# Distinct molecular epidemiology of resistances to extended-spectrum cephalosporins and carbapenems in Enterobacter hormaechei in cats and dogs versus horses in France

Marisa Haenni, Pierre Châtre, Antoine Drapeau, Géraldine Cazeau, Jonathan Troncy, Pauline François, Jean-Yves Madec

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Marisa Haenni, Pierre Châtre, Antoine Drapeau, Géraldine Cazeau, Jonathan Troncy, et al. Distinct molecular epidemiology of resistances to extended-spectrum cephalosporins and carbapenems in Enterobacter hormaechei in cats and dogs versus horses in France. Journal of Antimicrobial Chemotherapy, 2025, 80 (2), pp.567-575. 10.1093/jac/dkae448. anses-04926358

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# Distinct molecular epidemiology of resistances to extended-spectrum cephalosporins and carbapenems in *Enterobacter hormaechei* in cats and dogs versus horses in France

Marisa Haenni ID ¹*, Pierre Châtre¹, Antoine Drapeau¹, Géraldine Cazeau², Jonathan Troncy¹, Pauline François¹ and Jean-Yves Madec¹

¹ANSES—Université de Lyon, Unité Antibiorésistance et Virulence Bactériennes, Lyon, France; ²ANSES—Université de Lyon, Unité Epidémiologie et Appui à la Surveillance, Lyon, France

*Corresponding author. E-mail: marisa.haenni@anses.fr

Received 14 June 2024; accepted 20 November 2024

**Background:** *Enterobacter hormaechei* is an important pathogen in humans and animals, which, in addition to its intrinsic AmpC, can acquire a wide variety of genes conferring resistances to extended-spectrum cephalosporins (ESCs) and carbapenems (CPs). In France, human clinical outbreaks of *E. hormaechei* resistant to ESC or carbapenem were reported.

**Objectives:** To study *E. hormaechei* isolates from cats and dogs (=59) as well as from horses (n = 55) presenting a non-susceptible phenotype to beta-lactams in order to determine which clones, resistance genes and plasmids are circulating in France.

**Material and methods:** *E. hormaechei* isolates (n = 114) were short-read sequenced and five isolates were long-read sequenced to better characterize the plasmids carrying ESC- and CP-resistance determinants. Phenotypes were characterized by antibiograms using the disc diffusion method.

**Results:** A clear divergence in the molecular epidemiology was observed depending on the host. In cats and dogs, most of the isolates presented an overexpressed *ampC* gene or the *bla*<sub>CTX-M-15</sub> gene carried by an IncH12 plasmid, and eight isolates (8/59, 13.6%) presented the *bla*<sub>OXA-48</sub> carbapenemase gene. Thirty-two isolates (32/59, 54.2%) belonged to the human high-risk clones ST78, ST114 and ST171. Contrarily, in horses, ESC resistance was mostly due to the *bla*<sub>SHV-12</sub> and *bla*<sub>CTX-M-15</sub> genes carried by an IncH12 plasmid, and high-risk clones were rarely identified (5/55, 9.0%).

**Discussion:** Potential selection by antibiotic use (which is on an increasing trend in France for cats, dogs and horses), the dissemination capacities of both conjugative IncH12 plasmids and high-risk clones, and possible transfers of resistant bacteria between humans and animals strongly indicate that *E. hormaechei* should be closely monitored.

## Introduction

*Enterobacter cloacae* complex (ECC) members are important opportunistic pathogens in humans and animals. Their classification has considerably evolved in the last decade, and they currently encompass seven genetically diverse species, i.e. *E. asburiae*, *E. carcinogenus*, *E. cloacae*, *E. hormaechei*, *E. kobei*, *E. nimipressuralis* and *E. mori*,¹ themselves divided into subspecies and 19 phylogenomic groups (named A-S).²,³ *E. hormaechei*, with

its main subspecies *hormaechei*, *steigerwaltii*, *hoffmannii* and *xiangfangensis*, is a frequent cause of nosocomial infections.⁴ As all ECC members, *E. hormaechei* was placed by the WHO on the list of pathogens of critical importance because of its capacity to acquire resistance to extended-spectrum cephalosporins (ESCs) and carbapenems (CPs).

In both humans and animals, *E. hormaechei* isolates producing ESBLs have disseminated worldwide mostly due to the spread of *bla*<sub>SHV-12</sub> and *bla*<sub>CTX-M</sub> genes.⁴⁻⁷ More importantly,
*E. hormaechei* emerged as one of the main vehicles of carbapenemases, which, in addition to the intrinsic AmpC gene and frequent additional acquired resistances, are causing difficult to treat infections. In humans, a large variety of genes (*bla*<sub>VIM</sub>, *bla*<sub>IMP</sub>, *bla*<sub>KPC</sub>, *bla*<sub>NDM</sub> or *bla*<sub>OXA-48-like</sub>) have been identified internationally, sporadically or causing clinical outbreaks.<sup>4,8,9</sup> In animals, despite the restriction or ban of use of CPs, CP resistance is increasing, mostly mediated by the *bla*<sub>OXA-48</sub> gene<sup>10-12</sup> but also by other determinants such as *bla*<sub>NDM-1</sub>,<sup>13</sup> *bla*<sub>KPC-4</sub>,<sup>14</sup> or *bla*<sub>OXA-181</sub>.<sup>15</sup> While CP-resistant isolates were mostly found in companion animals, they were also identified in pigs in China and in beef-derived products in Egypt.<sup>16,17</sup>

Both ESBL and CP genes in *E. hormaechei* are often carried by plasmids, among which IncH12 are often reported.<sup>6,16,18,19</sup> These large plasmids (average size of 319.5 kb in *Enterobacter* spp.) carry a multiplicity of genes conferring resistances to many antibiotic families, including to critically important antibiotics such as colistin, ESC or CP,<sup>19</sup> to heavy metals or to biocides. *E. hormaechei* are also spreading through epidemic high-risk clones among which ST66, ST78, ST108, ST114 and ST171.<sup>20,21</sup> These two drivers, i.e. plasmids and clones, ensure the successful dissemination of this pathogen in all sectors, including the environment.<sup>22,23</sup>

In France, ECC members are the third most frequent species producing carbapenemases in humans.<sup>2</sup> VIM- and OXA-48-producing ECC members were consecutively reported since 2012 and 2014,<sup>24,25</sup> while the dissemination of NDM-1-producing *E. hormaechei* isolates has been reported recently in the Lyon area.<sup>26</sup> In the veterinary sector, a recent report of the Resapath network monitoring antimicrobial resistance in animals showed that, in dogs and horses, while ESC resistance has decreased in *Escherichia coli* below a 10% threshold, higher proportions (&gt;25%) of resistances were observed for ECC members.<sup>27</sup> Consequently, our goals were (i) to collect ECC members presenting a non-susceptible phenotype to beta-lactams from cats, dogs and horses and (ii) to molecularly characterize all circulating clones, genes and plasmids in order to understand their transmission dynamics on the French territory.

