Abstracts book: 9. Symposium on Antimicrobial
Resistance in Animals and the Environment - ARAE

Sylvie Baucheron, Sébastien Leclercq, Michel S Zygmunt, Axel Cloeckaert,
Benoît Doublet

► To cite this version:

Sylvie Baucheron, Sébastien Leclercq, Michel S Zygmunt, Axel Cloeckaert, Benoît Doublet. Abstracts book: 9. Symposium on Antimicrobial Resistance in Animals and the Environment - ARAE. 9th Symposium on Antimicrobial Resistance in Animals and the Environment, ARAE, Jul 2023, Tours, France. hal-04170355

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https://hal.inrae.fr/hal-04170355

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Abstracts book

ARAE

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9th Symposium on
Antimicrobial
Resistance in
Animals and the
Environment

2023

JULY 3-5

TOURS

FRANCE

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

PALAIS
DES CONGRÈS
TOURS

<figure><img src="image_5.png" /></figure>
Contents

Welcome page 2

Scientific programme page 7

Speaker abstracts page 15

Session 1 page 15

Session 2 page 25

Session 3 page 47

Session 4 page 58

Session 5 page 66

Sponsors page 74

Poster abstracts page 76

Session 1 page 76

Session 2 page 92

Session 3 page 123

Session 4 page 140

Session 5 page 160

Acknowledgements page 170
## ARAE 2023: An introduction

The emergence of antimicrobial resistance is a seminally important public health concern. Significant progress has been made in recent years regarding an understanding of the genetic and biochemical basis for antimicrobial resistance, the emergence of resistance genes, and factors promoting their widespread dissemination including the role of lateral gene transfer. Nevertheless, there is a dearth of information regarding the key ‘hotspots’ and genetic mechanisms responsible for resistance development, and the exposure routes leading to the failure of antimicrobial agents important in human and animal medicine. There is thus an urgent need for research to provide governments, public health stakeholders, and the agricultural sector the knowledge required to develop policies and practices that effectively mitigate resistance development. This, within a growing recognition that humans, animals and the environment must be considered as intimately linked together if any resistance management strategy is to be successful.

Livestock are in close contact with soil and water, natural reservoirs of microbiota harbouring resistance genes. In turn, the use of manures as fertilizers for crop production is a potentially important source for environmental contamination of resistance genes selected for and enriched in the animal. A better understanding of the significance of animal and environmental reservoirs of antimicrobial resistance, and factors leading to the emergence and dissemination of antibiotic-resistant bacteria in agricultural production systems is a priority.

The ARAE symposium, created by scientists from INRAE, is organized since 2005. Since the first edition in Lyon, ARAE has been a great success with many scientists from all over the world still participating in this symposium which is now in its ninth edition.

Benoît Doublet
*Chair*

Axel Cloeckaert
*Co-Chair*

Michel S. Zygmunt
*Co-Chair*
# Scope and aim of ARAE 2023

The aim of the ARAE conference is to present a global vision of the impact of antibiotic use and resistance in the animal world, its environment and consecutive repercussion on human health. During six sessions, all aspects related to epidemiology of antibiotic-resistant bacterial pathogens with a zoonotic potential, mobile elements containing resistance genes, emerging antimicrobial resistance mechanisms, resistome of microbiotas, and the role of the environment as dissemination routes and potential source of resistance genes transfer will be discussed.

## Themes

### 1- Roles of the environment in resistance evolution and transmission

While acquisition of new resistance factors in pathogens is uncommon, acquisition of resistance factors from other microbial species may occur in diverse natural microbial ecosystems, and may impact the prevention and cure of bacterial infections both in humans and animals. On the other hand, environmental pollution by already widespread resistances is common. Therefore, environmental health (water, sanitation, waste management, food hygiene and safety) appears essential to prevent the spread of antibiotic-resistant bacteria and reduce the global antibiotic resistance threat.
This session will consider the source, propagation, transfer and dissemination of resistance genes in the environment (aquatic, terrestrial, ...). The impact of agricultural practices, such as the veterinary use of antimicrobial agents and use of fertilizers of animal and human origins, will be emphasized.

### 2- Monitoring and molecular epidemiology of antimicrobial resistance

To determine the prevalence of antimicrobial resistance among the most relevant veterinary and zoonotic pathogens, monitoring programs are currently conducted worldwide either as large-scale national monitoring programs or as drug-specific monitoring studies. The results of such monitoring programs – in particular when supported by (a) molecular analysis of the resistance genes and resistance-mediating mutations, (b) studies on the transferability of the resistance properties, (c) genomic analysis of resistant strains, and (d) consumption data of antimicrobial agents – provide important information for the understanding of the development and spread of antimicrobial resistance. Examples of the results of such monitoring programs highlighting the molecular epidemiology of resistance properties will be presented and discussed in this session.

### 3- Mechanisms and dissemination of antimicrobial resistance in animal and zoonotic pathogens

Gaining insight into the mechanisms of antimicrobial resistance, long-term persistence, and successful spread, is fundamental to reduce the burden of antimicrobial resistance. This session will focus on emerging antimicrobial resistance mechanisms in animal and zoonotic pathogens. All mechanisms will be considered such as target modification, protection of the target, efflux, decreased permeability, enzymatic inactivation as well as induction of resistance mutagenesis... All mechanisms implicated in the successful spread and persistence of mobile genetic elements harbouring resistance genes (plasmids, integrative elements,...) will be also of particular interest for this session.

### 4- Understanding the connection of antimicrobial resistance between Animals and Humans

Antimicrobials used to treat infectious diseases in animals are often the same or similar to those used to treat human infections. Antibiotic-resistant bacteria may spread between animals and humans and reciprocally, and through their environment as well. It may in turn result in worldwide epidemics of
antibiotic resistant genes and their vehicle.

The spread of antibiotic-resistant bacteria and genes across ecosystems can occur through multiple ways, agriculture-related such as reuse of animal wastewater and manures, via the food chain (zoonotic or animal food contamination) and also by direct contact between animal and human (companion animals and wild life).

This session aims to gain insight on the origin and dissemination routes of antibiotic-resistant bacteria and genes in the animal-human-environment continuum. We welcome presentations providing new knowledge and insight in this field.

## 5- Novel approaches, methods and tools dedicated to antimicrobial resistance (detection, evolution, diagnostics, surveillance)

Monitoring antibiotic resistance dissemination in husbandry or in the environment, investigating the links between antimicrobial resistance in animals and humans, or exploring the diversity of mechanisms of resistance and mobile genetic elements, all rely on the production of an ever-growing amount of data. Specific tools and methodologies designed to manage such data are therefore constantly developed. The session is open for description of new methods and their diverse applications for exploring or monitoring antimicrobial resistance. Tools for genomic or metagenomic analyses as well as original molecular approaches, modeling approaches or laboratory/sampling procedures are all welcome in this session.
**International Scientific Committee**

Muna Anjum (UK)

Sylvie Baucheron (France)

Franck Biet (France)

Axel Cloeckaert (France)

Benoît Doublet (France)

Sabine Favre-Bonté (France)

Séamus Fanning (Ireland)

Sébastien Leclercq (France)

Jean-Yves Madec (France)

Vincent Perreten (Switzerland)

Laurent Poirel (Switzerland)

Etienne Ruppé (France)

Ashley Shade (USA)

Michel S. Zygmunt (France)

**Local Organizing Committee**

Sylvie Baucheron (France)

Franck Biet (France)

Axel Cloeckaert (France)

Benoît Doublet (France)

Anne-Lise Dumont (France)

Sébastien Leclercq (France)

Stéphanie Salmon (France)

Patrice Valade (France)

Michel S. Zygmunt (France)
**Host Institution**

INRA, UMR1282, Infectiologie et Santé Publique, ISP, Nouzilly, F-37380, France

**Scientific contact person:**

Dr. Benoît Doublet

Phone: +33 2 47 42 72 95

arae2023@inrae.fr; benoitdoublet@inrae.fr

https://arae2023.symposium.inrae.fr/

**Symposium Venue**

**Palais des congrès de Tours**
26 Boulevard Heurteloup
B.P.4225
37042 Tours Cedex 1 France

+33.(0)2.47.70.70.70

https://www.tours-evenements.com/palais-des-congres-de-tours
Monday July 3

08h00-08h50 Reception/Registration
08h50-09h00 Symposium opening

Session 1 - Roles of the environment in resistance evolution and transmission

Chair: Sabine Favre-Bonté

Co-chair & keynote speaker: **Ashley SHADE**

09h00-09h40: **Ashley SHADE**

Antibiotic resistance and production genes in the soil microbiome given anthropogenic disturbance: insights from the Centralia coal seam fire

09h40-09h55: **Ashlyn H. STRICKLAND**, Sarah A. MURRAY, Javier VINASCO, Jason E. SAWYER, Harvey M. SCOTT, Brent. W. AUVERMANN, Jack BUSH, and Keri N. NORMAN

Metagenomic analysis of the bacterial community structure within cattle feces, the feedyard environment, and particulate matter as a function of antibiotics, probiotics, and environmental manipulation

09h55-10h10: Gonçalo MACEDO, **Joost HORDIJK**, Pieter VAN VEELEN, Lucia HERNANDEZ LEAL, Alex BOSSERS, Asmus Kalckar OLESEN, Søren J. SØRENSEN, Dik HEEDERIK, Dik MEVIUS, and Heike SCHMITT

The effect of manure amendment on the soil grassland resistome

10h10-10h25: **Michaela RUZICKOVA**, Kristina NESPOROVA, Jana PALKOVICOVA, Simon KREJCI, Ivan LITERAK, and Monika DOLEJSKA

Dynamics of cephalosporin-resistant in *Escherichia coli* in gulls highlight the importance of longitudinal wildlife studies

10h25-10h40: **Dayana JARMA**, Oriol SACRISTÁN-SORIANO, Francisco HORTAS, Juan M. PERALTA-SÁNCHEZ, Alexandre SÁNCHEZ-MELSIÓ, José L. BALCÁZAR, Carles M. BORREGO, and Marta I. SÁNCHEZ

Spatial spreading of antibiotic resistance genes by gulls

10h40-11h10: Coffee break/Poster

11h10-11h25: Charlène SAGRILLO, Christophe MERLIN, and **Xavier BELLANGER**

Compartmentalization of antibiotic resistance between phages and bacteria in a river - wastewater treatment plant continuum

11h25-11h40: **Caroline WYBRANIEC**, Geneviève GAGNE, Cécile MOUSSARD, Marion BEAUPERE, Léa LUSURIER, Françoise LERICHE, Laurence MARJOLET1, Nicolas SERTILLANGES, Dominique PATUREAU, Benoit COURNOYER, and Wessam GALIA

Occurrence of 41 sanitary indicators in digestates derived from different anaerobic digestion processes and various raw organic wastes

11h40-11h55: **Brune JOANNARD**, Timothy M. VOGEL, and Concepcion SANCHEZ-CID

Microplastics increase the selective potential of antibiotics at sub-inhibitory concentrations

11h55-12h10: **Federica PIERGIACOMO**, Franck LEJZEROWICZ, Tam TRAN, Atif A. CHOWDURY, Luciano BENEDUCE, Odd-Gunnar WIKMARK, Leonardo PAGANI, Alexander EILER, and Lorenzo BRUSETTI

Do microplastics offer a suitable surface for antibiotic resistance spread into the open environment?
**12h10-12h25:** **Florent ROSSI**, Eloïse LEBRAS, Eric DEFERT, Nathalie TURGEON, Marc VEILLETTE, Jean-Luc BARAY, Pierre AMATO, and Caroline DUCHAINE
Can antimicrobial resistance genes travel across the Atlantic Ocean?

**12h30-14h00:** Lunch

**Session 2 - Monitoring and molecular epidemiology of antimicrobial resistance**

Chair: **Jean-Yves MADEC**

Co-chair & keynote speaker: **Muna ANJUM**

**14h00-14h40:** **Muna ANJUM**

Use of genomics to characterise antimicrobial resistances present within livestock on UK farms

**14h40-14h55:** **Olgica CERIC**, Jake GUAG, Sarah PELOQUIN, Amy MERRILL, Chih-Hao HSU, Claudine KABERA, and Gregory H. TYSON

The role of FDA's Veterinary Laboratory investigation and response network in monitoring antimicrobial resistance of animal pathogens

**14h55-15h10:** **Henrike KRÜGER-HAKER**, Xing JI, Dennis HANKE, Stefan FIEDLER, Andrea T. FEßLER, Nansong JIANG, Heike KASPAR, Yang WANG, Congming WU, and Stefan SCHWARZ

Genomic diversity of porcine LA-MRSA CC398 isolates collected in the German national resistance monitoring program GERM-Vet between 2007 and 2019

**15h10-15h25:** **Janine MULLER**, Yuhong LIU, Ilhan MOHAMMAD, Brendan RODONI, and Fiona CONSTABLE

Antimicrobial resistance in pathogenic bacteria of Victorian cattle

**15h25-15h40:** **Maud de LAGARDE**, John M. FAIRBROTHER, Marie ARCHAMBAULT, Simon DUFOUR, David FRANCOZ, Jonathan MASSÉ, and Jean-Philippe ROY

Clonal and plasmidic dissemination of critical antimicrobial resistance genes in dairy cattle in Québec, Canada

**15h40-15h55:** **Virginie GUÉRIN**, Nicolas CABANEL, Damien THIRY, Jacques MAINIL, Marc SAULMONT, and Philippe GLASER

A phylogeny study of β-lactam-resistant *E. coli* from Belgian calves

**15h55-16h05:** **Amelie BARTHELEMY** (StandardBio Tools)

Setting the new benchmarks in automated and cost-effective qPCR and NGS assay using a single platform

**16h10-16h40:** Coffee break/Poster

**16h45-17h00:** **Ahmad I. AL-MUSTAPHA**, Emmanuel AJ. AWOSANYA, Victoria O. ADETUNJI, and Annamari HEIKINHEIMO

Multidrug-resistant *Escherichia coli* isolated from slaughtered broilers in Ilorin, Nigeria

**17h00-17h15:** **Moniek RINGENIER**, Filip BOYEN, Sigrid C.J. DE KEERSMAECKER, Kevin VANNESTE, Bert BOGAERTS, Mathias DEVREESE, and Jeroen DEWULF

Fluoroquinolone resistance in *E. coli* in broilers: mapping the spread

**17h15-17h30:** **Alicia MANZANARES**, Florencia CORREA-FIZ, Teresa AYATS, Miquel NOFRARÍAS, and Marta CERDÀ-CUÉLLAR
In-vitro validation of AMR genes prevalence revealed by WGS in *Campylobacter jejuni* isolated from chicken livers in Spain

17h30-17h45: Ojas DIXIT, Aidan PREUSS, and **Claire L O'BRIEN**
Antimicrobial resistance determinants in bacteria isolated from retail chicken and pork meat across New South Wales, Australia

17h45-18h00: **Ramon P. MALUPING**, Remedios F. MICU, Thomas CHISNALL, Alistair DAVIES, Evelyn E. EMBESTRO, Mary Ann ESCOTO, Mildred A. PADILLA, and Roderick CARD
Prevalence of Extended-Spectrum Beta-Lactam (ESBL) and colistin resistance in *Escherichia coli* isolated from broiler chickens in dressing plants in the Philippines

18h00-18h15: Joel JAVID, Emmanuelle MOREAU, Lionel PINNEAU, Niki HAYATGHEIB, Claire MALTRET, Segolene CALVEZ, and **Nora NAVARRO-GONZALEZ**
Risk factors for antimicrobial resistant *Aeromonas* spp. and *Escherichia coli* in the rainbow trout farm environment in Brittany, NW France

**18h15-19h00: Poster session**

**19h00: Welcome reception**
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Tuesday July 4

Session 2 - continued

Chair: **Jean-Yves MADEC**

Co-chair & keynote speaker: **Muna ANJUM**

09h00-09h40: **Windi Muziasari**

Abundance and diversity of antibiotic resistance genes in different types of environmental samples: 5 years of Resistomap

09h40-9h55: **Lorcan O'NEILL**, Maria RODRIGUES da COSTA, Edgar GARCIA MANZANILLA, Fiona CRISPIE, Raul CABRERA-RUBIO, Rachel GRIMES, and Finola C. LEONARD¹

The evolution of the faecal resistome is shaped by age and antimicrobial use during the pig lifecycle

09h55-10h10: **Mike BROUWER**, Alieda van ESSEN-ZANDBERGEN, Yvon GEURTS, Frank HARDERS, and Kees VELDMAN

Genetic analysis of ampC promotor mutants of *E. coli* from livestock exhibiting resistance to extended-spectrum cephalosporins

10h10-10h25: **Sabine DELANNOY**, Corine HOFFER, Raphaëlle YOUF, Emilie DAUVERGNE, Hattie E. WEBB, Thomas BRAUGE, Mai-Lan TRAN, Graziella MIDELET, Sophie A. GRANIER, Marisa HAENNI, Patrick FACH, and Anne BRISABOIS

High throughput screening of antimicrobial resistance genes in Gram-negative seafood bacteria

10h25-10h40: **Sandra MARTÍNEZ-ÁLVAREZ**, Pierre CHÂTRE, Úrsula HÖFLE, Carla Andrea ALONSO, Pauline FRANÇOIS, Teresa CARDONA-CABRERA, Myriam ZARAZAGA, Jean-Yves MADEC, Marisa HAENNI, and Carmen TORRES

High occurrence of carbapenemase- and extended-spectrum-β-lactamase-producing *Escherichia coli* and *Klebsiella pneumoniae* from migratory birds (*Ciconia ciconia*) with detection of high-risk clones

**10h45-11h15: Coffee break/Poster**

11h10-11h25: **Maeve Louise FARRELL**, Georgios MILIOTIS, Firinne KELLY, Evan NAUGHTON, Brigid HOOBAN, Alexandra CHUEIRI, Mark MAGUIRE, Louise O' CONNOR, Martin CORMICAN, Niamh CAHILL, Kelly FITZHENRY, Aoife JOYCE, Sinead DUANE, Liam P. BURKE, and Dearbhaile MORRIS

Assessing the resistome and virulence of human and environmental *Escherichia coli* isolates

11h25-11h40: **Camille FAVIER**, Mylène TOUBIANA, Olivier COUILLEROT, Elodie PICHON, Patricia LICZNAR-FAJARDO, and Estelle JUMAS-BILAK

Wastewater-based Epidemiology (WBE): toward a relevant tool to monitor hospital antimicrobial resistance

11h40-11h55: **Andrea OTTESEN**, Brandon KOCUREK, Shawn BEHLING, Claudine KABERA, Patrick MCDERMOTT, and Errol STRAIN

Metagenomic survey of antimicrobial resistance in surface waters of Maryland with high and low human impact classifications

11h55-12h10: Iva SUKKAR, Adam VALCEK, Martin KLVANA, Jarmila LAUSOVA, Kristina NESPOROVA, **Jana PALKOVICOVA**, and Monika DOLEJSKA

Gram-negative bacteria resistant to last-line antibiotics from hospital and municipal wastewaters
12h10-12h25: **Mélanie PIMENTA**, Degrâce BATANTOU, Margaux GASCHET, Stéphanie GUYOMARD, Marie-Cécile PLOY, Sébastien BREUREC, and Christophe DAGOT on behalf of the ANR ACRAS-R consortium
One-Health AMR spread in a French Caribbean island

**12h30-14h00: Lunch**

## Session 3 - Mechanisms and dissemination of antimicrobial resistance in animal and zoonotic pathogens

Chair: **Axel CLOECKAERT**

Co-chair & keynote speaker: **Séamus FANNING**

14h00-14h40: **Séamus FANNING**

*Klebsiella* a hitherto underappreciated zoonotic pathogen expressing AMR phenotypes of importance to One Health

