# Identification of ESBL-producing enterobacterales from vegetable plants: Preliminary findings from a small cross-sectional study in a rural area of Madagascar

Adrien Rieux, Mamitina Alain Noah Rabenandrasana

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Adrien Rieux, Mamitina Alain Noah Rabenandrasana. Identification of ESBL-producing enterobacterales from vegetable plants: Preliminary findings from a small cross-sectional study in a rural area of Madagascar. Environmental Microbiology Reports, 2025, 17 (3), pp.e70130. 10.1111/1758-2229.70130. hal-05428475

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# Identification of ESBL-Producing Enterobacterales From Vegetable Plants: Preliminary Findings From a Small Cross-Sectional Study in a Rural Area of Madagascar

Adrien Rieux¹ | Mamitina Alain Noah Rabenandrasana²

¹CIRAD, UMR PVBMT, F-97410 St Pierre, La Réunion, France | ²Experimental Bacteriology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar

**Correspondence:** Adrien Rieux (adrien.rieux@cirad.fr)

**Received:** 19 December 2024 | **Revised:** 13 May 2025 | **Accepted:** 6 June 2025

**Funding:** This work was supported by European Regional Development Fund, GURDT I2016-1731-0006632, Conseil Régional de La Réunion.

## ABSTRACT

Extended-spectrum beta-lactamases (ESBL)-producing enterobacterales are considered a key indicator for antimicrobial resistance (AMR) epidemiological surveillance in animal, human, and environment compartments. In this study, we aim to investigate the presence and genetic diversity of ESBL-producing enterobacterales on vegetable plants. We isolated beta-lactam resistant enterobacterales from several vegetable plants and sequenced their whole genome. Utilising standard genomic and phylogenetic methods, we sought to (i) characterise the resistance genes and plasmid content of the plant-isolated strains, (ii) investigate their genetic structure, and (iii) determine their relationships with strains from other reservoirs. Among the 22 strains collected from vegetable plants, 6 showed resistance to beta-lactam antibiotics, with 5 of them identified as ESBL producers. Our results indicated the presence of multidrug-resistant (MDR) strains containing multiple antibiotic resistance genes (ARGs). Importantly, no host-specific lineages were identified among the plant-isolated ESBL-producing *E. coli* (ESBL-Ec). Instead, these strains exhibited genetic and epidemiological connections with strains isolated from animals, humans, and the environment, suggesting transfer of ESBL-Ec between plants and other sources in rural Madagascar. These preliminary findings suggest that vegetable plants are contaminated as a result of human activities, posing a potential risk of human and animal exposure to antibiotic-resistant bacteria and genes.

## 1 | Introduction

Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are a leading cause of human and animal infection, being categorised as critically important pathogens by the World Health Organization (Tacconelli et al. 2018). In this context, antimicrobial resistance (AMR) is recognised as a major One Health challenge because of the rapid emergence and dissemination of resistant bacteria and genes among humans, animals and the environment (McEwen and Collignon 2018). Aside from assessing AMR prevalence, there is an increasing need to better understand behaviours, customs and practices that drive

the evolution and transmission of resistance, both globally and in low-resource settings in which the AMR threat is of particular concern (Rousham et al. 2018). In this context, the use of antibiotics in agriculture has historically been described as a major contributor to AMR in humans due to a large variety of potential dissemination pathways, with most of them involving the influence of livestock (Thanner et al. 2016). The role of livestock in the emergence of AMR was shown to be multifaceted, involving both direct transmission to humans and environmental contamination (Bava et al. 2024). In contrast, little is known about the existence and prevalence of antimicrobial resistant bacteria and resistance genes (ARB & ARG, respectively) within

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Environmental Microbiology Reports* published by John Wiley & Sons Ltd.</footnote>
the cultivated plant reservoir, despite the fact that such food is often consumed raw, hence increasing the risk of transmission to humans (Hölzel et al. 2018). Herein, we aimed to characterise the prevalence and genetic diversity of beta-lactam resistant enterobacteriaceae isolated from vegetable plants in a rural area of Madagascar. To assess the genetic and epidemiological relationships between strains isolated from plants and other reservoirs, we integrated data from a cross-sectional population-based recent study performed within the same area on animals, humans and the environment (Gay et al. 2023).

