High-risk clonal groups of avian pathogenic Escherichia coli (APEC) demonstrate heterogeneous phenotypic characteristics in vitro and in vivo

Abstract

Avian Pathogenic Escherichia coli (APEC), a major bacterial pathogen of poultry, comprises a diverse range of high-risk clonal groups. However how these lineages interact with avian host cells remains poorly characterized. This study examined the ability of key APEC clonal groups to adhere to, invade, and survive within avian host cells, alongside assessing their virulence in the Galleria mellonella infection model. Genomic analysis of APEC from a UK turkey colibacillosis outbreak identified ST-101 as the dominant clonal group, carrying numerous virulence factors. ST-101 was compared to other high-risk APEC clonal groups (ST-23, ST-140, ST-95, ST-117). Utilizing in vitro cell culture models, APEC isolates displayed comparable adhesion to 8E11 chicken epithelial gut and HD11 chicken macrophage cell lines. APEC ST-95, ST-101, and ST-140 demonstrated increased invasion of 8E11 cells, and intracellular survival within HD11 macrophages, relative to ST-23 and ST-117, suggesting pronounced phenotypic differences between clonal groups. However, in HD11 cell assays, no difference in magnitude of elicited immune response was observed between lineages, indicating differing intracellular survival was not a result of immune response modulation. In vivo virulence in the Galleria mellonella infection model was also observed to differ between APEC genotypes, with ST-117 inducing the highest mortality, despite the comparatively lower epithelial invasion and intramacrophage survival for other lineages. Collectively, this suggests that diverse APEC genotypes have distinct phenotypic profiles in vitro and in vivo. These results highlight the need for intervention strategies that can simultaneously target a broad range of pathogenic lineages.

Keywords: APEC; Escherichia coli; Galleria mellonella; avian pathogenic; cell culture; sequence types.

Figure 1.

Phylogenetic reconstruction of 91 E. coli genomes (Table S1) recovered from a colibacillosis outbreak in Turkey poults. Whole genome sequences of E. coli isolated from the heart, liver, and caeca of turkeys suspected of colibacillosis were used to reconstruct core-genome phylogeny using ParSNP [48]. Health-associated metadata of bird fate (dead or culled) and health status (sick/terminal or healthy) denoted by the colour strip to the right of the cladogram. Presence of encoded APEC-associated virulence genes determined using a custom abricate (https://github.com/tseemann/abricate) database using an 80% minimum sequence identity threshold. The presence or absence of a virulence gene within the bacterial genome is denoted by a grey box in the heatmap (grey=present, white=absent). Sequence type (ST) was established using Warwick classification scheme (Seemann T, mlst, Github https://github.com/tseemann/mlst) and denoted to the right of the heatmap.

Figure 2.

Plasmid map of ColV plasmid harboured by ST-101 SAP 4026. Genes identified by Bakta (https://github.com/oschwengers/bakta) annotation of circular plasmid contig determined through hybrid sequencing. Dark red = virulence associated genes. Purple = colicin genes. Green = iron acquisition genes. Blue = conjugation machinery. Created with SnapGene software (www.snapgene.com).

Figure 3.

Gene cluster comparison of pSAP4026-ColV with previously reported plasmids from avian pathogenic and commensal E. coli. Figure generated using the clinker and cluster map software [35] with identity threshold set at 0.3. Gene clusters demonstrating similarity greater than the threshold share the same colour code. Additional labelling performed in biorender (www.biorender.com). Plasmid sequences were selected from comparison from the NCBI database; APEC-O2-211A-ColV (Assession number: CP030791.1), pAPEC-p10_578_1 (Assession number: CP087565.1), and pCh101 (Assession number: CP127318.1) accessed through GenBank.

Figure 4.

Comparison of ability of APEC isolates to (A) adhere to and (B) invade the 8E11 cell line. Quantification of adhesion of APEC isolates determined following challenge with MOI 100 bacterial inoculum followed by two hours incubation at 41°C prior to washing and lysing of eukaryotic cells. Invasion assayed by the inoculation of cells for two hours followed by the addition of cell culture media containing gentamicin for a further two hours, before washing and lysis of eukaryotic cells. Bacterial viability determined by the Miles and Misra method. Experiments performed independently three times, with triplicate wells used for each isolate. Data shows average CFU/ml ± SEM. Significance determined by Kruskal Wallis statistical test, followed by post-hoc Dunn’s test of multiple comparisons compared to SAP16 with significance denoted by *= p ≥ 0.05, ** = p ≤ 0.01. ST-101 APEC demonstrated increased intracellular survival within HD11 chicken macrophage cells compared to ST-117 APEC.

Figure 5.

Comparison of intracellular survival of APEC isolates in HD11 chicken macrophage cells at (A) 4 hours post infection, (B) 6 hours post infection, and (C) 18 hours post infection. HD11 cells challenged with a MOI 10 bacterial inoculum and incubated for two hours at 41°C. Intracellular survival was determined by the addition of gentamicin containing media for the described incubation period and quantification of bacteria viability. Experiments performed independently five times, with triplicate wells used for each isolate. Data shows average CFU/ml ± SEM. Production of (D) NO determined by Griess assay six hours post-infection. Experiment performed independently eight times with triplicate wells used for each isolate. Data shows average nitrate concentration ± SEM. Production of (E) ROS production by addition of DCFA and measurement of fluorescence intensity for 10 hours post infection. Experiment performed independently three times with triplicate wells used for each isolate. Data shows average fluorescence intensity (flu) ± SEM. Significance determined by Kruskal Wallis statistical test *** = p ≤ 0.001, followed by post-hoc Dunn’s test of multiple comparisons, with differing letters denoting significance (p ≥ 0.05).

Figure 6.

Survival of Galleria mellonella challenged with APEC clonal groups ST-23, ST-95, ST-117, and ST-101 24 hours, 48 hours, and 72 hours following challenge, expressed by Kaplan-Meier plots. Larvae challenged with 1x10³ CFU APEC or PBS mock and survival determined every 24 hours by confirming response to stimuli. Experiment performed with 10 larvae per group. Significance determined log-rank (Mantel-Cox) test *= p ≥ 0.05.