14h40-14h55: **Valentina DONÀ**, Sonja KITTL, Andrea ENDIMIANI, and Vincent PERRETEN

WGS-based characterization of emerging pathogenic OXA-48-producing *Enterobacter hormaechei* belonging to ST114 and ST418 in a Swiss companion animal clinic

14h55-15h10: **Jana PALKOVICOVA**, Adam VALCEK, and Monika DOLEJSKA

Massive plasticity of IncHI2-ST3 plasmids encoding clinically important IMP-4 in bacteria from silver gulls

15h10-15h25: **Laurent POIREL**, Otávio Hallal FERREIRA RARO, and Patrice NORDMANN

Determining the impact of veterinary antibiotics sub dosage on resistance transfer in animal gut microbiota - a One-Health approach

15h25-15h40: **Suad ALGARNI**, Jing HAN, Dereje GUDETA, Rajesh NAYAK, and **Steven L. FOLEY**

In silico analyses of Incompatibility group (Inc) HI2 plasmids from enteric bacteria

15h40-15h55: **Indre NAVICKAITE**, Harry HOLMES, Letizia DONDI, Luke RANDALL, Catherine FEARNLEY, Emma TAYLOR, Edward FULLICK, Manal ABUOUN, Christopher TEALE, and Muna F. ANJUM

Investigation of *rmtB*-harbouring *Salmonella* and *Escherichia coli* from a pig farm

**16h00-16h30: Coffee break**

16h35-16h50: **Xingyang DAI**, Li CHEN, Junjie SUN, Jinhu HUANG, and **Liping WANG**

Various mobile genetic elements involved in the dissemination of phenicol-oxazolidinone resistance gene *oprA* in *Streptococcus suis*

16h50-17h05: **Seiji YAMASAKI**, Ryosuke NAKASHIMA, Takayoshi SUZUKI, and **Kunihiko NISHINO**

Development of efflux pump MexB and MexY dual inhibitors against multi-drug resistant *Pseudomonas aeruginosa*

17h05-17h20: **Katie WALL**, Leonard KOOLMAN, Guerrino MACORI, and Séamus FANNING

Glyphosate induces changes to cellular metabolic pathways and selects for antimicrobial resistance in *Klebsiella* species

17h20-17h35: **Amandine CHAUVIAT**, Gwenaëlle GRAULIER, Sylvie NAZARET, and Sabine FAVRE-BONTE
<figure><img src="image_7.png" /></figure>

Quinolone resistance and expression of RND efflux pump in *Stenotrophomonas* spp. under exposure to sub-lethal concentration of nickel

17h35-17h55: **Emilie DEHON**, Alban MATHIEU, Arnaud DROIT, Timothy M. VOGEL, and Concepcion SANCHEZ-CID

Heavy metals may increase ARG persistence in the environment

**20h00-Midnight: Gala dinner**
## Wednesday July 5

### Session 4 - Understanding the connection of antimicrobial resistance between Animals and Humans

**Chair:** Laurent POIREL

**Co-chair & keynote speaker:** Vincent PERRETEN

**09h00-09h40:** Vincent PERRETEN

WGS-based relationship between multidrug-resistant bacteria from humans and animals: emphasis on CPE and MRSA

**09h40-9h55:** Rajesh NAYAK, Jing HAN, and Steven L. FOLEY

U.S. FDA's National Center for toxicological research AMR research: applications across the One Health Continuum

**09h55-10h10:** Juliette HAYER, Ella MARCY, Mallorie HIDÉ, Savatey HAK, Sivhour CHIEK, Navin SRENG, Meymey LEM, Rina DORK, Chiva MA, Gauthier DELVALLEZ, Patrice PIOLA, Véronique CHEVALIER, Anne-Laure BAÑULS, and Sokleaph CHENG

Investigating the circulation of AMR between animals, humans and their environment in Cambodia - ARCAHE

**10h10-10h25:** Sakib RAHMAN, and Aidan HOLLIS

The effect of antibiotic usage on resistance in humans and food-producing animals: a longitudinal, one-health study using European data

### 10h30-11h00: Coffee break/Poster

**11h05-11h20:** Michaela RUZICKOVA, Ivana JAMBOROVA, Tomas NOHEJL, Iva SUKKAR, Jana PALKOVICOVA, Ivo PAPOUSEK, Max CUMMINS, Steven DJORDJEVIC, and Monika DOLEJSKA

Comparative genomics of *Escherichia coli* ST131 of human, animal and environmental origin from the Czech Republic

**11h20-11h35:** Joyshri SARKER, Aylar SABA SHIRVAN, Céline DAVID, Stephane CORVEC, Eric BATARD, Maria UGARTE-RUIZ, Ségolène CALVEZ, and Nora NAVARRO-GONZALEZ

Understanding the risks associated with recirculating aquaculture systems: persistence of antimicrobial-resistant *Enterococcus* spp. and comparison with isolates from human infections

**11h35-11h50:** Marta ROZWANDOWICZ, Henrik HASMAN, Mike SM BROUWER, Muna F ANJUM, Stefan BÖRJESSON, Manuela CANIÇA, Jens A HAMMERL, Virginia CARFORA, Benoît DOUBLET, Antoni PA HENDRICKX, Emma ÖSTLUND, Jannice S SLETTEMEÅS, Magdalena ZAJĄC, Pieter-Jan CEYSSENS, Joost HORDIJK, and the Full Force consortium

Successful host adaptation of IncK2 plasmids

**11h50-12h05:** Ulrike BINSKER, Kathrin OELGESCHLÄGER, Annemarie KÄSBOHRER, and Jens A. HAMMERL

Indications of transmission of *mcr*-1.26 IncX4 plasmids along the poultry food chain to humans

### 12h10-13h40: Lunch
Session 5 - Novel approaches, methods and tools dedicated to antimicrobial resistance (detection, evolution, diagnostics, surveillance)

Chair: Sébastien LECLERCQ

Co-chair & keynote speaker: Etienne RUPPE

13h40-14h20: **Etienne RUPPE**

The present and future in antimicrobial resistance surveillance

14h20-14h35: **Carole KOWALEWICZ**, Michael TIMMERMANS, David FRETIN, Pierre WATTIAU, and Cécile BOLAND

An in-house 45-plex array for the detection of antimicrobial resistance genes in Gram-positive bacteria

14h35-14h50: **Yolanda GONZALEZ-FLORES**, Margaux GASCHET, Mélanie PIMENTA, Christophe DAGOT, and Marie-Cécile PLOY

Identification of novel antibiotic resistance integron gene cassettes in water samples with different anthropogenic levels

14h50-15h05: **Samuel J. BLOOMFIELD**, Aldert L. ZOMER, Justin O'GRADY, Gemma L. KAY, John WAIN, Nicol JANECKO, Raphaëlle PALAU, and Alison E. MATHER

Determination and quantification of microbial communities and antimicrobial resistance on food through host DNA-depleted metagenomics

15h05-15h20: **Ouléye SIDIBE**, Anne-Carmen SANCHEZ, Guillaume KON-KAM-KING, Fanny CALENGE, Benoit DOUBLET, Sylvie BAUCHERON, Sébastien LECLERCQ, and Anne-Laure ABRAHAM

Characterization and quantification of antibiotic resistance gene variants in the gut microbiota

15h20-15h35: **Sarah NAUDIN**, Aude FERRAN, Pedro IMAZAKI, Nathalie ARPAILLANGE, Camille MARCUZZO, Maïna VIENNE, Sofia DEMMOU, Felipe RAMON-PORTUGAL, Marlène LACROIX, Claire HOEDE, Maïalen BARRET, Véronique DUPOUY, Delphine BIBBAL

Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota

15h35-15h50: **Athina ANDREA**, Susanne KARLSMOSE PEDERSEN, Lauge HOLM SØRENSEN, Jette SEJER KJELDGAARD, and Rene S. HENDRIKSEN

From genotype to antimicrobial resistance (AMR) phenotype prediction: how genomic proficiency tests can facilitate the AMR laboratories' performance evaluation

15h50-16h05: **Miki IKEBE**, Kota AOKI, Mitsuko HAYASHI-NISHINO, and Kunihiko NISHINO

Correlation analysis of bacterial cell morphology and multidrug resistance

16h10-16h25: **Awards for the best oral and poster presentations**

and Symposium closure
**Session 1 - Roles of the environment in resistance evolution and transmission**

O1

**Antibiotic resistance and production genes in the soil microbiome given anthropogenic disturbance: insights from the Centralia coal seam fire**

Ashley Shade

CNRS, Université Lyon, University Claude Bernard Lyon 1, Ecologie Microbienne

Environmental disturbances can affect the diversity and persistence of microbial antibiotic resistance and production genes. We leveraged the unique, seven-decade-old underground coal seam fire ongoing in Centralia, Pennsylvania, USA, to examine surface soil bacterial microbiome response to an unnatural, long-term heat disturbance. As the fire slowly advances along coal seams, formerly fire-affected soils cool to ambient temperature enabling geographically localized comparisons of impacted soils across a gradient of fire intensities with recovered and reference soils. We executed a seven-year field study, annually sampling sites selected to capture the range of disturbance intensities. We used amplicon gene sequencing to assess changes in bacterial microbiome structure in response to the fire and untargeted metagenome sequencing to assess changes in the soil functional potential, including assessment of antibiotic resistance and production. Over the years, the fire-affected sites cooled, and their microbiome structures recovered towards those of reference and recovered soils. We present unpublished data on the diversity and patterns of antibiotic resistance and production genes over time and with fire cooling in the impacted sites compared to the ambient-temperature sites. This work contributes to understanding how disturbance affects the functional potential of environmental antibiotic resistance and production.
O2

# Metagenomic analysis of the bacterial community structure within cattle feces, the feedyard environment, and particulate matter as a function of antibiotics, probiotics, and environmental manipulation

Ashlyn H. Strickland<fnref n="1" />, Sarah A. Murray<fnref n="2" />, Javier Vinasco<fnref n="2" />, Jason E. Sawyer<fnref n="3" />, Harvey M. Scott<fnref n="2" />, Brent. W. Auvermann<fnref n="4" />, Jack Bush<fnref n="4" />, and Keri N. Norman<fnref n="1" />

The cattle feedyard environment has been shown to harbor antimicrobial resistant (AMR) bacteria and is implicated as a reservoir capable of spreading AMR to nearby animals and humans via effluent water, airborne particulate matter, and manure [1]. Multiple studies have investigated the potential for airborne particulate matter originating from feedyards to transfer bacteria, AMR determinants [2], and antibiotics [3] to surrounding environments; however, little work has been done to characterize the bacterial communities present within particulate matter. Moreover, information regarding bacterial population dynamics within the feedyard environment during the finishing period is limited. It is important to understand the dynamics of bacterial communities that persist in cattle and the environment to determine the ability of particulate matter to transmit AMR determinants to nearby environments and explore potential mitigation strategies. Our study aimed to characterize and compare the metagenomes of cattle feces and the feedyard environment as a function of tylosin and/or probiotic administration, and pen environment change and artificially model environmental manure pack desiccation to dust to characterize the metagenomic changes experienced in the cattle feedyard environment over time. We performed 16s rRNA amplicon sequencing on environmental and fecal samples from a 2x2x2 full factorial longitudinal study in which tylosin, an *Enterococcus faecium/Saccharomyces cerevisiae* probiotic, and pen change were investigated. Bioinformatic analyses of sequence data were performed using the QIIME2 pipeline and associated plug-ins to evaluate and compare microbiomes. Bacterial communities present within cattle feces, the environment, and airborne particulate matter exhibited discordant bacterial communities. Day and pen change were frequently associated with changes in bacterial populations within the same sample type. Treatment with tylosin and/or probiotics was not associated with alterations in the bacterial community structure within cattle feces, the feedyard environment or particulate matter during the finishing period. The environment likely plays a role in the persistence and transmission of certain bacterial taxa in the feedyard. Understanding bacterial community changes as a result of antibiotic and probiotic administration allows for a better understanding of the effects of these commonly administered therapies in cattle and the environment.

## References

[1] Noyes, N.R., et al., Characterization of the resistome in manure, soil and wastewater from dairy and beef production systems. *Sci Rep*, 2016. **6**: p. 24645.

[2] McEachran, A.D., et al., Antibiotics, bacteria, and antibiotic resistance genes: aerial transport from cattle feed yards via particulate matter. *Environ Health Perspect*, 2015. **123**(4): p. 337-43.

[3] Wooten, K.J., G.D. Mayer, and P.N. Smith, Persistence of elevated concentrations of PM, affiliated pharmaceuticals, and tetracycline resistance genes downwind of feedyards. *Environ Pollut*, 2019. **247**: p. 467-473.

<footnote n="1">¹Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA</footnote>

<footnote n="2">²Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, USA.</footnote>

<footnote n="3">³Department of Animal Sciences, Texas A&M University, College Station, TX, USA</footnote>

<footnote n="4">⁴Texas A&M AgriLife Research, Amarillo, TX, USA</footnote>
O3

The effect of manure amendment on the soil grassland resistome

Gonçalo MACEDO<fnref n="1" />,<fnref n="5" />, Joost HORDIJK<fnref n="4" />, Pieter VAN VEELEN<fnref n="1" />, Lucia HERNANDEZ LEAL<fnref n="1" />, Alex BOSSERS<fnref n="2" />, Asmus Kalckar OLESEN<fnref n="3" />, Søren J. SØRENSEN<fnref n="3" />, Dik HEEDERIK<fnref n="2" />, Dick MEVIUS<fnref n="2" />, and Heike SCHMITT<fnref n="1" />, 2, 4, 5

Manure fertilisation is one of the routes through which animal fecal bacteria enter the environment and can act as source of resistance genes. In a field study in the Northern part of the Netherlands, the influence of manure fertilisation on the soil microbiome and resistome of grasslands was investigated, and linked to studies on horizontal gene transfer. As observed from analysis of resistance genes by qPCR, manuring initially increased levels of erm(B) and tet(W) by ~1 log copies/kg across soil textures, after which levels gradually declined. An increase was also seen in adjacent surface waters. The gene decay in soils was strongly dependent on the type of ARG. Recovery of ARG levels was predicted to occur after 29–42 days. Similarly, targeted metagenomics showed a substantial increase of ARGs in soils and adjacent waters, while no change in the overall bacterial community was found despite an increase in specific families. Targeted metagenomics also was able to demonstrate that not all resistance genes increase after manuring – some genes potentially hosted by soil bacteria rather declined in their relative abundance. Last, we studied whether horizontal gene transfer could occur under conditions representative of manure application, by use of fluorescently labelled strains. In model systems for manure-amended soils, HGT could indeed be detected. However, the time window for (observable) HGT events was limited by the survival of manure-borne donor bacteria. All in all, manure-incuded changes were observed in all different test systems used, however these changes were transient in nature.

References

[1] G. Macedo, L Hernandez-Leal, P van der Maas et al. (2020). *Sci Total Environ* 737:139563

[2] G. Macedo, P van Veelen, L Hernandez-Leal et al. (2021). *Sci Total Environ* 770:1458399.

[3] G. Macedo, AK Olesen, L Maccario, et al. (2022). *Env Sci Technol* 56: 11398-11408.

<footnote n="1">¹Wetsus - European Centre of Excellence for Sustainable Water Technology, Leeuwarden, The Netherlands</footnote>

<footnote n="2">²Utrecht University, Utrecht, The Netherlands</footnote>

<footnote n="3">³University of Copenhagen, Copenhagen, Denmark</footnote>

<footnote n="4">⁴National Institute for Public Health and the Environment, Bilthoven, The Netherlands</footnote>

<footnote n="5">⁵TU Delft, Delft, The Netherlands</footnote>
O4

# Dynamics of cephalosporin-resistant in *Escherichia coli* in gulls highlight the importance of longitudinal wildlife studies

Michaela Ruzickova<fnref n="1" />,<fnref n="2" />, Kristina Nesporova<fnref n="1" />, Jana Palkovicova<fnref n="1" />,<fnref n="2" />, Simon Krejci<fnref n="2" />, Ivan Literak<fnref n="1" />,<fnref n="2" />, Monika Dolejska<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="4" />

Wildlife is generally overlooked as part of the environment but plays an important role in relation to antimicrobial resistance (AMR). The impact of migrating and vagrant species on the dissemination of the AMR remains unclear. To resolve this, it is crucial to understand whether wild animals are just temporary carriers of the AMR or if the resistant bacteria can be maintained in their gut. We performed a longitudinal experiment to analyse the dynamics of cephalosporin-resistant *Escherichia coli* in Caspian gulls (*Larus cachinnans*).

Five non-flying chicks were sampled at their nesting site and then transferred into captivity for 10 weeks where they were held together till the end of the experiment. The set of samples consisted of cloacal swabs obtained periodically every two weeks. Selective cultivation was conducted, from which five colonies were taken, and resistant *E. coli* isolates were tested for the presence of resistance genes and sequenced via whole-genome sequencing (Illumina, MinION) to obtain their genomic profiles and complete plasmid sequences.

The data showed significant changes in the presence of various sequence types (STs) over time and their transmission between individual birds. At the first sampling, most birds were colonized by *E. coli* ST11893 with *bla*<sub>CMY-2</sub> disrupting colicin-encoding gene on F34:A:-B:- plasmid. Only one gull carried CTX-M-1-producing *E. coli*. The *bla*<sub>CTX-M-1</sub> gene was carried by a multi-drug resistance colicin-associated IncI1/ST3 plasmid. In next samplings, the original predominating *E. coli* disappeared and IncI1/ST3 plasmid started to spread in various *E. coli* genotypes in all birds with a single ST predominating over time. The carriage of cephalosporin-resistant isolates decreased two months after the beginning of the experiment.

Our results point out that gulls can carry cephalosporin-resistant bacteria for over a month which leads to further spread of AMR. We observed that birds can exchange bacteria among each other which may result in the emergence of new and possibly more successful STs. Association of AMR plasmids with colicin production seems to be an important feature in their successful spread. Our data prove the importance of longitudinal studies, providing information on dynamic changes in the gut resistome over time.

