Molecular characterization of ESBL- and carbapenemase-producing Enterobacteriaceae isolated from cats in Lebanon

Abstract

Background

Antimicrobial resistance (AMR) represents a pressing global health challenge, influencing human, animal, and environmental health within the interconnected One Health framework. ESBL- and carbapenemase-producing Enterobacteriaceae constitute a major global public health threat. In Lebanon, their presence is particularly concerning, as they are detected across diverse environments, including hospitals, surface water, wastewater, poultry, and livestock. Despite close contact between pets and their owners, the role of companion animals, particularly cats, in spreading AMR determinants has been overlooked. Based on the One Health approach, the study addresses this gap by presenting the first Whole Genome Sequencing (WGS)-based report of multidrug-resistant (MDR) Enterobacteriaceae isolated from cats in Lebanon.

Results

A total of 13 ESBL-producing Enterobacteriaceae, including 11 Escherichia coli and two Enterobacter hormaechei, were isolated from fecal samples of domestic and stray cats. The isolates were among those classified as World Health Organization (WHO) Critical Priority Pathogens, highlighting their public health importance. Whole-genome characterization and antimicrobial susceptibility testing revealed alarmingly high resistance rates to multiple antibiotics, including carbapenems and tigecycline. In silico resistome analysis identified over 37 diverse resistance determinants, including bla_CTX-M-15, bla_TEM-1B, bla_OXA-1, and bla_NDM-5. Plasmid analysis uncovered 17 distinct Inc groups, markedly IncU, IncFII, and IncFIB. Phylogenetic analysis demonstrated high genetic similarity among isolates of the same sequence type (ST), irrespective of their isolation source or geographical location. E. coli ST167, a high-risk clone, carried the bla_NDM-5 gene, associated with carbapenem resistance, on an IS26-flanked composite transposon. It’s noteworthy that bla_CTX-M-15 was chromosomally encoded in one E. coli isolate within a rare genetic cassette, co-localized with qnrS1, Tn2, ISEcp1, and ISKpn19.

Conclusions

This study provides the first whole-genome sequencing-based evidence of multidrug-resistant ESBL- and carbapenemase-producing Enterobacteriaceae in cats in Lebanon, highlighting their overlooked role as reservoirs of antimicrobial resistance. The detection of high-risk clones, along with a diverse resistome and mobilome linked to multidrug resistance, reinforces the urgent need for integrated national AMR surveillance within a One Health framework.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04255-2.

Introduction

The World Health Organization (WHO) has identified antimicrobial resistance (AMR) as one of the top ten public health threats facing humanity. As bacteria increasingly develop resistance, there is a need to adopt a collaborative and multisectoral approach to combat the spread of these pathogens [1]. The One Health approach (OHA), a transdisciplinary initiative, aims at mitigating public health concerns at a national and global scale by addressing environmental issues and mobilizing various sectors to create sustainable solutions [2]. The OHA aims to achieve optimal health outcomes by examining how interactions between humans, animals, and the environment influence the spread of infectious diseases [3]. The transmission and exchange of resistance determinants among humans, animals, and the environment are aggravated by increased mobility through international trade and travel, human expansion into new geographic areas, the growing dependence on animals as sources of companionship and nutrition, and climate and land changes [4]. However, international surveillance agencies do not adequately recognize the role of companion animals in the emergence and spread of resistance determinants. Consequently, they are often overlooked in studies implementing the OHA [5]. A distinctive factor contributing to AMR in pets is their close proximity to humans, which facilitates the transfer of resistant organisms from the skin, saliva, and fecal material between the two hosts [6]. Additionally, the considerable rise in the number of household pets and increased use of antimicrobial agents to treat sick pets, are important factors contributing to and accelerating AMR development [1, 7]. Stray and domestic cats have been found to harbor pathogens of public health concern, such as extended-spectrum β-lactamase (ESBL) producers and carbapenem- resistant Enterobacteriaceae (CRE) [2], but are not well characterized compared to those recovered from humans and dogs [8].

In Lebanon, the misuse of antibiotics, driven by self-medication to avoid costly medical consultations, over-the-counter access, limited public awareness, and weak stewardship among both prescribers and patients, has significantly contributed to high rates of antimicrobial resistance in hospital and community settings [9, 10]. A national study found widespread gaps in knowledge, attitudes, and practices related to antibiotic use in both human and veterinary contexts, which correlate with the increased incidence of multidrug-resistant (MDR) bacteria across various environments [10]. The national prevalence of ESBL-producing E. coli among hospitalized patients has steadily increased over the past decade [11]. Environmental surveillance has also uncovered multidrug-resistant E. coli and Klebsiella pneumoniae in surface water, pointing to the spread of resistance determinants through sewage-contaminated waterways [51,69]. In parallel, studies in livestock have identified carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii in cattle and poultry, emphasizing the circulation of AMR organisms across clinical, environmental, and agricultural settings [12, 13]. In veterinary settings, frequent, and at times inappropriate, use of third- and fourth-generation cephalosporins has been reported, particularly in the dairy sector, where empirical treatment without diagnostic confirmation is common [14]. Although specific data on carbapenem use in pets is lacking, the limited regulatory oversight raises concerns about the potential misuse of last-resort antibiotics in companion animals. The national misuse of antibiotics in clinical, veterinary, and environmental settings, coupled with the high incidence of resistant bacteria amongst different niches, highlights the urgent need to address antimicrobial resistance across various sectors to safeguard public health [9, 10].