# Materials and methods

## Sampling, bacterial isolation, and identification

Between 2019 and 2022, 119 *Enterobacter* spp. isolates from cats (n=30), dogs (n=34) and horses (n=55) presenting a non-susceptible phenotype to beta-lactams and originating from all over the French territory were collected from the Resapath (https://resapath.anses.fr/; Table S1, available as *Supplementary data* at JAC Online). All samples were collected from sick animals. All isolates were confirmed as *Enterobacter* spp. by API galleries.

## Antimicrobial susceptibility testing

Susceptibility testing was performed using disc diffusion on Mueller-Hinton agar (BioRad, Marne-la-Coquette, France), according to the guidelines and clinical breakpoints (human and veterinary) of the Antibiogram Committee of the French Society for Microbiology (https://www.sfm-microbiologie.org/2023/06/15/casfm-veterinaire-2023/ for veterinary-related antibiotics and https://www.sfm-microbiologie.org/wp-content/uploads/2023/06/CASFM2023_V1.0.pdf for human-related antibiotics). Antibiotics (Mast Diagnostics) of human and/or veterinary interest were tested (Table S1). *E. coli* ATCC 25922 was included for quality

control both for disc diffusion and broth micro-dilution. MICs were determined (i) for colistin by broth microdilution, according to the EUCAST, and (ii) for ertapenem using Etests® (BioMerieux). All isolates presenting resistance to three or more antibiotic family were considered MDR.<sup>28</sup>

## WGS using short-read Illumina

DNA was extracted using the NucleoSpin Microbial DNA extraction kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer's instructions. Library preparation (Nextera XT technology) and sequencing (NovaSeq-6000 instrument) were outsourced (Eurofins, Germany). After sequencing, reads were quality trimmed and *de novo* assembled using Shovill v1.0.4, and the quality of assemblies was assessed using QUAST v5.0.2 (Table S3). Subspecies identification was performed on the JSpecies webserver (https://jspecies.ribohost.com/jspeciesws/) and considering the highest average nucleotide identity value attributed to each isolate (&gt;98%). STs were determined using the CGE online tool MLST v2.0.4 (http://www.genomicepidemiology.org/), while resistance genes and replicon content were inferred using ABRicate v1.0.1 (https://github.com/tseemann/abricate). The presence of virulence determinants was inferred using the CGE tool VirFinder 2.0.

## Phylogenetic analysis

The cgMLST was extracted from the WGS data. For all French data, the pan-genome was determined, and core gene alignments were generated with Roary v. 3.13.0 using a Protein BLAST identity of 80% and a core definition of 90%. All assemblies were annotated *de novo* with Prokka v1.14.6 using default settings. The Prokka annotations were provided to Roary as input. Recombination was removed with gubbins v2.4.1, and pairwise SNP distances (Table S2) were calculated using snp-dists v0.7.0 (https://github.com/tseemann/snp-dists). The resulting tree was visualized using iTol v.6 (http://itol.embl.de/itol.cgi).

ST78, ST114 and ST418 genomes were further compared with genomes of the same STs collected either from cats and dogs in Switzerland or from humans. Genomes were retrieved from the NCBI dataset v15.13.1, an annotation was performed using Bakta v1.9.3, and the pangenome was constructed using the PPanGGOLIN pipeline v2.1.0. SNP distances (Table S2) were calculated using snp-dists v0.8.2.

## Characterization of the genetic determinants carrying resistance genes

The plasmidic content was determined from the WGS data using PlasmidFinder 2.0.1 (http://www.genomicepidemiology.org/). Plasmids carrying the *bla*<sub>ESBL/OXA-48</sub> gene were assigned *in silico* when the *bla*<sub>ESBL/OXA-48</sub> gene was located on the same contig as the plasmidic marker. *In silico* data showed co-occurrence on the same contig of replicons and resistance gene markers for only a few isolates. For the majority of the isolates, the plasmid carrying the *bla*<sub>ESBL/OXA-48</sub> gene was detected using S1-PFGE gels followed by Southern blots, using probes corresponding to the *bla*<sub>ESBL/OXA-48</sub> gene and the replicon identified *in silico* in the same isolate, as previously described.<sup>29</sup> When the probes detecting the resistance gene and the one detecting the replicon were co-localized, the resistance gene was considered as carried by the plasmid type identified by the replicon. When plasmidic location could not be evidenced, the chromosomal location was looked for by PFGE on *I-Ceu1*-digested DNA, followed by Southern blot hybridization using a 16S rDNA probe and adequate probes for the detection of resistance genes.

## Long-read sequencing

To get better insight into a subset of plasmids, MinION long-read sequencing libraries were prepared according to the Oxford Nanopore Technologies (ONT, Oxford, UK) using the ligation sequencing kit
(SQK-LSK114). Sequencing was performed on an Mk1B MinION sequencer using an R10.4.1 version flow cell (FLO-MIN114), and the baseCALLing was performed with Dorado v0.4.2. Hybrid assembly of both Illumina short reads and Nanopore long reads was performed using Unicycler v0.4.8. Annotation was performed using Bakta 1.9.1 and the genetic environments of *bla*<sub>ESBL</sub> and carbapenemase genes were explored using BLASTn. Circular plasmid maps were drawn using BLAST Ring Image Generator version 0.95.1. Data were manually curated using the Artemis software, IS finder (https://www-is.biotoul.fr), and Swiss-Prot database (http://www.uniprot.org). Sequence comparison was done using NCBI Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

## Statistics

To explore differences in the susceptibility patterns, a chi-squared test (or Fisher exact test) was performed per antibiotic for all three animal species. When this test was significant (P < 0.05), a Marascuilo procedure was used to compare all pairs and assess which proportion was statistically different from the other ones.³⁰ Since we never observed differences between cats and dogs, the results from these two hosts were aggregated, and a chi-squared test (or Fisher exact test) was performed on the ‘horses’ versus ‘cats + dogs’ groups. Proportions were considered as statistically different when P < 0.05.

## Accession number

The project was deposited in the NCBI database under the BioProject accession number PRJNA1123620.