<footnote n="1">¹Central European Institute of Technology, University of Veterinary Sciences Brno, Brno, Czech Republic</footnote>

<footnote n="2">²Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Brno, Czech Republic</footnote>

<footnote n="3">³Department of Clinical Microbiology and Immunology, Institute of Laboratory Medicine, The University Hospital Brno, Czech Republic</footnote>

<footnote n="4">⁴Biomedical Centre, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic</footnote>
Spatial spreading of antibiotic resistance genes by gulls

Dayana JARMA¹, Oriol SACRISTÁN-SORIANO², Francisco HORTAS¹, Juan M. PERALTA-SÁNCHEZ³,⁴, Alexandre SÁNCHEZ-MELSIÓ², José L. BALCÁZAR², Carles M. BORREGO²,⁵ and Marta I. SÁNCHEZ⁶,⁷

¹Instituto Universitario de Investigación Marina (INMAR); Universidad de Cádiz, Puerto Real, Cádiz, Spain

2 Water Quality & Safety, Institut Català de Recerca de l'Aigua (ICRA), Girona, Spain

3 Department of Zoology, Faculty of Biology, University of Seville, Seville, Spain

4 Department of Microbiology, Faculty of Sciences, University of Granada, Granada, Spain

5 Grup d'Ecologia Microbiana Molecular, Institut d'Ecologia Aquàtica, Universitat de Girona, Girona, Spain

6 Department of Plant Biology and Ecology, Faculty of Biology, University of Seville, Seville, Spain

7 Wetland Ecology Department, Estación Biológica de Doñana, EBD-CSIC, Seville, Spain

Due to their opportunistic behaviour (feeding on human waste in landfills or roosting in wastewater treatment plants), migratory gulls play a key ecological role in the dissemination of antibiotic resistance (AR) at the interface of human-domestic-wild environments. However, the factors determining AR spread by these birds are complex and not fully understood. We used the lesser black-backed gull (Larus fuscus, the second most abundant migratory waterbird in Andalusia, Spain) as a model and compared their faecal bacterial communities and their associated antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) between functional units (areas that gulls use for roosting and feeding consistently during their wintering) and within different habitats (wetlands vs. landfills). The richness and diversity of faecal bacterial communities were higher in faeces collected from more polluted sites. The gull faecal carriage of ARGs conferring resistance to aminoglycosides, chloramphenicol, and vancomycin varied spatially but were similar across wetlands and landfills, probably because of the high connectivity between these two types of habitats. The comparison between the abundance of ARGs and MGEs in gull faeces with that in the surrounding environment (soil and surface water) did not reveal the same pattern for all antibiotic families; ARGs conferring resistance to quinolones, vancomycin, and genes coding for integron-integrase gene, were higher on water and soil than in gull faeces, while for tetracyclines, cloramphenicoles, macrolides and genes encoding for other MGEs (insertion sequences) were the opposite. Our results demonstrate that the faecal microbiota of birds and their associated ARGs changed spatially and that birds using landfills for feeding and

roosting are more prone to disseminate antibiotic resistance determinants and potential pathogens
across different biomes.
# Compartmentalization of antibiotic resistance between phages and bacteria in a river - wastewater treatment plant continuum

Charlène SAGRILLO¹, Christophe MERLIN¹ and Xavier BELLANGER¹

¹LCPME UMR7564, CNRS-Université de Lorraine, Nancy, France

Transduction is probably one of the major horizontal transfer mechanisms allowing antibiotic resistance to spread between commensal, pathogenic and environmental bacteria [1]. If this mechanism is well described *in vitro*, its frequency is still difficult to evaluate in the environment and the parameters and actors that govern it remain poorly known [2,3]. In this work, we studied the compartmentalization of few antibiotic resistance genes and mobile genetic elements in bacteria and phages of microbial communities present in the water of the Meurthe River, and in the effluents of a wastewater treatment plant discharging into it over time. Bacterial and phage DNAs were extracted separately, carefully checking for the latter that no free/non-encapsidated DNA had contaminated the extracts. The 16S rDNAs and a panel of 10 antibiotic resistance genes and 9 mobile genetic elements selected for their environmental and/or clinical relevance were then quantified by quantitative or digital PCR in the extracts. Quantification of 16S rDNA in DNA purified from phage fractions confirmed the presence of transducing phages containing chromosomal DNA in all the different microbial communities. While in river water, transducing phages are not very abundant and only carry a maximum of 0.08% of the 16S rDNA present in a whole microbial community, this value can reach 16.3% in wastewater treatment plant effluents, thus demonstrating the very high proportion of transducing phages released in the environment through this medium. This trend was also observed with the different antibiotic resistance markers monitored where, for example, a maximum of 0.01% of class 1 integrons were carried by phages from river microbial communities while up to 2% of them were carried by phages from effluent microbial communities. In extreme cases, effluents even had more than half of the *sull* genes and more than a quarter of the *vanA* genes they contained that were vehiculated in the phage fraction! Overall, the tendency of antibiotic resistance genes and mobile elements to be encapsidated was variable from marker to marker and from medium to medium. While the effluents were usually much concentrated in antibiotic resistance determinants than the river water, no significant increase in antibiotic resistance could be detected in the river water downstream the treatment plant, likely because of the dilution effect. These results, however, arises the question of the impact of bacteria and especially that of the numerous transducing phages released in the river through the effluents with respect to their respective roles in disseminating antibiotic resistance in the environment.

## References

[1] M. Woegerbauer, X. Bellanger and C. Merlin (2020). *Front Microbiol* 11:671.

[2] E. González-Villalobos and J.L. Balcázar (2022). *Trends Microbiol* 30:1022-1024.

[3] C. Sagrillo, F. Changey and X. Bellanger (2022). *Environ Microbiol* 24:4317-4328.
O7

Occurrence of 41 sanitary indicators in digestates derived from different anaerobic digestion processes and various raw organic wastes

Caroline WYBRANIEC<fnref n="1" />, Geneviève GAGNE<fnref n="2" />, Cécile MOUSSARD2, Marion BEAUPERE<fnref n="1" />, Léa LUSURIER<fnref n="1" />, Françoise LERICHE<fnref n="2" />, Laurence MARJOLET<fnref n="1" />, Nicolas SERTILLANGES<fnref n="3" />, Dominique PATUREAU<fnref n="3" />, Benoit COURNOYER<fnref n="1" />, Wessam GALIA<fnref n="1" />

To date, there is no scientific consensus on the fate of pathogenic bacteria present in raw organic wastes and the sanitary quality of digestates. This work aims to characterize the influence of the nature of raw wastes as well as the types of processes on the sanitary quality of digestates. For this purpose, the occurrence and the fate of 41 sanitary indicators including pathogenic bacterial species, antimicrobial resistance genes as well as new indicators of health hazard such as virulence factors carried by mobile genetic elements were monitored during anaerobic digestion (AD). Three full-scale plants were selected to study the effect of the key parameters of the AD process (type of alimentation, number of steps, temperature, pH). While the impact of the nature of raw wastes has been studied with 3 lab-scale reactors where the AD process was similar (semicontinuous, mesophilic) but the proportions of inputs have been varied (mainly fecal matters supplemented or not with straw or straw and zeolite). First, the prevalence of these indicators was measured in digestates and compared with that measured in raw wastes. Of the 13 species studied, only one (Streptococcus agalactiae) was completely removed, regardless of the process and nature of the waste used. For virulence factors and mobile genetic elements, no significant decrease in prevalence could be detected. In a second step, absolute quantification of the indicators displayed a high prevalence in raw wastes has been done using a Digital Droplet PCR system. Regarding the effect of the nature of raw waste, the reactors fed with fecal matter supplemented with straw (Lab2) or with straw and zeolite (Lab3) have a higher hygienization efficiency compared to reactor fed uniquely with fecal matter (Lab1). Thus, 2 pathogenic species (Enterococcus faecalis, Enterococcus faecium) and 3 mobile genetic elements (intI1, intI2 and IS26) were significantly decreased in both reactors (Lab2 and Lab3). Nevertheless, 1 mobile genetic element (intI1) and 1 antibiotic resistance gene (tetO) have been significantly increased in Lab1. Interestingly, of 13 polycyclic aromatic hydrocarbons (PAHs) and 13 pharmaceutical products measured in this study, the quantities of 11 PAHs and 11 pharmaceutical products, were significantly higher in Lab1 samples (treated or not) than in Lab2 and Lab3 samples. The high concentration of some micropollutants such as triclosan found in Lab1 samples could explain the lower hygienization efficiency of AD process in this reactor compared to the other reactors (Lab2 and Lab3). Regarding the impact of the type of process, the fed-batch reactor had a better hygienization effect with a total reduction of a virulence factor (ybtA), involved in the production of siderophores, and a significant reduction of enterotoxigenic Escherichia coli. This study showed that the sanitary quality of digestate is first related to the sanitary quality of raw waste and then to the employed process.

<footnote n="1">¹UMR Écologie Microbienne Lyon (LEM), Université Claude Bernard Lyon 1, VetAgro Sup, INRAE 1418, CNRS 5557, Marcy l'Étoile, France</footnote>

<footnote n="2">²UMR Fromage, Université Clermont Auvergne, INRAE, VetAgro Sup, F-15000 Aurillac, France</footnote>

<footnote n="3">³Laboratoire de Biotechnologie de l'Environnement, UR0050, INRAE, Université Montpellier, 11100 Narbonne, France</footnote>
# Microplastics increase the selective potential of antibiotics at sub-inhibitory concentrations

Brune Joannard¹, Timothy M. Vogel¹, Concepcion Sanchez-Cid¹

¹Université de Lyon, Université Claude Bernard Lyon 1, UMR 5557, UMR INRAe 1418, VetAgro Sup, Écologie Microbienne, équipe BEER, F-69622 Villeurbanne, France

Microplastics (MPs) are major pollutants that are massively released into the environment. In urban waters, biofilms can form on plastic surfaces and thus MP particles might affect environmental bacteria and their associated resistome. In addition, antibiotics may adsorb onto plastic surfaces and have a greater effect on plastic-associated bacteria than on planktonic bacteria. Therefore, the presence of MPs in the environment could pose a risk for antibiotic resistance development and dissemination in environmental settings. The goal of this study was to determine the impact of microplastics in combination with antibiotics on environmental bacterial growth and antibiotic resistance. We hypothesized that the presence of MPs could increase the selective potential of antibiotics at sub-inhibitory concentrations on environmental bacteria and their associated antibiotic resistance genes (ARGs). DNA was extracted from urban river bacteria enriched in TSB medium or sterile water over 72 hours, with and without microplastics and gentamicin and ciprofloxacin at sub-inhibitory concentrations. The 16S rRNA and gentamicin resistance genes were amplified by qPCR to determine pollutant effects on growth and ARG selection. In addition, the influence of MPs and antibiotics on river water communities was evaluated by sequencing the 16S rRNA gene. Bacterial abundance was lower in the MP fraction than in the liquid fraction (both with or without MPs). Antibiotics had no effect on bacterial abundance in any fraction and where thus overall sub-inhibitory. Bacterial exposure to both gentamicin and ciprofloxacin at sub-inhibitory concentrations induced a larger shift in bacterial community composition in the MP fraction than in the liquid fraction (with or without MPs), with an increase in the relative abundance of *Citrobacter*, *Klebsiella* and *Pseudomonas*. Gentamicin and ciprofloxacin selected for gentamicin resistance genes in the MP fraction, both in TSB medium and in sterile water. Therefore, our results are consistent with antibiotics at sub-inhibitory concentrations having a larger impact on environmental antibiotic resistance in the presence of MPs. This study adds to the concerns related to the role of microplastic pollution on the emergence of antibiotic resistance in the environment.
O9

Do microplastics offer a suitable surface for antibiotic resistance spread into the open environment?

Federica PIERGIACOMO<fnref n="1" />, Franck LEJZEROWICZ<fnref n="2" />, Tam TRAN<fnref n="3" />, Atif A. CHOWDURY<fnref n="1" />, Luciano BENEDUCE<fnref n="4" />, Odd-Gunnar WIKMARK<fnref n="3" />, Leonardo PAGANI<fnref n="5" />, Alexander EILER<fnref n="2" /> and Lorenzo BRUSETTI<fnref n="1" />

Plastic pollution represent an anthropogenic stressor with presumably large ecological implications. In particular, microplastics (<5 mm particles) collectively constitute an abundant artificial surface for biofilm formation, where Horizontal Gene Transfer (HGT) of Antibiotic Resistance Genes (ARGs) could be enhanced. However, still little is known about the contributions of microplastics to microbial community change and ARGs spread. In this context, we propose a multidisciplinary study to address these potential contributions, in relation to agricultural practices, natural environments, food webs and human health. We set-up microcosms with microplastics (seven most common plastics; 10 % of the samples' volume), antibiotics (ciprofloxacin, vancomycin, cefotaxime - 1100 mg/L) and three environmental matrices (irrigation water and sediments; compost; manure). Shotgun metagenomics and microbiome data analysis revealed the effects of sample type, microplastic and antibiotic additions on phylogenetic community compositions and on the metabolic potential of assembled genomes(Fig.1). Resistome analyses accounting for chromosomic or plasmidic genomic location allowed us to suggest for ARGs spread mechanisms within environments. An overrepresentation of antimicrobial gene copies was found, especially for streptomycin, tetracycline, vancomycin, cefotaxime and ciprofloxacin. The microplastic additions affected copy numbers of a few antibiotic resistance genes such as those encoding resistance to e.g., streptomycin and tetracycline. Antibiotics addition was associated with differentially abundant genera such as *Achromobacter*. To conclude, interaction effects between microplastics and antibiotics may promote antibiotic resistance spread in a wide range of natural and anthropogenic environments.

<figure><img src="image_8.png" /><figcaption>**Fig.1.** Phylogenetic tree with barplots showing the sample type, treatments and the log ratio of ARGs abundance.</figcaption></figure>

<footnote n="1">¹Faculty of Agricultural, Environmental and Food sciences, Free University of Bolzano-Bozen, Bolzano, Italy</footnote>

<footnote n="2">²Department of Biosciences, University of Oslo, Oslo, Norway</footnote>

<footnote n="3">³NORCE, SIVA Innovation Centre, Tromsø, Norway</footnote>

<footnote n="4">⁴DAFNE, University of Foggia, Foggia, Italy</footnote>

<footnote n="5">⁵Infectious Diseases Unit, Central Hospital of Bolzano-Bozen, Bolzano, Italy</footnote>
Can Antimicrobial Resistance Genes Travel Across the Atlantic Ocean?

Florent Rossi<fnref n="1" />,<fnref n="2" />, Eloïse Lebras<fnref n="3" />, Eric Defert<fnref n="3" />, Nathalie Turgeon<fnref n="1" />,<fnref n="2" />, Marc Veillette<fnref n="1" />,<fnref n="2" />, Jean-Luc Baray<fnref n="5" />,<fnref n="6" />, Pierre Amato<fnref n="4" /> and Caroline Duchaine<fnref n="1" />,<fnref n="2" />,<fnref n="7" />

The presence of antimicrobial resistance genes (ARGs) in the air has been the topic of a flourishing literature in the past years. Recently, they have been detected in clouds at mid-altitude, attesting to their potential transport over long distances (Rossi et al., 2023). In this work, marine sources were highlighted as significant contributors of ARGs, especially regarding quinolone resistance. So far, however, the emissions of ARGs from seas and oceans remain under-studied, in particular because of the numerous sampling constraints.

In this study, the presence of ARGs over the Atlantic Ocean's surface was specifically addressed through a 20-days transatlantic campaign from Brest, France, to Woods Hole, on the west coast of the USA, aboard a sailing ship specially equipped with a microbiology lab. Every day and night, aerosols were collected for periods of 1 hour using a high-throughput point source sampler (SASS 3100), at a rate of ~300 L air/min. Additional sampling apparatuses (filter-holders connected to individual pumps) were deployed to collect samples over periods of 24h, at a flow rate of ~7 L/min, in triplicate. For each air samples, a panel of 33 different subtypes of ARGs (including quinolones, β-lactams, macrolides, tetracyclines, sulfonamides, aminoglycosides, vancomycins and one mobile genetic element) was monitored by qPCR and related to bacterial diversity (MiSeq Illumina) and concentration (qPCR 16S). These were further related to the backward trajectories of the corresponding air masses reaching the sampling site, using the ECMWF ERA-5 CAT 3D kinematic trajectory model.

Preliminary results highlight a high variability in the concentration of airborne bacteria at the ocean surface, averaging ~10³ cells.m⁻<fnref n="3" /> of air based on 16S gene abundance; this tended to increase as the boat moved away from the coasts. Regarding antibiotic resistance, 22 different subtypes were recurrently detected, dominated by quinolone, macrolide and tetracycline resistance genes. Concentrations were highly fluctuating, ranging from few copies to ~10⁴ copies per m<fnref n="3" /> of air, and ARGs' profiles differed between day/night sampling, with values that tended to be higher during the night. The data are currently being analyzed with respect to air masses geographical origins and bacterial diversity. This work highlights that ocean surfaces could contribute significantly to ARGs inputs in the atmosphere and to their global spread.

**Reference**

Rossi, F., Péguilhan, R., Turgeon, N., Veillette, M., Baray, J.-L., Deguillaume, L., Amato, P., Duchaine, C., 2023. Quantification of antibiotic resistance genes (ARGs) in clouds at a mountain site (puy de Dôme, central France). *Science of The Total Environment* 865, 161264. https://doi.org/10.1016/j.scitotenv.2022.161264

<footnote n="1">¹ Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, Canada</footnote>

<footnote n="2">² Centre de recherche de l'institut de cardiologie et de pneumologie de Québec, Québec, Canada</footnote>

<footnote n="3">³ Blue Observer, 165 quai Eric Tabarly, Brest, France</footnote>

<footnote n="4">⁴ Université Clermont Auvergne, CNRS, Institut de Chimie de Clermont-Ferrand, Clermont-Ferrand, France</footnote>

<footnote n="5">⁵ Université Clermont Auvergne, CNRS, Observatoire de physique du Globe de Clermont-Ferrand, Clermont-Ferrand, France</footnote>

<footnote n="6">⁶ Université Clermont Auvergne, CNRS, Laboratoire de Météorologie physique, Clermont-Ferrand, France</footnote>

<footnote n="7">⁷ Canada Research Chair on Bioaerosols</footnote>
**Session 2 - Monitoring and molecular epidemiology of antimicrobial resistance**

O11

**Use of genomics to characterise antimicrobial resistances present within livestock on UK farms**

Prof. Muna Anjum

*Department of Bacteriology, APHA*

The rise in antimicrobial resistance (AMR) in bacteria due to overuse or continued misuse of antibiotics in agriculture, human and veterinary medicine is a global threat. The food chain and the environment are also affected, resulting in widespread dissemination of multi-drug (MDR) bacteria, which is affecting our ability to treat bacterial infections. The global burden of AMR has continued to increase, and it has been estimated that there are 75,000 human fatalities a year due to MDR bacteria, and an increased morbidity that is affecting patient outcomes. To tackle the risk of AMR the United Kingdom has developed a National Action Plan which aims to evaluate and control it in the UK. The Animal and Plant Health Agency (APHA) has been central in its implementation, conducting research and surveillance within UK livestock, working in a One-Health context, as humans, animals and the environment are inherently linked. To survey and gain insight into the routes and mechanisms of transmission of AMR within the livestock compartment we have used genomics, so new and emerging AMR threats can be identified promptly, the flow of AMR genes can be tracked accurately and the risk to human health assessed in a One-Health context. Such work is helping to protect public and animal health through better control of AMR in bacteria occurring in livestock, their environment, and the food chain.
# The role of FDA's Veterinary Laboratory Investigation and Response Network in monitoring antimicrobial resistance of animal pathogens

Olgica CERIC<fnref n="1" />, Jake GUAG<fnref n="1" />, Sarah PELOQUIN<fnref n="1" />, Amy MERRILL<fnref n="2" />, Chih-Hao HSU<fnref n="2" />, Claudine KABERA<fnref n="2" />, Gregory H. TYSON<fnref n="1" />

**Background:** The Veterinary Laboratory Investigation and Response Network (Vet-LIRN) is an FDA program created in 2010 to coordinate a network of veterinary diagnostic laboratories. In 2017, Vet-LIRN initiated an Antimicrobial Resistance (AMR) Monitoring Program focused on monitoring AMR profiles in veterinary and zoonotic pathogens isolated from animals at veterinary clinics and diagnostic laboratories in the U.S. and Canada. Vet-LIRN's AMR program supports the national initiative to Combat Antibiotic Resistant Bacteria (CARB) and FDA's Center for Veterinary Medicine's multi-pronged, multi-year strategy designed to slow the emergence of resistance arising from the use of antibiotics in animals. It supplements existing efforts by the National Antimicrobial Resistance Monitoring System (NARMS), which monitors AMR of foodborne pathogens in humans, retail meats, and food animals at slaughter.