Stray and domestic cats form a high-density population in Lebanon, yet their role in the spread of ESBL-producing and carbapenem-resistant Enterobacteriaceae has not been previously studied. This study aims to perform a detailed molecular characterization of ESBL- and carbapenemase-producing Enterobacteriaceae found in cat feces, focusing on the role of cats in the emergence and spread of resistance determinants.

Materials and methods

Sample collection

Fecal samples from three domestic and four stray cats undergoing treatment for their respective illnesses were collected between June 2022 and January 2023 from two veterinary clinics in Beirut, while keeping a record of the breed and classification. The samples were collected from cat litter boxes and were preserved in sterile falcon tubes at 4 °C until further processing. The cats were designated as CA-1 to 7.

Isolation and identification of ESBL-producing Enterobacteriaceae

Serial dilutions in sterile PBS [15] were prepared from each fecal sample, and 100 μl from each dilution was inoculated on CHROMagar-ESBL plates [16] for the isolation of ESBL-producing bacteria. Following incubation at 37 °C for 24 h, without pre-enrichment, colonies were examined and classified according to the following scheme: dark pink to reddish colonies were classified as potential ESBL-producing E. coli, and metallic blue colonies as ESBL-producing Klebsiella, Enterobacter, or Citrobacter. Colonies were further purified on MacConkey agar [17], and Tryptone Soy Agar (TSA) [18]. The recovered organisms were designated after their identification as EC or ENT, E. coli and Enterobacter hormaechei, respectively. All isolates were maintained in glycerol stocks and stored at −20 and −80 °C until further use.

Antimicrobial susceptibility testing

Antibiotic susceptibility was determined using the Kirby–Bauer disk agar diffusion assay on Mueller–Hinton agar [17] plates against eight classes of antibiotics: tetracyclines (tetracycline), sulfonamide/folate pathway inhibitor (sulfamethoxazole-trimethoprim combination), folate pathway inhibitors (trimethoprim), quinolones (ciprofloxacin), fourth generation cephalosporins (cefepime), monobactams (aztreonam), penicillin/β-lactamase inhibitor (amoxicillin-clavulanic acid combination), and carbapenems (ertapenem, imipenem, and meropenem) [17]. Additionally, E-test strips [19] were used with four carbapenem resistant isolates (EC23, EC24, EC26, and EC28) to determine the minimal inhibitory concentration (MIC) of tigecycline and confirm resistance to meropenem, ertapenem, and imipenem. All results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [20].

DNA extraction

For Illumina-sequencing and PCR-based replicon typing (PBRT), DNA was extracted using the GenElute Bacterial Genomic DNA Kit, following the manufacturer's instructions [21]. For Nanopore sequencing, DNA was extracted using the MagAttract HMW DNA kit [22] following the manufacturer’s instructions.

PCR-Based Replicon Typing (PBRT)

Plasmid characterization was carried out using the DIATHEVA PBRT kit [23] that covers 25 replicons: A/C, B/O, FIA, FIB, FIB-M, FIC, FII, FIIK, FIIS, HI1, HI2, HIB-M, I1, I2, K, L/M, N, P, R, T, U, W, X1, X2, and Y, that are commonly found in Enterobacteriaceae [24]. All reactions included positive controls and adhered to the manufacturer’s instructions. The PCR products were visualized on a 2.5% agarose gel stained with ethidium bromide.

Genome sequencing

Illumina sequencing and assembly

All the isolates were sequenced using the Illumina MiSeq platform. Genomic libraries were constructed using the Nextera XT DNA library preparation kit and subsequently sequenced on an Illumina MiSeq for paired-end reads [25]. De novo genome assembly was performed using Spades Genome Assembler v3.15.3 [26], and the raw sequence data underwent quality control checks using FastQC [27].

