Draft genome of an extended-spectrum β-lactamase-producing multidrug-resistant Escherichia coli sequence type 131 strain isolated from the Msunduzi River in South Africa

ABSTRACT

The genome (length 4,949,272 bp, G+C content 50.70%) of the multidrug-resistant Escherichia coli strain 11.2.3 isolated from the Msunduzi River (South Africa) enabled assignment to sequence type 131, phylogroup B2, and serotype O16:H5. Antibiotic resistance genes (e.g., bla_CTX-M15) and extraintestinal pathogenic E. coli-associated virulence factors (e.g., fyuA) were detected.

ANNOUNCEMENT

Escherichia coli strains of sequence type (ST) 131 are well-known extraintestinal pathogenic E. coli (ExPEC), causing human blood and urinary tract infections (1–3). Strains of the pandemic clonal lineage ST131 frequently exhibit resistance against clinically relevant antibiotics (e.g., β-lactams, fluoroquinolones) and produce extended-spectrum-β-lactamases (ESBL) (1, 2, 4, 5). They are usually associated with humans or animals, but were also detected in waste or river water (2, 3, 5, 6).

E. coli strain 11.2.3 was isolated in 2022 when analyzing water samples collected from the Msunduzi River in Pietermaritzburg (KwaZulu-Natal, South Africa) for the presence of E. coli, employing a well-established MPN standard method (7) followed by confirmation using an E. coli-specific diagnostic amplicon PCR targeting the gadA gene (8). Antibiotic susceptibility testing using the EUCAST disk diffusion procedure (V14, https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_14.0_Breakpoint_Tables.pdf) and the MAST D67C ESBL kit revealed that the strain was multidrug-resistant, exhibiting an ESBL phenotype (Table 1; Fig. 1). Additionally, cells of strain 11.2.3 hydrolyzed the β-lactam ring of the chromogenic cephalosporin nitrocefin to the expected red reaction product, verified by spectrophotometric analysis against a negative control (Fig. 1) (9).

TABLE 1.

Genome characteristics of an ESBL-producing multidrug-resistant E. coli strain from the Msunduzi River in South Africa

Characteristic	Detail
Strain and basic features	E. coli strain 11.2.3, rod-shaped Gram-negative motile cells, oxidase-negative, gas produced from lactose at 44.5°C, indole produced from tryptophan, positive methyl red test, and β-glucuronidase positive
Isolation year, origin, location, province, and country	2022, Msunduzi River water sample, Pietermaritzburg, KwaZulu-Natal, and South Africa
GenBank accession number	JBJXCV000000000
Number of reads (input) and average short-read coverage	3 727 182 and 97
Number of contigs, largest contig, N50, and L50	115, 575 749 bp, 219 220 bp, and 9
Genome length and G+C content	4 949 272 bp and 50.70%
Completeness (BUSCO V5, enterobacterales_odb10)	100%
Protein coding sequences, tRNA, and rRNA	4821, 76, and 4
Identity (rMLST)	E. coli (100%)
Strain typing (sequence typeEnterobase/Pasteur, phylogroup, serotype, and fimH type)	ST131/506, B2, O16:H5, and fimH41
Digital DNA:DNA hybridization (dDDH, d4), best type strain genome match	E. coli DSM 30083T (GCF_000690815.1) (86%)
dDDH (d4), best ST131-O16:H5 strain genome match	E. coli ST131-O16:H5 UNQD01 (GCF_900536525.1) (99.70%)
ANIb best type strain genome match	E. coli DSM 30083T (GCF_000690815.1) (98.11%)
ANIb best ST131-O16:H5 strain genome match	E. coli ST131-O16:H5 UNQD01 (GCF_900536525.1) (99.89%)
Antibiotic-resistance genes	aac(3)-IId, aph(3'')-Ib, aph(6)-Id, blaCTX-M15, blaTEM-1B, dfrA17, mph(A), qacE, sul1, sul2, and tet(A)
Known chromosomal mutations enabling antibiotic resistance	gyrA (p.S83L), gyrA (p.D87N), parC (p.S80I), and parC (p.E84V)
Phenotypic antibiotic resistance profile	Ampicillin, aztreonam, cefalexin, cefotaxime, ceftaroline, ceftazidime, ciprofloxacin, gentamicin, levofloxacin, moxifloxacin, norfloxacin, tobramycin, trimethoprim, and trimethoprim-sulfamethoxazole
Prophages	3
Plasmid replicon types detected	FIA, FIB, and FII
Human pathogen probability	92.7%
Representative virulence factor encoding genes associated with ExPEC pathotypes	afaA/C/D, aslA, chuA, fimH, fyuA, iha, iucC, iutA, kpsE, kpsMII_K5, nlpI, ompT, sat, senB, sitA, traT, and yfcV

Fig 1.