**Methods:** Vet-LIRN laboratories provided antimicrobial susceptibility testing (AST) data from clinically relevant bacterial isolates from animals: *Salmonella enterica* from any animal host, and *Escherichia coli* and *Staphylococcus pseudintermedius* isolates from dogs, using a commercially available testing platform. Laboratories followed Clinical and Laboratory Standards Institute AST testing methods. Laboratories sequenced a subset of isolates and submitted the whole genome sequencing (WGS) data to the National Center for Biotechnology Information (NCBI) submission portal, where they were assembled and analyzed using NCBI's bioinformatics pipelines. All sequencing data are publicly available. Vet-LIRN has partnered with NARMS to include animal pathogen AMR data into the NARMS integrated report and create publicly available data dashboards.

**Results:** Since 2017, the Vet-LIRN AMR program has provided a wealth of information to detect emerging antimicrobial resistance, understand the effectiveness of FDA-regulated products over time, and foster antimicrobial stewardship in veterinary settings. For example, in 2019 the carbapenem resistance gene, blaNDM-5, was identified for the first time in an animal pathogen in the U. S., in a 2018 isolate of *E. coli* from a dog in Philadelphia. Over 15,000 isolates have undergone antimicrobial susceptibility testing, and more than 4,000 isolates have been sequenced with data publicly available under NCBI BioProject PRJNA314609. Vet-LIRN AMR data dashboards include resistance mechanisms from genomics data, along with the percent resistance and MIC distributions for each of the antibiotics included in the panels.

**Conclusions:** By making AMR monitoring data publicly accessible, the Vet-LIRN AMR monitoring program facilitates international One Health research and monitoring.

<footnote n="1">¹Veterinary Laboratory Investigation and Response Network (Vet-LIRN), Center for Veterinary Medicine, Office of Applied Science, US Food and Drug Administration, Laurel, MD, USA</footnote>

<footnote n="2">²Division of Animal and Food Microbiology, Office of Applied Science, Center for Veterinary Medicine, US Food and Drug Administration, Laurel, MD, USA</footnote>
<figure><img src="image_9.png" /></figure>

O13

# Genomic diversity of porcine LA-MRSA CC398 isolates collected in the German national resistance monitoring program GERM-Vet between 2007 and 2019

Henrike KRÜGER-HAKER<fnref n="1" />,<fnref n="2" />, Xing JI<fnref n="3" />, Dennis HANKE<fnref n="1" />,<fnref n="2" />, Stefan FIEDLER<fnref n="4" />, Andrea T. FEßLER<fnref n="1" />,<fnref n="2" />, Nansong JIANG<fnref n="5" />, Heike KASPAR<fnref n="4" />, Yang WANG<fnref n="5" />, Congming WU<fnref n="5" /> and Stefan SCHWARZ<fnref n="1" />,<fnref n="2" />,<fnref n="5" />

**Background and objectives:** The livestock-associated methicillin-resistant *Staphylococcus aureus* (LA-MRSA) clonal complex (CC) 398 lineage is known for its frequent multiresistance to antimicrobial agents and a low host specificity [1]. Colonized pigs represent a risk of LA-MRSA CC398 colonization or infection for occupationally exposed people, through which such isolates might be spread further within the human community [1]. Here, we investigated a collection of unrelated LA-MRSA CC398 isolates for evolutionary relationships and genomic diversity with focus on their virulence and antimicrobial resistance (AMR) properties.

**Materials and methods:** In total, 178 unrelated LA-MRSA CC398 were collected in the German national resistance monitoring program GERM-Vet from diseased swine between 2007 and 2019. Whole-genome sequencing was carried out, followed by molecular typing and sequence analysis. A minimum spanning tree based on core-genome multilocus sequence typing was constructed and antimicrobial susceptibility testing was performed according to CLSI standards [2].

**Results:** The LA-MRSA CC398 isolates showed close phylogenetic relationships and a wide molecular variety, including 13 *spa* types as well as 19 known and four novel *dru* types. Several toxin-encoding genes, including *eta*, *seb*, *sek*, *sep* and *seq*, were detected. The isolates harbored a wide range of AMR properties mirroring the proportions of the classes of antimicrobial agents applied in veterinary medicine in Germany. Several novel or rare AMR genes were identified, including the novel macrolide-lincosamide-streptogramin B resistance gene *erm* (54), the lincosamide-pleuromutilin-streptogramin A resistance gene *vga* (C), and the phenicol-lincosamide-oxazolidinone-pleuromutilin-streptogramin A resistance gene *cfr*. Many AMR genes were part of small transposons or plasmids. Clonal and geographical correlations of molecular characteristics, AMR and virulence genes were more frequently observed than temporal relations.

**Conclusion:** This study provided insight into diversity and population dynamics of the epidemic porcine LA-MRSA lineage in Germany over a 13-year-period. In order to detect new emerging, possibly more dangerous clones and to prevent LA-MRSA transmission between livestock farms and entries into the human community, a large-scale LA-MRSA monitoring is essential.

**References**

[1] P. Butaye, M. A. Argudín and T. C. Smith (2016). *Curr Clin Microbiol Rep* 3:19-31.

[2] Clinical and Laboratory Standards Institute (CLSI) (2022): CLSI supplement M100, 32nd Ed.

<footnote n="1">¹Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="2">²Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany</footnote>

<footnote n="3">³Jiangsu Key Laboratory for Food Quality and Safety, State Key Laboratory, Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, China</footnote>

<footnote n="4">⁴Federal Office of Consumer Protection and Food Safety (BVL), Berlin, Germany</footnote>

<footnote n="5">⁵Key Laboratory of Animal Antimicrobial Resistance Surveillance, MARA, College of Veterinary Medicine, China Agricultural University, Beijing, China</footnote>
O14

Antimicrobial resistance in pathogenic bacteria of Victorian cattle

Janine Muller<fnref n="1" />, Yuhong Liu<fnref n="1" />, Ilhan Mohammad<fnref n="1" />, Brendan Rodoni<fnref n="1" /> and Fiona Constable<fnref n="1" />

Australia has one of the most conservative approaches to the use of antimicrobials in food producing animals in the world. Registration of antibiotics for use in animals is tightly controlled, prescribed by veterinarians and the use of fluoroquinolones, colistin and fourth generation cephalosporins are prohibited for use in food producing animals to minimise their impact on human medicine. In Australia, surveys have been undertaken for antimicrobial resistance (AMR) in commensal bacteria from live cattle at slaughter, however little knowledge is shared on the AMR status of pathogenic bacteria isolated from cattle in Australia. Understanding the presence of AMR and particularly multi-drug resistant (MDR) pathogens on farm is a critical step in understanding the risks of AMR/MDR to the farm and the broader industry, to support biosecurity and public health.

This project focused on 11 pathogenic bacterial isolates (10 *Salmonella* spp and 1 *E. coli*) taken from significant disease investigations of cattle in Victoria, submitted to the Agriculture Victoria Research (AVR) Veterinary Diagnostics Laboratory (VDL) over a 3-year period (Feb 2020 - Feb 2023). The isolates were selected based on their phenotypic resistance to 2 to 5 antibiotics, sequenced using the Illumina NovaSeq and bioinformatic analysis conducted. Antimicrobial resistance genes (ARGs), mobile genetic elements and heavy metal resistance genes were identified on the genomes of all 11 isolates. Five isolates that carried ARGs for different antibiotic classes were selected and further sequenced on MinION to complete the genome and investigate the genetic links between the genes and the mobile genetic elements. The presence of ARGs identified in these isolates may not be directly related to the particular antibiotics used on farm and may be the result of co-selection of other antibiotics or heavy metals.

<footnote n="1">¹ Agriculture Victoria Research, Department of Energy Environment and Climate Action, Melbourne, Australia</footnote>
# Clonal and plasmidic dissemination of critical antimicrobial resistance genes in dairy cattle in Québec, Canada.

Maud de Lagarde<fnref n="1" />,<fnref n="2" />, John Morris Fairbrother<fnref n="3" />,<fnref n="4" />,<fnref n="5" />, Marie Archambault<fnref n="2" />,<fnref n="4" />,<fnref n="5" />, Simon Dufour<fnref n="2" />,<fnref n="5" />, David Francoz<fnref n="1" />,<fnref n="2" />, Jonathan Massé<fnref n="2" />,<fnref n="5" />, Jean-Philippe Roy<fnref n="1" />,<fnref n="2" />.

Tackling AMR should be a priority for all public health stakeholders. Increasing the judicious usage of antimicrobials (AM) in food production has proven efficient to limit AMR. However, its effect on resistance gene dissemination in animal populations is not known. The province of Québec (Canada) enacted a new legislation in February 2019, to limit usage of critical AM in production animals.

The aim of our study was to investigate the putative link between ESBL/AmpC *E. coli* isolated pre- and post-legislation and to determine the presence of plasmid carrying genes responsible for resistance to crucial AM in these isolates.

We collected fecal samples from calves, cows, and the manure pit in 87 dairy farms in Quebec approximately 2 years before and two years after the enactment of the legislation. The whole genomes of 183 (91 before and 93 after) putative ESBL/AmpC *E. coli*, isolated after cefotaxime enrichment, were sequenced. Their phylogenetic characteristics (MLST, serogroup, cgMLST) and the presence of virulence and resistance genes and replicons were examined. A maximum likelihood phylogenetic tree was constructed based on SNP data. We identified 10 clonal lineages (based on cgMLST) and 6 clones (SNPs < 35). Isolates belonging to these clones could be found on different farms pre- and post- legislation, strongly suggesting clonal dissemination of antimicrobial resistance genes in the population during this 4-year period (cf Figure1). All clones were MDR, Clone 2 being noteworthy, due to the presence of resistance genes for macrolides, fluoroquinolones and third generation cephalosporines and of virulence genes *afa* and *papC*. Our data also strongly suggest the presence of at least two epidemic plasmids belonging to the incompatibility groups IncH12 and IncY and carrying *qnrs1* and *bla*<sub>CTX-M</sub>, and one IncC plasmid carrying *bla*<sub>CMY-2</sub> gene. These different plasmids were identified pre- and post- legislation, suggesting that they are persistant despite the changes in antimicrobial usage (data not shown).

Clonal and plasmidic dissemination of resistance genes persist in the dairy population in Quebec despite limiting the use of critical antimicrobials. Further monitoring is essential to better understand the emergence and persistence mechanisms of these genes. In particular, it will be important to determine the capacity of Clone 2 to become a high risk clone.

<figure><img src="image_10.png" /><figcaption>**Fig. 1.** Phylogenetic tree of 183 ESBL/AmpC *E. coli* isolates based on the SNP differences. Isolates with an ID ending in CTX belongs to the “pre-legislation” period all other isolates belong to the post-legislation period. The two digits in the 3rd and 4th position of the ID represent the farm number.</figcaption></figure>

<footnote n="1">¹ Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, Saint-Hyacinthe, QC, Canada.</footnote>

<footnote n="2">² Regroupement FRONT Op+lait, Saint-Hyacinthe, QC, Canada.</footnote>

<footnote n="3">³ WOAH Reference Laboratory for Escherichia coli, Faculty of Veterinary Medicine, Université de Montréal, Saint-Hyacinthe, QC, Canada.</footnote>

<footnote n="4">⁴ Swine and Poultry Infectious Research Center (CRIPA-FQRNT), Faculty of Veterinary Medicine, Université de Montréal, Saint-Hyacinthe, QC, Canada.</footnote>

<footnote n="5">⁵ Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Université de Montréal, Saint-Hyacinthe, QC, Canada.</footnote>
A phylogeny study of β-lactam-resistant *E. coli* from Belgian calves

Virginie GUÉRIN<fnref n="1" />*, Nicolas CABANEL<fnref n="1" />, Damien THIRY<fnref n="2" />, Jacques MAINIL<fnref n="2" />, Marc SAULMONT<fnref n="3" /> and Philippe GLASER<fnref n="1" />

In a One Health context, some antimicrobials classified by WHO as “critically important for human medicine” have a restrictive use in veterinary medicine in the European Union, among which 3rd and 4th generation cephalosporins (3GC and 4GC), from β-lactam, a family widely used in human and veterinary medicine. To evaluate the impact of such restrictions, we have characterized the epidemiology of β-lactam resistance in *Escherichia coli* isolated from diseased calves in Belgium. During three calving seasons (2017–2020) *E. coli* were collected from young diarrheic or septicemic calves and their phenotypic resistance profiles were determined, showing a constant high rate of β-lactam resistance, but a decrease of 3GC/4GC resistance [1]. To characterize these observations at the genomic level, we sequenced 642 *E. coli* from our collection, including 522 3GC/4GC resistant isolates. Their evolution during the three seasons and their relationships with other animal and human *E. coli* were studied.

The 642 calf *E. coli* showed a broad diversity of β-lactamase patterns, but are consistently multidrug resistant. Phylogenetic analysis shows the striking importance of phylogroup C, with 30% of ST88, and none isolate belongs to the B2 phylogroup, associated with human extraintestinal infections. The distribution is similar during the three seasons. Most of the *E. coli* (78%) belong to 13 different STs, which were analyzed jointly with genome sequences from the same STs downloaded from Enterobase. We identified some lineages showing a local dissemination (Figure 1). On the other hand, the intermixed human and animal isolates in some STs reveal its zoonotic potential.

<figure><img src="image_11.png" /><figcaption>**Fig. 1.** ST88 phylogeny (A), with three major lineages identified (red, blue and green). Focus on the ST88-red lineage (B) and the geographical repartition of isolates in this lineage (C). The colors in B and C are consistent.</figcaption></figure>

In

conclusion, the reduction of C3G resistance was not associated with changes in resistance mechanisms and population structure. In the 13 dominant STs, isolates clustering reflected either local transmissions or/and endemicity. For some of them, a zoonotic potential was observed. This is to our knowledge the first large scale study in diseased calves, as most studies in livestock are focusing on carriage isolates, possibly explaining the specificity of our findings.

**Reference:** [1] V. Guérin, A. Farchi, D. Thiry, F. Cawez, P.S. Mercuri et al. (2022) *Vet. Sci.* 9:45.

<footnote n="1">¹Ecology and Evolution of Antibiotics Resistance Unit, Université Paris Cité, Institut Pasteur, Paris, France</footnote>

<footnote n="2">²Bacteriology, Department of Infectious and Parasitic Diseases, FARAH and Faculty of Veterinary Medicine, ULiège, Liège, Belgium</footnote>

<footnote n="3">³Bacteriology, Regional Animal Health and Identification Association (ARSIA), Ciney, Belgium</footnote>

<footnote>*Current affiliation: École nationale vétérinaire d'Alfort (EnvA), Université Paris Est Créteil, UMR-1161 Virologie, Maisons-Alfort, France</footnote>
# Multidrug-resistant *Escherichia coli* isolated from slaughtered broilers in Ilorin, Nigeria

Ahmad I. AL-MUSTAPHA<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Emmanuel AJ. AWOSANYA<fnref n="2" />, Victoria O. ADETUNJI<fnref n="2" />, and Annamari HEIKINHEIMO<fnref n="3" />,<fnref n="4" />

The occurrence of multidrug-resistant *Escherichia coli* in food-producing animals including poultry poses economic losses and public health hazards. Genomic information on the molecular diversity of E. coli will aid public health decision-making, increase food safety, improve understanding of the molecular basis of antimicrobial resistance (AMR), and could provide vital data for the improvement of E. coli vaccines. Hence, we conducted genomic characterization of *E. coli* isolated from broilers in Ilorin, Kwara State, Nigeria. A structured questionnaire was interviewer-administered to 125 poultry farmers in Ilorin to determine their knowledge, attitude, and perception towards prudent antibiotic use and its implications. Also, broiler faecal samples (n=1000) were screened for *E. coli*. Phenotypic antibiotic susceptibility testing was done using both the disc diffusion assay and molecular characterization was done using whole genome sequencing. Many poultry farmers (63.0%) used unprescribed antibiotics, there was zero compliance with antibiotic withdrawal periods, and 69.6% of them were aware of AMR. Older farmers, male respondents, those with tertiary education, and those with larger flock sizes were more likely to have satisfactory knowledge of AMR. *E. coli* was isolated from 403 samples. A total of 189 samples were multidrug resistant. WGS of the 31 ESBL-producing multi-antibiotic resistant isolates revealed that revealed high genomic diversity. The resistome revealed that the isolates harboured resistance determinants to fluoroquinolone (n=8), aminoglycoside (n=3), tetracycline (n=3), beta-lactam antibiotics (n=11), chloramphenicol (n=4), foliate pathway antagonist (n=4), fosfomycin (n=3), macrolides (n=3), and two disinfectant resistance genes which conferred resistance to hydrogen peroxide and quaternary ammonium compounds. Mutations in the *gyrA* gene conferring resistance to fluoroquinolones were also detected. There was a positive correlation between phenotypic resistance patterns and the antibiotic resistance genes (ARGs) that were detected in the sequenced isolates. Isolates were of diverse sequence types (ST) with 12.9% belonging to ST117 and the virulome comprised of diverse virulence factors such as adhesins, iron acquisition genes, toxins, and protectins. The mobilome consisted of several Col-plasmids (Col440I, ColRNAI, and ColpVC) and the predominant IncF plasmids (IncFIB, IncFII) belonged to the IncF64:A-B27 sequence type. The isolates were of diverse serotypes with O17:H18 being the most prevalent (9.7%) and were classified into five phylotypes (A, B1, B2, E, and F) with phylotype F being the most prevalent (32.3%). Intact phages belonging to the *Myoviridae* (77.4%), *Siphoviridae* (51.6%), *Inoviridae* (29%), and *Podoviridae* (6.5%) viral families were detected. No antibiotic RGs were identified on any of the phages. The isolates were clustered into five different clades and 71% of them had >80% similarity to human pathogens. The occurrence of multi-antibiotic-resistant *E. coli*, co-occurrence of antibiotic- and disinfectant-resistant genes, and ESBL-*E. coli* isolates that were potential human pathogens in poultry were established in Kwara State. Our findings suggest that poultry are potential carriers of clonally diverse and pathogenic pathogens that pose a public health threat. Hence, critical control points in the poultry value chain are urgently needed.

## References

[1] Al-Mustapha, A. I., Raufu, I. A., Ogundijo, O. A., Odetokun, I. A., Tiwari, A., et al., (2023). *Int. J. Food Microbiol.* **110086**.

[2] Al-Mustapha, A. I., Alada, S. A., Raufu, I. A., Lawal, A. N., Eskola, K., Brouwer, M. S., et al. (2022). *J Glob Antimicrob Resist*, 31, 337-344.

[3] Al-Mustapha A. I., Adetunji VO, Heikinheimo A. (2020). *Antibiotics*. 9(7):E378.

<footnote n="1">¹Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland.</footnote>

<footnote n="2">²Department of Veterinary Services, Kwara State Ministry of Agriculture and Rural Development, Ilorin, Kwara State, Nigeria.</footnote>

<footnote n="3">³Department of Veterinary Public Health and Preventive Medicine, Faculty of Veterinary Medicine, University of Ibadan, Oyo State, Nigeria.</footnote>

<footnote n="4">⁴Finnish Food Authority, Laboratory and Research Division, Microbiology Unit, Helsinki, Finland.</footnote>
O18

Fluoroquinolone resistance in *E. coli* in broilers: mapping the spread

Moniek RINGENIER<fnref n="1" />, Filip BOYEN<fnref n="2" />, Sigrid C.J. DE KEERSMAECKER<fnref n="3" />, Kevin VANNESTE<fnref n="1" />, Bert BOGAERTS<fnref n="3" />, Mathias DEVREESE<fnref n="2" /> and Jeroen DEWULF<fnref n="1" />

**Objective**

Although restrictions on the use of fluoroquinolones in livestock have been imposed in Belgium since 2016, high levels of fluoroquinolone (FQ) resistance are still found in commensal *E. coli* in broilers. The purpose of this study was to describe the FQ resistance prevalence in *E. coli* and to investigate the role of day-old chicks and the environment in the dynamics of the spread within different flocks.