Nanopore sequencing and assembly

EC28 was resistant to all tested antibiotics and exhibited diverse plasmid content and was thus additionally chosen for Nanopore sequencing. Sequencing was performed using the ligation Sequencing (SQK-LSK109; ONT, Oxford, England) and Native Barcoding Kits (EXP-NBD104 and EXP-NBD114; ONT, Oxford, England). End-prep was performed using a 100–200 fmol sample diluted in 65 μL nuclease-free water, according to the Native Barcoding Kit amplicon protocol. Adapter ligation and cleaning step was performed using NEB ligation and AMPure XP beads (Beckman Coulter, USA) with the final adapter-ligated DNA library used being 50–100 fmol. The library was loaded into R9.4 flow cell (ONT) and sequenced using the MinION (ONT). Base calling was performed using Guppy software v5.0.17 (ONT) with a Q-score threshold of 9 [28]. The minimum read length was set at 1000 bp. Read assembly was conducted using Flye v2.9.2 [29]. The quality and genome completeness were checked using the Quality Assessment Tool for Genome Assemblies (QUAST) v5.2.0 [30] and Busco v5.4.7 [31], respectively. Hybrid assembly using the short and long reads was performed using Unicycler [32], and the resulting assembly was checked again with Quast and Busco.

Genome annotation and analysis

The genomes were annotated using the RASTk online server and viewed using the Genome Seed Viewer [33]. A preliminary molecular characterization was performed using the Center for Genomic Epidemiology (CGE) website [34]. The genus and species of all the sequenced isolates were identified using KmerFinder v3.2 [35]. The Sequence types (STs) were determined in silico using PubMLST [36]. E. coli serotypes and phylogroups were determined in silico using SeroTypeFinder v2.0.1 [37] and EzClermont [38], respectively. Screening for intrinsic and acquired resistance genes was performed using ResFinder v4.1 [39]. PlasmidFinder v2.1 [40] was used to identify the plasmid Inc groups.

To characterize the genetic environments of bla_NDM-5 (isolates EC23, EC24, EC26, and EC28) and bla_CTX-M-15 (isolate EC8), assembled contigs were annotated and visualized using Bandage v0.8.1 [41], the RASTk online server [33], SnapGene [42], and PROKKA v1.14.5 [43]. The final schematic was manually assembled using Gene Graphics [44].

Phylogenetic analysis

To assess the genetic relatedness and evolutionary patterns within individual E. coli STs from our study with those from diverse environments and geographical locations, we chose twelve distinct publicly available E. coli genomes for phylogenetic analysis, including a reference genome, and compared them with the genomes from this study (Fig. 3); the availability of WGS data was a primary inclusion criterion. The accession numbers are as follows: U00096.3, NZ_CP048376.1, NZ_SGIX01000100.1, AYNJ01000001.1, NZ_MUKK01000100.1, NZ_CP093919.1, NZ_JAAXNW010000100.1, MUFZ01000001.1, NZ_NEDL01000100.1, NZ_PPCU02000001.1, NZ_MUKD01000100.1, NZ_NBCL02000001.1 [11, 45–53]. The genomes were annotated using the rapid prokaryotic genome annotation pipeline (Prokka) v1.13.4 with a similarity cutoff e-value 10⁻⁶ and minimum contig size of 200 bp [43]. The output GFF files were piped into the rapid large-scale prokaryote pan-genome analysis (Roary) pipeline v3.11.2 [54]. The command was run on 8 threads, involving gene alignment with MAFFT using the following flags: roary -p 8 -f -e -n -v -s. The output was then visualized using the Interactive Tree of Life (iTol) v6.8.1 [55]. Phylogroups and STs were elucidated using EzClermont [38] and PubMLST [36], respectively.

Fig. 3.

Phylogenetic Analysis of E. coli Showing STs, Phylogroups, Isolation Source/Country, bla_NDM-5 Status, and References. A Maximum-Likelihood Tree With 1000 Number Seed Bootstrap Showing the Phylogenetic Relationships Between the Isolates Based on Variations in the Core Genes Generated Using Roary and Visualized Using iTol

Global tracking of STs

To track the global occurrence of the E. coli sequence types detected in this study across various niches, we conducted a comprehensive analysis of existing data (Table 3). A structured search was performed using the PubMed database during December 2024. Search terms included combinations of “E. coli ST167,” “ST410,” “ST90,” “ST38,” “ST46,” and “ST345” alongside keywords such as “NDM-5,” “animal,” “environment,” “human,” and “carbapenem-resistant”. We prioritized studies that described molecular characterizations of the different sequenced strains while highlighting their isolation sources. Preference was given to peer-reviewed articles published between 2010 and 2023.

Table 3.

