Characterization of a carbapenem-resistant Escherichia coli from dairy cattle harbouring bla_NDM-1 in an IncC plasmid

NDM-producing Enterobacteriaceae have increased worldwide in human infections, but are still rare in food-producing animals.¹ The only NDM type described in cattle so far was NDM-5,^2–4 always carried by IncX plasmids. To the best of our knowledge, this study represents the first description of a bla_NDM-1 gene in Escherichia coli isolated from cattle, carried in an IncC plasmid.

In July 2020, E. coli isolates were recovered on ChromID^® Carba Smart plates (bioMérieux) from rectal faeces collected from dairy calves in the Basque Country (Spain) within a longitudinal study conducted to monitor antimicrobial resistance (AMR) in commensal E. coli. MICs determined by broth microdilution (Thermo Scientific™ Sensititre™ AST plates EUVSEC1 and EUVSEC2) and interpreted using epidemiological cut-off values as developed by EUCAST (http://www.eucast.org) showed microbiological resistance to all β-lactams tested, including temocillin and carbapenems, and sulfamethoxazole and trimethoprim (Table S1, available as Supplementary data at JAC Online). Genomic DNA from E. coli strain EC1110 was extracted using a NZY Microbial gDNA Isolation Kit (NZYTech) and Illumina WGS was performed as previously described⁵ and further complemented by Oxford Nanopore–ONT sequencing (MinION Mk1C, SQK-LSK109 library and R9.4.1 flow-cell). Base-calling of ONT reads on Guppy (HAC mode) was followed by adapter removal with Porechop⁶ and discharge of shorter reads (<1000 bp) by Filtlong retaining only the best 1000 Mbp. A hybrid assembly (Illumina-ONT) was generated with Unicycler⁷ and analysed as described elsewhere.⁵ Details on bioinformatic tools are available in Table S2.

The Illumina-ONT assembly produced two contigs, i.e. the chromosome (4 816 563 bp) and an IncC plasmid (145 165 bp) that contained the bla_NDM-1 gene and nine other AMR genes (GenBank JADWPF000000000, BioProject PRJNA680938 and BioSample SAMN16926619) (Figure 1). E. coli EC1110 belonged to serogroup O74: H23 and was assigned to a novel MLST type (ST-11626; cgST-151275). It carried a fimH60 fimbrial adhesion allele and harboured virulence-associated genes related to adhesion (CFA/I and Type 1 fimbriae), iron uptake (ent and fep) and invasion of brain endothelial cells (ompA, ibeB and ibeC). The IncC plasmid, named pEC1110_NDM-1, was assigned to pST-3 and included genes for the initiation of replication (repA), conjugative transfer (tra) and plasmid partitioning (stb and par) (Figure 1a). AMR genes were located in accessory modules of AMR islands, ARI-A and ARI-B. The bla_NDM-1 gene was in ARI-A flanked upstream by ISAba125 and downstream by the bleomycin resistance gene ble_MBL, followed by a truncated Δbla_DHA-1 gene (Figure 1b). Other AMR genes in ARI-A were the sulphonamide-resistance sul1 gene (two copies), the trimethoprim-resistance dfrA12 gene, genes that confer resistance to amikacin [aph(3')-VI] and streptomycin (aadA2), the qacEΔ1 gene (quaternary ammonium compound resistance) and a mercury-resistance operon (merDACPTR). The sulphonamide-resistance gene sul2 was present in ARI-B, downstream of IS91 (Figure 1b).

Figure 1.

(a) Circular representation of the pEC1110_NDM-1 IncC plasmid from E. coli strain EC1110 isolated from dairy cattle. (b) Schematic representation of the genetic context of the bla_NDM-1 gene in the ARI-A region of the pEC1110_NDM-1 plasmid and the sul2 gene in the ARI-B region. The position of the bla_NDM-1 gene is highlighted by a red square. Coding sequences, represented by arrows indicating the translational direction, are named above and coloured according to the key. IS designations are followed by the family name in brackets. Annotations were graphically depicted using SnapGene (v.5.2.4) (http://www.snapgene.com/).

A BLAST search showed that pEC1110_NDM-1 was related to IncC bla_NDM-1-harbouring plasmids in Providencia stuartii (pMR0211; 94% coverage, 99.9% identity), Salmonella enterica subsp. enterica serovar Corvallis (pSE12-01738–2; 92% coverage, 99.9% identity) and E. coli (pM214_AC2; 88% coverage, 99.9% identity). Moreover, it was also similar to IncC plasmids in Enterobacteriaceae that do not carry bla_NDM genes, isolated from food-producing animals, like S. enterica subsp. enterica serovar Newport p34530-1 isolated from cattle in the USA (92% coverage, 99.9% identity) and E. coli pEC3-1/2a from a chicken in China (91% coverage, 99.9% identity), or from human cases, like pRMH760 from Klebsiella pneumoniae (89% coverage, 99.8% identity), the first IncC plasmid described in detail (Figure S1).

The genetic environment of bla_NDM-1 in pEC1110_NDM-1 had features present in other bla_NDM-1-harbouring strains. The ISAba125-bla_NDM-1-ble_MBL-trpF-Δbla_DHA-1-ampR region was conserved in the partial sequence of the plasmid of E. coli DVR22 (JF922606; 4036 bp), the first description of an NDM-1 carbapenemase-producing E. coli in Spain.⁸ Homology with the PGI1-PmPEL genomic island of Proteus mirabilis was nearly 100% in the segment that extended from the ISCR1 upstream of bla_NDM-1 to the ampR gene (Figure S2). Insertion of bla_NDM-1 from a circular molecule mediated by ISCR1 was proposed and ISCR1 was also associated with acquisition of the bla_DHA-ampR gene region as part of a class 1 integron.⁹ On the other hand, the gene synteny of ARI-A in pEC1110_NDM-1 was highly conserved compared with the same region in p34530-1 (Figure S2). Despite lacking the 9639 kb fragment from the ISCR1 element to the ampR gene where the bla_NDM-1 gene is located in pEC1110_NDM-1, both plasmids shared a sul1-type class 1 integron structure (intI1-dfrA12-gcuF-aadA2-qacEΔ1-sul1), suggesting a common origin. Whereas only sul2 was present in pEC1110_NDM-1 ARI-B, other IncC plasmids usually contain several additional AMR genes, such as floR, strA, strB and tet(A).¹⁰ Different-sized deletions reported in ARI-B are considered potentially useful evolutionary and epidemiological markers.¹⁰ pEC1110_NDM-1 ARI-B showed an IS26-mediated deletion of 12 451 bp upstream of parA and parB genes that removed part of the plasmid backbone. The presence of several different mobile genetic elements suggests that a series of recombination events was likely at the origin of the different resistance-gene arrays identified on pEC1110_NDM-1.

In conclusion, to the best of our knowledge, this is the first description of a bla_NDM-1-harbouring plasmid in an MDR E. coli isolated from cattle. This IncC plasmid also carried genes for aminoglycoside, sulphonamide and trimethoprim resistance. The occurrence of NDM-1 plasmid-mediated carbapenem resistance in E. coli in cattle is worrisome since it might pose a risk for resistance spread in food-producing animals. However, this was the only detection after monitoring the herd for over 2 years, suggesting a sporadic event.