# Impacts of chemicals and microbiota from a hospital on the emergence of resistance in sewer systems

INRAE

envt

École
nationale
vétérinaire
toulouse

EDAR7
(Environnement et Santé)
(École d'Études en Environnement et Santé)

Sarah Naudin¹, Pedro Henrique Imazaki¹, Véronique Dupouy¹, Aude A. Ferran¹, Nathalie Arpaillange¹, Natalia Klimova², Claire Hoede³, Felipe Ramon-Portugal¹, Claire Gibson², Maialen Barret⁴, Dominic Frigon² and Delphine Bibbal¹

¹ INTHERES, Université de Toulouse, INRAE, ENVT, Toulouse, France ; ² Department of Civil Engineering, McGill University, Montreal, QC, Canada ; ³ Université de Toulouse, INRAE, UR 875 MIAT, BioinfOmics, Castanet-Tolosan, France ; ⁴ Laboratoire Ecologie Fonctionnelle et Environnement, Université de Toulouse, CNRS, Toulouse, France

From a One Health perspective, understanding the role of the environment in the rise of antimicrobial resistance is critical. Hospital wastewaters (HWW) stand out as critical hotspots to be addressed, as they harbor a complex mix of antibiotic resistant bacteria (ARB), pathogenic bacteria and a cocktail of chemical agents exerting a selective pressure. The impact of HWW and its different constituents (chemicals and microbiota) when mixed with domestic wastewater (DWW) was explored in this study.

## What is the respective role of the microbiota and chemicals originating from HWW in the emergence and dissemination of AMR ?

### Materials and methods

* Wastewater sampling:

* Exposition experiments for 96h:

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* Measure of the percentage of ARBs resistant to ciprofloxacin (CIP), cefotaxime (CTX) and erythromycin (ERY)

* Characterization of antibiotic resistance genes (84 genes) and mobile genetic elements (MGEs) (11 genes) with high-throughput qPCR (Fluidigm)

* Sequencing of 30 ARG variants from microcosms at 24h

### Wastewater characterization

* Percentage of resistance to CIP and CTX were **significantly higher** in HWW than DWW or MWW.

<figure><img src="image_2.png" /><figcaption>Percentage of ARB resistant to ciprofloxacin (CIP), cefotaxime (CTX) and erythromycin (ERY) in wastewater samples</figcaption></figure>

* 30 ARGs across several classes are significantly different between wastewater types, **most of them higher in HWW**.

<figure><img src="image_3.png" /><figcaption>Scaled relative quantification of ARGs and MGEs with a significant difference between environmental samples (Kruskal-Wallis, p<0,05) (top) and corresponding sum of relative abundance for all samples (bottom)</figcaption></figure>

* Resistance is consistently higher in HWW compared to DWW and MWW.

* The impact of HWW on DWW is complex: evidence suggests immigration and/or selective pressure, warranting further investigation.

* ✓ Microcosms experiment will be repeated

* ✓ 16S rRNA sequencing will be performed

### Microcosm experiments

* Monitoring of percentage of resistance to CIP showed similar resistance profiles between DWW exposed to HWW and DWW exposed only to its chemical part → **consistent with selection by HWW chemicals**

<figure><img src="image_4.png" /><figcaption>Percentage of ARB resistant to ciprofloxacin (CIP) and cefotaxime (CTX) in microcosms over 96 hours</figcaption></figure>

* When ARGs were grouped by drug family, only two classes showed an increase in DWW exposed to HWW → **consistent with impact of HWW**

* Differences unexplained by selection or immigration alone

<figure><img src="image_5.png" /><figcaption>Mean relative abundance of genes belonging to aminoglycosides (n = 4 genes) and macrolides (n= 6 genes) antibiotics in microcosms over 96 h</figcaption></figure>

* Identification of several variant profiles

<table><thead><tr><th rowspan="2">Single variant in all microcosms</th><th colspan="2">Unique variant(s) in HWW</th><th rowspan="2">Multiple variants in all microcosms</th></tr><tr><th>Not conserved when mixed with DWW</th><th>Conserved when mixed with DWW</th></tr></thead><tbody><tr><td>sul2, bla<sub>VEB</sub>, qnrD, dfrA12, tetW, mdtH, bla<sub>PDC</sub>, qnrV4</td><td>bla<sub>FOX</sub>, ermB, bla<sub>CMY-2</sub></td><td>bla<sub>MIR</sub>, mdtg</td><td>bla<sub>CTX-M-1</sub>, mcr-3, tetM</td></tr></tbody></table>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Number of reads of gene variants for *bla*<sub>FOX</sub> and *bla*<sub>MIR</sub> in microcosms</figcaption></figure>

This work was funded by the French National Research Program for Environmental and Occupational Health of Anses (Anses-22-EST-073). Antibiotic resistance gene sequencing was supported by an additional fund from Dominic Frigon's Lab. Sarah Naudin was partly funded by a Doctoral grant from La Région Occitanie (arrêté N°00137923/22009716). Doctoral mobility was supported by fund from Université Toulouse 3 and Walter-Zellidja's grant.

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sarah.naudin@envt.fr

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