Epidemiological tracking of study STs detected globally, including isolation sources and referenced studies

The absence and presence ofbla_NDM-5has been noted for the ST167, 410, and 90. The highlighted isolates have been used in the phylogenetic analysis. N/A Not Applicable. “blaNDM-5 (+)” indicates the strain was harboring bla_NDM-5. “bla_NDM-5(-)” indicates the strain was not harboring bla_NDM-5

Studies from both high- and low-income countries were included to reflect diverse geographic and healthcare settings. Reference tracing was also performed manually from key articles to identify additional relevant reports. Duplicates, incomplete records, or studies lacking ST confirmation were excluded. For each identified isolate, we included the isolation source and country, and for E. coli ST167, ST410, and ST90, the presence or absence of NDM-5 based on the reported data was additionally noted.

Results

Bacterial cultures and host information

A total of 13 ESBL-producing Enterobacteriaceae were recovered from the collected fecal samples. A sample recovered from CA-1, a domestic cat, had four distinct ESBL-producers (Table 1). Most of the ESBL-producing Enterobacteriaceae were E. coli, and only two were Enterobacter hormaechei. The sample from CA-7, a stray cat, harbored three different E. coli strains.

Table 1.

Breed, Collection Site, and Cat Types, with Phylogroups, Serotypes, and STs for Recovered Isolates

Host	Collection Site	Type	Breed	Isolate	Phylogroup	Serotype	ST
CA-1	2	Domestic	Himalayan	ENT5	NA	NA	ST946
				ENT9			ST946
				EC1	B1	H21:O134	ST345
				EC8	A	H10:O9	ST46—ST46 Cplx
CA-2	1	Stray	Domestic shorthair	EC11	D	H30:O99	ST38—ST38 Cplx
CA-3	1	Domestic	Domestic shorthair	EC21	D	H30:O99	ST38—ST38 Cplx
CA-4	1	Stray	Domestic shorthair	EC22	B2	H1:O6	ST1735*
				EC23	A	H9:O101	ST167—ST10 Cplx
CA-5	1	Domestic	Domestic shorthair	EC24	C	H9:O untypabale	ST410—ST23 Cplx
CA-6	2	Stray	Mixed breed	EC25	A	H9:O101	ST167—ST10 Cplx **
CA-7	2	Stray	Domestic shorthair	EC26	C	H9:O8	ST90—ST23 Cplx
				EC27	C	H9:O8	ST410—ST23 Cplx
				EC28	A	H9:O101	ST167—ST10 Cplx

1, Canicat veterinary clinic; 2, Petcare veterinary clinic; Cplx complex, NA not applicable; *novel allele fumC; **novel allele icd. EN Enterobacter hormaechei, EC Escherichia coli

Antimicrobial susceptibility testing

All the isolates were resistant to the 4th generation cephalosporin cefepime, demonstrating extended-spectrum activity (Table 2). Resistance was also detected to sulfamethoxazole-trimethoprim (92.3%), tetracycline (84.6%), ciprofloxacin (84.6%), aztreonam (84.6%), amoxicillin-clavulanic acid (76.9%), and trimethoprim (46.1%). EC23 and EC28, isolated from stray cats at two different veterinary clinics, exhibited resistance to all the tested antibiotics. Resistance patterns among isolates from stray cats were more pronounced, with three isolates exhibiting carbapenem resistance (EC23, EC26, EC28) and two also showing tigecycline resistance (EC23 and EC28). Additionally, EC24 was the only domestic cat exhibiting resistance to all tested carbapenems. Inhibition zone diameters (measured in mm) for meropenem were 14 (EC23), 16 (EC24), 19 (EC26), and 13 (EC28), for ertapenem: 13 (EC23), 15 (EC24), 11 (EC26), and 10 (EC28), and for imipenem: 18 (EC23 and EC24), 11 (EC26), and 16 (EC28). The results suggest a potentially larger ARG reservoir in stray cats compared to domestic cats. Apart from tigecycline and carbapenem resistance, no notable difference was observed between the resistance profiles of isolates from stray and domestic cats or between the Enterobacter and Escherichia isolates.

Table 2.

Kirby-Bauer Disk Diffusion Assay and E-test Results

The inhibition zone diameters (mm) against the carbapenem for EC23, EC24, EC26, and EC28 have been noted. Light blue, Resistant; Light yellow, Intermediate resistance; White, Susceptible; NT Not tested

Genome statistics

All the isolates were sequenced using the Illumina MiSeq platform. The number of contigs ranged from 77 to 268, and genome sizes from 4,709,195 bp to 5,318,796 bp. The mean GC-content was 50.6% for E. coli and 55.5% for Enterobacter. The EC28 hybrid assembly encompassed 5,248,400 bp distributed across eight contigs, with a GC content of 50.8%. Detailed information on the genome size, GC content, and number of contigs, subsystems, coding sequences, and RNAs for the isolates can be found in Supplementary Table S1.

