Escherichia coli is a natural inhabitant of and the most common bacterial species colonizing the gastrointestinal tract of warm-blooded animals, including humans. It is classified as a Gram-negative commensal bacterium and facultative pathogen, since it is an important cause of morbidity and mortality in humans and animals worldwide, associated with both subclinical disorders and a wide range of clinical conditions, including enteric, urinary, and systemic infections (García and Fox, 2021). E. coli can also be considered a potential zoonotic pathogen, being ubiquitous in most food animals and their environment, including wastewater and vegetable products. The exceptionally versatile genome of the members of the E. coli genus reflects their multifaceted nature, supported by the presence of several different strains showing diverse combinations of resistance genes and virulence factors. Depending on the virulence factors encoded and expressed, E. coli strains are classified into specific pathotypes. There are two main classifications of pathogenic E. coli: diarrheagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Among DEC, there are at least nine well-recognized pathotypes: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), Shiga toxin (Stx)-producing E. coli (STEC), enteroinvasive E. coli (EIEC), diffusely adhering E. coli (DAEC), adherent-invasive E. coli (AIEC), and cell-detaching E. coli (CDEC) (Braz et al., 2020). All these pathotypes are common among both humans and animals.

ExPEC can cause a wide variety of infections, and the main pathotypes among this group are uropathogenic E. coli (UPEC), sepsis-causing E. coli (SEPEC), and neonatal meningitis-associated E. coli (NMEC). Following a One Health perspective, the avian pathogenic E. coli (APEC) has also been included in the ExPEC group causing diverse local and systemic infections in poultry, such as chickens, turkeys, ducks, and many other avian species. APEC are now recognized as foodborne zoonotic pathogens as well as a source or reservoir of extraintestinal infections in humans (Kathayat et al., 2021).

Antimicrobial resistance (AMR), a global issue contributing to untreatable infections, is driven by antibiotic exposure in healthcare, agriculture (animals, plants, or food-processing technology), and the environment (sea, soil, drinking water, and wastewater) (Smit et al., 2023). Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing E. coli are of great concern worldwide, considering also that both resistance genes and virulence traits can be carried on chromosome and on mobile genetic elements, as plasmids, the latter being transferable intra- and inter-species. In addition to third-generation cephalosporin and carbapenem resistance, fluoroquinolone and aminoglycoside resistance is also particularly worrisome in E. coli (Kocsis et al., 2022). The dissemination of these elements, and the convergence of wide resistomes and virulomes, is associated with and contributed to the successful expansion of international high-risk clones of critical priority, often shared between different hosts and environments (Fuga et al., 2022). Many of the high-risk E. coli pandemic clones are classified into eight phylogroups (A, B1, B2, C, D, E, F, and G) and in different sequence types (STs). This classification of E. coli, together with the analysis of virulence and resistance traits, has been very useful to investigating the distribution of E. coli clones among hospital, community, animal and environment settings. It has been suggested that group B2 strains may be specific to humans and that some group B1 strains harboring the hly gene may be specific to animals (Clermont et al., 2011). Phylogroup A, considered one of the most ancestral groups, is primarily associated with commensal, non-pathogenic strains and commonly found in the environment. Phylogroup C strains of E. coli are less common; they can be found in both humans and animals, even though evidence of their association with specific human diseases is scarce. The boundary between settings has long been crossed, and it is increasingly frequent to identify E. coli clones, previously typically of one, causing epidemics in others. Nowadays, some multidrug-resistant (MDR) high-risk clones, such as ST131, ST10, ST69, ST73, ST405, ST410, and ST457, have been established, but several less frequent clones overlapping human and non-human sources are emerging.

This study aims to characterize at the genomic level four carbapenem-resistant E. coli (CR-Ec) isolated from human samples, collected at the Fondazione IRCCS Policlinico San Matteo hospital in Pavia (Italy), to evaluate their resistance, virulence traits, and association with high-risk clones.