## 2 | Experimental Procedures

### 2.1 | Sampling, Bacterial Culture and Sequencing

In September 2018, 22 vegetable plants (spinach, lettuce, tomato and cabbage) were sampled within 6 backyard gardens in Andoharanofotsy, Madagascar (Figure S1). Sampling was performed using sterile equipment. Plant leaves were transported back to the laboratory in individual envelopes before being finely cut and soaked in LB broth for 24h at 35°C ± 2°C with continuous shaking. 100 µL of the enriched suspension was directly streaked onto selective chromogenic agar plates (CHROMagar ESBL; CHROMagar, Paris, France) and incubated overnight at 35°C ± 2°C under aerobic conditions. All presumptive ESBL-producer morphotypes were subcultured individually on LB agar plates.

In vitro antimicrobial susceptibility testing was performed on one isolate according to the standard disc methods described in the 2024 'Comité de l'Antibiogramme de la Société Française de Microbiologie' (CASFM)-EUCAST guidelines (Cattoir 2024). The presence of ESBL enzymes was confirmed by synergy of cefotaxime, ceftazidime and cefepime with amoxicillin/clavulanate or ticarcillin/clavulanate.

One bacterial isolate per sample was randomly selected for genetic analysis. DNA extraction was performed using the Cador Pathogen Extraction Kit (INDICAL Bioscience) from 5 mL of liquid cultures grown overnight at 37°C in LB broth medium. Library preparation was performed using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) and sequencing was performed on a NextSeq 500 platform (Illumina) using 2×150 bp runs.

### 2.2 | Core Genome Analyses

Raw reads were trimmed with Trimmomatic (Bolger et al. 2014) to remove adapters and low-quality sequences before being *de novo* assembled using the Unicycler pipeline (Wick et al. 2017) (default parameters). Taxonomic assignment was performed on assembled bacterial genomes both by computing ANI using FastANI (Jain et al. 2018) and by running Kraken on the microbial reference database (Wood and Salzberg 2014). In order to investigate the phylogenetic structure of the plant-isolated ESBL genomes within a global One-health framework, we aligned the bacterial genomes generated within the course of this study with a public dataset of 510 ESBL strains also isolated in 2018 from human, animal and water in the same rural area of Madagascar

(Gay et al. 2023). Genomes were aligned using *parsnp* (Treangen et al. 2014) and regions acquired via horizontal gene transfers were detected using *Gubbins* (Croucher et al. 2015). From the recombination-free SNP alignment, a maximum likelihood phylogeny was constructed using *RAxML* (Stamatakis 2014) with a rapid bootstrap analysis, general time-reversible model of evolution with a four rate categories γ distribution (GTRGAMMA) and 1000 iterations. Transmission clusters were inferred from the reconstructed phylogeny using the dedicated Phydelity tool (Han et al. 2019).

### 2.3 | Genotyping, Mobilome and Resistome Analysis

Sequence types and phylogroups were estimated *in silico* using staram (Bharat et al. 2022) and ClermonTyping (Beghain et al. 2018) softwares, respectively. Staramr was also used to scan bacterial genome contigs against the ResFinder, PointFinder, and PlasmidFinder databases to detect the presence of plasmids and antimicrobial resistance genes. RFPlasmid (van der Graaf-van Bloois et al. 2021) was used to predict whether the detected ARGs were located on plasmids or chromosomes.

## 3 | Results

### 3.1 | Prevalence of Beta-Lactam Resistant and ESBL Producing Enterobacterales From Vegetable Plants

We identified beta-lactam resistant enterobacterales from 6 out of 22 collected plants (average prevalence of 27%, Figure 1A). Resistant isolates were isolated from 5 out of the 6 sampled backyard gardens and originated from lettuce (n = 2), spinach (n = 2), cabbage (n = 1) and tomato (n = 1) (Figure 1B).

In vitro antimicrobial susceptibility testing yielded variable results according to the strains and antibiotics tested (Figure S2). All 6 strains were sensitive to Ertapenem & Imipenem, resistant to Amoxicillin, Ceftazidim & Cefotaxim and showed variable sensitivity patterns towards the other tested antibiotics. Five strains (P4, P7, P12, P15 and P16) passed the synergy test and were hence considered as ESBL producers. Strain P18, although resistant to cefotaxime, ceftazidime, ticarcillin/clavulanate & amoxicillin/clavulanate did not produce any ESBL.

### 3.2 | Bacterial Sequencing

Sequencing generated around 8 million paired-end reads with a majority (99.90%–99.96%) of bases scoring Q30 and above. *Denovo* assembly statistics are given in Table S1.