In silico typing

Both E. hormaechei isolates belonged to sequence type ST946. E. coli sequence typing revealed the presence of ST167 (n = 3), with equal prevalence of ST410 (n = 2) and ST38 (n = 2) (Table 1). Several singletons were also detected, including ST345, ST90, ST1735, and ST46. Two isolates, EC22 and EC25, exhibited novel housekeeping alleles and were typed as the closest matches, ST1735 and ST167, respectively. Correlations between E. coli STs, serotypes, and phylogroups were established through in silico typing using SerotypeFinder and EzClermont, respectively. Five distinct phylogroups were identified, with phylogroup A (n = 4) being the most prevalent, followed by C (n = 3), and D (n = 2). Phylogroups B1 (n = 1) and B2 (n = 1) were also detected. Isolates typed as ST167 (EC23, EC25, and EC28) belonged to serotype H9:O101 and phylogroup A.

Resistance determinants

In silico analysis revealed the presence of 37 distinct genes conferring resistance to aminoglycosides, sulfonamides, β-lactams, fosfomycin, tetracyclines, quinolones, trimethoprim, chloramphenicol, and macrolides (Fig. 1). Most isolates carried at least two β-lactamase encoding genes, with bla_CTX-M-15 (n = 10) being the most common ESBL variant, followed by an equal prevalence (n = 6) of bla_TEM-1B and bla_OXA-1. The occurrence of bla_CTX-M-15 was accompanied by either bla_TEM-1B (ENT5, ENT9, EC1), or bla_OXA-1 (EC23, EC25, and EC28). In some cases, all three were co-harbored (EC11, EC21, and EC27). The only identified carbapenemase gene, bla_NDM-5, was detected in four isolates (EC23, EC24, EC26, and EC28) that exhibited carbapenem resistance. It’s noteworthy that three β-lactamase variants, detected in EC22, EC23, and EC28, could only be annotated to their closest matches: bla_TEM-141, bla_TEM-214, bla_TEM-1B, or bla_TEM-206. For the AmpC β-lactamases, three bla_CMY variants, bla_CMY-27, bla_CMY-42, and bla_CMY-148, were identified, with one occurrence of each (n = 1), and each found in a different isolate. ENT5 and ENT9 additionally harbored the bla_ACT-7 and fosA genes while lacking resistance determinants for chloramphenicol and macrolide.

Fig. 1.

Antimicrobial Resistance Genes and Plasmid Profiles. (i) Distribution of Antibiotic Classes and Associated Resistance Genes in Study Isolates, (ii) Prevalence and Types of ESBL and Carbapenemase Genes Detected, (iii) Plasmid Inc Group Profiles Identified through PBRT and in silico Analysis. The Legend Under (A) Contains All the Needed Information, Resistance genes, and Plasmids

Plasmid typing and analysis of genetic environments

The combined results from PBRT and in silico genome-based analysis revealed the presence of 17 distinct Inc groups (Fig. 1, Table S2). IncU (n = 9) was the most common, followed by an equal prevalence of IncFII and IncFIB. Other plasmids such as IncFIIS, IncI1, and Col were also identified. The highest number of Inc groups (n = 7) was detected in isolate EC27 of sequence type ST410, which was recovered from a stray cat. Interestingly, EC28 (ST167) was isolated from the same cat and harbored 12 distinct resistance determinants including bla_NDM-5, bla_CTX-M-15, and bla_OXA-1, and five different Inc groups (IncFIA, IncFIB, IncFII, IncFIIS, and IncI(gamma)) (Fig. 1). EC8 was plasmid-negative both in silico and by PBRT, yet it tested positive for six distinct resistance genes, including bla_CTX-M-15.

The contigs carrying bla_NDM-5 were associated with multi-replicon IncF-type plasmids, specifically IncFIA/IncFIB in EC23, IncFIA/IncFIB/IncFII in EC24, and IncFIA/IncFII in both EC26 and EC28. In all four isolates, bla_NDM-5 was embedded within a composite transposon flanked by IS26 elements and located downstream of a conserved TnAS1 transposase, suggesting IS26-mediated mobilization (Fig. 2A). An ISCR1 element consistently preceded a highly conserved downstream region comprising dsbD–trpF–ble–bla_NDM-5–IS30–IS26. Upstream of ISCR1, aminoglycoside and trimethoprim resistance genes were detected in variable combinations (aadA2/dfrA12 or aadA5/dfrA17), often accompanied by recombinases such as xerC/xerD and pinR (Fig. 2A).

Fig. 2.