**Materials and methods**

On 29 broiler farms, antibiotic use was monitored and both water and environmental samples were collected prior to arrival of the chicks. Thirty broilers were sampled per farm on days 0 (before entering the stable), 3 and 35 of the production round. In all samples, total *E. coli* and FQ resistant *E. coli* isolates were quantified by plating on MacConkey agar without and with 0.25 µg/ml enrofloxacin (ECOFF). All isolates were identified using MALDI-TOF MS. A selection of the isolates was submitted for whole genome sequencing to investigate their phylogenetic relatedness using core-genome MLST (cgMLST) analysis, and to detect resistance determinants.

**Results**

Before they entered the stable, the day-old chicks carried FQ resistant *E. coli* in 82.8% of the farms and FQ resistant *E. coli* were found in the environment on boots (10.3%), hygiene locks (24.1%), drinking cups (10,3%), feeding pans (20.7%), and stable floors (24.1%). According to cgMLST, overall there was a large diversity of FQ resistant *E. coli* strains. However, identical FQ resistant isolates were found on day 0 and day 35, suggesting that FQ resistant isolates present in the environment at the start of a production round or in day-old chicks, remained present until slaughter, even though no FQs were used.

**Conclusion**

The continued presence of FQ resistant *E. coli* in Belgian broiler farms is likely the result of both a historical contamination at the farm level and a continuous influx along the production chain.

<footnote n="1">¹Department of Internal Medicine, Reproduction and population medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium</footnote>

<footnote n="2">²Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium</footnote>

<footnote n="3">³Transversal activities in Applied Genomics, Sciensano, Brussels Belgium</footnote>
O19

In-vitro validation of AMR genes prevalence revealed by WGS in *Campylobacter jejuni* isolated from chicken livers in Spain.

Alicia MANZANARES<fnref n="1" />,<fnref n="2" />, Florencia CORREA-FIZ<fnref n="1" />,<fnref n="2" />, Teresa AYATS<fnref n="1" />,<fnref n="2" />, Miquel NOFRARÍAS<fnref n="1" />,<fnref n="2" />, Marta CERDÀ-CUÉLLAR<fnref n="1" />,<fnref n="2" />

*Campylobacter* is the most common cause of foodborne human gastroenteritis worldwide. The largest contributors of human campylobacteriosis in developed countries are poultry and poultry products. There have been outbreaks of campylobacteriosis attributed to undercooked chicken livers. In previous studies, we determined the prevalence of *Campylobacter* in chicken livers, highlighting their potential relevance as a source of human campylobacteriosis. On the other hand, the high prevalence of antimicrobial resistant (AMR) strains in *Campylobacter* spp. from poultry has become a growing concern since these strains can be transferred to humans causing an antibiotic-resistant infection. The aim of this study was to assess the AMR prevalence of a selection of *C. jejuni* isolates from chicken livers and the characterization of the AMR gene presence through whole-genome sequencing (WGS) and comparison with Resfinder database. A total of 33 liver isolates obtained from carcasses at slaughter from healthy chicken from 21 different farms from Catalonia (north-eastern Spain) were tested, obtaining valid results from 24 isolates since the remaining nine were excluded due to poor growth. AMR to at least one antimicrobial was revealed by the broth microdilution method with EUCAMP3 plates, in 91,67% of the isolates. The most common resistances found were to ciprofloxacin (79,17%) and tetracycline (66,7%), and the AMR profile ciprofloxacin-tetracycline was the most frequent (50%). One isolate was multidrug resistant (profile ciprofloxacin-tetracycline-ertapenem) while two isolates were pansusceptible. DNA was extracted from all the isolates and submitted to shot-gun sequencing (Illumina HiSeq). The raw sequences were processed to be quality filtered and the genomes were *de-novo* assembled (with SPADES) using PATRIC. The contigs obtained were inspected for AMR gene presence using the ResFinder 4.1 tool (https://cge.cbs.dtu.dk/services/ResFinder). All the isolates showing ciprofloxacin resistance *in-vitro* carried the point mutation in the gyrase gene (T86I) which confers resistance to quinolones. For the 16 tetracycline-resistant isolates, all but one carried the Tet(O) gene. All isolates carried the genes coding for the CmeABC multidrug efflux pump which has been shown to contribute to both intrinsic and acquired resistance to tetracycline and might explain the tetracycline-resistance profile of the remaining isolate. The isolate showing ertapenem resistance was the only carrying the blaOXA-185 gene, while all isolates carried the blaOXA-61 gene. These genes are associated with b-lactam resistances, however, since other carbapenems apart of ertapenem are not included in the EUCAMP3 plates, it could not be confirmed *in vitro*. Additionally, the information provided by WGS allowed to predict a variety of AMR profiles that included resistances to antibiotics not included in the EUCAMP3 plates, particularly to ampicillin and nalidixic acid. Altogether, these valuable results show the risk posed by chicken livers in the transmission of AMR *C. jejuni* and the power of WGS in providing additional meaningful information on the AMR potential of those isolates.

<footnote n="1">¹Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia. Spain.</footnote>

<footnote n="2">²IRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193, Catalonia. Spain.</footnote>
O20

# Antimicrobial resistance determinants in bacteria isolated from retail chicken and pork meat across New South Wales, Australia

Ojas Dixit<fnref n="1" />,<fnref n="2" />, Aidan Preuss<fnref n="1" />, and Claire L O'Brien<fnref n="1" />

**Introduction and aims.** Few studies have been conducted in Australia to determine the diversity of antimicrobial resistance in bacteria isolated from retail meat. The aim of this study was to utilize whole genome sequencing to determine the diversity of resistance genes in bacteria isolated from retail meat samples across the most populous state of Australia; New South Wales (NSW).

**Methods.** A total of 404 meat samples (244 chicken and 160 pork) were purchased from 55 supermarkets across NSW, Australia. All samples were streaked onto Brilliance™ CRE/VRE/ESBL (CRE/VRE/ESBL), CAMPY (CAM), XLD (XLD), and MacConkey (MAC) agar. Whole genome sequencing was performed for 288 isolates, with all isolates that grew on CRE, VRE, ESBL, CAM, and XLD agar being prioritized, the remainder were isolates that grew on MAC agar. DNA libraries were prepared using plexWell™ 96 Kits (seqWell™) and sequencing performed on an Illumina NovaSeq™ platform (Illumina, Inc.) in a 150 bp paired-end format. Sequences were assembled using SPAdes, then annotated using RASTtk. Antibiotic resistance genes, plasmids, and virulence genes were identified using the Mobile Genetic Element finder tool from the Center for Genomic Epidemiology (CGE). The CGE's PathogenFinder 1.1 tool was used to predict the likelihood of isolates being pathogenic to humans. Antibiotic susceptibility testing was performed using Gram-negative and CAMPY Sensititre® plates containing critically- or highly-important antibiotics (CIA/HIA), as defined by WHO<fnref n="1" />. All plates were automatically inoculated and automatically read using Sensititre™ SWIN™ software.

**Results.** The 288 genomes were found to belong to 17 different genera and we detected an average of 1.53 antimicrobial resistance genes per genome (range: 0-11). Three isolates; one Serratia strain, one Aeromonas strain, and one Chromobacter strain, harbored a mobilizable colistin resistance (*mcr*) gene, identified as variants *mcr*-9, *mcr*-3.15, and *mcr*-5.1, respectively. A number of plasmid-associated quinolone resistance genes were also detected. Based on gene content, many of the isolates met the criteria for pathogenic pathogens of *E. coli*, including EPEC, ExPEC, and UPEC. Of the *E. coli* isolates, 98% were predicted to be pathogenic to humans, while 85% of the *Campylobacter* isolates were. Antibiotic susceptibility testing revealed that 12% (35/288) of isolates were deemed to be multiple drug resistant, of which 19% were cultured on CRE agar. Resistance to ciprofloxacin was observed for 14/288 (5%) of the isolates.

**Conclusions.** A vast diversity of bacterial species, antimicrobial resistance and virulence genes can be detected in bacteria isolated from retail chicken and pork meat in NSW. The identification of gene variants conferring resistance to CIA or HIAs not previously detected in Australian meat samples highlights the need for regular surveillance and monitoring of antimicrobial resistance.

References

[1] World Health Organisation, 'Critically important antibiotics for human medicine: 6th revision' (webpage 2023)
https://www.who.int/publications/i/item/9789241515528.

<footnote n="1">¹Centre for Research in Therapeutic Solutions, Faculty of Science and Technology, University of Canberra, Canberra, Australia</footnote>

<footnote n="2">²School of Medicine and Health, University of Wollongong, Wollongong, Australia</footnote>
O21

# Prevalence of Extended-Spectrum Beta-Lactam (ESBL) and Colistin Resistance in *Escherichia coli* Isolated from Broiler Chickens in Dressing Plants in the Philippines

Ramon P. MALUPING<fnref n="1" />, Remedios F. MICU<fnref n="2" />, Thomas CHISNALL<fnref n="1" />, Alistair DAVIES<fnref n="1" />, Evelyn E. EMBESTRO<fnref n="2" />, Mary Ann ESCOTO<fnref n="2" />, Mildred A. PADILLA<fnref n="3" /> and Roderick CARD<fnref n="1" />

Antimicrobial resistance (AMR) in *Escherichia coli* from animals is a public health concern because of potential for transfer of antibiotic resistant bacteria and resistance genes to humans/ bacteria in humans via the food chain. Moreover, *E. coli* is commonly used as a sentinel indicator organism for monitoring AMR in enteric bacteria as it is found frequently in a wide range of hosts; may be exposed to antimicrobials in the intestine; acquires resistance easily; and provides potential indication of transferable resistance genes present in other intestinal Enterobacterales. The WHO categorises 3rd and 4th generation cephalosporins and colistin as critically important antimicrobials (CIAs) to human health. The European Medicines Agency (EMA) has also categorised resistance to these antibiotics as higher risk to public health (Category 2). A recent study on the antimicrobials used in backyard and commercial poultry farms in the Philippines, indicated that CIAs are frequently used in poultry farms [1]. However, published data on AMR of commensal *E. coli* from chickens raised for food are scarce; with only one publication indicating a resistance of 38.33% to nalidixic acid and 25% to ciprofloxacin but published data on resistance to cephalosporins and colistin are lacking [2].

In the present study, resistance to 3rd generation cephalosporins (ceftazidime & cefotaxime) and colistin and the prevalence and types of genes conferring resistance to these compounds were determined from 101 *E. coli* isolated from apparently healthy broiler chickens at slaughter. These isolates were obtained from chicken caecal content from National Meat Inspection Service (NMIS) accredited poultry dressing plants representing several regions of the Philippines. *E. coli* isolation was performed using standard microbiological methods and identification was carried out using VITEK 2 System (BioMerieux) and MALDI-TOF spectrometer (Bruker). Broth microdilution using the Sensititre™ System (Thermo Fisher Scientific) was used to determine the minimum inhibitory concentration (MIC) of 15 antibiotics representing nine antibiotic classes. MIC values were interpreted using EUCAST ECOFFs and whole-genome sequencing was carried out to determine the presence of AMR genes using NCBI AMR Finder Plus & APHA SeqFinder tools. Cephalosporin resistance was identified in 23/101 (23%) isolates. This resistance was due to the presence of *bla*DHA-1, *bla*CMY-2, *bla*CTX-M-55, *bla*CTX-M-15, or *bla*CTX-M-123 genes with *bla*DHA-1 the most common of these genes, 12/23 (52%). In addition, 10/101 (10%) isolates were resistant to colistin and harboured the *mcr*-1 resistance gene, six of which were also resistant to cephalosporins harbouring both the *bla*DHA-1 and *mcr*-1 genes. This study supports previous findings that apparently healthy farm animals can be reservoirs of ESBL and colistin resistant *E. coli*. This is of public health significance as the genes that encode for critically important antimicrobial resistance can spread to other bacterial species by horizontal gene transfer and can be transmitted to humans via food chain.

## References

[1] Barroga TRM, Morales RG, Benigno CC, Castro SJM, Caniban MM, Cabullo MFB, Agunos A, de Balogh K, Dorado-Garcia A. (2020). *Front Vet Sci.* 2020 Jul 8;7:329.

[2] Jiao S, Fami RML, Pedernal VAD and Cabrera EC. (2007). *The Philippine Agricultural Scientist* 90(1): 64-70.

<footnote n="1">¹FAO Reference Centre for Antimicrobial Resistance, Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB United Kingdom</footnote>

<footnote n="2">²National Meat Inspection Service (NMIS), Quezon City, 1101 Philippines</footnote>

<footnote n="3">³University of the Philippines, Los Baños, College, Laguna, 4031 Philippines</footnote>
O22

# Risk factors for antimicrobial resistant *Aeromonas* spp. and *Escherichia coli* in the rainbow trout farm environment in Brittany, NW France

Joel Javid¹, Emmanuelle Moreau¹, Lionel Pinneau, Niki Hayatgheib¹, Claire Maltret¹, Segolene Calvez¹, Nora Navarro-Gonzalez¹

¹ BIOEPAR UMR 1300, Oniris - INRAE, Nantes, France

Fish farms typically operate with surface water, which can be polluted with fecal bacteria from humans and livestock. As antimicrobial resistance (AMR) becomes widespread in humans, animals, and the environment, bacterial pollution of water used in food-producing environments is a rising concern. *Escherichia coli* is an indicator of faecal contamination in surface water and an indicator of AMR from the environment, thus the prevalence of antimicrobial resistant- *E. coli* is an interesting tool to monitor pollution in surface water, and in freshwater aquaculture in particular. In addition, *Aeromonas* spp., a ubiquitous aquatic bacterium that easily acquires antimicrobial resistance elements, can be an indicator of AMR from the aquatic environment and provide with a complementary overview of AMR in aquaculture.

This longitudinal study aims at identifying risk factors for AMR in *Aeromonas* spp. and *E. coli* from the environment of two flow-through rainbow trout farms in Brittany, France [1]. Farm A was located downstream from a wastewater treatment plant, whereas Farm B was located downstream from livestock. Both farms had a history of furunculosis outbreaks (*Aeromonas salmonicida*) and use of antibiotics. 309 samples were collected from fish, biofilm and water over the course of seven months (January to July 2020). Additionally, faecal samples of the farmers were collected. *Aeromonas* spp. and *E. coli* were isolated on GSP and EMB agar respectively; presumptive isolates were confirmed with the use of MALDI-TOF. 257 confirmed *Aeromonas* spp. and 67 *E. coli* were tested for their antimicrobial susceptibility with the broth microdilution method. The antibiotics tested were flumequine (0.016-256 µg/mL), oxalinic acid (0.016-64 µg/mL), enrofloxacin (0.016-64 µg/mL), oxytetracycline (0.016-512 µg/mL), florfenicol (0.25-32 µg/mL), trimethoprim-sulfamethoxazole (0.015/0.3-64/1216 µg/mL), and colistin (0.781-400 µg/mL). Epidemiological cut-off values were used to classify an isolate as either “wild type” or “resistant”. The percentage of AMR ranged from 13% to 60% depending on the bacterium and the antibiotic. Farm surveys were conducted in the form of questionnaires concurrently with each sampling event. Farm-level information and event-level information were collected about weather events, farm management practices, season (spring vs summer), and disease occurrence, among others. Odds ratio and 95% Confidence Intervals will be calculated to detect risk factors that can explain the presence of *E. coli* in rainbow trout farms, the presence of AMR-*E. coli*, and the presence of AMR-*Aeromonas*. Statistical analyses will be performed with the use of R Software.

## Reference

[1] N. Hayatgheib, S. Calvez, C. Fournel, L. Pineau, H. Pouliquen, and E. Moreau, “Antimicrobial susceptibility profiles and resistance genes in genus aeromonas spp. Isolated from the environment and rainbow trout of two fish farms in France,” *Microorganisms*, vol. 9, no. 6, 2021, doi: 10.3390/microorganisms9061201.
O23

# Abundance and diversity of antibiotic resistance genes in different types of environmental samples: 5 years of Resistomap

Windi I. MUZIASARI

Resistomap Oy, Helsinki, Finland

Resistomap is the first and frontrunner company for antibiotic resistance monitoring in the environment. We provide universities, research centers, governments, water utilities and industries with an end-to-end service to monitor genes associated with antibiotic resistance, mobile genetic elements, and pathogens from any type of environmental samples, including wastewater, surface water, sediment, sludge, soil, manure, stool, and others. We combine culture-independent laboratory and high-throughput gene quantification methods using the SmartChip qPCR system with our digital platform for data analysis and visualization. Since we fully operated in 2019, we have analyzed over 10,000 environmental samples across 45 countries. Figure 1 shows the medians of antibiotic resistance levels in wastewater from 28 countries. With more samples and genes analyzed, the data shown in Figure 1 could change. Besides regions, our data show that the most detected and abundant genes also differ based on the sample types. This suggests the need for a routine monitoring of antibiotic resistance in its hotspots such as wastewater treatment plants and agricultural soils, to evaluate the resistance discharge to surface waters and other environmental compartments. With routine monitoring, the data can also be used to measure the risk of resistance prevalence and spread, and as an early warning for potential outbreaks of resistant bacteria.

<figure><img src="image_12.png" /><figcaption>**Fig. 1.** Heatmap of antibiotic resistance levels in wastewater samples</figcaption></figure>
# The evolution of the faecal resistome is shaped by age and antimicrobial use during the pig lifecycle

Lorcan O'NEILL<fnref n="1" />,<fnref n="2" />, Maria RODRIGUES da COSTA<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Edgar GARCIA MANZANILLA<fnref n="1" />,<fnref n="2" />, Fiona CRISPIE<fnref n="4" />, Raul CABRERA-RUBIO<fnref n="4" />, Rachel GRIMES<fnref n="5" />, Finola C. LEONARD<fnref n="1" />

Although new European Union regulations aim to restrict antimicrobial use (AMU) in livestock, antimicrobials remain an essential tool in ensuring animal health and welfare. In many countries, the pig sector represents the largest consumer of veterinary antimicrobials and thus pigs are a potential reservoir for antimicrobial resistance genes (ARG). Field studies investigating how different levels of AMU affect the evolution of antimicrobial resistance during the production cycle are lacking. This study investigated the dynamics of the porcine faecal resistome in batches of pigs on Irish farms with differing levels of AMU throughout their entire lifecycle.

Twelve Irish farrow-to-finish farms were selected according to their level of AMU in medicated feed (low, moderate, high). Ten litters of piglets were selected from each farm; pooled faecal samples were collected during each production stage (piglet, weaner, grower and finisher) and once from their dams at the initial visit. Metagenomic sequencing and read mapping against the Resfinder database was used to characterise and quantify the acquired resistome at each time point.

Genes conferring resistance to tetracyclines, macrolides, aminoglycosides and beta-lactams were the most abundant ARGs on all farms. The abundance of ARGs in growing pigs peaked during the weaner stage decreasing thereafter while the lowest abundance was observed in sows. Abundance was lower on low AMU farms compared to moderate or high AMU farms. Alpha diversity (Shannon diversity and richness) was highest in piglets and lowest in sows and finishers and was higher on the moderate and high AMU farms compared to low AMU farms. The composition of the resistome was strongly influenced by the composition of the microbiota in an age dependent manner.

