Complete genome sequences of a Canadian strain of enteroaggregative Escherichia coli (EAEC) with multiple metals and antimicrobial resistance genes isolated from municipal waste-activated sludge

ABSTRACT

Enteroaggregative Escherichia coli (EAEC) is an emerging food-borne pathogen causing acute or persistent diarrhea in humans. Here, we report the complete genome sequence of a strain of EAEC with multiple metals and antimicrobial resistance genes isolated from a waste-activated sludge collected from a Canadian municipal wastewater treatment plant.

ANNOUNCEMENT

Enteroaggregative Escherichia coli (EAEC) causes diarrhea in humans and is responsible for both acute and persistent diarrhea worldwide (1). The current study reports the genome sequence of an EAEC strain (HH35) isolated from a waste-activated sludge collected from a Canadian municipal wastewater treatment plant in 2010. The strain was isolated by enrichment in lauryl sulfate tryptose broth (35°C, 24 hours) and then in E. coli broth (45°C, 24 hours), and isolation using Levine’s eosin methylene blue agar (35°C, 24 hours) (2). It was identified as EAEC, based on the presence of the aggR gene and a complete AAF/V gene cluster within its genome (3).

Genomic DNA (gDNA) extract, used for both sequencing procedures below, was prepared from an overnight culture grown from a single colony in Tryptic Soy Broth using NanoBind CBB Kit (PacBio, US), followed by treatment with Short Read Eliminator XS (PacBio, US), according to the manufacturer’s instructions. Illumina sequencing was conducted by library preparation using Illumina DNA Prep Kit (Illumina, US), and sequencing on the MiSeq platform (Illumina, US) using MiSeq reagent kit v3 with a total of 598,140 paired-end (300 bp) reads generated, followed by filtration and trimming with Fastp v.0.23.2 (4). Nanopore sequencing was performed by MinION library preparation using a ligation sequencing gDNA-native barcoding kit (SQK-NBD112.24) (Oxford Nanopore Technologies, UK) without shearing, and sequencing using an FLO-MIN112 (R10.4.1) flow cell on a MinION Mk1B device. A total of 2,191,450 reads (N50 of 2,128 bp) were obtained, followed by base-calling using Guppy v6.1.2, trimming using Porechop v0.2.4, and filtering using NanoFilt v2.8.0 (5). Assembly of MinION reads was performed using flye v2.9.1 (6) and polished with medaka v1.7.2 (https://github.com/nanoporetech/medaka), followed by short-read polishing using NextPolish v1.4.1 (7), ntEdit v1.3.5 (8), and Polypolish v0.5.0 (9). The circularity of the genome and genome rotation using dnaA as the starting point was determined by Circlator v1.5.5 (10). The sequencing coverage depth (474×) was determined and assessed using Samtools v1.13 (11). Gene predictions and annotations were performed using NCBI Prokaryotic Genome Annotation Pipeline v6.4 (12). Metal, acid, virulence, and antimicrobial resistance genes were identified using AMRFinderPlus v3.11.18 with database v2023-08-08.2 (13). The plasmids were identified by PlasmidFinder v2.0.1 with database v2023-01-18 (14), and prophage sequences were analyzed using the PHASTER web server (15). Its serotype was identified using ECTyper v1.0.0 (16), and pathogenicity was predicted using PathogenFinder v1.1 (17). Default parameters of bioinformatics tools were used except where otherwise noted.

The HH35 isolate was predicted as serotype O99:H10 EAEC. Its genome contains a single chromosome with one plasmid. Table 1 demonstrates detailed information for total length, chromosome size, GC%, protein count, prophage, antibiotics, heavy metal, acid resistance, and virulence genes. On average, the median total length, CDS, and GC% of E. coli genome assemblies in GenBank are similar to those of this E. coli strain (Table 1).

TABLE 1.

Genomic characteristics of the EAEC strain (HH35) isolated from a municipal waste-activated sludge

Strain ID	Contigs	Plasmid	Total length (Mb)	Chromosome size (bp)	GC%	Protein counts	Genes related to AMRa	Genes related to metal-resistancea	Genes related to acid-resistancea	Virulence genesa	Intact prophage
HH35	2	1	5.02	4,916,664	50.7%	4572	10 mdtM blaEC emrD acrF dfrA17 tet(A) aph (6)-Id aph(3'')-Ib sul2 blaTEM-1	3 fieF arsC arsR	2 asr ariR	13 fdeC espX1 capU aaiC pic aatA aar aap aggR agg5A agg3B agg3C agg3D	3
Median GenBank sequencesb			5.10		50.6%	4727

^a Antimicrobial, heavy metal, and acid resistance, and virulence genes were predicted by AMRFinderPlus.

^b Data summarized on 11–14-2023 using 36,469 E. coli genome assemblies available on GenBank.

DATA AVAILABILITY

The genome and plasmid sequences of strain HH35 have been deposited at GenBank under accession numbers CP136993.2 and CP136994.1. MinION and MiSeq raw data are available in the NCBI Sequence Read Archive through accession numbers SRR26967109 and SRR26407666, respectively.