Escherichia ruysiae May Serve as a Reservoir of Antibiotic Resistance Genes across Multiple Settings and Regions

ABSTRACT Gut colonization with multidrug-resistant Enterobacterales (MDR-Ent) has reached worrisome levels worldwide. In this context, Escherichia ruysiae is a recently described species mostly found in animals. However, its spread and impact on humans is poorly understood. A stool sample from a healthy individual living in India was screened for the presence of MDR-Ent using culture-based methods. Colonies were routinely identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and phenotypically characterized by broth microdilution. Illumina and Nanopore whole-genome sequencing (WGS) platforms were implemented to generate a complete assembly. E. ruysiae genomes deposited in international databases were used for a core genome phylogenetic analysis. An extended-spectrum β-lactamase (ESBL)-producing E. coli strain (S1-IND-07-A) was isolated from the stool. WGS confirmed that S1-IND-07-A was indeed E. ruysiae, belonged to sequence type 5792 (ST5792), core genome (cg) ST89059, serotype O13/O129−:H56-like, clade IV phylogroup, and possessed five virulence factors. A copy of blaCTX-M-15 and five other antimicrobial resistance genes (ARGs) were detected in a conjugative IncB/O/K/Z plasmid. A database search identified 70 further E. ruysiae strains from 16 countries (44, 15, and 11 strains isolated from animals, the environment, and humans, respectively). The core genome phylogeny revealed five major STs: ST6467, ST8084, ST2371, ST9287, and ST5792. Three out of the seventy strains possessed important ARGs: OTP1704 (blaCTX-M-14; ST6467), SN1013-18 (blaCTX-M-15; ST5792), and CE1758 (blaCMY-2; ST7531). These strains were of human, environmental, and wild animal origin, respectively. E. ruysiae may acquire clinically important ARGs and transmit them to other species. Due to its zoonotic potential, further efforts are needed to improve routine detection and surveillance across One Health settings. IMPORTANCE Escherichia ruysiae is a recently described species of the cryptic clades III and IV of the genus Escherichia and is commonly found in animals and the environment. This work highlights the zoonotic potential of E. ruysiae, as it has been shown to colonize the human intestinal tract. Importantly, E. ruysiae may be associated with conjugative plasmids carrying clinically relevant antibiotic resistance genes. Therefore, it is important to closely monitor this species. Overall, this study highlights the need for improved identification of Escherichia species and continued surveillance of zoonotic pathogens in One Health settings.

these infections can be challenging because of the possible presence of antibiotic resistance genes (ARGs), such as those encoding extended-spectrum b-lactamases (ESBLs; e.g., bla CTX-Ms ), which compromise the treatment (5).
E. ruysiae has been recently reported from animal and environmental sources, but it is still very rarely isolated in humans (1,6,7). Moreover, routine identification and differentiation from its counterpart E. coli is very complex using standard laboratory methods (e.g., matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]), unless a PCR-based approach (e.g., targeting the chuA and aes genes) or whole-genome sequencing (WGS) are used (1,2,8,9). Overall, very little is known about the role of E. ruysiae in humans, in particular about its potential to spread ARGs. Of note, a plasmid-associated (IncI) bla CTX-M-14 -possessing Escherichia cryptic clade IV strain (OPT1704) was recently isolated from the stool of a traveler to Asia (10). This strain was later characterized and proposed as E. ruysiae sp. nov., which also includes cryptic clade III (1).
In this work, we characterized a bla CTX-M-15 -harboring E. ruysiae strain (S1-IND-07-A) isolated from a fecal sample of a healthy individual living in India. To investigate the spread of E. ruysiae in humans, we searched for other E. ruysiae genomes in international databases and compared the results using core genome phylogenetic analysis. Furthermore, we investigated the distribution of the associated ARGs, plasmids, virulence factors, and serotypes characteristic of strains isolated from various human and nonhuman samples.