To the authors' knowledge, this is the first multi-farm longitudinal study to investigate the dynamics and evolution of the faecal resistome throughout the pig production cycle and gives important insight into how age and antimicrobial use impact the resistome in pig farming.

<footnote n="1">¹ School of Veterinary Medicine, University College Dublin, Dublin, Ireland</footnote>

<footnote n="2">² Pig Development Department, Teagasc, Fermoy, Ireland</footnote>

<footnote n="3">³ Veterinary & Animal Science, Centre for Epidemiology and Planetary Health (CEPH), Scotland's Rural College, Inverness, Scotland</footnote>

<footnote n="4">⁴ Food Research Centre, Teagasc, Fermoy, Ireland</footnote>

<footnote n="5">⁵ Department of Clinical Microbiology, Trinity College Dublin, Dublin 2, Ireland</footnote>
O25

# Genetic analysis of *ampC* promotor mutants of *E. coli* from livestock exhibiting resistance to extended-spectrum cephalosporins.

Mike Brouwer, Alieda van Essen-Zandbergen, Yvon Geurts, Frank Harders, Kees Veldman

Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands

**Background:** Extended-spectrum cephalosporins (ESCs) are considered critically important for human clinical use.¹ There is a high focus on resistance to these antimicrobials through ESBL and plasmid-encoded AmpC (pAmpC) genes in *Escherichia coli* in both veterinary and human monitoring and surveillance programs, and many studies have focussed on these resistance mechanisms since WGS has become commonly adapted. Resistance to ESCs can also occur through mutations in the promotor region of the chromosomal *ampC* gene.² As this type of resistance is not transmissible on plasmids via horizontal gene transfer, the mechanism is considered a smaller risk compared to ESBL or pAmpC genes and less is known about the epidemiology of this resistance type in livestock.

**Objective:** To describe the epidemiology of ESC-resistant *E. coli* containing *ampC* promotor-mutants from livestock in the Netherlands using WGS.

**Methods:** ESC-resistant *E. coli* were selectively isolated between 2014-2021 according to EU legislation from faecal samples of broiler chickens, pigs, veal calves and dairy cattle. Isolates for which no ESBL or pAmpC genes could be detected were sequenced on Illumina NextSeq.

**Results/Discussion:** The proportion of ESC-resistant *E. coli* suspected to contain *ampC* promotor mutations without known ESBL pAmpC genes differs between the different livestock species. In broilers, this was 1.7% of cephalosporin-resistant *E. coli*, 5.4% in veal calves, 18.8% in dairy cattle and 35.8% in pigs.

A total of 248 *E. coli* isolates was shown to contain mutations in the *ampC* promotor region. Other commonly found mechanisms in this set of isolates include resistance to fluoroquinolones, sulfamethoxazole, trimethoprim and tetracycline, which were detected using antimicrobial susceptibility testing and were confirmed using WGS. While there was a large diversity of *E. coli* ST present in the collection, certain lineages appear to have transmitted through the production sectors.

**References**

[1] Collignon *et al*. World Health Organization Ranking of Antimicrobials According to Their Importance in Human Medicine: A Critical Step for Developing Risk Management Strategies to Control Antimicrobial Resistance From *Food Animal Production*. DOI: 10.1093/cid/ciw475

[2] Peter-Getzlaff *et al*. Detection of AmpC beta-lactamase in *Escherichia coli*: comparison of three phenotypic confirmation assays and genetic analysis. DOI: 10.1128/JCM.00091-11
High Throughput Screening of Antimicrobial Resistance Genes in Gram-
Negative Seafood Bacteria

Sabine Delannoy<fnref n="1" />, Corine Hoffer<fnref n="1" />, Raphaëlle Youf<fnref n="1" />, Emilie Dauvergne<fnref n="1" />, Hattie E. Webb<fnref n="2" />, Thomas Brauge<fnref n="3" />, Mai-Lan Tran<fnref n="1" />, Graziella Midelet<fnref n="3" />, Sophie A. Granier<fnref n="4" />, Marisa Haenni<fnref n="5" />, Patrick Fach<fnref n="1" /> and Anne Brisabois<fnref n="6" />

Seafood and the marine environment are often considered as potential reservoirs of antimicrobial
resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and
sparse results on this sector. This study aimed at providing new data and insights regarding the content
of resistance markers in various seafood samples and sources, and therefore the potential exposure to
humans in a global One Health approach.

An innovative high throughput qPCR screening was developed and validated in order to
simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons,
integrons, and insertion sequences associated to Gram-negative bacteria.

Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat
fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) showed the occurrence of sul-1,
ant(3")-Ia, aph(3')-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9-group genes in Pseudomonas spp.,
Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates. MGEs were identified
in all bacterial species investigated. We found that the occurrence of MGE may be associated with
the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE
were detected in half of the seafood isolates investigated, association with ARG was only identified
for twelve isolates.

Our results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria and ARGs
decreases with increasing distance from potential sources of fecal contamination. Moreover, we
provide unique and original high throughput micro-array designed for the screening of ARGs and
MGE in Gram-negative bacteria, which is easily implementable for monitoring antimicrobial
resistance gene markers in diverse contexts.

<footnote n="1">¹ COLiPATH Unit & Genomics Platform IdentyPath, Laboratory for Food Safety, ANSES, 94700 Maisons-Alfort,
France;</footnote>

<footnote n="2">² Department of Animal and Food Sciences, International Center for Food Safety Excellence, Texas Tech University, Lubbock, TX 79409, USA;</footnote>

<footnote n="3">³ Bacteriology and Parasitology of Fishery and Aquaculture Products Unit, Laboratory for Food Safety, ANSES, 62200 Boulogne sur Mer, France;</footnote>

<footnote n="4">⁴ Antibiotics, Biocides, Residues and Resistance Unit, Fougères Laboratory, ANSES, 35306 Fougères, France;</footnote>

<footnote n="5">⁵ ANSES - Université de Lyon, Unité Antibiorésistance et Virulence Bactériennes, 69364 Lyon, France;</footnote>

<footnote n="6">⁶ Strategy and Programs Department, ANSES, 94700 Maisons-Alfort, France.</footnote>
O27

High occurrence of carbapenemase- and extended-spectrum-β-lactamase-
producing *Escherichia coli* and *Klebsiella pneumoniae* from migratory birds
*(Ciconia ciconia)* with detection of high-risk clones

**Sandra MARTÍNEZ-ÁLVAREZ**<fnref n="1" /> Pierre CHÂTRE<fnref n="2" />, Úrsula HÖFLE<fnref n="3" />, Carla Andrea ALONSO<fnref n="4" />, Pauline FRANÇOIS<fnref n="2" />, Teresa CARDONA-CABRERA<fnref n="2" />, Myriam ZARAZAGA<fnref n="1" />, Jean-Yves MADEC<fnref n="2" />, Marisa HAENNI<fnref n="2" />, Carmen TORRES<fnref n="1" />

**Background:** The presence of Enterobacterales producing carbapenemases (CP) and extended-spectrum β-lactamases (ESBL) is closely monitored in humans and animals, but the potential of migratory birds as carriers of resistance genes remains poorly understood. The aim of the study was to detect and characterize CP- and ESBL-producing *E. coli* (EC) and *K. pneumoniae* (KP) obtained from storks feeding on two landfills in Spain.

**Methods:** ESBL and CP-producing EC/KP were isolated from 211 stork faecal samples using chromogenic culture media, and collected isolates were sequenced using NovaSeq6000 (Illumina) and MinION (Oxford Nanopore) technologies. Resistome, virulome, sequence types (ST) and replicon profiles were determined using bioinformatic tools. Localization of ESBL/CP genes was performed using Southern blots on S1-PFGE gels.

**Results:** ESBL-EC/KP were detected in 71 samples (33.6%; 28.4%-EC and 5.2%-KP), while 28 samples (13.3%) contained CP-producing EC/KP (11.8%-EC and 1.4%-KP). Different sequence types (ST) (EC, n=33; KP, n=3) were identified, including high-risk clones associated with humans (EC: ST131, ST58 or ST69; KP: ST307), potential high-risk clones (EC: ST10 and ST48) and more ubiquitous clones (EC: ST46, ST155, ST117, ST617). A wide range of ESBL/pAmpC-conferring genes (blaCTX-M-1/blaCTX-M-14/blaCTX-M-15/blaCTX-M-27/blaCTX-M-32/blaCTX-M-55/blaCTX-M-65/blaSHV-12/blaCMY-2/blaDHA-1) was identified, as well as a large variety of CP-genes (blaKPC-2, blaKPC-3, blaNDM-1, blaNDM-7, blaOXA-48, blaVIM-1 and blaGES-7), including in some cases a combination of up to three types of CP (blaKPC-2, blaNDM-7 and blaVIM-1) and/or with co-detection of ESBL/pAmpC genes. Likewise, a variety of ESBL/pAmpC- and CP-carrying plasmids were identified, such as IncY (n=30), IncF (n=60), IncX3 (n=25) IncN (n=19) or IncL (n=8). The genetic characterization of these plasmids is ongoing.

<footnote n="1">¹Area of Biochemistry and Molecular Biology, OneHealth-UR Research Group, University of La Rioja - Logroño (Spain)</footnote>

<footnote n="2">²ANSES – Université de Lyon, Unité Antibiorésitance et Virulence Bactériennes - Lyon (France)</footnote>

<footnote n="3">³Health and Biotechnology (SaBio) Research Group, Institute for Game and Wildlife Research IREC (CSIC-UCLM) - Ciudad Real (Spain)</footnote>

<footnote n="4">⁴Hospital San Pedro, Logroño (Spain)</footnote>
Assessing the Resistome and Virulome of Human and Environmental
*Escherichia coli* isolates.

Maeve Louise Farrell<fnref n="1" />,<fnref n="2" />, Georgios Miliotis<fnref n="1" />,<fnref n="2" />, Firinne Kelly<fnref n="1" />,<fnref n="2" />, Evan Naughton<fnref n="1" />,<fnref n="2" />, Brigid Hooban<fnref n="1" />,<fnref n="2" />, Alexandra Chueiri<fnref n="1" />,<fnref n="2" />, Mark Maguire<fnref n="1" />,<fnref n="2" />, Louise O' Connor<fnref n="1" />,<fnref n="2" />, Martin Cormican<fnref n="1" />,<fnref n="2" />,<fnref n="4" />, Niamh Cahill<fnref n="1" />,<fnref n="2" />, Kelly Fitzhenry<fnref n="1" />,<fnref n="2" />, Aoife Joyce<fnref n="1" />,<fnref n="2" />, Sinead Duane<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Liam P. Burke<fnref n="1" />,<fnref n="2" />, Dearbhaile Morris<fnref n="1" />,<fnref n="2" />.

**Introduction:** In order to understand the dynamics of Antimicrobial Resistance (AMR), it is essential to take a One Health approach. The One Health concept recognizes the interconnectedness between human, animal and environmental health. This study aimed to assess the relationships between *Escherichia coli* isolates collected from human and environmental sources.

**Methods:** *E. coli* isolates (n=156) collected as part of a study investigating AMR in anthropogenically contaminated aquatic sources from 2018-2020 (n=113) were compared with those isolated from healthy participants of a cross sectional colonisation study in 2020-2021 (n=43). Total DNA was extracted from ESBL-producing *E. coli* (n = 118) and carbapenem resistant *E. coli* (n=14), and whole genome sequencing was performed on Illumina (PE150). Bioinformatic analysis was carried out to characterise isolates and to assess sequence type (MLST), phylogroup (ezclermont), resistome (Resfinder), virulome (VFDB), plasmidome (Plasmidfinder) and mobilome (Platon) of the isolates. Core Genome MLST (cgMLST) and dRep of the isolates mobilome were compared to assess similarity and clustering, with cluster hits of ≤10 allelic differences considered.

**Results:** In silico analysis revealed a high level of diversity, with 40 sequence types (STs) detected. The most prevalent were ST131, ST38 and ST10, which were detected in 24% (n=27), 7% (n=8), and 7% (n=8) of environmental isolates and 16% (n=7), 11% (n=5) and 11% (n=5) of human isolates, respectively. The most prevalent phylogroup was B2 (n=50), which was primarily associated with environmental isolates (n=36). The ESBL gene *bla*<sub>CTX-M</sub> was detected in both human (n=28, 65%) and water (n=85, 75%) isolates, while CPE remained exclusive to environmental isolates (n=14, 12%). Overall, 423 virulence factors (VF) were detected, with 83 isolates carrying 24 VF types on mobile genetic elements (MGEs). While both sample types had a median of approximately three VF, the overall proportion of isolates carrying predicted plasmid-borne VF was significantly higher in human isolates (67%) than water isolates (47%) (p=0.028, χ<fnref n="2" /> 4.8326). In contrast, water isolates had a higher overall number of VF (x̄ 80.74 ± 20.33). Overall, 19 clusters were identified, of which 16 contained only environmental (n=11) or human (n=4) isolates. The remaining four clusters contained both environmental and human isolates (n=11) all of which had the same ST, phylogroup and *bla*<sub>CTX-M</sub> gene detected in each.

**Conclusions:** The abundance of VFs and antimicrobial resistance genes, including those associated with resistance to last-resort antibiotics, in isolated *E. coli* strains suggests the potential for widespread dissemination of these genes in both aquatic ecosystems and the community.

<footnote n="1">¹ Antimicrobial Resistance and Microbial Ecology Group, School of Medicine, University of Galway, Ireland.</footnote>

<footnote n="2">² Centre for One Health, Ryan Institute, University of Galway, Ireland.</footnote>

<footnote n="3">³ J.E. Cairns School of Business and Economics, University of Galway, Ireland.</footnote>

<footnote n="4">⁴ National Carbapenemase-producing Enterobacterales Reference Laboratory Service, Ireland</footnote>
O29

# Wastewater-based Epidemiology (WBE): toward a relevant tool to monitor hospital antimicrobial resistance

Camille FAVIER<fnref n="1" />, Mylène TOUBIANA<fnref n="1" />, Olivier COUILLEROT<fnref n="2" />, Elodie PICHON<fnref n="2" />, Patricia LICZNAR-FAJARDO<fnref n="3" /> and Estelle JUMAS-BILAK<fnref n="3" />

Resistance to antibiotics can lead to therapeutic failures of infectious diseases but also can slow down advances in intensive medicine or surgery. Among extremely resistant bacteria (XDR), carbapenemase-producing enterobacteria (CPE) are of particular concern because of their capacities to hydrolyze most of the β-lactam antibiotics including the last resort antibiotic carbapenems [1]. In France, *bla*OXA-48 and *bla*NDM are the most widespread carbapenemases. Another kind of XDR of concern are vancomycin-resistant Enterococci (VRE) associated with *vanA* gene [2]. Based on current knowledge, these 3 antimicrobial resistance genes (ARG) are emerging in France and spread through an epidemic mode. However, data on XDR carriage is patchy in general population because it is monitored only in high risk-hospitalized patients. New tools are needed to make surveillance of emerging resistances more comprehensive.

During SARS-CoV-2 pandemic, efficiency of wastewater-based epidemiology (WBE) has been showed [3]. Because CPE and VRE are gastrointestinal bacteria, we propose WBE to improve the surveillance in human population. To achieve this goal, we developed a strategy to monitor *bla*OXA-48, *bla*NDM and *vanA* genes in wastewater from three hospital wards. Two wards were at high-risk for XDR carriage in patients. The last one was considered at low-risk and was used as a standard. *bla*CTX-M gene was also monitored as “endemic standard” because its endemic character both inside and outside the hospital. Genes’ quantifications were performed by qPCR and dPCR from hospital wastewater samples. CPE and VRE were cultured on selective media, enumerated and identified by MALDI-TOF-MS.

Molecular signals were observed both for endemic (*bla*CTX-M) and emerging ARG (*bla*OXA-48, *bla*NDM and *vanA*) with space-time variations. It was supported by significant positive correlations between results obtained by qPCR and dPCR. For the low-risk ward, a stable baseline signal was observed, with quantities of genes significantly different from those of the 2 high-risk wards. This suggests the possibility to determine a basal state of ARG quantifications in hospital wastewater to set up alert thresholds. *bla*NDM and *vanA* had epidemic dynamics as expected. Furthermore, *bla*OXA-48 signal was either similar or superior to *bla*CTX-M signal in all the wards, suggesting an undervaluation of OXA-48 diffusion in France, or a possible amplification of the signal in the hydric bacterial community of wastewater pipes. Finally, there was no taxonomic overlap between resistant bacterial communities from patients and from wastewater. These results could be in favor of ARG exchanges between patients’ and waterborne bacteria.

This study highlights the feasibility of AMR monitoring with WBE in a hospital. To validate this approach, further studies had to be performed considering ARG transfers and persistence.

References

[1] A. Teban-Man, E. Szekeres, P. Fang, U. Klümper, A. Hegedus, et al. (2022). *Microbiol. Spectr.* 10 : e02711-21

[2] M.O. Ahmed and B.E. Keith (2018). *Microb. Drug Resist.* 24: 590-606

[3] P. M. Choi, B. J.Tscharke, E. Donner, J. W. O'Brien, S. C. Grant, et al. (2022). *Trends Anal.Chem.*105:453-469

<footnote n="1">¹HSM, Univ. Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote n="2">²Ingéniérie et Analyses en Génétique Environnementale (IAGE), Montpellier, France</footnote>

<footnote n="3">³HSM, Univ. Montpellier, CNRS, IRD, CHU de Montpellier, Montpellier, France</footnote>
# Metagenomic Survey of Antimicrobial Resistance in Surface Waters of Maryland with High and Low Human Impact Classifications

Andrea OTTESEN<fnref n="1" />,<fnref n="3" />, Brandon KOCUREK<fnref n="1" />,<fnref n="3" />, Shawn BEHLING<fnref n="2" />, Claudine KABERA<fnref n="1" />,<fnref n="3" />, Patrick MCDERMOTT<fnref n="1" />,<fnref n="3" />, and Errol STRAIN<fnref n="1" />,<fnref n="3" />*

Antimicrobial resistance (AMR) is recognized as one of the most critical threats to public health world-wide. In alignment with One Health strategic planning, the National Antimicrobial Resistance Monitoring System (NARMS) has added an environmental monitoring component that will focus on surface waters. Surface waters are demonstrated AMR reservoirs and key integrators across human, animal, and natural environments. Use of the National Land Cover Database of the United States Geological Survey (USGS) for sampling site selection, provides an approach by which to examine AMR by anthropogenic impact. Land use designations were used to select 30 sites representing high (n=15) and low (n=15) human impact. Dead-end ultrafiltration was used to collect 20 liters of water from each of the 30 sites and metagenomic (culture-independent) and quasimetagenomic (enriched) shotgun data were used with multiple annotation pipelines (AMRplusplus, AMRFinderplus, and CARD pipelines) to profile AMR across Maryland surface waters. Thirty-three ‘critically important’ ARGs (antimicrobial resistance genes) from the NARMS list were identified using quasimetagenomic data in 86% (26/30) of sampling sites. These belonged to AMR classes: Colistin (1), Macrolide (5), β-lactam (15), and Fluoroquinolone (12). Metagenomic sequencing identified 5 critically important ARGs in 20% (6/30) of sampling sites contrasted to low impact (Figure1). This study provides a valuable baseline survey of AMR across high and low human impact classifications of Maryland surface waters and has provided a robust methodology for future monitoring efforts to support improved stewardship of antimicrobials.