From November 2022 to September 2023, four CR-Ec strains were isolated from the rectal swabs of as many patients and sequenced. All four isolates, named 7521, 7926, 8501, and 9008, were resistant to aztreonam, amoxicillin/clavulanate, cephalosporins, ertapenem, and ceftolozane/tazobactam and susceptible to colistin and fosfomycin ( Supplementary Table 1 ). Resistance to a higher number of antibiotics was observed for the two CR-Ec strains 8501 and 9008, being resistant also to ceftazidime/avibactam, imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, gentamicin, and trimethoprim/sulfamethoxazole. The immunochromatographic assay detected the production of Klebsiella pneumoniae carbapenemase (KPC) for strains 7521 and 7926, VIM and NDM for 8501, and VIM for 9008.

Table 1 shows the results of the WGS analyses. In silico MLST identified ST401 for both CR-Ec 9008 and CR-Ec 7521, ST355 for CR-Ec 7926, and ST3564 for CR-Ec 8501. The four studied strains revealed a heterogeneous phenotype: CR-Ec 9008 and CR-Ec 7521 belonged to phylogroup A and serotypes O21:H33 and O15:H11, respectively; CR-Ec 7926 to phylogroup B2 and serotype O1:H5; and CR-Ec 8501 to phylogroup C and serotype O8:H19 ( Table 1 ).

Concerning carbapenem resistance genes, CR-Ec 7926 and CR-Ec 7521 carried blaKPC-3, CR-Ec 9008 carried blaVIM-1, and CR-Ec 8501 co-harbored blaNDM-7 and blaVIM-1. Based on the short and long sequencing, the four strains had a heterogeneous plasmidome. CR-Ec 7521 contained an IncFIB(pQiL)-IncFII(k) plasmid of 107,213 bp, an IncX5 plasmid of 39,317 bp, and a p0111 plasmid of 57,904 bp; CR-Ec 7926 showed an IncFII-IncFIB(pQiL)-IncFII(k) plasmid of 87,125 bp, an IncFIA-IncFIB(AP001918) of 114,034 bp, and a small Col156 plasmid of 6,646 bp. Differently, CR-Ec 8501 carried an IncFIB(k)-IncFII(k) plasmid of 175,363 bp, an IncN plasmid of 54,469 bp, and an IncX₃ plasmid of 45,122 bp; while CR-Ec 9008 included an IncFIA(HI1)-IncFIB(k) plasmid of 154,767 bp and an IncA plasmid of 152,625 bp.

Here we report the identification of three E. coli STs (ST401, ST355, and ST3564), usually found in animal reservoirs, harboring several carbapenemases (including the first Italian case of NDM-7), from patients admitted in critical wards, including the Intensive Care Unit.

E. coli is an important cause of morbidity and mortality in humans and animals worldwide. The fine ability of E. coli to adapt to different lifestyles and settings reflects its versatile nature and should be prioritized within the One Health framework involving humans, animals, and the environment. Although some E. coli STs have evolved to adapt to specific environments, others are found in both humans and animals, circulating through shared environments, food sources, or direct contact. In general, the STs identified in humans and animals differ for virulence and resistance determinants harbored, with the human-associated strains usually displaying a higher amount of resistance than virulent genes, and vice versa. Resistance to cephalosporins, fluoroquinolones, and aminoglycosides is reported as the most frequent phenotype in human E. coli isolates, while in animals, ESBL-producing E. coli isolates and resistance to penicillins, cephalosporins, and trimethoprim/sulfamethoxazole are more frequent (Giufrè et al., 2021). Resistance to carbapenems in E. coli remains uncommon in Europe in both humans and animals (EARS-Net, 2023; European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2024).

E. coli ST401 has been described to be associated with both poultry and aquatic environments (Vincent et al., 2010; Behruznia and Gordon, 2022; Constantinides et al., 2020). In particular, the poultry strains are often recognized as APEC and showed several virulence factors, primarily adhesins, invasins, and toxins (Kathayat et al., 2021), and fewer resistance determinants, with aminoglycoside, sulfonamide, and beta-lactam resistance genes as the mainly reported ones (Kathayat et al., 2021). The occurrence of clinically relevant carbapenemases, such as KPC and NDM types, in such strains possessing potential virulent features, is a worrying event. Here we report the first cases of blaKPC-3 and blaVIM-1 determinants harbored in ST401 E. coli strains from human sources. The different plasmidome content and the conjugation rate pointed out the ability of ST401 to acquire and fit several AMR traits. These data, together with a wide armamentarium of virulence genes, increase its infective potential in the human environment. These data suggest the ability of ST401 to fit in different environments.