RESULTS AND DISCUSSION
The first aim of this study was to characterize a rare ESBL-producing E. ruysiae strain isolated from a person living abroad. To contextualize our findings, the second aim was to search global databases for other reported E. ruysiae strains isolated from different sources to perform a core genome phylogeny analysis. Finally, we examined the associated ARGs, plasmid replicons, virulence, and serotype distribution of all E. ruysiae strains included in this study to evaluate the potential of E. ruysiae as a human pathogen.
Core genome phylogeny of E. ruysiae strains derived from human and nonhuman sources. To investigate the origin of S1-IND-07-A, we performed a database search for other E. ruysiae strains of the same sequence type and core genome sequence type (ST5792 and cgST89059). We also included strains isolated from different sources to identify potential ARG hot spots and evaluate the pathogenic potential of E. ruysiae in humans. Overall, we included a total of 70 E. ruysiae genomes with complete metadata (collection date, country of isolation, host, sample/BioSample accession number) from the NCBI (n = 13: clade III, n = 9; clade IV, n = 4) and Enterobase (n = 57: clade III, n = 27; clade IV, n = 30) databases.
Most of the other ARGs were detected only in S1-IND-07-A, SN1013-18, OPT1704, and 1-176-05_S3_C2, while the rest of the strains carried only mdf(A) and (sporadically) sitABCD. Furthermore, many replicon sequence types were detected in both clades III and IV, most frequently Col and IncFI type ( Fig. 1; Table S2). These data suggest that E. ruysiae can be associated not only with resistance plasmids but also with many other plasmid backbones that have the potential to acquire ARGs (19).
Notably, all strains carried fimH-type variants, while 4 strains were positive for both estB and astA (pig strain FSIS12215121) and pic and sat (human strains 1-176-05_S3_C2, B37_6, and S1-IND-07-A [our strain]). Both estB and astA genes have been reported in enterotoxigenic E. coli strains in both diseased pigs and pigs with diarrhea, while pic and sat have been found in enteroinvasive E. coli strains (20-23). Therefore, the presence of novel serotype variants and virulence factors and the association with a wide range of hosts in E. ruysiae suggests that this species may have pathogenic and zoonotic potential that requires close monitoring.
Conclusions. International travel and living in high regions of endemicity increase the risk of acquiring emerging MDR-Ent in the gut, which may include zoonotic diseases that can be transmitted between humans, animals, and the environment (28). Species with such zoonotic potential are of concern because they can silently colonize human and animal intestinal tracts.
In the present work, we showed that E. ruysiae, which is mainly found in animals and the environment, has the ability to acquire worrisome ARGs (e.g., bla ESBL ) and transfer them to other clinically important bacteria. Therefore, the application of WGS surveillance methods (e.g., species ID and SNV analysis) is essential for the discovery and characterization of novel Escherichia species with zoonotic potential and the ability to serve as reservoirs for resistance plasmids. In this context, it is crucial to develop and improve routine laboratory identification techniques such as MALDI-TOF MS to distinguish between Escherichia species.  Table S5 for the gene annotation coordinates shown.

MATERIALS AND METHODS
Stool screening. A healthy 62-year-old Swiss man living in India for 1.3 years was screened for intestinal carriage of multidrug-resistant Enterobacterales (MDR-Ent) on his return to Switzerland in 2022 as part of an ongoing prospective screening study (https://data.snf.ch/grants/grant/192514). The stool sample was collected by the volunteer in a sterile container and sent within 1 day to the Institute for Infectious Diseases in Bern for processing (see below). No hospitalization or diarrhea was reported, but previous antibiotic use (not specified) was noted in two independent episodes 2 and 4 months before arrival in Switzerland. The study participant provided written consent, and the study was approved by the ethical committee of the Canton Bern, Switzerland (2020-01683).
Conjugation experiments. The rifampicin-resistant E. coli recipient strain J53d-R1 was conjugated with the donor S1-IND-07-A as previously described (30). In short, bacterial conjugation was conducted in liquid LB broth for 16 h at 36 6 1°C. The resulting transconjugants (TCs) were selected on MAC agar plates supplemented with rifampicin (50 mg/mL) and ampicillin (100 mg/mL). Phenotypic and genotypic confirmation of TCs was performed by broth microdilution as described above and by PCR using previously published bla CTX-M group I primers (32). Experiments were performed in duplicate.
Data availability. The complete genome sequence of S1-IND-07-A has been deposited at GenBank (accession numbers CP112983 to CP112985) and under BioProject accession number PRJNA903117.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, XLSX file, 0.1 MB.