### 3.3 | Taxonomic Assignment, Mobilome and Resistome

Out of the 6 beta-lactam resistant enterobacterales, 5 were assigned to *Escherichia coli* and 1 to *Enterobacter asburiae* species. The five *E. coli*, all ESBL producers, were assigned to three
A

<table><thead><tr><th>Host plant</th><th>Sampling (N)</th><th>ESBL+ (N)</th><th>Prevalence</th></tr></thead><tbody><tr><td>Lettuce</td><td>7</td><td>2</td><td>0,29</td></tr><tr><td>Spinach</td><td>8</td><td>2</td><td>0,25</td></tr><tr><td>Tomato</td><td>4</td><td>1</td><td>0,25</td></tr><tr><td>Cabbage</td><td>3</td><td>1</td><td>0,33</td></tr></tbody><tfoot><tr><th>Total</th><td>22</td><td>6</td><td>0,27</td></tr></tfoot></table>

B

<table><thead><tr><th>Sample</th><th>Host</th><th>Species</th><th>ST</th><th>Phylogroup</th></tr></thead><tbody><tr><td>P4</td><td>Lettuce</td><td><i>E. coli</i></td><td>1727</td><td>B1</td></tr><tr><td>P7</td><td>Lettuce</td><td><i>E. coli</i></td><td>354</td><td>F</td></tr><tr><td>P12</td><td>Cabbage</td><td><i>E. coli</i></td><td>12821</td><td>B1</td></tr><tr><td>P15</td><td>Spinach</td><td><i>E. coli</i></td><td>46</td><td>A</td></tr><tr><td>P16</td><td>Tomato</td><td><i>E. coli</i></td><td>8680</td><td>A</td></tr><tr><td>P18</td><td>Spinach</td><td><i>E. asburiae</i></td><td>-</td><td>-</td></tr></tbody></table>

C

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

D

<caption>**FIGURE 1** | (A) Sampled plants and prevalence of isolated beta-lactam resistant enterobacterales. (B) Taxonomic assignment of isolated enterobacterales. (C) Pairwise SNP number between the five ESBL-Ec. (D) Phylogenetic maximum likelihood tree of 515 ESBL-Ec genomes built from 329,817 core and non-recombinant SNPs. Hosts for each isolate (human, animal, plant, water) are depicted using different colours, with plant isolates highlighted in green. Transmission clusters and their composition are indicated with red frames and pie charts, respectively. The number of isolates in each cluster (N) and their average SNP count (SNP) are shown below.</caption>

different phylogroups (A; n=2, B1; n=2 and F; n=1) and 5 STs (1727, 354, 12,821, 46 & 880), respectively.

A total of 14 different ARGs, 5 point mutations and 5 plasmids were identified among the six plant beta-lactam resistant enterobacterales (Figure 2 and Table S2). In total, the six isolated strains harboured ARGs against seven different antibiotic families. The five ESBL-Ec strains were predicted to be resistant to at least three antibiotic families and hence considered as multiple drug resistant (MDR). All six strains were predicted to be resistant to several antibiotics from the Beta-lactams family. When comparing resistance patterns predicted *in silico* with those determined in vitro, we observed consistent results for 94% (74/78) of the strains/antibiotics tested (Figure S3).

The most frequently detected ESBL-gene was blaCTX-M-15, and a total of 5 different plasmids (IncY, IncFIB, IncFIC, IncFII and IncI1-I) were shown to segregate within the sampled strains. Detected ARGs were predicted to be carried either by chromosomes or plasmids (Table S2) except for bla<sub>CTX-M-15</sub>, which was detected both on a chromosome (strain P7) and a plasmid (strain P4). Most detected ARGs displayed very high nucleotide identity rates with reference genes (Table S2).

## 3.4 | Genomic Diversity and Phylogenetic Structure

As illustrated by their assignment to unique STs, the five ESBL-Ec strains isolated from plants displayed high levels of genomic diversity with a mean pairwise SNP number = 54,851 (Figure 1C). In order to decipher their genetic relationships

with strains isolated from other reservoirs, we built a phylogenetic tree using a dataset composed of previously published ESBL-Ec genomic resources (Gay et al. 2023) (Total of 329,817 SNPs in between 515 ESBL-Ec strains). Interestingly, plant ESBL-Ec strains did not cluster together within the phylogenetic tree but were instead intermixed within other human/animal/environment-isolated strains (Figure 1D). Furthermore, among the five plant ESBL-Ec strains, four were assigned to transmission clusters with other strains originating from other compartments (Figure 1D and Table S3). Strain P16, isolated from tomato leaf, was not assigned to any transmission cluster.

