Whole genome sequence analysis of Australian avian pathogenic Escherichia coli that carry the class 1 integrase gene

Abstract

Avian pathogenic Escherichia coli (APEC) cause widespread economic losses in poultry production and are potential zoonotic pathogens. Genome sequences of 95 APEC from commercial poultry operations in four Australian states that carried the class 1 integrase gene intI1, a proxy for multiple drug resistance (MDR), were characterized. Sequence types ST117 (22/95), ST350 (10/95), ST429 and ST57 (each 9/95), ST95 (8/95) and ST973 (7/95) dominated, while 24 STs were represented by one or two strains. FII and FIB repA genes were the predominant (each 93/95, 98 %) plasmid incompatibility groups identified, but those of B/O/K/Z (25/95, 26 %) and I1 (24/95, 25 %) were also identified frequently. Virulence-associated genes (VAGs) carried by ColV and ColBM virulence plasmids, including those encoding protectins [iss (91/95, 96 %), ompT (91/95, 96 %) and traT (90/95, 95 %)], iron-acquisition systems [sitA (88/95, 93 %), etsA (87/95, 92 %), iroN (84/95, 89 %) and iucD/iutA (84/95, 89 %)] and the putative avian haemolysin hylF (91/95, 96 %), featured prominently. Notably, mobile resistance genes conferring resistance to fluoroquinolones, colistin, extended-spectrum β-lactams and carbapenems were not detected in the genomes of these 95 APEC but carriage of the sulphonamide resistance gene, sul1 (59/95, 63 %), the trimethoprim resistance gene cassettes dfrA5 (48/95, 50 %) and dfrA1 (25/95, 27 %), the tetracycline resistance determinant tet(A) (51/95, 55 %) and the ampicillin resistance genes blaTEM-1A/B/C (48/95, 52 %) was common. IS26 (77/95, 81 %), an insertion element known to capture and mobilize a wide spectrum of antimicrobial resistance genes, was also frequently identified. These studies provide a baseline snapshot of drug-resistant APEC in Australia and their role in the carriage of ColV-like virulence plasmids.

Keywords: Escherichia coli; antimicrobial resistance; avian pathogenic E. coli; genomic epidemiology; microbial genomics; whole genome sequencing.

Fig. 1.

Phylogenetic relatedness of the APEC isolates and nine reference strains (accession numbers: 1, CP006830; 2, CP007442; 3, NC_008563; 4, CP013048; 5, CP004009; 6, AE014075; 7, CP006784; 8, U00096; 9, CP007275), as determined by PhyloSift in combination with FastTree. The tree is midpoint rooted. Red tip labels indicate isolates from the present study, while those shown in black are the reference strains. The STs and e-serotypes of isolates have been appended to their names (e.g. A5_ST117_O111:H4). The coloured bands encircling sections of the tree indicate the phylogroups into which isolates were categorized. Asterisks in sample names indicate low read depth at one or more loci for a given MLST/O-type/H-type (see https://www.github.com/maxlcummins/ARIBAlord for more information).

Fig. 2.

Genotypic profiles of APEC isolates, clustered on the basis of the PhyloSift tree in Fig. 1, with the tip labels indicating the ST and e-serotype (in text) and phylogroup (indicated by text colour, where A is red, B1 is green, B2 is purple and D is mustard). Carriage of mobile genetic element-associated genes (teal), AMR genes (purple), VAGs (red) and plasmid repA genes (blue) are shown adjacent to the tree in a hit-table, with a white square indicating the absence of a specific gene.

Fig. 3.

High-resolution phylogenetic comparison of ST117 APEC isolates, as determined by Snippy in combination with Gubbins, SNP-sites and FastTree. The tree is midpoint-rooted. The ST and e-serotype are shown on the tip labels, while the colour of the labels indicates the state of origin (brown, QLD; green, VIC; red, WA; blue, NSW; black, unknown). A gene hit map is also shown; mobile genetic element-associated genes (teal), AMR genes (purple), VAGs (red) and plasmid repA genes (blue) are shown adjacent to the tree in a hit-table, with a white square indicating the absence of a specific gene.

Fig. 4.

Mapping of short-reads indicating the presence of ColV-like virulence plasmids. Purple colour indicates a median depth of 10 or greater at a given 250 bp bin, whereas white space indicates the inverse. Clustering of rows on this heatmap is based on similarity between the coverage profiles of the isolates, while a schematic of pCERC4 is shown above the heatmap to provide an overview of the genetic elements that were present or absent based on this analysis. Key: repA1^, IncFII repA1 gene; repA1*, IncFIB repA1 gene.