## Results

### Characterization of the collection of beta-lactam non-susceptible isolates

Isolates (n=119) presenting a non-susceptible phenotype to beta-lactams were collected and identified as *E. hormaechei* (n=114), *E. cloacae* complex (n=2), *E. bugandensis* (n=1), *E. kobei* (n=1) and *E. ludwigii* (n=1). Given the over-representation of *E. hormaechei* isolates, only isolates belonging to this species were further studied, thus leading to a collection of 114 *E. hormaechei* from cats (n=28), dogs (n=31) and horses (n=55).

In addition to intrinsic resistances to amoxicillin, amoxicillin + clavulanic acid, cefalotin and cefoxitin, all 114 collected *E. hormaechei* isolates were resistant to cefuroxime, and most of them were also resistant to cefotaxime (92.7%–100% depending on the host), ceftazidime (96.4%–100%) and aztreonam (92.9%–98.2%; Table 1). Proportions of resistance to ceftiofur and cefquinome were statistically higher in cats and dogs compared with horses. Three isolates from cats and five from dogs were resistant to ertapenem (MICs 1–8 mg/L).

Among all animals included, 72 presented a *bla*<sub>ESBL</sub> gene (72/114, 63.2%), seven the *bla*<sub>OXA-48</sub> gene (6.1%) and eight an acquired *bla*<sub>AmpC</sub> gene (7.0%; Table S1, Figure 1). The most frequently identified genes were *bla*<sub>SHV-12</sub> (n=39) and *bla*<sub>CTX-M-15</sub> (n=31). These genes were sometimes found in combination, such as the *bla*<sub>OXA-48</sub>/*bla*<sub>CTX-M-15</sub> genes found in 3 isolates or the *bla*<sub>DHA-1</sub>/*bla*<sub>SHV-12</sub> gene found in 3 other isolates, so that 92 genes conferring resistance to ESC and CP were identified. Apart from these acquired genes, 34 animals presented an over-expression of the intrinsic *ampC* gene with no further acquired *bla*<sub>ESBL/AmpC</sub> gene, which conferred resistance to ceftazidime.

The over-expression of the *ampC* gene was observed in 14 cats (14/28, 50%), 17 dogs (17/31, 54.8%) and 3 horses (3/55, 5.5%), while acquired *bla*<sub>ESBL/AmpC</sub> genes were identified in 14 cats (14/28, 50%), 11 dogs (11/31, 35.5%) and 52 horses (52/55, 94.5%).

### Resistances to non-beta-lactam antibiotics

Among the 114 isolates, 99 (86.8%) were MDR. The 15 non-MDR isolates originated from 6 cats, 6 dogs and 3 horses, of which 2 carried a *bla*<sub>ESBL</sub> gene and 13 overexpressed the *ampC* gene (Table S1). Otherwise, proportions of resistances were high in the three hosts for nalidixic acid (70.9–82.1%), tetracyclines (67.9–78.2%) and enrofloxacin (65.5–78.6%; Table 1). Proportions of resistances were statistically higher in horses versus cats and dogs for aminoglycosides, chloramphenicol, sulphonamides and trimethoprim, ranging from 70.9% to netilmicin up to 94.5% for sulphonamides. On the contrary, the proportion of resistances to florfenicol was higher in cats and dogs versus in horses. Resistance to amikacin was only found in horses (n=4), while no resistance was found to apramycin and colistin.

Genes responsible for the phenotypic resistances differed between horses on the one hand, and cats and dogs on the other hand. For example, resistances to tetracyclines, sulphonamides, trimethoprim and aminoglycosides were preferentially conferred by *tet(A)* (n=27/59 versus 13/55), *sul1* (n=17/59 versus 4/55), *dfrA14* (n=31/59 versus 12/55) and *aadA1* (n=27/59 versus 13/55) in cats and dogs, while they were more frequently conferred by *tet(D)* (n=10/59 versus 29/55), *sul1/sul2* (n=13/59 versus 37/55), *dfrA19* (n=2/59 versus 35/55) and *aadA2b* (n=3/59 versus 34/55) in horses. Amikacin resistance was conferred by the *rmtB* gene in three isolates co-harbouring the *bla*<sub>CTX-M-65</sub> gene and by *armA* in one isolate also presenting the *bla*<sub>SHV-12</sub> gene.