Genetic Environments of (A) bla_NDM-5 in EC23, EC24, EC26, and EC28, and (B) bla_CTX-M-15 in EC8. Arrows Represent Annotated Open Reading Frames, Color-coded by Functional Category; Red Indicates AMR Genes, Yellow Indicates MGEs, Green Indicates Metabolic Genes, and Blue Indicates Recombination/Regulatory Genes. Scale Bars (1 kb, 5 kb, 10 kb) are Included for Reference

In isolates EC23 and EC28, bla_CTX-M-15 was associated with IncFIA/IncFIB replicons, while in EC25 and EC27, it was linked to an IncFII replicon. In EC1, ENT5, ENT9, EC11, and EC21, bla_CTX-M-15 was detected, but the corresponding plasmid replicon could not be identified, likely due to the incomplete nature of the draft genome assemblies. In EC8, the chromosomally encoded bla_CTX-M-15 was part of a gene cassette that also carried qnrS1, mobilized by ISEcp1, Tn2, and ISKpn19, and flanked by elements such as relB–relE, integrases, and IS2-associated transposases, suggesting horizontal acquisition and stable chromosomal integration (Fig. 2B).

Phylogenetic analysis

To assess the relatedness of the recovered isolates to others from diverse ecological and geographical backgrounds, publicly available genomes sharing the same sequence types (STs) were included in a core genome phylogenetic analysis using Roary (Fig. 3) [11, 45–53]. The resulting tree shows six distinct clades, with clustering driven primarily by ST and clonal complexes (CCs) rather than by source of isolation, country of origin, or presence of bla_NDM-5. Short branch lengths within clades reflect high genetic similarity among isolates of the same ST/CC, while longer branches separating clades indicate substantial genomic divergence. These patterns support the presence of stable, globally distributed ST-specific lineages across diverse settings.

EC11 and EC21, isolated from a stray and a domestic cat respectively, were genetically identical, suggesting shared evolutionary events despite differences in host environment. Isolates of ST410 and ST90 clustered together within a single clade, consistent with their classification under clonal complex 23 (CC23). ST410 isolates from this study clustered closely with publicly available genomes from diverse sources, including humans, animals, sewage, and freshwater, and included both bla_NDM-5-positive and -negative profiles. This highlights the broad ecological distribution of the ST410 lineage and its role in the spread of carbapenem resistance. Similarly, ST167 isolates (EC28, EC25, EC22) clustered with human- and animal-derived strains from multiple countries, also spanning both bla_NDM-5-positive and -negative variants. These patterns suggest interspecies transmission and the global dissemination potential of these high-risk clones, further reinforcing the genetic stability observed within each ST across varied ecological settings.

Global tracking of STs

We identified 30 relevant studies [11, 45, 47–53, 56–76] that reported the global occurrence of E. coli sequence types detected in this study, across a range of host and environmental sources (Table 3). These reports cover multiple continents and ecosystems, including clinical, veterinary, wildlife, freshwater, wastewater, and soil settings. Among the identified sequence types, ST167, ST410, and ST90 have been found in both human and non-human reservoirs across Europe, Asia, and South America. Several of these studies also documented the presence or absence of the bla_NDM-5 gene in isolates belonging to ST167, ST410, and ST90. All six sequence types identified in our study have been reported globally, though to varying extents, with some showing broader ecological and geographic spread than others. These findings offer valuable context for understanding the wider epidemiological trends and One Health significance of the sequence types detected in feline isolates.

Discussion and conclusion

AMR poses a major global health threat, affecting human, animal, and environmental sectors of the One Health framework. While previous studies in Lebanon have examined resistant bacteria in clinical, animal, and environmental settings [9], the role of companion animals, specifically cats, in the spread of resistance determinants remains unaddressed. To fill this gap, we conducted molecular characterization of ESBL- and carbapenemase-producing Enterobacteriaceae isolated from cat fecal samples to evaluate their contribution to AMR emergence and dissemination in Lebanon.

A total of nine ESBL-producing and four carbapenemase-producing Enterobacteriaceae were isolated from seven different fecal samples collected from diseased cats in Beirut. Differences at the level of the resistome, plasmidome, and AST patterns were detected, including ESBL and carbapenemase gene content (Fig. 1). Among the recovered isolates, several were untypable and were assigned novel sequence types (Table 1). All isolates were resistant to the fourth-generation cephalosporin cefepime, and four also showed resistance to all tested carbapenems (Table 2). Based on these resistance profiles, they meet the WHO’s criteria for classification as critical priority bacterial pathogens [77], given the limited treatment options, high potential for transmission, and their impact on global health systems. In silico analysis identified distinct combinations of resistance genes and plasmid incompatibility (Inc) types, which correlate with the antimicrobial resistance patterns observed in susceptibility testing.