E. coli ST355 is often identified among UPEC isolates, widely reported in the poultry environment and associated with fluoroquinolone resistance traits (Myrenås et al., 2018; Kaspersen et al., 2020). Based on the available metadata and on the literature, ST355 is rarely associated with beta-lactam resistance, while the E. coli 7926 isolated in this study present blaKPC-3 carbapenemase gene. These data highlighted the worrying entrance of successful carbapenemases, such as blaKPC-3, in E. coli ST355 with high virulence potential.

E. coli ST3564 belongs to the clonal complex ST23 and has not been characterized in the literature, yet. The only data reporting this ST are present in an annual report of the Danish surveillance program of AMR and consumption in humans and animals drafted in 2016 (Borck Høg et al., 2016). In that document was reported one isolate of ST3564 E. coli collected from a pig, harboring the blaCTX-M-1 gene. No carbapenemases have been detected, to date, in such an ST. Here, the 8501 strain, belonging to ST3564, presented, besides different β-lactamases, two metallo-β-lactamases, blaVIM-1 and blaNDM-7, here reported, to the best of our knowledge, for the first time in Italy. This allelic variant has already been reported in several regions, such as Japan (Mizuno et al., 2014), India (Rahman et al., 2014; Hammerum et al., 2015; Devanga Ragupathi et al., 2017; Paul et al., 2017; Ramakrishnan et al., 2021), China (Wang et al., 2016; Xu and He, 2019; Xia et al., 2022; Liu et al., 2024), Nepal (Shrestha et al., 2017), the Arabian Peninsula (Pál et al., 2017), Myanmar (Sugawara et al., 2019), Brazil (Rodrigues et al., 2021; de Oliveira Alves et al., 2022), Nigeria (Tickler et al., 2021), Fiji (Young-Sharma et al., 2024), and Europe in Enterobacterales, mostly K. pneumoniae and E. coli, and in different plasmids, mainly IncX3. In Europe, NDM-7 has been reported since 2013 and in Germany and France in two E. coli isolates belonging to ST599 and ST167, respectively (Göttig et al., 2013; Cuzon et al., 2013). In the same year, an interhospital outbreak of an ST437 NDM-7-producing K. pneumoniae clone occurred in Spain (Seara et al., 2015). In the following years, other sporadic cases of NDM-7 were described, mainly in Spain, in E. coli belonging to different STs (ST399, ST679, and ST167) and in plasmids of different types (IncX3 and IncX4) (Pérez-Moreno et al., 2016; Espinal et al., 2018; Pérez-Vázquez et al., 2019; Marí-Almirall et al., 2021). The fact that NDM-7-producing isolates are often found in strains from surveillance rectal swabs (van der Mee-Marquet et al., 2016; Solgi et al., 2017) highlights that more attention should be paid to the silent circulation of these clones, since they can asymptomatically colonize patients and be introduced into hospitals, thereby posing a major risk of infections in fragile patients, also considering that the blaNDM-7 gene is often driven by conjugative plasmids, such as the case of the IncX3 plasmid here reported, which can easily be transferred to other bacterial species. Moreover, E. coli 8501 was of serotype O8:H19, characteristic of porcine stx2-producing E. coli (Saupe et al., 2017), which is associated with mild disease and has occasionally caused hemolytic uremic syndrome in the Netherlands (Friesema et al., 2015). This report suggested a peculiar evolutionary turning point of ST3564, with a reduction in its virulence content and, on the other hand, an increase in its ability to acquire clinically relevant carbapenemases.

The above set of results confirms the thick bond between veterinary and human compartments in the diffusion of high-risk pathogens and emphasizes how the epidemiology of carbapenemases is constantly evolving, also highlighting the importance of genome-wide studies for better understanding of circulating clones and plasmids.