### Genetic determinants carrying beta-lactam-resistance genes

Most of the *bla*<sub>CTX-M-15</sub> (26/31, 83.9%) and *bla*<sub>SHV-12</sub> (36/39, 92.3%) genes were carried on large (>250 kb) IncHI2 plasmids, which all belonged to the DLST ST1 (Table 2, Table S1). All three long-read sequenced IncHI2 plasmids co-harbouring the *mer* and *ter* operons conferring resistance to mercury and tellurite (Figure S1). The five remaining *bla*<sub>CTX-M-15</sub> genes were located on the chromosome, while the three last *bla*<sub>SHV-12</sub> genes were identified on small (around 50 kb) IncX3 plasmids. The *bla*<sub>SHV-2</sub> gene was carried by an IncF/R plasmid. The *bla*<sub>CTX-M-65</sub> gene were all three located on an IncF/F33:A:-B- that co-harbouring the *rmtB* gene, together with the *fosA3* gene in one isolate. Two plasmids (p61840 and p61864) that were long-read sequenced shared 100% identity and 99% coverage, but also 99.9% identity/94% coverage with a CTX-M-55-carrying plasmid from *E. coli* collected from human in China (Genebank: CP025949.1) or 99.9% identity/91% coverage with a plasmid from Klebsiella pneumoniae (CP113188.1) originating from poultry in China. In four isolates, the *bla*<sub>DHA-1</sub> gene was identified on an IncHI2 plasmid, either alone or co-harbouring with the *bla*<sub>SHV-12</sub> gene and even with the *armA* gene in one isolate (57283). The three remaining *bla*<sub>DHA-1</sub> genes were located on IncF/F:-A:-B47 plasmids, and the *bla*<sub>CMY-2</sub> gene was identified on an IncC
<table><caption>**Table 1.** Antimicrobial susceptibility patterns of all *E. hormaechei* by animal species (N=114)</caption><thead><tr><th rowspan="2"></th><th colspan="2">Cats (n=28)</th><th colspan="2">Dogs (n=31)</th><th colspan="2">Horses (n=55)</th></tr><tr><th>n</th><th>%</th><th>n</th><th>%</th><th>n</th><th>%</th></tr></thead><tbody><tr><td>Cefuroxime</td><td>28</td><td>100.0</td><td>31</td><td>100.0</td><td>55</td><td>100.0</td></tr><tr><td>Cefotaxime</td><td>28</td><td>100.0</td><td>31</td><td>100.0</td><td>51</td><td>92.7</td></tr><tr><td>Ceftiofur</td><td>26</td><td>92.9</td><td>28</td><td>90.3</td><td>28</td><td>50.9</td></tr><tr><td>Ceftazidime</td><td>27</td><td>96.4</td><td>31</td><td>100.0</td><td>54</td><td>98.2</td></tr><tr><td>Cefquinome</td><td>18</td><td>64.3</td><td>16</td><td>51.6</td><td>16</td><td>29.1</td></tr><tr><td>Cefepime</td><td>12</td><td>42.9</td><td>10</td><td>32.3</td><td>15</td><td>27.3</td></tr><tr><td>Aztreonam</td><td>26</td><td>92.9</td><td>28</td><td>90.3</td><td>54</td><td>98.2</td></tr><tr><td>Ertapenem<sup>a</sup></td><td>2</td><td>7.1</td><td>5</td><td>16.1</td><td>0</td><td>0.0</td></tr><tr><td>Streptomycin</td><td>18</td><td>64.3</td><td>20</td><td>64.5</td><td>47</td><td>85.5</td></tr><tr><td>Kanamycin</td><td>14</td><td>50.0</td><td>18</td><td>58.1</td><td>46</td><td>83.6</td></tr><tr><td>Amikacin</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>4</td><td>7.3</td></tr><tr><td>Apramycin</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td>Gentamicin</td><td>11</td><td>39.3</td><td>12</td><td>38.7</td><td>46</td><td>83.6</td></tr><tr><td>Tobramycin</td><td>15</td><td>53.6</td><td>15</td><td>48.4</td><td>50</td><td>90.9</td></tr><tr><td>Netilmycin</td><td>11</td><td>39.3</td><td>11</td><td>35.5</td><td>39</td><td>70.9</td></tr><tr><td>Chloramphenicol</td><td>16</td><td>57.1</td><td>18</td><td>58.1</td><td>45</td><td>81.8</td></tr><tr><td>Florfenicol</td><td>11</td><td>39.3</td><td>13</td><td>41.9</td><td>2</td><td>3.6</td></tr><tr><td>Tetracycline</td><td>19</td><td>67.9</td><td>22</td><td>71.0</td><td>43</td><td>78.2</td></tr><tr><td>Colistin<sup>b</sup></td><td>0</td><td>0.0</td><td>0</td><td>0.0</td><td>0</td><td>0.0</td></tr><tr><td>Sulphonamides</td><td>20</td><td>71.4</td><td>22</td><td>71.0</td><td>52</td><td>94.5</td></tr><tr><td>Trimethoprim</td><td>21</td><td>75.0</td><td>24</td><td>77.4</td><td>51</td><td>92.7</td></tr><tr><td>Nalidixic acid</td><td>23</td><td>82.1</td><td>24</td><td>77.4</td><td>39</td><td>70.9</td></tr><tr><td>Enrofloxacin</td><td>22</td><td>78.6</td><td>24</td><td>77.4</td><td>36</td><td>65.5</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Proportion of genes responsible for resistance to ESCs or CPs in *E. hormaechei* from cats, dogs and horses. h-AmpC, overexpressed *ampC* gene. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.</figcaption></figure>

<footnote>*Indicates a statistically significant difference (P < 0.05, according to the chi-squared test or Fisher exact) between horses on the one hand and cats/dogs on the other hand.</footnote>

<footnote>aOnly resistance phentotypes confirmed by Etests were reported here (also see Table S1).</footnote>

<footnote>bColistin resistance was assessed by broth microdilution.</footnote>
<table><caption>**Table 2.** Genetic determinants carrying the ESBL, AmpC and carbapenemases genes in *E. hormaechei*</caption><thead><tr><th></th><th>IncHI2</th><th>IncFII</th><th>IncFIB</th><th>IncF/R</th><th>IncX3</th><th>IncC</th><th>IncL</th><th>CHR</th><th>ND</th><th>Total</th></tr></thead><tbody><tr><td>bla<sub>CTX-M-15</sub></td><td>26</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>5</td><td>—</td><td>31</td></tr><tr><td>bla<sub>CTX-M-9</sub></td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>1</td></tr><tr><td>bla<sub>CTX-M-65</sub></td><td>—</td><td>3</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>3</td></tr><tr><td>bla<sub>CTX-M-32</sub></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>1</td><td>1</td></tr><tr><td>bla<sub>SHV-12</sub></td><td>36</td><td>—</td><td>—</td><td>—</td><td>3</td><td>—</td><td>—</td><td>—</td><td>—</td><td>39</td></tr><tr><td>bla<sub>SHV-2</sub></td><td>—</td><td>—</td><td>—</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>1</td></tr><tr><td>bla<sub>OXA-48</sub></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>8</td><td>—</td><td>—</td><td>8</td></tr><tr><td>bla<sub>DHA-1</sub></td><td>4</td><td>—</td><td>3</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>7</td></tr><tr><td>bla<sub>CMY-2</sub></td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>1</td><td>—</td><td>—</td><td>—</td><td>1</td></tr><tr><td>Total</td><td>67</td><td>3</td><td>3</td><td>1</td><td>3</td><td>1</td><td>8</td><td>5</td><td>1</td><td>92</td></tr></tbody></table>

CHR, chromosomal carriage; ND, not determined.

plasmid. Finally, the *bla*<sub>OXA-48</sub> gene was always identified on small IncL plasmids (ca. 63 kb). The plasmid identified in isolate 53887 (ST114, Figure S1) carried no additional resistance genes and presented 100% identity with 99% coverage with the 21KM1498 plasmid (whose isolate belonged to ST78) identified in a dog in Switzerland,¹² which was in turn highly similar to a plasmid from the same Swiss study identified in an ST114 human isolate.

## Genetic diversity of the E. hormaechei population

Four subspecies were identified, namely *E. hormaechei* subsp. *hoffmannii* (n=43), *steigerwaltii* (n=39), *xiangfangensis* (n=31) and *oharae* (n=1, from a dog). *E. hormaechei* subsp. *xiangfangensis* was more frequent in cats and dogs than in horses (53.6% and 48.4% versus 1.8%), while the opposite trend was observed for subsp. *steigerwaltii* (17.9% and 16.1% versus 52.7%; Table S1). All isolates carried the virulence gene *nlpL*, while the *iroN* gene was found in all *E. hormaechei* subsp. *steigerwaltii* and in none of the other subspecies. The *terC* gene was identified in 78 isolates, while only sporadic isolates carried the *clpK1* (n=8), *traT* (n=4), *traJ* (n=3), *irp2/fyuA* (n=1) and *mcbA* (n=1) genes.