E. coli ST167 harboring bla_NDM-5 is a high-risk global clone detected in a number of countries, including Italy [45, 50], Finland [62], and Switzerland [47], and is the most commonly detected ST in our study (n = 3) (Table 1, Table 3). Consistent with our findings, this clone extends beyond clinical settings and appears in diverse ecological niches such as companion animals, surface waters, and wildlife [45, 47] (Table 3). It was previously isolated from companion animals in Italy and Finland [45, 62] and hospitalized patients in Lebanon [60] (Table 3). In this study, EC23, EC25, EC28 were typed as ST167 (H9:O101), with EC23 and EC28 harboring bla_NDM-5 (Table 1, Table 3). Phylogenetic analysis showed that these isolates clustered with ST167 strains recovered from hospitalized patients in Lebanon and Italy, as well as from a cat in Italy, supporting the potential for interspecies transmission and international dissemination of this lineage (Fig. 3). The identification of this high-risk clone in cat fecal material reinforces its prevalence and role in the mobilization of resistance genes, highlighting the urgent need for a comprehensive national surveillance program.

E. coli belonging to clonal complex CC23, particularly ST410 and ST90, are recognized as endemic carbapenemase-producing isolates [78], and clustered together phylogenetically (Fig. 3). E. coli ST410 is a global high-risk clone associated with resistance to third-generation cephalosporins and carbapenems, posing a significant threat due to its link with severe and recurrent infections [79]. Moreover, E. coli of this sequence type have demonstrated the ability to cross boundaries between wildlife, humans, companion animals, and environmental reservoirs [74, 76, 79] (Table 3). E. coli ST410 carrying bla_NDM-5 was previously isolated from a cat and a dog residing in the same veterinary hospital in South Korea [64]. In this study, EC24 and EC27 were identified as ST410, with EC24 carrying bla_NDM-5. These isolates clustered closely with ST410 strains from diverse ecological and geographical sources, regardless of their bla_NDM-5 status (Fig. 3). Although ST410 has previously been reported in hospitalized patients in Lebanon [11, 60], an estuary in Tripoli [71], and surface waters [51], there were no documented instances of these clones harboring bla_NDM-5 (Table 3, Fig. 3). Additionally, there are no previous reports of E. coli ST410 being isolated from livestock, poultry, or companion animals in Lebanon, revealing the role of companion animals as potential reservoirs and vectors in the mobilization and spread of critical resistance determinants. Another E. coli isolate carrying bla_NDM-5, EC26 (H9:O8), also belonged to clonal complex CC23 and sequence type ST90 (Table 1). This ST has been previously reported in hospitalized patients [66], as well as in wastewater and a refugee camp in Lebanon [69], but not in animals, indicating potential new routes of transmission. This observation extends to other sequence types identified in this study (Table 3), where conserved clustering within STs, regardless of host or geographic origin, suggests that these clones are highly successful at maintaining their genetic structure across varied environments (Fig. 3). This adaptability is likely supported by MGEs that facilitate the acquisition and dissemination of resistance genes.

CTX-M-15 is considered the most widespread β-lactamase among hospitalized patients both globally and in Lebanon [9]. Nationally, this enzyme has also been predominant in isolates recovered from estuaries [71], wastewater [69], and cattle [80]. Our findings further demonstrate its predominance in E. coli isolates from cats (Fig. 1). In contrast, previous studies have reported CTX-M-1 as the most common β-lactamase in companion animals [81, 82]. The widespread presence of CTX-M-15 in Lebanese patients, livestock, water sources, and now cats may be linked to the use of untreated natural spring water for both human and animal consumption, particularly in rural areas [9]. Consistent with this pattern, most isolates in this study belonged to phylogenetic group A. This contrasts with findings from other studies, where ESBL- and carbapenemase-producing E. coli from cats were more frequently assigned to phylogroups B2 [83] and C [84]. The predominance of phylogroup A has also been observed in E. coli from hospitalized patients [11, 60] and various water samples in Lebanon [51, 71]. The spectrum of ESBL-encoding genes identified in our study, including bla_CTX-M-15, bla_TEM-1B, bla_OXA-1, bla_NDM-5, bla_CTX-M-3, and bla_CMY-TYPE, reveals the resistance gene profiles reported in isolates from clinical, environmental, and agricultural sources [9, 51, 53, 69, 71]. The high prevalence of CTX-M-15 and the dominance of phylogroup A across these different niches further support the occurrence of antimicrobial resistance gene spillover between environmental reservoirs, clinical settings, and companion animals. These findings emphasize the role of companion animals as reservoirs of resistance determinants, contributing to cross-transmission to humans either directly or through environmental pathways.

