Complete genome sequence of an avian pathogenic Escherichia coli strain isolated from poultry

ABSTRACT

Avian pathogenic Escherichia coli found in the avian intestinal tract can cause systemic disease in birds and act as a foodborne zoonotic pathogen associated with human disease. Here, we report the complete genome sequence of E. coli strain H1998 isolated from a chicken with colisepticemia.

ANNOUNCEMENT

Avian pathogenic Escherichia coli (APEC) is a subset of extraintestinal pathogenic E. coli, which can infect outside the intestinal tract (1). APEC can cause respiratory and systemic diseases in poultry and zoonotic, foodborne infections associated with human urinary tract infection, sepsis, and meningitis (2–4). APEC can result in significant economic losses in the poultry industry due to mortality and reduced productivity (5). However, the contribution of APEC genotype toward virulence is not well understood (6). To gain insight into its biology for effective treatments and prevention, we sequenced strain H1998, a serotype O2 APEC isolate (7). Although it was used extensively in infection studies, the genetic makeup remained undisclosed (8–12). We analyzed the complete genome to identify genes, virulence factors, and antimicrobial resistance (AMR) associated markers.

The E. coli strain was isolated from the blood of a chicken with colisepticemia (7). Following growth on a lactose-negative agar, a serotype O2, non-motile colony was selected. For sequencing, a single colony was recovered from frozen stock and cultured overnight aerobically in lysogeny broth (Thermo-Fisher Scientific) at 37°C. Genomic DNA was purified using the AquaPure Genomic DNA Isolation Kit (Bio-Rad) without shearing. The Circulomics Short Read Eliminator Kit (Circulomics) was used for depleting reads shorter than 30 kb. Library was prepared using the Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies) with the Native Barcoding expansions (EXP-NBD104 and EXP NBD114; Oxford Nanopore Technologies). Whole-genome sequencing was conducted using a Nanopore GridION sequencer (Oxford Nanopore Technologies) with FLO-MIN106: SQK-LSK109 flow cell kit. Sequencing quality was monitored using MinKNOW v21.11.7 (Oxford Nanopore Technologies) and NanoPlot v1.41.0 (13). Guppy v5.1.13 (Oxford Nanopore Technologies) was used under the high-accuracy mode for base calling. A total of 64,276 reads were obtained, averaging 6,491.7 bp in length with an N₅₀ of 10,127. Reads of at least 10,000 bp length were assembled using de novo long sequence assembler Canu v1.7.1 (14), achieving 115× coverage. The assembled genome was annotated using BV-BRC platform v3.28.21. The strain’s serotype was identified using SerotypeFinder v2.0 (15) and CHTyper (16). AMR genes and associated single-nucleotide polymorphisms (SNPs) were identified using AMRFinderPlus v3.10.24 (17) and ResFinder 4.0 (18). Standard protocols and default parameters were used unless otherwise specified.

The assembly identified three contigs, totaling 5.2 Mb, with a genome coverage of 78.0×, G + C content of 50.5%, and N₅₀ of 5 Mb. The chromosome and two plasmids were identified, with sizes of 4,986,890 bp, 147,176 bp, and 115,852 bp, respectively.(Table 1) Genome analysis revealed H1998 is an O2:H4 serotype strain and a C38:H27 CH type (15, 16). Plasmid replicons IncFIB(AP001918), IncFIT, and p0111 were identified on the two plasmid contigs using PlasmidFinder (19). The H1998 strain carries several genes predicted to encode virulence factors such as adhesins, iron acquisition, toxins, and quorum-sensing (6). Three genetic changes associated with AMR were found on the chromosome contig, predicting resistance against colistin (20), fosfomycin (21), ampicillin, chloramphenicol, quinolone, rifampin, and tetracycline (22, 23). Additionally, sitABCD_AY598030, conferring resistance to hydrogen peroxide (24), was identified on both the chromosome and plasmid contigs.

TABLE 1.

Overview of the avian pathogenic Escherichia coli genome assembly

Contig	Size (bp)	Avg coverage (×)	GC content (%)	No. of Coding Sequence (CDS)	No. of RNAs	AMR gene(s)	AMR-associated SNP(s)	Predicted resistance phenotype	Replicon
APEC-H1998 (chromosome)	4,986,890	78	50.5	5,651	108	sitABCD_A Y598030	pmrB_E123D, glpT_E448K, and marR_53N	Hydrogen peroxide, colistin, fosfomycin, ampicillin, chloramphenicol, quinolone, rifampin, and tetracycline	–a
pH1998-IncF (plasmid)	147,176	25.6	49.5	214	0	sitABCD_A Y598030	–	Hydrogen peroxide	IncFIB(AP001918), IncFIT
pH1998-p0111 (plasmid)	115,852	19.5	47.5	181	0	–	–	–	p0111

^a The symbol “–” represents no discovery.

DATA AVAILABILITY

All assembled genomes have been published in GeneBank under accession no. CP121347.1 (chromosome), CP121348.1 (pH1998-IncF), and CP121349.1 (pH1998-p0111). The raw sequences have been submitted in NCBI Sequence Read Archive (SRA) under BioProject accession number SRR24008056. The annotation file has been submitted to GitHub and is publicly accessible (https://github.com/jiatuya/APEC-H1998-genome/blob/main/APEC_annotation.xls).