The population structure of *E. hormaechei* isolates was globally diverse, with 44 different STs identified among the 114 isolates. Apart from ST114 found in 18 isolates (8 cats, 9 dogs and 1 horse), ST78 found in 16 isolates (5 cats, 7 dogs and 4 horses) and ST113 found in 6 isolates (all from horses), all other STs were found in 1-3 isolates (Table S1). Twenty-eight STs were unique to horses, 14 unique to cats and 13 unique to dogs, while only ST114 and ST78 (though over-represented in cats and dogs) were identified in all 3 hosts.

The phylogenetic tree presented four clades (named C1-C4) corresponding to the four subspecies identified and differed mostly according to the host (Figure 2). Clade C1 (subspecies *xiangfangensis*) comprised 93.3% (n=31/33) of isolates originating from cats and dogs, clade C2 (subspecies *steigerwaltii*) encompassed 72.5% (29/40) isolates from horses and clade C3 (subspecies *hoffmannii*) was more heterogeneous with 58.5% of isolates originating from horses. Clade C4 consisted in a unique isolate collected from a dog (subspecies *oharae*). Only two clusters of clonal

isolates were observed, i.e. isolates 57305 and 53875 that belonged to ST62, and isolates 53883, 57310 and 61291 that belonged to ST113 (Table S3). These isolates were all collected from horses, but they were sampled in different regions of France and animals had no known epidemiological links. All other isolates sharing the same ST were similar but not clonal.

A phylogenetic comparison was performed with Swiss isolates belonging to ST78 (n=1), ST114 (n=6) and ST418 (n=2) collected from cats and dogs in the same period. Isolates from ST78 and ST114 were genetically distant from the French isolates (>80 SNPs differences). On the contrary, the two ST418 isolates from Switzerland were highly similar (<10 SNPs differences) to the French isolates (Table S2). Similar comparisons with human genomes retrieved from the NCBI database (ST78, n=347; ST114, n=143; ST418, n=52) showed that none of the human ST418 genome shared more similarities with the French animal genomes than the Swiss ones originating from cats and dogs. For ST78, multiple human genomes shared more similarities with the French animal genomes (<100 SNPs) than the Swiss animal ones. A similar situation was observed for ST114, with French animal genomes closer to those of several humans than to those of Swiss animals. Of note, the human genome (GCF_003289465) sharing the highest similarity with dog genomes (<50 SNPs) came from a hospital in Paris. In all other cases, no epidemiological link between animal and humans genomes could be established.

## Discussion

Our results indisputably identified *E. hormaechei* as the main ECC pathogen of French companion animals. Four different subspecies were identified, which partially segregated according to the host, with *E. hormaechei* subsp. *xiangfangensis* preferentially identified in cats and dogs and *E. hormaechei* subsp. *steigerwaltii* and *hoffmannii* preferentially identified in horses. All *E. hormaechei* subsp. *steigerwaltii* carried the *iroN* virulence gene, as already reported.³¹ Otherwise, only a few virulence determinants were sporadically observed, such as the *mrkA* gene, the major component of Type 3 fimbriae usually found in *K. pneumoniae*. The five non-*hormaechei* isolates originated from cats and dogs. The two *E. cloacae* complex isolates belonged to ST873,
<figure><img src="image_4.png" /><figcaption>**Figure 2.** SNP-based phylogeny of the 114 *E. hormaechei* isolates from cats, dogs and horses. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.</figcaption></figure>

an ST described in France as VIM-4-carrier and founder of the phylogenomic group S.² Here, the two isolates identified from a cat and a dog presented an overexpression of the *ampC* gene with no further acquired genes.

Resistance to beta-lactams also diverged depending on the host: the ESBL phenotype conferred by the *bla*<sub>SHV-12</sub> gene was mostly observed in horses (38/55, 69.1%; present only in one dog isolate), while the overexpression of the intrinsic *ampC* gene was mostly identified in cats and dogs (31/59, 52.5%; present in only three horse isolates). The *bla*<sub>CTX-M-15</sub> gene was less prevalent than the two other mechanisms but was more evenly distributed between horses (13/55, 23.6%), cats (11/28, 39.2%) and dogs (7/31, 22.6%). The *ampC* gene is highly inducible by antibiotics such as imipenem, cefoxitin or clavulanate,³² and its overexpression is selected by the use of last-generation cephalosporins.³³ As clavulanate is increasingly prescribed to treat cats and dogs in France since 2016³⁴ and not used in horses, this might be one explanation for the higher proportion of overexpression of the *ampC* gene in cats and dogs in France. This mechanism has often been disregarded, so that its importance is difficult to estimate, especially in animals. However, a recent

multicentre study in European hospitals showed that *ampC* over-expression was the most prevalent mechanism among bacteria harbouring a chromosomally encoded inducible AmpC beta-lactamase,³⁵ potentially because of the absence of associated biological cost.³⁶

Most of the ESBL-producing genes described in this study (63/ 76, 82.9%), among which 36 *bla*<sub>SHV-12</sub> and 26 *bla*<sub>CTX-M-15</sub>, were carried by IncH12 plasmids. These large conjugative plasmids carry multiple genes conferring resistances to antibiotics but also heavy metals or biocides and ensure intra- and inter-species transmission.¹⁹,³⁷ Here, the high prevalence of these plasmids, together with the fact that 12/15 non-MDR isolates were AmpC overproducers from cats and dogs, explained why proportions of resistance to non-beta-lactam antibiotics were higher in horses than in cats and dogs. Consequently, there is a major risk of co-selection of these isolates by the use of non-beta-lactam antibiotics. In France, antibiotic use in horses is on an increasing trend, mainly due to the use of penicillins, sulphonamides and trimethoprim,³⁴ and resistances to these last two antibiotic classes reached >90% in our study. The horse sector also has the particularity to use amikacin in specific cases such
as septic arthritis. Consequently, four amikacin-resistant isolates were identified, which were carrying the *rmtB* (*n* = 3) and *armA* (*n* = 1) methylase genes. The three *rmtB* genes were carried by IncF/F33:A:-B- plasmids highly similar to the IncF33 that emerged in China as *rmtB/CTX-M-55/65* carriers and further disseminated worldwide.³⁸ Conversely, the *armA* gene was carried together with the *bla*<sub>SHV-12</sub> gene on an IncHI2 plasmid, a plasmid that has only sporadically been identified as a methylase carrier.³⁹

