# Dissemination of antibiotic resistance in a constructed wetland

Florian Giudici, Estelle Saras, Marie-Noëlle Pons, Aïcha Hamieh, Agnese

Lupo, Nouceiba Adouani, Marisa Haenni, Xavier Bellanger

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## To cite this version:

Florian Giudici, Estelle Saras, Marie-Noëlle Pons, Aïcha Hamieh, Agnese Lupo, et al., Dissemination of antibiotic resistance in a constructed wetland. Antimicrobial Resistance in Animals & the Environment (ARAE), Jun 2025, Berlin, Germany. hal-05147716

HAL Id: hal-05147716

https://hal.science/hal-05147716v1

Submitted on 7 Jul 2025

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# Dissemination of antibiotic resistance in a constructed wetland

Florian GIUDICI<fnref n="1" />,<fnref n="2" /> (florian.giudici@univ-lorraine.fr), Estelle SARAS<fnref n="3" />, Marie-Noëlle PONS<fnref n="2" />, Aïcha HAMIEH<fnref n="3" />, Agnese LUPO<fnref n="3" />, Nouceiba ADOUANI<fnref n="2" />, Marisa HAENNI<fnref n="3" />, Xavier BELLANGER<fnref n="3" />

LCPME LRP
LABORATOIRE REACTIONS DES PROCESSES

cnrs

anses

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Zones
Ateliers

LTER FRANCE BASSIN DE LA MOSEBLE

## CONTEXT

An increasing number of constructed wetlands (CW) are being used to receive treated water from Wastewater treatment plants (WWTPs) before its final discharge into the environment. This nature-based treatment primarily aims to improve the physical and chemical properties of the water, particularly by reducing the levels of certain chemical elements and pharmaceutical products. However, WWTPs are not designed to eliminate all antibiotic resistance genes (ARGs) in wastewater. These genes are then commonly released in CWs downstream of WWTPs. Nevertheless, the impact of flowing treated water in a CW on antibiotic resistance remains unknown. Therefore, analysing the fate and behaviour of ARGs and the mobile genetic elements (MGEs) that carry them when released into CWs appears to be necessary.

This study aims to determine whether discharging treated water into the CW would decrease the abundance of certain markers or, conversely, increase it in the receiving aquatic environment. Monthly sampling campaigns are performed in the Grand Reims CW, which receives treated water from the local WWTP. Located upstream of the La Vesle River, this CW comprises three ponds, each with a surface area of two hectares, making it a large-scale pilot infrastructure containing various plant species. Animals such as swans, coypus and foxes have taken up residence on the CW, meaning they could be exposed to the discharge of ARGs and MGEs in the water.

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## METHODS

### Sampling site

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### Collected samples

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Faeces from swans are compared to samples collected on a protected area near Reims (Lac du Der: LDD).

Water

Faeces

### Culture-based analyses

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Total coliforms
counting

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Third-generation
cephalosporin- and
carbapenem-resistant
Enterobacteriales counting

→ Antibiograms

### Molecular biology & Bioinformatics-based analyses

<figure><img src="image_11.png" /></figure>

Markers quantification
(qPCR):

* 16S rDNA,

* MGE: IncF (tral) & IncP (trfA) plasmids, class 1 integrons (intI1),

* Integron cassettes: aadA, aadB, aacA4.

Total DNA extraction

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Shotgun
metagenomic

Taxonomic
classification of
bacterial families

Relative abundance

MGE:
MGE copies

16S rDNA copies

Cassette copies

intI1 copies

## RESULTS

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### Taxonomic classification of bacterial families

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### Escherichia coli counting

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(Nov. 2023 - April 2025)

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**Legend:**

E<sub>wwTP</sub>: WWTP entry,

P<sub>J</sub>E: Pond 2 entry,

Ex<sub>intI1</sub>: WWTP exit,

P<sub>J</sub>Ex, P<sub>J</sub>Ex, P<sub>J</sub>Ex: Ponds 1, 2 & 3 exits

→ A clear shift in the bacterial community composition is observed between the Entry and Exits of the ponds. Anthropogenic bacteria (e.g. E. coli) from the WWTP entry tended to disappear in favor of environmental (aquatic, terrestrial) species.

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### Class 1 integrons (intI1) relative abundance

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### IncP plasmids relative abundance

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→ For the MGEs, there is a significant decrease between the Entry and the Exits only on few campaigns (4/20 for the relative abundance of Class 1 integrons and 8/20 for IncP plasmids)

→ Low to moderate impact of flowing the treated water through the Constructed Wetland.

