the APEC plasmid PAI among Turkey Clinical (N = 397), Turkey Cecal (N = 562), and Turkey Retail (N = 1,468) E. coli. Data are displayed using a stacked bar graph depicting population prevalence (%). Genes of the pentaplex APEC typing scheme (Johnson et al., 2008) are boxed in red.

Figure 3

Patterns of prevalence of Clermont phylogenetic groups among Turkey Clinical (N = 397), Turkey Cecal (N = 562), and Turkey Retail (N = 1,468) E. coli. Data are displayed using a bar graph depicting population prevalence (%) above each bar.

Figure 4

Minimal spanning trees of Turkey Clinical, Turkey Cecal, and Turkey Retail isolates based upon the 7-gene E. coli MLST scheme. A: Sequence types (STs) are colored by source and dashed lines indicate membership in Clermont phylogenetic groups. B: STs are colored proportionally by percentage containing the APEC plasmid, and major STs are noted in either green (Cecal-associated) or red (Clinical-associated).

Figure 5

Patterns of prevalence of selected genes of the APEC plasmid PAI among Chicken Cecal (N = 440) and Chicken Retail (N = 611) E. coli. Data are displayed using a stacked bar graph depicting population prevalence (%).

Figure 6

Patterns of prevalence of Clermont phylogenetic groups among Chicken Cecal (N = 440) and Chicken Retail (N = 611) E. coli. Data are displayed using a bar graph depicting population prevalence (%) above each bar.

Figure 7

Core single nucleotide polymorphism-based phylogenetic tree depicting relationships between ST117 isolates analyzed in this study (N = 201). The inner ring lists isolate name or NCBI SRA accession number. The second two rings depict isolate source and serogroup, colored by host source (red = chicken and blue = turkey). The outer ring displays presence (green) or absence (white) of 46 APEC-associated fitness or virulence factors.

Figure 8

Scheme for revised typing of high-risk avian pathogenic E. coli (APEC).

Figure 9

Agarose gel electrophoresis depicting the revised 9-plex PCR that detects high-risk APEC clones. Lane 1 = 100-bp ladder; Lane 2 = PP0878 (ompT, hlyF, O78, ST23); Lane 3 = PP0507 (ompT, hlyF, ST117); Lane 4 = PP0293 (ompT, hlyF, O78, ST117); Lane 5 = PP0178 (ompT, hlyF, ST428); Lane 6 = PP0171 (ompT, hlyF, O78, ST131); Lane 7 = PP0209 (ompT, hlyF, ST355); Lane 8 = PP0902 (ompT, hlyF); Lane 9 = DNA pool of isolates from Lanes 2-8; Lane 10 = E. coli K-12 MG1655; Lane 11 = blank; Lane 12 = 100-bp ladder.