IncHI2 are primary vehicles of ESBL genes in *Enterobacter* spp in both humans and animals ⁴,⁶,⁴⁰ but, even more importantly, they are also disseminating carbapenemase genes. In humans, they were identified internationally, causing outbreaks due to the *bla*<sub>IMP-1</sub>,⁴¹ *bla*<sub>IMP-4</sub>,⁸,¹⁸ or *bla*<sub>VIM-4</sub> genes.²⁶,⁴²⁻⁴⁴ In animals also, carbapenemase-producing *Enterobacter* spp are emerging. In the USA, where off-label use of CPs is tolerated in companion animals, *E. hormaechei* subspecies *xianfangensis* carrying the *bla*<sub>KPC-4</sub> gene on an IncHI2 plasmid were reported in 2016 from two dogs attending the same clinic.¹⁴ A *bla*<sub>VIM-1</sub> gene also carried by an IncHI2 plasmid was identified in beef-derived product in Egypt.¹⁶ These reports are still exceptions since most carbapenemase genes identified in animals are *bla*<sub>OXA-48</sub> carried by an IncL or an untypable plasmid¹⁰⁻¹² and more sporadically *bla*<sub>OXA-181</sub> and *bla*<sub>NDM-1</sub> genes carried by IncX3 plasmids.¹⁵,¹⁷ In our study, three IncX3 plasmids were also identified, but carrying the *bla*<sub>CTX-M-15</sub> gene. We also reported for the first time in *E. hormaechei* from French companion animals the emergence of a *bla*<sub>OXA-48</sub> gene carried by an IncL plasmid, which was highly similar to plasmids recently reported in Swiss companion animals as well as humans.¹² In the same Swiss study, the *bla*<sub>DHA-1</sub> gene was identified on an IncFIB plasmid, which shared the same formula (F:-A:-B47) as three *bla*<sub>DHA-1</sub>-carrying plasmids here. Further studies are needed to understand the transmission routes of these plasmids since French and Swiss animals shared no epidemiological links, except that they were all sampled in 2021–22. The occurrence of *bla*<sub>OXA-48</sub>-carrying Enterobacterales is globally increasing in France, even though the use of CPs is prohibited by law in Europe (https://eur-lex.europa.eu/legal-content/FR/TXT/?uri=CELEX:32022R1255). At some point, OXA-48-positive Enterobacterales most probably reached animals through contacts with humans. However, since OXA-48 is highly transmissible through the dissemination of the IncL plasmid, these pathogens were most probably amplified in the animal host. They can then be disseminated through contamination in veterinary clinics where they are carried by attending dogs or where they can contaminate the environment of the clinic²³ or through direct contacts between dogs, amplified due to their licking habits or their tendency to coprophagy.⁴⁵

Apart from plasmid transfers, clonal spread is the second driver of resistance transmission in *E. hormaechei*. High-risk clones such as ST66, ST78, ST108, ST114 and ST171 were reported internationally in humans and are responsible for the dissemination of clinically important resistances.²⁰,⁴⁶ Interestingly, the same STs are often reported in animals, such as CTX-M-15-producing ST114 in companion animals in France ⁶ and ST78 in a calf omphalitis in Brazil,⁵ SHV-12-producing ST114 in Switzerland and¹² KPC-4- or OXA-181-producing ST171 in dogs in the USA and in cats and dogs in Thailand.¹⁴,¹⁵ In this study, the 114 *E. hormaechei* isolates presented 44 different STs, among which the most widespread were ST114, found in 18 isolates, and

ST78, identified in 16 isolates. Both STs were clearly associated with cats and dogs since only one ST114 and four ST78 were identified in horses. Genomes of these two STs but originating from humans shared some similarities (<100 SNPs) with animals genomes, suggesting that they can disseminate without host barriers. ST66 (*n* = 2; one cat and one horse) and ST171 (*n* = 3; three dogs) were also identified. Of note, all eight *bla*<sub>OXA-48</sub> genes identified in this study were found in high-risk clones. Also, three ST418 isolates reported in cats and dogs in this study were genetically highly similar (<10 SNPs) with two Swiss isolates found in a cat and a dog,¹² while human genomes were genetically more distant. As for plasmids, no epidemiological link could be found, so that it will be interesting to assess whether this ST is particularly adapted to the canine/feline hosts. On the contrary, STs found in horses were more diverse, with 55 different STs reported, but rarely belonged to high-risk clones. This strongly suggests that transfers between cats/dogs and humans can occur through direct contacts, whatever the direction of transfer, at a much higher frequency than between horses and humans.

## Conclusion

Our results proved that *E. hormaechei* is an important pathogen in companion animals and that both the clones and mechanisms of resistance at play clearly diverged between cats and dogs on the one hand, and horses on the other one. We mostly observed overexpression of the *ampC* gene and the emergence of the carbapenemase *bla*<sub>OXA-48</sub> in high-risk clones in cats and dogs, and the dissemination of the IncHI2/*bla*<sub>SHV-12</sub> plasmid in a wide variety of STs in horses. Taken together, these two drivers are increasing the possibilities of resistance spread in the veterinary sector and beyond, indicating that the importance of *E. hormaechei* as an animal pathogen should not be underestimated.

## Acknowledgements

We would like to thank all veterinary laboratories from the Resapath network that provided the studied isolates. We thank Sandra Martínez Álvarez (group of Prof. Carmen Torres, Universidad de La Rioja, Spain) for her help with the BRIG vizualization of the plasmids.

## Funding

This research was funded by institutional support from ANSES.

## Transparency declarations

None to declare.

## Ethical approval

No ethical approval was needed since samples were sent to veterinary for diagnostic purposes.