## 4 | Discussion

In this study, we report for the first time the presence of ESBL-producing enterobacterales from vegetable plants sampled in Madagascar. In this regard, the occurrence of ESBL producers in various crops and fresh vegetables has previously been described in other countries (Hölzel et al. 2018; Lopes et al. 2021; Reuland et al. 2014; Zeynudin et al. 2025; Tresch et al. 2024; Zurfluh et al. 2015). Interestingly, members of the enterobacterales order have previously been shown to exhibit both an endophytic (i.e., colonise internal tissues) and epiphytic (i.e., live and multiply on the outside of aerial surfaces) lifestyle on vegetable plants (Lopes et al. 2021). Unfortunately, the methodology used in this study did not allow for a distinction between the two lifestyle modes. Among the five *E. coli* strains recovered, isolate P15 sampled from spinach was assigned to ST46, which encompasses pathogenic strains responsible for septicaemia, diarrhoea, meningitis, and urinary tract infections (Vignaroli et al. 2016). In addition, P18, also isolated from spinach, was
<figure><img src="image_5.png" /><figcaption>**FIGURE 2** | Distribution of AMR genotypes and predicted resistance phenotypes among the six plant-isolated beta-lactam resistant enterobacterales strains. Filled boxes indicate the presence of ARGs, point mutations, plasmids, or a predicted antibiotic-resistant phenotype, with colours corresponding to different antibiotic families.</figcaption></figure>

assigned to *E. asburiae*, a bacterial species previously shown to be associated with humans and animals while also causing disease in plants such as rice and radish (Xue et al. 2021; Wang et al. 2023). One limitation of this study is that only a single bacterial isolate was analysed per sample, which, while practical for a preliminary investigation, may introduce selection bias and potentially overlook the presence of other co-existing resistant strains within the same sample.

Determinants of antimicrobial resistance and plasmids were in silico detected from the genomic data. Among the detected genes providing resistance to beta-lactams, *bla*<sub>CTX-M-15</sub> and *bla*<sub>CTX-M-55</sub> are both members of the CTX-M-1 group, considered as one of the most predominant enzymes in the widespread dissemination of ESBLs (Yang et al. 2023). Interestingly, those ESBL genes

have previously been detected within different compartments in Madagascar (Gay et al. 2023; Milenkov et al. 2021), highlighting their relevance to One Health concerns. In this context, a recent study revealed frequent and multiple transmission events between ESBL-Ec strains isolated from humans, animals, and the environment (drinking water) in a suburban rural area of Antananarivo, Madagascar (Gay et al. 2023). Through a dedicated phylogenetic analysis, we demonstrated that the newly isolated ESBL-Ec strains from vegetable plants were interspersed within the genomic diversity of strains from the other compartments, as illustrated by the inference of shared transmission clusters. The lack of genomic diversity within the detected clusters supports a likely recent epidemiological connection between strains from vegetable plants and other reservoirs. Although the origins of ESBL-producing enterobacterales
found in this study on vegetable plants were not investigated,
they could originate from human (as sewage), animal (manure),
and/or environmental (such as contaminated soil and irrigation
water) sources that come into contact with crops (Beuchat 2002;
Araújo et al. 2017). Therefore, our pilot results suggest that the
consumption of raw vegetables should be seen as a public health
problem and a potential risk of human exposure to antibiotic-
resistant bacteria and/or their resistance genes. Future studies
performed on a higher number of samples are required to deci-
pher more accurately the drivers and pathways of ARB & ARG
transmission between plants and other compartments, in order
to gather data for risk assessment.

Author Contributions

A.R. designed the study and performed field sampling. M.A.N.R. per-
formed lab work and assisted with the sequencing work. A.R. and
M.A.N.R. both performed genomic analyses and wrote the manuscript.

Acknowledgements

We thank Noellie Gay/the Andoharanofotsy community (mayors, heads of fokontanys) for help with sampling and the staff of the Plateforme de Microbiologie Mutualisée (P2M) at Institut Pasteur Paris for their help with whole-genome sequencing. We are also grateful to Tania Crucitti and Olivier Pruvost for valuable comments and discussions. Computational work was performed on the MESO@LR-Platform at the University of Montpellier (https://hal.umontpellier.fr/MESO). This work was financially supported by the European Regional Development Fund (ERDF contract GURDT I2016-1731-0006632) and Région Réunion.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are openly avail-
able in ncbi at https://www.ncbi.nlm.nih.gov/, reference number
PRJNA1147432.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section.