Phylogenomic comparison of the multidrug-resistant, ESBL-producing E. coli strain 11.2.3 and representative antibiotic resistance-related strain features. (A) Midpoint-rooted phylogenetic tree inferred with FastME (2.1.6.1) from genome blast distance phylogeny (GBDP) distances calculated from type and reference strain genome sequences using the Type Strain Genome Server (TYGS) and default parameters. The five most closely related (ANIb) genomes of E. coli ST131-O16:H5 strains were additionally included. Branch lengths are scaled based on the GBDP distance formula d5; numbers shown are GBDP pseudo-bootstrap support values ≥ 60% (100 replications, average branch support = 60.7%). (B) E. coli strain 11.2.3 exhibiting an ESBL phenotype with enlarged zones of inhibition for three representative third-generation cephalosporins (ceftazidime, cefotaxime, and cefpodoxime) in the presence of the ESBL inhibitor clavulanate compared to matching cephalosporin-only disks when using the MAST D67C ESBL test. (C) β-Lactam ring hydrolysis of the chromogenic cephalosporin nitrocefin (yellow) to yield the red hydrolysis product by cells of strain 11.2.3 but not cells of a negative control (C = E. coli ATCC 25922). (D) Spectroscopic demonstration of β-lactam ring hydrolysis of the chromogenic cephalosporin nitrocefin by cells of E. coli strain 11.2.3. Negative control (broken line, E. coli ATCC 29522, λ_max~390 nm, yellow) and ESBL phenotype E. coli strain 11.2.3 (solid line, λ_max~486 nm, red). The resulting bathochromic shift was captured by recording spectra (cell OD₆₀₀ = 1 in 50 mM phosphate buffer, pH 7, 30 min incubation at 37°C, 50 µM nitrocefin) using cell-free supernatant.

DNA was extracted using the Quick-DNA miniprep kit (Zymo, USA) from washed cells harvested by centrifugation from an overnight culture (37°C, 100 rpm, Nutrient broth, Neogen). Before shotgun sequencing, DNA was quantified using a NanoDrop 2000 spectrophotometer, followed by preparing a library using the FS DNA Library Prep Kit with enzymatic fragmentation (NEB, USA) and sequencing (Illumina NextSeq) to establish 2 × 150 bp paired-end reads (Inqaba Biotech, South Africa). Raw reads were quality checked (FastQC V0.12.1), trimmed (TrimGalore V0.6.5, Cutadapt V4.2), assembled (Unicycler V0.4.8, 500 bp minimum contig size), polished (Pilon V1.23), and quality controlled (Quast V5.2.0) using the BV-BRC analysis pipeline (10–16). Annotation was done using PGAP (V6.9) (17). Antibiotic resistance and virulence genes, plasmid replicons, and prophages were detected using ResFinder 4.6, VirulenceFinder 2.0, PlasmidFinder 2.1, and Phigaro 2.0 (18–21). Default parameters were used for all software tools unless otherwise specified.

The genome of strain 11.2.3 (4,949,272 bp) was 100% complete according to the benchmarking tool BUSCO V.5 (22). The essential genomic features are summarized in Table 1. Multilocus sequence (PubMLST-tools) (23) and additional strain typing tools (ECTyper 2.0, ClermonTyper 22.03, FimTyper 1.0, and PathogenFinder 2.1) (24–27) confirmed strain 11.2.3 as E. coli ST131-B2-O16:H5-FimH41. Sequence similarity (ANIb) (28) and phylogenetic analyses using the TYGS-LPSN database with genome blast distance phylogeny and automatically selected reference genomes (29) demonstrated that strain 11.2.3 clusters with the five most similar (ANIb) genomes of E. coli ST131-O16:H5 isolates from France (Fig. 1). Furthermore, the strain exhibited an ExPEC-like virulence gene profile (Table 1) (19). Antibiotic resistance genes detected (e.g., aph(6)-Id, bla_CTX-M-15, and sul1) generally matched the phenotypic antibiotic resistance profile of strain 11.2.3, with resistance against β-lactams (e.g., aztreonam and cefotaxime), fluoroquinolones (e.g., ciprofloxacin), aminoglycosides (e.g., tobramycin), and sulfonamide (e.g., trimethoprim-sulfamethoxazole) (Table 1).

Our results are concerning as third-generation cephalosporin-resistant E. coli are regarded as critical priority pathogens by the World Health Organization (30). Such ESBL multidrug-resistant pathogenic bacteria in the Msunduzi River are a potential health risk when the water is used for recreational, domestic, or agricultural purposes.

DATA AVAILABILITY

This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBJXCV000000000. The version described in this paper is version JBJXCV00000000.1. The genome assembly was deposited under accession number GCF_046638585.1 (bioproject number PRJNA843922). The raw reads were deposited under accession number SRR31667184.