Draft Genome Sequences of Antibiotic-Resistant Escherichia coli Isolates from U.S. Wastewater Treatment Plants.

The spread of antibiotic-resistant microorganisms is a major public health concern. Here, we report the draft genome sequences of three Escherichia coli isolates from primary effluent collected from geographically dispersed U.S. wastewater treatment plants (WWTPs). Genomic analysis confirms the presence of genes encoding resistance to a broad spectrum of antibiotics.

ANNOUNCEMENT

A survey for antibiotic-resistant (AR) Escherichia coli was undertaken by collecting samples of primary effluents from geographically dispersed U.S. wastewater treatment plants (WWTPs) (1). E. coli, a Gram-negative bacterium and member of the family Enterobacteriaceae, is a commensal inhabitant of the gastrointestinal tract (2) and is commonly used as an indicator of fecal pollution (1). AR E. coli in wastewater could provide information on the occurrence and dissemination of sequence types (ST) within a given community or population (1, 3).

Strains EPA165, EPA233, and EPA336 were isolated from effluent collected from primary clarifiers at WWTPs located in New Jersey, California, and Maryland, as previously described by Hoelle et al. (1). Briefly, samples were filtered and transferred to membrane fecal coliform (m-FC) agar (Becton, Dickinson, Franklin Lakes, NJ) supplemented with imipenem (1 mg/liter), ciprofloxacin (4 mg/liter), cefotaxime (4 mg/liter), or ceftazidime (16 mg/liter) and incubated at 44.5°C. Single colonies were transferred to m-FC plates supplemented with 4-methylumbelliferyl-β-d-glucuronide (MUG). The DNA from MUG-positive colonies was extracted using the UltraClean DNA microbial isolation kit following the manufacturer’s instructions (Mo Bio Laboratories, Solana Beach, CA). Genomic libraries were prepared using the Nextera XT index kit v2 set A and sequenced on the HiSeq 4000 platform (Illumina, Inc., San Diego, CA) with a HiSeq 3000/4000 PE cluster kit (2 × 150 bp).

A total of 29,806,503 paired-end reads (EPA165, 9,001,282; EPA223, 11,064,325; EPA336, 9,740,896) were generated. Prior to assembly, libraries were cleaned from adapters and phiX artifacts, error corrected, normalized (≤100×), and filtered to a minimum length of 100 nucleotides (nt) using the software package BBMap v38.22 (ktrim=r k=23 mink=11 hdist=1 tbo tpe maxns=0 trimq=10 qtrim=r maq=12 minlength=100 ecco=t eccc=t ecct=t target=100) (4). A reference-assisted de novo assembly approach was used to assemble the processed reads using Unicycler v0.4.7 (5). Average nucleotide identity (ANI), a similarity index between two genomes (6), was determined using FastANI v1.1 (7). The in silico multilocus sequence type (MLST) based on seven alleles (adk, fumC, gyrB, icd, mdh, purA, and recA) was obtained using mlst v2.16.1 (8, 9) and phylotyping was performed with the EzClermont Web tool (10), serotyping (O-antigen and flagellin genes) with SerotypeFinder v2.0 (11), subtyping (fumC and fimH alleles) with CHTyper v1.0 (12), antibiotic resistance gene determination with ResFinder v3.1 (13), and chromosomal point mutation determination with PointFinder v3.1 (14). Default parameters were used for all software unless otherwise specified. Genome quality and statistics were estimated with BBMap and annotated with Prokka v1.13.1 (15) (Table 1).

TABLE 1

Summary statistics of whole-genome assemblies

Straina	Coverage (×)	Genetic elementb	No. of contigs	Assembly size (bp)	Contig N50 (bp)	G+C content (%)	Gene annotation data (no.)c				PTd	STe	Serotypef	Subtypeg	Reference genome	GenBank accession no.
							Genes	CDS	rRNAs	tRNAs
EPA165	255	Chromosome	110	5,040,423	288,542	50.53	4,816	4,714	15	86	D	973	O21:H15	187:95	GCA_001542545.1	SJTD00000000
		Plasmid 1	1	4,936	4,936	47.81
		Plasmid 2	1	4,074	4,074	49.93
		Plasmid 3	4	72,427	58,169	50.21
EPA233	328	Chromosome	59	4,669,471	271,035	50.79	4,559	4,460	14	84	B1	156	O9:H9	29:38	GCA_000010385.1	SJTC00000000
		Plasmid 1	1	36,144	36,144	42.66
		Plasmid 2	10	126,415	22,867	50.95
EPA336	293	Chromosome	66	4,712,839	477,434	50.83	4,599	4,501	10	87	B1	205	O100:H12	23:54	GCA_001007915.1	SJTB00000000
		Plasmid 1	1	8,379	8,379	55.48
		Plasmid 2	1	113,422	113,422	46.17

The numbers of paired-end reads were 9,001,282 (EPA165), 11,064,325 (EPA223), and 9,740,896 (EPA336).

Plasmid contig identifiers for EPA165 are contig000025 (plasmid 1), contig000027 (plasmid 2), and contig000022, contig000024, contig000015, and contig000034 (plasmid 3); for EPA223, contig000027 (plasmid 1) and contig000038, contig000036, contig000032, contig000033, contig000023, contig000053, contig000054, contig000037, contig000048, and contig000031 (plasmid 2); and for EPA336, contig000019 (plasmid 1) and contig000013 (plasmid 2).

CDS, coding sequences.

PT, phylotype.

ST, sequence type (in silico MLST; adk, fumC, gyrB, icd, mdh, purA, recA).

O, O-antigen; H, flagellin gene.

fumC:fimH alleles.

ANI calculations revealed an average genome similarity of 99.10% between strains EPA233 and EPA336, which were both closely related to EPA165, with 97.30% similarity. Despite their high genome similarity, further analysis confirmed the clustering of the three strains to a different sequence type, serotype, and subtype complex (Table 1). EPA233 and EPA336 were identified as members of phylotype B1 and EPA165 was identified as phylotype D. Pangenome analysis confirmed the presence of genes encoding resistance to a broad spectrum of antibiotics, such as β-lactams (blaCMY-2 and blaTEM-1B), the multiple drug resistance (MDR) family (mdfA), aminoglycoside [aac(6′)-Ib3, aph(6)-Id, aadA1, and aph(3″)-Ib], fluoroquinolone [aac(6′)-Ib-cr], phenicol (catA1 and catB3), sulfonamide (sul1 and sul2), tetracycline (tetA and tetB), and trimethoprim (dfrA1). Furthermore, chromosomal point mutations associated with antimicrobial resistance in gyrA and parC genes were detected. The plasmids carried antimicrobial resistance genes and contained putative conjugal transfer modules, including an oriT-like region, relaxase, and components of the type IV coupling protein (T4CP) and type IV secretion system (T4SS) (16).

Data availability.

This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession numbers listed in Table 1. The raw sequence reads have been submitted to the NCBI SRA under the accession numbers SRR8648405, SRR8648406, and SRR8648407. The versions described in this paper are the first versions.