## Supplementary data

Figure S1 and Tables S1 to S3 are available as Supplementary data at JAC Online.
## References

**1** Davin-Regli A, Lavigne J-P, Pagès J-M. *Enterobacter* spp. update on taxonomy, clinical aspects, and emerging antimicrobial resistance. *Clin Microbiol Rev* 2019; **32**: e00002-19. https://doi.org/10.1128/CMR.00002-19

**2** Beyrouthy R, Barets M, Marion E et al. Novel *Enterobacter* lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains. *Emerg Infect Dis* 2018; **24**: 1505-15. https://doi.org/10.3201/eid2408.180151

**3** Chavda KD, Chen L, Fouts Derrick E et al. Comprehensive genome analysis of carbapenemase-producing *Enterobacter* spp.: new insights into phylogeny, population structure, and resistance mechanisms. *mBio* 2016; **10**: 1128. https://doi.org/10.1128/mbio.02093-16

**4** Yeh T-K, Lin H-J, Liu P-Y et al. Antibiotic resistance in *Enterobacter hormaechei*. *Int J Antimicrob Agents* 2022; **60**:106650. https://doi.org/10.1016/j.ijantimicag.2022.106650

**5** Cardoso B, Sellera FP, Sano E et al. Phylogenomic analysis of CTX-M-15-producing *Enterobacter hormaechei* belonging to the high-risk ST78 from animal infection: another successful One Health clone? *J Glob Antimicrob Resist* 2022; **29**: 113-5. https://doi.org/10.1016/j.jgar.2022.02.010

**6** Haenni M, Saras E, Ponsin C et al. High prevalence of international ESBL CTX-M-15-producing *Enterobacter cloacae* ST114 clone in animals. *J Antimicrob Chemother* 2016; **71**: 1497-500. https://doi.org/10.1093/jac/dkw006

**7** Harada K, Shimizu T, Mukai Y et al. Phenotypic and molecular characterization of antimicrobial resistance in *Enterobacter* spp. isolates from companion animals in Japan. *PLoS One* 2017; **12**: e0174178. https://doi.org/10.1371/journal.pone.0174178

**8** Roberts LW, Harris PNA, Forde BM et al. Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing *Enterobacter hormaechei*. *Nat Commun* 2020; **11**: 466. https://doi.org/10.1038/s41467-019-14139-5

**9** Hadjirin NF, van Tonder AJ, Blane B et al. Dissemination of carbapenemase-producing Enterobacter cloacae in Ireland from 2012 to 2017: a retrospective genomic surveillance study. *Microb Genom* 2023; **9**: mgen000924. https://doi.org/10.1099/mgen.0.000924

**10** Yousfi M, Touati A, Muggeo A et al. Clonal dissemination of OXA-48-producing Enterobacter cloacae isolates from companion animals in Algeria. *J Glob Antimicrob Res* 2018; **12**: 187-91. https://doi.org/10.1016/j.jgar.2017.10.007

**11** Pulss S, Stolle I, Stamm I et al. Multispecies and clonal dissemination of OXA-48 carbapenemase in Enterobacteriaceae from companion animals in Germany, 2009–2016. *Front Microbiol* 2018; **9**: 1265. https://doi.org/10.3389/fmicb.2018.01265

**12** Donà V, Nordmann P, Kittl S et al. Emergence of OXA-48-producing *Enterobacter hormaechei* in a Swiss companion animal clinic and their genetic relationship to clinical human isolates. *J Antimicrob Chemother* 2023; **78**: 2950-60. https://doi.org/10.1093/jac/dkad337

**13** Kobs VC, de Medeiros F, Fernandes PP et al. Healthcare-associated NDM-1-producing *Enterobacter hormaechei* subsp. *xiangfangensis* clone ST136 emerging as pathogen of companion animals in Brazil. *J Antimicrob Chemother* 2023; **78**: 1553-6. https://doi.org/10.1093/jac/dkad124

**14** Daniels JB, Chen L, Grooters SV et al. *Enterobacter cloacae* complex sequence type 171 isolates expressing KPC-4 carbapenemase recovered from canine patients in Ohio. *Antimicrob Agents Chemother* 2018; **62**: e01161-18. https://doi.org/10.1128/AAC.01161-18

**15** Leelapsawas C, Sroithongkham P, Payungporn S et al. First report of *bla*<sub>OXA-181</sub>-carrying IncX3 plasmids in multidrug-resistant *Enterobacter*

hormaechei and *Serratia nevei* recovered from canine and feline opportunistic infections. *Microbiol Spectr* 2024; **12**: e0358923. https://doi.org/10.1128/spectrum.03589-23

**16** Sadek M, Nariya H, Shimamoto T et al. First genomic characterization of *bla*<sub>VIM-1</sub> and *mcr-9*-coharbouring *Enterobacter hormaechei* isolated from food of animal origin. *Pathogens* 2020; **9**: 687. https://doi.org/10.3390/pathogens9090687

**17** Li R, Liu Z, Li Y et al. Characterization of *bla*<sub>NDM</sub>-positive Enterobacteriaceae reveals the clonal dissemination of *Enterobacter hormaechei* coharboring *bla*<sub>NDM</sub> and tet(X4) along the pork production chain. *Int J Food Microbiol* 2022; **372**: 109692. https://doi.org/10.1016/j.ijfoodmicro.2022.109692

**18** Macesic N, Blakeway LV, Stewart JD et al. Silent spread of mobile co-listin resistance gene *mcr-9.1* on IncH12 ‘superplasmids’ in clinical carbapenem-resistant Enterobacterales. *Clin Microbiol Infect* 2021; **27**: 1856.e7-e13. https://doi.org/10.1016/j.cmi.2021.04.020

**19** Algarni S, Gudeta DD, Han J et al. Genotypic analyses of IncH12 plasmids from enteric bacteria. *Sci Rep* 2024; **14**: 9802. https://doi.org/10.1038/s41598-024-59870-2

**20** Lumbreras-Iglesias P, de Toro M, Vázquez X et al. High-risk international clones ST66, ST171 and ST78 of *Enterobacter cloacae* complex causing blood stream infections in Spain and carrying *bla*<sub>OXA-48</sub> with or without *mcr-9*. *J Infect Public Health* 2023; **16**: 272-9. https://doi.org/10.1016/j.jiph.2022.12.015

**21** Girlich D, Poirel L, Nordmann P. Clonal distribution of multidrug-resistant *Enterobacter cloacae*. *Diagn Microbiol Infect Dis* 2015; **81**: 264-8. https://doi.org/10.1016/j.diagmicrobio.2015.01.003

**22** Pot M, Guyomard-Rabenirina S, Couvin D et al. Dissemination of extended-spectrum-β-lactamase-producing *Enterobacter cloacae* complex from a hospital to the nearby environment in Guadeloupe (French West Indies): ST114 lineage coding for a successful IncH12/ST1 plasmid. *Antimicrob Agents Chemother* 2021; **65**: e02146-20. https://doi.org/10.1128/AAC.02146-20

**23** Haenni M, Boulouis HJ, Lagrée AC et al. Enterobacterales high-risk clones and plasmids spreading *bla*<sub>ESBL/AmpC</sub> and *bla*<sub>OXA-48</sub> genes within and between hospitalized dogs and their environment. *J Antimicrob Chemother* 2022; **77**: 2754-62. https://doi.org/10.1093/jac/dkac268