<figure><img src="image_13.png" /><figcaption>**Fig. 1.** β-lactam genes in high and low impact surface waters across the state of Maryland, USA.</figcaption></figure>

<footnote n="1">¹Center for Veterinary Medicine, Food and Drug Administration / Laurel, MD, United States</footnote>

<footnote n="2">²Washington Fish and Wildlife / Seattle, WA, United States</footnote>

<footnote n="3">³National Antimicrobial Resistance Monitoring System (NARMS)</footnote>

<footnote>*current affiliation Center for Food Safety and Applied Nutrition / College Park, MD, United States</footnote>
# Gram-negative bacteria resistant to last-line antibiotics from hospital and municipal wastewaters

Iva SUKKAR<fnref n="1" />, Adam VALCEK<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Martin KLVANA<fnref n="1" />,<fnref n="4" />, Jarmila LAUSOVA<fnref n="1" />,<fnref n="4" />, Kristina NESPOROVA<fnref n="1" />, Jana PALKOVICOVA<fnref n="1" />, Monika DOLEJSKA<fnref n="1" />,<fnref n="4" />,<fnref n="5" />,<fnref n="6" />

Wastewaters are considered as an important player in the spread of antimicrobial resistance. Here, we focused on hospital and municipal wastewaters, and river water in the city of Brno (Czech Republic) as a possible source of carbapenemase-producing Gram-negative bacteria for the environment.

Selective cultivation on antibiotic-supplemented media followed by PCR was used to obtain a set of 124 isolates carrying *bla*GES or *bla*VIM carbapenemase-encoding genes among 160 Gram-negative bacteria from hospital and municipal wastewaters, and river sampled in the Czech Republic. The isolates were species identified and tested for carbapenemase production and susceptibility to 24 antibiotics. Selected isolates were subjected to short- (Illumina) and long- (MinION) read sequencing. The obtained sequencing data were analysed for the content of resistance genes, plasmids and other genetic determinants using the BLAST algorithm and CGE tools. The phylogenetic tree including publicly available genomes of *Enterobacter asburiae* was conducted using Prokka, Roary and RAxML.

The beta-lactamase GES was produced by 121 strains of different genera (*Enterobacter*, *Escherichia*, *Citrobacter*, *Klebsiella*, etc.). The predominance of the *bla*GES-1 (51%, no carbapenemase activity) and *bla*GES-5 (49%, carbapenemase activity) variants was observed among 68 sequenced isolates. The isolates carried resistance genes to other antimicrobial groups such as aminoglycosides, fosfomycin, sulphonamides, etc. The conjugation transfer of *bla*GES was successful only in 18% of isolates and was mainly associated with ColE2- like plasmids (n=28).

Three VIM-1-producing *E. asburiae* strains originated from hospital and municipal wastewaters. The *bla*VIM-1 gene was located within class 1 integron on different types of plasmids including IncFIB(K)/IncFIB(pQil), IncFIB/IncFII and one non-typeable plasmid. One of the isolates carried *mcr-10* conferring resistance to colistin while in another isolate *mcr-9* gene without the colistin resistance phenotype was detected. In addition, phylogenetic analysis of 360 publicly available *E. asburiae* genomes was performed, showing high genetic diversity but also high prevalence of carbapenemases, alerting the increasing importance of this species for human health.

The dissemination of Gram-negative bacteria with clinically important resistance via wastewaters was observed. These findings pointed out important contribution of hospitals and community wastewaters in transmission of multi-drug resistant pathogens with resistance to last-line drugs.
The study was funded by the grant number NU20J-09-00040.

<footnote n="1">¹Central European Institute of Technology, VETUNI Brno, Brno, Czech Republic</footnote>

<footnote n="2">²Microbial Resistance and Drug Discovery, VIB-VUB Center for Structural Biology, VIB, Flanders Institute for Biotechnology, Brussels, Belgium</footnote>

<footnote n="3">³Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium</footnote>

<footnote n="4">⁴Department of Biology and Wildlife Diseases, VETUNI Brno, Brno, Czech Republic</footnote>

<footnote n="5">⁵Biomedical Center, Faculty of Medicine, Charles University, Pilsen, Czech Republic</footnote>

<footnote n="6">⁶Department of Clinical Microbiology and Immunology, Institute of Laboratory Medicine, The University Hospital Brno, Czech Republic</footnote>
One-Health AMR spread in a French Caribbean island

Mélanie PIMENTA<fnref n="1" />, Degrâce BATANTOU<fnref n="2" />, Margaux GASCHET<fnref n="1" />, Stéphanie GUYOMARD<fnref n="2" />, Marie-Cécile PLOY<fnref n="1" />, Sébastien BREUREC<fnref n="2" />,<fnref n="3" />, Christophe DAGOT<fnref n="1" /> on behalf of the ANRACRAS-R consortium

Antimicrobial resistance (AMR) is considered as a major health issue. AMR needs to integrate different ecosystems such as humans, animals and the environment, called the “One Health” approach. It remains knowledge gaps in this field, particularly concerning the environmental side, among which is the role of environmental conditions (exposome, diversity of the matrices and their microbiota) on the transmission of AMR. Furthermore, the impact of touristic activities in a specific geographical area has not been fully addressed. In this context, the ACRAS-R project aims to characterize the transmission pathways of AMR in a continuum from the clinical settings to the natural receiving environment in the French Caribbean island, Guadeloupe, and assess the contribution of the exposome and tourism on AMR spread. Four sampling campaigns have been done in Guadeloupe during wet and dry seasons. Clinical isolates from patients were collected in addition to water samples (hospital wastewater, influent and effluent of wastewater treatment plants (WWTPs) and their receiving environment), animal feces and the contents of aircraft toilets from metropolitan France to Guadeloupe. DNA extraction for microbiome and resistome analysis was performed on these samples as well as genomic/plasmid analysis on a selection of 3GCsusceptible and ESBL-producing Escherichia coli. The exposome was also analyzed. Preliminary results showed a prevalence of 7% of ESBL E. coli in clinical samples, whereas the prevalence in environmental samples was 32%. We showed the variations in terms of resistome in the different sampling sites, revealing a wide diversity of resistance genes to antibiotics, heavy metals and mobile genetic elements, with a higher relative abundance of these genes in hospital water samples. Concerning aircraft toilets, the relative abundance of resistance genes was variable depending on the company and the day of sampling, but we observed the same

resistome/microbiome signature (proportional abundance). The most abundant phyla in water samples were the same as those usually found in the human gut microbiota: Firmicutes, Bacteroidetes and Actinobacteria, illustrating the anthropogenic impact. In animal samples, the main phyla were Firmicutes, Proteobacteria and Bacteroidetes. Exposome analysis revealed the presence of antibiotic residues (Sulfamethoxazole, Ciprofloxacin, ...) mostly in hospital samples and biocides (Levamisole, Pyrantel pamoate, ...) in WWTP. The results collected will help develop a mathematical model to predict resistance and understand the transmission routes of AMR.

<footnote n="1">¹ Univ. Limoges, Inserm, CHU Limoges, RESINFIT, U 1092, F-87000 Limoges, France.</footnote>

<footnote n="2">²Transmission, Reservoir and Diversity of Pathogens Unit, Pasteur Institute of Guadeloupe, Les Abymes, France</footnote>

<footnote n="3">³University of the Antilles, Faculty of Medicine Hyacinthe Bastaraud, Pointe-à-Pitre, France,</footnote>

<footnote n="3">³INSERM, Center for Clinical Investigation 1424, Pointe-à-Pitre/Les Abymes, France</footnote>

<footnote>Corresponding authors: melanie.pimenta@unilim.fr & dbatantou@pasteur-guadeloupe.fr</footnote>
**Session 3 - Mechanisms and dissemination of antimicrobial resistance in animal and zoonotic pathogens**

O33

**Klebsiella a hitherto underappreciated zoonotic pathogen expressing AMR phenotypes of importance to One Health**

Séamus Fanning

UCD-Centre for Food Safety, School of Public Health, Physiotherapy & Sports Science, University College Dublin, Belfield, Dublin D04 N2E5, Ireland

The threat posed by multidrug resistant (MDR) members of the genus *Klebsiella* can be magnified due to the convergence of antibiotic resistance and virulence phenotypes. These bacteria are now responsible for severe community (CA) - and hospital (HA) - acquired human infections and increasing reports have epidemiologically linked members of this genus to animal infections and within the broader environment. Furthermore, bacterial isolates have been cultured from niches in all three axes of One Health; humans, animals and the environment.

This presentation will review the current understanding of the taxonomy of the genus *Klebsiella*, using comparative genomics, in addition to commenting on a recent study characterising a collection of MDR *K. pneumonia* cultured from a nosocomial setting. The nature of the associated AMR-encoding genotypes will be described along with the virulence factors in these isolates. Furthermore, data commenting on the prevalence of *Klebsiella* species isolated from food and food-producing animals, a hitherto infrequently recognised ecological niche, will be explored. Finally in the context of the environment and *One Health*, the impact that commonly used agrochemicals, such as the herbicide glyphosate, exerts on the antimicrobial susceptibility profiles of these bacteria and associated mechanisms of resistance will be presented. This paper will highlight the relevance of contextualising genomic data to extend our understanding of the risks posed to *One Health*, associated with emerging *K. pneumoniae* and how predictive genomic data can be used to inform and interpret surveillance information.
# WGS-based characterization of emerging pathogenic OXA-48-producing *Enterobacter hormaechei* belonging to ST114 and ST418 in a Swiss companion animal clinic

Valentina Donà<fnref n="1" />, Sonja Kittl<fnref n="1" />, Andrea Endimiani<fnref n="2" />, and Vincent Perreten<fnref n="1" />

**Background.** *E. hormaechei* producing the carbapenemase OXA-48 was repeatedly identified in infections of companion animals hospitalized at a Swiss veterinary clinic, where an outbreak with OXA-48-producing *K. pneumoniae* occurred in the previous months.

**Objectives.** To determine the genetic relatedness of the *E. hormaechei*, and assess the possible acquisition of mobile genetic elements from *K. pneumoniae*.

**Methods.** Hybrid assemblies for phylogenetic and comparative analysis of the isolates (n=9) collected between May 2021 and August 2022 from infectious material of 5 dogs and 3 cats treated at a veterinary hospital in Switzerland were obtained by sequencing with Illumina, PacBio and Oxford Nanopore Technologies. Antimicrobial susceptibility testing was performed by broth microdilution. Phylogenetic relatedness and molecular characteristics were analyzed using publically available bioinformatic tools (e.g., Unicycler v.0.4.8 for hybrid assemblies) and web platforms, such as the Center of Genomic Epidemiology for plasmid and resistance-gene typing, and JSpeciesWS for average nucleotide identity (ANI) calculation, as well as software included in seqSphere v7.7.5 (Ridom) and Geneious v2023.03 (Dotmatics) for the development of a custom core-genome MLST (cgMLST) scheme and core-genome SNP analysis, and for read-mapping and (whole-genome and whole-plasmid) alignments, respectively.

**Results.** The strains were identified as *E. hormaechei* subsp. *xiangfangensis* belonging to sequence type (ST) 114 (n=6) and ST418 (n=2), and as *E. hormaechei* subsp. *hoffmannii* of ST78 (n=1). Core-genome SNP analysis confirmed the clonality of the ST114 and ST418 isolates (0 to 10 SNPs). The strains contained genes associated with decreased susceptibility to meropenem (*bla*<sub>OXA-48</sub>), cephalosporins (*bla*<sub>TEM-1</sub>, *bla*<sub>OXA-1</sub>, *bla*<sub>SHV-12</sub>, *bla*<sub>DHA-1</sub>, *bla*<sub>LAP</sub>), aminoglycosides (*aac*(6')-*Ic*-cr5, *ant*(2")-*Ia*, *aadA*, *aph*(3")-*Ib*, *aph*(3')-*Ia*, *aph*(6)-VIb), tetracyclines [*tet*D], phenicolis (*cat*A2, *cat*B3, *floR*), quinolones (*oqxB*, *qnrA*, *qnrB*, *qnrS*), trimethoprim (*dfrA*), sulfonamides (*sul*1, *sul*2), macrolides [*mph*A], fosfomycin (*fosA*), and rifamycin (*arr*). Five ST114 strains carried a 250 kb IncHI2 multidrug resistance plasmid harboring the colistin resistance gene *mcr*9.1, but were phenotypically susceptible to colistin, as already observed for this species. All strains harbored the *bla*<sub>OXA-48</sub> on identical 63 kb IncL plasmids, but one ST114 and both ST418 isolates showed an inverted variant of the *bla*<sub>OXA-48</sub>-carrying transposon Tn1999.2. This specific transposon was also present in the plasmids carried by the *K. pneumoniae* clones previously responsible for an outbreak in the same animal clinic. Additionally, the *bla*<sub>DHA-1</sub>-carrying *ISCR1* element located on 140 kb IncFIB plasmids in one ST114 and in both ST418 clones was identical to that found in the *K. pneumoniae* outbreak strains, suggesting the transfer of the complete element to an endogenous plasmid backbone.

**Conclusion.** OXA-48-producing *Enterobacter hormaechei* has emerged in companion animals in Switzerland. Common ecological niches could favor the spread of plasmid-borne carbapenemases among *Enterobacterales* and the emergence of MDR *E. hormaechei* clones.

<footnote n="1">¹Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland</footnote>

<footnote n="2">²Institute for Infectious Diseases, University of Bern, Bern, Switzerland</footnote>
# Massive plasticity of IncHI2-ST3 plasmids encoding clinically important IMP-4 in bacteria from silver gulls

Jana PALKOVICOVA<fnref n="1" />, Adam VALCEK<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> and Monika DOLEJSKA<fnref n="1" />,<fnref n="4" />,<fnref n="5" />,<fnref n="6" />

Even though the transmission of carbapenem-resistant *Enterobacteriaceae* (CPE) is mostly associated with clinical facilities, the number of reports of the CPE dissemination outside of healthcare settings is increasing [1]. Mobilization of *bla*IMP-4 and other resistance genes via plasmids was observed in a silver gull colony during our previous study [2]. The most prevalent group, IncHI2 plasmids, showed changes in size during conjugation transfer. In this study, we focus on microevolutionary changes of large IncHI2 plasmids of sequence type 3 (IncHI2-ST3) carrying *bla*IMP-4 that are prone to rearrangements and fusions with other plasmids during a horizontal transfer.

One *Klebsiella pneumoniae* and 42 *Escherichia coli* isolates carrying IncHI2-ST3 and -ST3-like plasmids encoding IMP-4 carbapenemase were short-read sequenced. Out of these, 21 representative wild-type isolates and 12 transconjugants carrying plasmids of diverse size compared to the original plasmid, were selected for long-read sequencing. Complete plasmids were reconstructed and deep bioinformatical analyses on the plasmids were performed.

All studied IncHI2-ST3 and -ST3-like plasmids provide resistance to multiple antibiotics (e.g. carbapenems, beta-lactams) and non-antibiotic resistance determinants (e.g. heavy metals). IncHI2-ST3-like plasmids carry additional genes, e.g. encoding heat resistance or transporters, compared to IncHI2-ST3 plasmids. Analysed plasmids tend to change size through the horizontal transfer during which they acquire genes for ethanol utilization, ATP synthesis, conjugative transfer, virulence, and resistance to antibiotics, heavy metals and formaldehyde. Conjugation of HI2 plasmids lead to fusion with other plasmid types (IncX5, IncP1, IncF-type). The fusions were driven by homologous recombination of MGEs.

Studied IncHI2-ST3 are highly variable, dynamically changing plasmids that are linking resistance to last-line carbapenems with other antimicrobials. Their plasticity represents a threat to public health due to the ability to cointegrate with other plasmids and incorporate additional genes. The changes we observed may provide novel traits beneficial for their bacterial hosts, leading to persistence and further spread of AMR.

## References

[1] J. Xu, W. Lin, Y. Chen, F. He (2020). *Infect Drug Resist* 13: 773-779.

[2] M. Dolejska, M. Masarikova, H. Dobiasova, I. Jamborova, R. Karpiskova, M. Havlicek, N. Carlile, D. Priddel, A. Cizek, I. Literak (2016). *J Antimicrob Chemother* 71(1): 63-70.

<footnote n="1">¹Central European Institute of Technology, University of Veterinary Sciences Brno, Czech Republic,</footnote>

<footnote n="2">²Microbial Resistance and Drug Discovery, VIB-VUB Center for Structural Biology, VIB, Flanders Institute for Biotechnology, Brussels, Belgium,</footnote>

<footnote n="3">³Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium,</footnote>

<footnote n="4">⁴Department of Biology and Wildlife Diseases, University of Veterinary Sciences Brno, Czech Republic,</footnote>

<footnote n="5">⁵Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic,</footnote>

<footnote n="6">⁶Department of Clinical Microbiology and Immunology, Institute of Laboratory Medicine, The University Hospital Brno, Czech Republic</footnote>
# Determining the impact of veterinary antibiotics sub dosage on resistance transfer in animal gut microbiota - a One-Health approach

Laurent Poirel¹,²,³, Otávio Hallal Ferreira Raro¹, Patrice Nordmann¹,²,³,⁴

Medical and Molecular Microbiology, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland¹
Swiss National Reference Center for Emerging Antibiotic Resistance (NARA), Universities of Fribourg and Lausanne, Switzerland²

INSERM European Unit (IAME), University of Fribourg, Fribourg, Switzerland³

Institute for Microbiology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland⁴

## Background

Antimicrobials are an important global resource and have been widely used over the years. From treating humans and animals to ensuring livestock production. However, excessive usage of antibiotics has been driven by pressure and development of bacterial resistance worldwide. Resistance-associated genes can be genetically transmitted and exchanged between pathogenic species co-habiting environments, human and animal communities. Our aim was to evaluate whether sub dosage of widely administered antibiotics in veterinary medicine could enhance plasmid transfer, and consequently, resistance genes exchange in animal gut microbiota.

## Materials

*Escherichia coli* carrying pOXA-48a (*bla*OXA-48) plasmid was submitted to minimum inhibitory concentration for ceftiofur, enrofloxacin, florfenicol, tiamulin, colistin, neomycin, amoxicillin, erythromycin, apramycin, oxytetracycline, narasin, lincomycin, sulfamethazine, zinc oxide, and copper. Treatments in sub-inhibitory dosage were performed with and without antioxidant (edaravone), before moving to i) the conjugation assays to access conjugation frequency; ii) RT-qPCR to access the genomic expression of SOS response associated genes; iii) and fluorescence ROS detection assay to access stress oxidative response. Statistical analyses were performed using Graph Prism 9.3.1.

## Results

Increased conjugation frequencies were observed when treating the isolate with florfenicol (13.7x higher, *p* = 0.0172) and oxytetracycline (97.2x higher, *p* < 0.0001) compared to the control. Increased expression of the SOS-associated *recA* gene for both treatments was also observed (florfenicol, 2,20 ± 0,61, *p* = 0.0057; oxytetracycline, 1.24 ± 0.16, *p* < 0.05). The ROS experiment reinforced previous results for both antibiotics with an intensity of fluorescence 3.7x (florfenicol, *p* < 0.001) and 2.5x (oxytetracycline, *p* = 0.0009) higher when compared with the control. Addition of edaravone reduced plasmid conjugation frequencies when supplemented with the inducing antibiotic (florfenicol, 7.6x lower, *p* = 0.0251; oxytetracycline, 2.4x lower, *p* = 0.0002).