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### aadA relative abundance

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### aadB relative abundance

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→ For the integron cassettes, there is a significant decrease between the Entry and the Exits only in half of the campaigns (10/20) for the relative abundance of aadA and in just a few campaigns for aadB (3/20)

→ Low to moderate impact of flowing the treated water through the Constructed Wetland.

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### Abundances of resistant-Enterobacteriales

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#### Frequency of positive water samples
(whole months counted)

<table><thead><tr><th></th><th>E<sub>wwTP</sub></th><th>Ex<sub>wwTP</sub></th><th>P<sub>J</sub>E</th><th>P<sub>J</sub>Ex</th><th>P<sub>J</sub>Ex</th></tr></thead><tbody><tr><td>CTXM-1</td><td>94%</td><td>82%</td><td>71%</td><td>12%</td><td>12%</td></tr><tr><td>CTXM-9</td><td>59%</td><td>35%</td><td>24%</td><td>0%</td><td>6%</td></tr><tr><td>ESBL</td><td>100%</td><td>100%</td><td>76%</td><td>12%</td><td>12%</td></tr><tr><td>OXA-48</td><td>65%</td><td>59%</td><td>12%</td><td>0%</td><td>0%</td></tr><tr><td>NDM</td><td>29%</td><td>12%</td><td>12%</td><td>0%</td><td>0%</td></tr><tr><td>MDR</td><td>0%</td><td>6%</td><td>6%</td><td>0%</td><td>0%</td></tr><tr><td>CHN</td><td>0%</td><td>6%</td><td>0%</td><td>0%</td><td>0%</td></tr></tbody></table>

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#### Frequency of positive
faeces samples

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<table><thead><tr><th></th><th>Résistance</th><th>Proportion</th></tr></thead><tbody><tr><td>CTXM-1</td><td>18,55%</td></tr><tr><td>CTXM-9</td><td>4,84%</td></tr><tr><td>ESBL</td><td>24,19%</td></tr><tr><td>OXA</td><td>2,42%</td></tr><tr><td>MDR</td><td>2,42%</td></tr><tr><td>CHN</td><td>3,23%</td></tr><tr><td>VIM</td><td>0,81%</td></tr><tr><td>LDD</td><td>ESBL</td><td>3,45%</td></tr></tbody></table>

→ Resistant bacteria tend to disappear while the water flows through the WWTP and CW. There are few to no resistant bacteria found at the Exits of the ponds, even though they are sometimes present at the entry of the CW.

→ There are higher proportions of resistant bacteria, and a greater variety of resistances in swans faeces from Reims than in those from Lac du Der.

<figure><img src="image_32.png" /></figure>

### MGE relative abundances in swan faeces

### Relative abundances of integron gene
cassettes in swan faeces

<figure><img src="image_33.png" /></figure>

→ For the MGE, there is a significant difference between the two sampling sites for class 1 integrons relatives abundances. Indeed, their abundance is higher in Reims' samples. For the cassettes, there is qualitatively less cassettes in the integrons in the swans faeces from Reims.

## CONCLUSIONS

Changes in the composition of bacterial communities during water flow in constructed wetlands, coupled with the stable relative abundances of MGEs/ARGs, suggest horizontal gene transfer between anthropogenic and environmental bacteria and/or coalescence of anthropogenic and environmental bacterial communities. These analyses also demonstrate the limited effectiveness of the CW in reducing antibiotic resistance markers (MGEs and ARGs), with only a few campaigns showing a significant decrease.

Nevertheless, the amount of resistant *Enterobacteriales* decreases as water flows through the WWTP-CW ecosystem, supporting the hypothesis of community coalescence. Regarding swan faeces, the high load of resistant *Enterobacteriales* in the CW strongly suggest contamination by anthropogenic bacteria. In addition, in a genetic point of view, swans microbiota seems impacted by the origin of the water near which they live.

In the short term, we plan to sequence the whole genome of some resistant *Enterobacteriales* in order to characterize them, as well as to analyze the collected samples using high-throughput quantitative PCR on a large panel of MGEs/ARGs.

<footnote n="1">¹ LCPME UMR7564, Université de Lorraine - CNRS, Vandoeuvre-lès-Nancy, France;</footnote>

<footnote n="2">² LRGP UMR7274, Université de Lorraine - CNRS, Nancy, France;</footnote>

<footnote n="3">³ Unité Antibiorésistance et Virulence Bactériennes, Université de Lyon - ANSES, Lyon, France</footnote>