**24** Emeraud C, Petit C, Gauthier L et al. Emergence of VIM-producing *Enterobacter cloacae* complex in France between 2015 and 2018. *J Antimicrob Chemother* 2022; **77**: 944-51. https://doi.org/10.1093/jac/dkab471

**25** Dortet L, Cuzon G, Ponties V et al. Trends in carbapenemase-producing Enterobacteriaceae, France, 2012 to 2014. *Euro Surveill* 2017; **22**: 30461. https://doi.org/10.2807/1560-7917.ES.2017.22.6.30461

**26** Rezzoug I, Emeraud C, Rodriguez C et al. Regional dissemination of NDM-1 producing *Enterobacter hormaechei* ST1740, with a subset of strains co-producing VIM-4 or IMP-13, France, 2019 to 2022. *Euro Surveill* 2024; **29**: 2300521. https://doi.org/10.2807/1560-7917.ES.2024.29.11.2300521

**27** ANSES. French Surveillance Network for Antimicrobial Resistance in Bacteria From Diseased Animals. https://www.anses.fr/en/system/files/LABO-Ra-Resapath2022EN.pdf, 2022.

**28** Magiorakos AP, Srinivasan A, Carey RB et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. *Clin Microbiol Infect* 2012; **18**: 268-81. https://doi.org/10.1111/j.1469-0691.2011.03570.x

**29** Saidani M, Messadi L, Chaouechi A et al. High genetic diversity of Enterobacteriaceae clones and plasmids disseminating resistance to extended-spectrum cephalosporins and colistin in healthy chicken in Tunisia. *Microb Drug Resist* 2019; **25**: 1507-13. https://doi.org/10.1089/mdr.2019.0138
30 Wagh ST, Razvi NA. Marascuilo method of multiple comparisons (an analytical study of caesarean section delivery). *Int J Contemp Med* 2016; **3**: 1137-40.

31 Qiu X, Ye K, Ma Y *et al*. Genome sequence-based species classification of *Enterobacter cloacae* complex: a study among clinical isolates. *Microbiol Spectr* 2024; **12**: e0431223. https://doi.org/10.1128/spectrum.04312-23

32 Jacoby GA. Ampc beta-lactamases. *Clin Microbiol Rev* 2009; **22**: 161-82. https://doi.org/10.1128/CMR.00036-08

33 Choi S-H, Lee Jung E, Park Su J *et al*. Emergence of antibiotic resistance during therapy for infections caused by Enterobacteriaceae producing AmpC β-lactamase: implications for antibiotic use. *Antimicrob Agents Chemother* 2008; **52**: 995-1000. https://doi.org/10.1128/AAC.01083-07

34 ANSES. Sales Survey of Veterinary Medicinal Products Containing Antimicrobials in France in 2022. https://www.anses.fr/en/system/files/ANMV-Ra-Antibiotiques2022EN.pdf, 2023.

35 Boattini M, Bianco G, Llorente LI *et al*. Enterobacterales carrying chromosomal AmpC β-lactamases in Europe (EuESCPM): epidemiology and antimicrobial resistance burden from a cohort of 27 hospitals, 2020-2022. *Int J Antimicrob Agents* 2024; **63**: 107115. https://doi.org/10.1016/j.ijantimicag.2024.107115

36 Barceló IM, Escobar-Salom M, Jordana-Lluch E *et al*. Filling knowledge gaps related to AmpC-dependent β-lactam resistance in *Enterobacter cloacae*. *Sci Rep* 2024; **14**: 189. https://doi.org/10.1038/s41598-023-50685-1

37 Allen JL, Doidge NP, Bushell RN *et al*. Healthcare-associated infections caused by chlorhexidine-tolerant *Serratia marcescens* carrying a promis-cuous IncH12 multi-drug resistance plasmid in a veterinary hospital. *PLoS One* 2022; **17**: e0264848. https://doi.org/10.1371/journal.pone.0264848

38 Gao G, He W, Jiao Y *et al*. The origin and evolution of IncF33 plasmids based on large-scale data sets. *mSystems* 2023; **8**: e0050823. https://doi.org/10.1128/msystems.00508-23

39 Kang HY, Kim KY, Kim J *et al*. Distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae isolates collected in 1995 to 1998 and 2001 to 2006 at a university hospital in South Korea and identification of conjugative plasmids mediating dissemination of 16S rRNA methylase. *J Clin Microbiol* 2008; **46**: 700-6. https://doi.org/10.1128/JCM.01677-07

40 Borjesson S, Greko C, Myrenas M *et al*. A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying *bla*<sub>SHV-12</sub> from horses in Sweden. *J Glob Antimicrob Resist* 2020; **20**: 285-9. https://doi.org/10.1016/j.jgar.2019.08.007

41 Aoki K, Harada S, Yahara K *et al*. Molecular characterization of IMP-1-producing *Enterobacter cloacae* Complex isolates in Tokyo. *Antimicrob Agents Chemother* 2018; **62**: e02091-17. https://doi.org/10.1128/AAC.02091-17

42 Chavda KD, Westblade LF, Satlin MJ *et al*. First report of *bla*<sub>VIM-4</sub>- and mcr-9-coharboring *Enterobacter* species isolated from a pediatric patient. *mSphere* 2019; **4**: e00629-19. https://doi.org/10.1128/mSphere.00629-19

43 Soliman AM, Maruyama F, Zarad HO *et al*. Emergence of a multidrug-resistant *Enterobacter hormaechei* clinical isolate from Egypt co-harboring mcr-9 and *bla*VIM-4. *Microorganisms* 2020; **8**: 595. https://doi.org/10.3390/microorganisms8040595

44 Izdebski R, Biedrzycka M, Urbanowicz P *et al*. Genome-based epidemiologic analysis of VIM/IMP carbapenemase-producing *Enterobacter* spp., Poland. *Emerg Infect Dis* 2023; **29**: 1618. https://doi.org/10.3201/eid2908.230199

45 Haenni M, Métayer V, Lupo A *et al*. Spread of the *bla*<sub>OXA-48</sub>/IncL plasmid within and between dogs in city parks, France. *Microbiol Spectr* 2022; **10**: e0040322. https://doi.org/10.1128/spectrum.00403-22

46 Izdebski R, Baraniak A, Herda M *et al*. MLST reveals potentially high-risk international clones of *Enterobacter cloacae*. *J Antimicrob Chemother* 2015; **70**: 48-56. https://doi.org/10.1093/jac/dku359