To explore the potential mechanisms behind ARG spillover across ecological niches, we analyzed the mobilome of the study isolates. A diverse range of plasmid Inc groups was identified, with IncU and various IncF replicons (IncFIA, IncFIB, IncFII, IncFIC, IncFIIS) being the most common, followed by IncQ, IncX1, and IncI types (Fig. 1). Among these, IncU, rarely reported in clinical or environmental isolates in Lebanon, was predominant among the bla-type gene carriers in cat-derived isolates. Plasmids belonging to the IncU group are known for their broad host range, conjugative potential, and capacity to carry multiple resistance gene cassettes [85]. Likewise, IncI1 plasmids are increasingly recognized for their role in disseminating resistance genes, frequently associated with bla_CTX-M-15, and are widely distributed among Enterobacteriaceae from human, animal, and environmental sources [86]. IncF replicons are prevalent among Enterobacteriaceae in Lebanon across multiple niches and are globally recognized as major vectors for the dissemination of bla-type genes [53, 71, 87, 88]. In this study, IncF plasmids were frequently associated with the co-localization of bla_CTX-M-15 alongside either bla_TEM-1B, bla_OXA-1, or, in some cases, all three β-lactamase genes. These plasmids often also carried aminoglycoside resistance genes, consistent with previous reports from clinical isolates in Lebanon [53] and with global observations of IncF-mediated multidrug resistance [87]. The four carbapenem-resistant Enterobacteriaceae (CRE) isolates, EC23, EC24, EC26, and EC28, carried bla_NDM-5 on multi-replicon IncF plasmids, with mobilization attributed to an IS26-mediated composite transposon (Fig. 2A). Discrepancies were observed between in silico plasmid replicon detection and PBRT results, particularly for IncU plasmids, likely due to limitations of short-read assemblies and primer mismatches affecting PCR amplification [40]. in silico tools may fail to detect replicons split across multiple contigs, while mutations at primer binding sites can result in false negatives in PBRT [40]

All four genetic environments carrying bla_NDM-5 shared a conserved segment consisting of dsbD–trpF–ble–bla_NDM-5–IS30–IS26, with an upstream TnAS1 transposase potentially facilitating the mobilization of this IS26-flanked transposon. This conserved arrangement is associated with efficient dissemination and expression of bla_NDM-5, as the IS26 element is known to promote mobilization through recombination and transposition mechanisms [89].The presence of MDR gene cassette on IncF plasmids has been previously reported in human isolates from India [89] and Italy [50], and NDM-carrying IncF plasmids continue to raise concern, especially when found in high-risk clones such as ST167 [47].

In contrast, the bla_CTX-M-15 gene in EC8 was chromosomally integrated and co-localized with qnrS1, ISEcp1, ISKpn19, and Tn2 (Fig. 2B). Similar configurations have been reported in human isolates from Saudi Arabia and Zambia [90, 91], reflecting a broader trend of chromosomally encoded resistance cassettes supported by mobile genetic element insertions. These structures provide a stable, yet potentially mobilizable, platform for the dissemination of ARGs, particularly when linked to integrative elements or recombination systems [91].

Collectively, these findings highlight the complex and dynamic nature of the resistome in both companion and stray animals. The diversity of MGEs and plasmid incompatibility types, especially those associated with high-risk clones and resistance genes such as bla_NDM-5 and bla_CTX-M-15, emphasizes the critical role of the mobilome in facilitating resistance dissemination across ecological niches.

This study highlights the role of companion animals, particularly cats, as reservoirs of ESBL and carbapenemase-producing Enterobacteriaceae. Novel sequence types were identified along with diverse resistance genes, susceptibility profiles, and mobilome structures, with particular focus on the dissemination of bla_NDM-5. The detection of resistance determinants shared with clinical, environmental, and animal sources in Lebanon, along with high phylogenetic relatedness to globally reported isolates, suggests a common pool of antimicrobial resistance genes circulating across ecological niches. All isolates in this study are classified as WHO critical priority pathogens, emphasizing their public health relevance and the need for a national surveillance system that includes companion animals. Importantly, no significant differences were observed between isolates from stray and domestic cats, indicating similar levels of antimicrobial resistance risk across different animal lifestyles. These findings support a One Health approach by demonstrating the interconnectedness of human, animal, and environmental health in the spread of resistance. Given the study’s limited geographic scope and sample size, future research should include larger and more diverse samples to enable broader population-level insights.

Authors’ contributions

Study conception and design: ST, ME, CA; investigation and data collection: ME; analysis and interpretation of results: ST, ME, CA; draft manuscript preparation: ST, ME, CA; Supervision: ST and CA; Resources: ST. All authors reviewed the results and approved the final version of the manuscript.

Funding

Not applicable.

Data availability

All genomes generated and analyzed in this study have been deposited in the NCBI repository under BioProject accession number PRJNA1028851. The additional genomes used for comparison are available through previously published studies, as referenced. Accordingly, all data from this study are publicly accessible.

Declarations

Ethics approval and consent to participate

The Institutional Animal Care and Use Committee (IACUC) of the Lebanese American University reviewed the study and granted it exempt status, as the sample collection was non-invasive and involved no direct interaction with animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.