## Conclusions

Induction of resistance dissemination through sub-inhibitory dosage seems to be a key factor in terms of public-health control. In the current One Health concept, we believe that environment, animals, and human health are interlinked and exchanging genetic determinants, mainly critical antimicrobial resistance. Our findings reinforce the commitment to this concept, detecting at least two widely used antibiotics in veterinary medicine, oxytetracycline, and florfenicol, as horizontal resistance genes transfer enhancers.
<figure><img src="image_14.png" /></figure>

O37

# A *In silico* Analyses of Incompatibility Group (Inc) HI2 Plasmids from Enteric Bacteria

Suad ALGARNI<fnref n="1" />,<fnref n="2" />, Jing HAN<fnref n="1" />, Dereje GUDETA<fnref n="1" />, Rajesh NAYAK³, and Steven L. FOLEY<fnref n="1" />,<fnref n="2" />

Antimicrobial resistance (AMR) plasmids play key roles in the dissemination of resistance genes among bacterial pathogens. One interesting group of resistance plasmids is the incompatibility (Inc) HI2 plasmids which are typically large in size (>200 kb), often encode for AMR, heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR) and can be transmissible potentially facilitating resistance dissemination among enteric bacteria. To better understand the distribution and diversity of AMR, DBR and HMR-encoding genes among the IncHI2 plasmids, we utilized multiple computational approaches to evaluate resistance elements and transfer-associated genes among plasmid sequences. A total of 667 IncHI2 plasmid sequences (all available at the time of the search) were identified using the IncHI2 replicon sequence<fnref n="1" /> for a BLAST searching against “Complete Plasmids” available in the Microbial Genomes database. FASTA files for each plasmid were extracted from GenBank and analysed using AMRFinderPlus<fnref n="2" /> web-based tools to detect various resistance genes, IntegronFinder³ to identify class 1 integrons and Plasmid Transfer Factor database to identify transfer (type 4 secretion system, T4SS) genes within the plasmid sequences. Among the plasmid population the most common genera identified as carrying the plasmids include *Enterobacter* (N=209), *Escherichia* (N=208), and *Salmonella* (N=204) isolated across the globe. In many cases, the distribution of the AMR and HMR genes were diverse with plasmids from *Escherichia* and *Salmonella* showing similarity in comparison to the *Enterobacter* and other taxa, which group together. For the HMR, the plasmids from *Enterobacter* and other taxa, had a significantly higher (p<0.05) prevalence of many of the mercury resistance genes (*merA*,*B*,*D*,*E*,*G*,*P*,*R*,*T*) and the arsenic resistance gene, *arsC*, compared to those from *Escherichia* and *Salmonella*. For the AMR genes, there was also diversity, for example, among the sulfonamide resistance genes, *sul1* was more common among the plasmids from *Enterobacter* and other taxa, while *sul2* and *sul3* were more common among those from *Escherichia* and *Salmonella*. Similar diverse AMR genetic trends were displayed for several of the other antimicrobial classes as well. Integron sequences were common among the plasmids and most plasmids contained most IncHI2-associated T4SS genes. These findings provide insights into the diversity of resistance genes among bacteria and allow for the understanding of the dissemination of AMR across different enteric bacterial taxa and geographical locations. The results also underscore the value of computational-based approaches for the assessment of AMR and mobile genetic elements, which are important to understand the molecular epidemiology and public health impact of these large plasmids.

**References**

[1] Carattoli, A., A. Bertini, L. Villa, et al. (2005). *J. Microbiol. Methods* 63:219-28.

[2] Feldgarden, M., V. Brover, B. Fedorov, et al. (2022). *Microb. Genom.* 8:e000832.

[3] Cury, J., T. Jove, M. Touchon, et al. (2016). *Nucleic Acids Res.* 44:4539-50.

<footnote n="1">¹Division of Microbiology and ³Regulatory Compliance and Risk Management, FDA National Center for Toxicological Research, Jefferson, Arkansas, USA</footnote>

<footnote n="2">²Cell and Molecular Biology Program, University of Arkansas, Fayetteville, Arkansas, USA</footnote>
O38

# Investigation of *rmtB*-harbouring *Salmonella* and *Escherichia coli* from a pig farm

Indre NAVICKAITE¹, Harry HOLMES¹, Letizia DONDI¹, Luke RANDALL¹, Catherine FEARNLEY¹, Emma TAYLOR¹, Edward FULLICK¹, Manal ABUOUN¹, Christopher TEALE¹ and Muna F. ANJUM¹

¹ANIMAL AND PLANT HEALTH AGENCY, Addlestone, United Kingdom

Aminoglycosides are antibiotics with broad-spectrum and rapid bactericidal activity making them critically important in human and veterinary medicine as recognized by World Health Organisation (WHO) and World Organization for Animal Health (WOAH), respectively. However, the usage of these antibiotics in humans, livestock, and companion animals has led to the emergence of resistance with post-transcriptional methylation of the 16S rRNA prompting greatest concerns. Methylation of bases key in the binding between 16S rRNA and aminoglycosides leads to decrease or loss of affinity of the antibiotic to its target, thus resulting in high-level resistance to many clinically important aminoglycosides¹. A frequently detected 16S rRNA methylase gene in Enterobacteriaceae isolates is *rmtB*, however this gene is not common in bacteria of veterinary origin. This study aimed to investigate and characterise the spread of *rmtB*-harbouring Enterobacteriaceae isolates from environmental samples on a pig farm as well as to determine the effect of disinfection to mitigate risk of bacterial spread within the farm. Two visits were carried out, where samples were collected between farm cleaning and disinfection procedures. Short- and long-read whole-genome sequencing (WGS) was used to analyse recovered isolates to investigate mobility and spread of *rmtB*-harbouring element(s) in *Salmonella enterica* and commensal *Escherichia coli*. Amikacin resistant isolates were found at both farm visits suggests that disinfection efforts were not effective in removing resistant bacteria. WGS analysis identified a stable ~9kb sequence containing *rmtB* to be chromosomally located in resistant *S. enterica* isolates and on IncFII type plasmids in resistant *E. coli* isolates. Furthermore, this sequence contained insertion (IS)26 element, which is known to promote dissemination of *rmtB*², thus posing a risk of *rmtB* to be spread horizontally to other strains and species. *rmtB* emergence in a livestock environment highlights the need for aminoglycoside resistance to be closely monitored.

References

[1] V.S. Lioy, S. Goussard, V. Guerineau et al. (2014). *RNA* **20**, 382-391.

[2] J. Wang, ZY. Wang, Y. Wang et al. (2020). *Front Microbiol* **11**, 604278.
O39

Various mobile genetic elements involved in the dissemination of phenicol-oxazolidinone resistance gene *oprA* in *Streptococcus suis*

Xingyang DAI¹, Li CHEN¹, Junjie SUN¹, Jinhu HUANG¹, Liping WANG¹*

¹MOE Joint International Research Laboratory of Animal Health and Food Safety, Centre for Risk Assessment of Veterinary Drug Residue and Antimicrobial Resistance, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

Oxazolidinones represent the last-resort antimicrobial agents against infections caused by multidrug-resistant Gram-positive pathogens. Antimicrobial resistance to phenicol and oxazolidinone (PhO) by the dissemination of *oprA* gene leads to treatment failure in both veterinary and human medicine. However, genetic mechanisms for the dissemination of *oprA* gene in streptococci was limited, especially for the zoonotic pathogen *Streptococcus suis*. In this study, we observed a rapid increase of PhO resistance in *S. suis* is attributable to the transferable resistance gene *oprA* (from ~2.5% to 56.4% between ~2011 and 2021) in 601 isolates from eastern China. we selected *oprA*-positive *S. suis* isolates for whole genome sequencing and analysis. IS*1216E* element was presented in 85% of the *oprA*-carrying contigs despite genetic variation observed in the flanking region. IS*1216E*-oprA-carrying segments could be inserted into larger mobile genetic elements (MGEs), including integrative and conjugative elements (ICEs), plasmids, prophages and antibiotic-resistance-associated genomic islands. IS*1216E*-mediated circularization occurred to form the IS*1216E*-oprA-carrying translocatable units, suggesting a crucial role of IS*1216E* in *oprA* spreading. Three types of *oprA*-carrying MGEs (ICE, plasmid and prophage) were successfully transferred via conjugation at different transfer frequencies. In addition, conjugative transfer of *oprA*-carrying plasmid and prophage in streptococci was validated for the first time. In conclusion, different *oprA*-carrying MGEs are associated with the rapid increase of PhO resistance in *S. suis*. Considering the abundance of MGEs in *S. suis* and the mobility of IS*1216E*-oprA-carrying translocatable units, attention should be paid to the potential risks to public health by the emergence and spread of PhO-resistant *S. suis*.

<figure><img src="image_15.png" /><figcaption>Fig. 1. Transfer of *oprA*-carrying MGEs contributes to the rapid increase of PhO resistance.</figcaption></figure>

References

[1] Huang J, Sun J, Wu Y, Chen L, Duan D, et al. (2019). *Int J Antimicrob Agents* 54:43-48.

[2] Du F, Lv X, Duan D, Wang L, Huang J. (2019). *Front Microbiol* 10:2026.

[3] Schwarz S, Zhang W, Du X, Kruger H, Fessler AT, et al. (2021). *Clin Microbiol Rev* 34:e0018820.
# Development of efflux pump MexB and MexY dual inhibitors against multi-drug resistant *Pseudomonas aeruginosa*

Seiji YAMASAKI<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Ryosuke NAKASHIMA<fnref n="1" />, Takayoshi SUZUKI<fnref n="1" /> and Kunihiko NISHINO<fnref n="1" />,<fnref n="2" />,<fnref n="4" />

Multi-component type (RND type) multidrug efflux pumps are important in the multidrug resistance of Gram-negative bacteria. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. This study is aimed to develop drugs for multi-drug resistant *Pseudomonas aeruginosa* (MDR-PA) infections targeting the drug efflux pump. We focused on two RND-type multidrug efflux pumps, MexB and MexY, and decided to discover a dual inhibitor against both pumps (Fig. 1). We have discovered an advanced compound that demonstrates the potent and dual inhibition activities both MexB and MexY and enhances the antibacterial activity of the combination antimicrobial agent against *P. aeruginosa* through rational structure-based drug design. The combination of drug efflux pump inhibitors can restore the antimicrobial activity of various chemotypes of antimicrobials and is expected to be highly effective in the treatment of MDR-PA infections. In addition, the drug efflux pump inhibitor inhibits the efflux of the antimicrobials and increases its concentration in the bacteria, and thus is likely to contribute not only to the recovery of antimicrobial activity but also to the prevention of resistance to the antimicrobials itself. Furthermore, since RND-type multidrug efflux pumps is involved in the resistance mechanism of not only *P. aeruginosa* but also many Gram-negative bacteria, the findings from this development of the drug efflux pump inhibitors can be applied to the development of the efflux pump inhibitors against several drug-resistant bacteria.

<figure><img src="image_16.png" /><figcaption>**Fig. 1.** Development of Universal Inhibitors for the MexB and MexY Multidrug Efflux Pumps in *P. aeruginosa*.</figcaption></figure>

## References

[1] S. Yamasaki, M. Zwama, R. Nakashima, K. Nishino, et al. (2022). *Antimicrob Agents Chemother* 66:e0067222.

[2] M. Fujiwara, S. Yamasaki, Y. Morita, and K. Nishino. (2022). *J Infect Chemother* 28:595-601.

[3] R. Nakashima, K. Sakurai, S. Yamasaki, K. Hayashi, C. Nagata, K. Nishino, et al. (2013). *Nature* 500:102-106.

<footnote n="1">¹SANKEN, Osaka University, Osaka, Japan</footnote>

<footnote n="2">²Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan</footnote>

<footnote n="3">³Institute for Advanced Co-Creation Studies, Osaka University, Osaka, Japan</footnote>

<footnote n="4">⁴Center for Infectious Disease Education and Research, Osaka University, Osaka, Japan</footnote>
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# Glyphosate Induces Changes to Cellular Metabolic Pathways and Selects for Antimicrobial Resistance in *Klebsiella* species.

Katie WALL¹, Leonard KOOLMAN¹, Guerrino MACORI¹ and Séamus FANNING¹

¹UCD Centre for Food Safety, University College Dublin, Dublin, Ireland

The shikimic acid metabolic pathway supports the biosynthesis of aromatic amino acids in plants and microorganisms. Glyphosate is a competitive inhibitor of this pathway and a key ingredient in broad-spectrum herbicides such as RoundUp®. *Klebsiella pneumoniae* is associated with nitrogen fixation in plant roots and has recently been identified as a hitherto unrecognised zoonotic pathogen in the food chain being characterised by its hypervirulence and multidrug resistance in clinical settings. Transient exposure to glyphosate in the environment could lead to changes in susceptibility to antimicrobial compounds in root-associated *K. pneumoniae* as has been identified in other bacteria and these changes could lead to more severe community acquired infections, that have an impact on public health (1).

In this study, the minimum inhibitory concentration (MIC) of glyphosate was determined by broth microdilution for five *Klebsiella* isolates (including the type strain MGH 78578), along with clinical (*K. pneumoniae* and *K. variicola*), environmental (*K. variicola*), and an animal (*K. variicola*) strain. A sub-inhibitory concentration of glyphosate (0.25 X MIC) was then used to determine whether it exerted any changes on antimicrobial susceptibility against a panel of antibiotics using Sensititre plates. Phenotype microarrays (PM) were also used to further explore any metabolic effects using the same sub-inhibitory selection. Changes in protein expression in the presence of sub-inhibitory glyphosate were assessed by LC-MS/MS.

When the *Klebsiella* species were exposed to glyphosate, a significant (P < 0.05) increase in resistance to tigecycline was recorded in all study isolates, and to imipenem in all but one strain (a *K. pneumoniae* strain with a RamA deletion). PM results describing bacterial metabolism (carbon, nitrogen, phosphorous and sulphur utilisation), chemical sensitivity and effects of pH, osmosis, and ionic effects were studied. showed that metabolic pathways were affected following exposure to glyphosate leading to reduced metabolism of carbon sources and redirecting flux towards the purine salvage pathway. Cellular metabolism in glyphosate-exposed strains was also significantly increased at pH 9.5, and this was mirrored by increased crystal violet staining suggestive of an increase in biofilm formation at this pH. Preliminary proteomic results appear to confirm the emphasis on purine metabolism.

This study reports that sub-inhibitory glyphosate can extend the antimicrobial resistance phenotype, unmasking resistance to tigecycline and carbapenems, as well as inducing virulence traits such as biofilm formation.

## References

[1] Hertel, R., J. Gibhardt, M. Martienssen, R. Kuhn & F. M. Commichau (2021) Molecular mechanisms underlying glyphosate resistance in bacteria. *Environmental Microbiology*, 23, 2891-2905.
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Quinolone resistance and expression of RND efflux pump in *Stenotrophomonas* spp. under exposure to sub-lethal concentration of nickel

Amandine CHAUVIAT, Gwenaëlle GRAULIER, Sylvie NAZARET, and Sabine FAVRE-BONTE

« Bacterial Efflux and Environmental Resistance » research group, UMR CNRS 5557, INRAE 1418, Ecologie Microbienne, University Lyon 1 ; 69622 Villeurbanne Cedex, France.

The worldwide use of antibiotics in human and animal medicine and in food animal production has contributed to the emergence of antibiotic resistant bacteria. Anthropogenic activities have participated to the continuous input of antibiotics, antibiotic resistant bacteria and antibiotic resistance genes into aquatic and terrestrial environments. Along to antibiotics, toxic chemical stressors like metals might be present in contaminated environments and exert a co-selection pressure that contributes to the maintenance and proliferation of antibiotic resistance in the environment (1). A positive relationship between antibiotic and metal resistance has been described in *S. maltophilia* soil strains (2). This species is responsible for healthcare-associated infections and has the ability to resist numerous antibiotic families due to efflux pumps. However, regardless of the origin of the strains (clinical or environmental), the efflux pump content is similar (3) to that of *S. indicatrix*, a phylogenetically related species never isolated from humans (4).

To evaluate whether the exposure to anthropogenic pressures could lead to the evolution and the induction of the emergence of antibiotic resistance in an environmental species, representing a risk to human health, we performed a phylogenetic study in the *Stenotrophomonas* genus using two environmental strains, a *S. maltophilia* one and a *S. indicatrix* one. We then conducted an experimental evolution assay comparing both strains under sub-MIC NiCl₂ exposure every two days for one month. Throughout the experiment, ~1100 isolates were collected and submitted to antibiotic phenotyping. The results showed an increase in the number of quinolone resistant colonies of *S. indicatrix* (MIC > 2 mg/L and MIC > 4 mg/L for ciprofloxacin and pefloxacin respectively). We screened for the gene expression of 8 Sme efflux pumps and showed an overexpression of smeVWX gene from day 9 in *S. indicatrix* strain exposed to MIC/8 of NiCl₂. All these data suggest the potential for emergence of efflux-mediated antibiotic resistance among environmental bacteria exposed to metals, even if they are only slightly available in the environment, and consequently the risk for humans to be in contact with them.

References

[1] Baker-Austin et al. (2006). *Trends Microbiol.* 14(4):176-82.

[2] Deredjian et al. (2016) *Res Microbiol.* 167(4):313-324.

[3] Youenou et al. (2015) *Genome Biol Evol.* 14(9):2484-505.

[4] Weber et al. (2018) *Int J Syst Evol Microbiol.* 68(6): 1830-1838.
Heavy metals may increase ARG persistence in the environment

Emilie DEHON<fnref n="1" />,<fnref n="2" />, Alban MATHIEU<fnref n="2" />, Arnaud DROIT<fnref n="2" />, Timothy M. VOGEL<fnref n="3" /> and Concepcion SANCHEZ-CID<fnref n="1" />

The OneHealth approach requires determining the risk that antibiotic resistance genes (ARGs) in the environment pose to human health. ARGs in the environment provide opportunities for human microbiome bacteria and human pathogens to acquire resistance to antibiotics through horizontal gene transfer. Heavy metals can impose a selective pressure on environmental bacteria and select for antibiotic resistance, since ARGs can co-occur with metal resistance genes. In addition, crossresistance mechanisms between metals and antibiotics such as efflux pumps have been observed. Since metals persist longer than antibiotics in the environment, they may exert a selective pressure on environmental bacteria for longer periods than antibiotics and could cause an increase in the persistence of ARGs in the environment. In our study, river water microcosms were exposed to heavy metals (copper and zinc), fluoroquinolone antibiotics (ciprofloxacin and ofloxacin) and both for 30 days to evaluate the persistence of ARGs using shotgun metagenomic analysis of the extracted DNA. Higher abundances of efflux pumps conferring resistance to ciprofloxacin and ofloxacin were observed in samples exposed to metals, both in the presence and absence of antibiotics than in controls. Therefore, copper and zinc could select for and maintain fluoroquinolone resistance in the environment by cross-resistance. The ARGs that showed an increase in abundance in heavy metal-contaminated river water depended on whether antibiotics were present or not. Therefore, both metals and antibiotics conferred resistance, although the resistance profiles were not the same. Thus, our results are consistent with heavy metal persistence leading to the maintenance of ARGs in the environment.

<footnote n="1">¹Environmental Microbial Genomics, CNRS UMR 5005, Laboratoire Ampère, École Centrale de Lyon, Université de Lyon, Écully, France</footnote>

<footnote n="2">²CHU de Québec-Université Laval Research Center, Endocrinology and Nephrology Axis, Québec City, Québec, Canada</footnote>

<footnote n="3">³Université de Lyon, Université Claude Bernard Lyon 1, UMR 5557, UMR INRAe 1418, VetAgro Sup, Écologie Microbienne, équipe BEER, F-69622 Villeurbanne, France</footnote>
WGS-based relationship between multidrug-resistant bacteria from humans
and animals: emphasis on CPE and MRSA

Division of Molecular Bacterial Epidemiology & Infectious Diseases, Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland

The increasing number of bacteria exhibiting resistance the clinically important antibiotics in humans, animals and environment has raised questions about their origin and their dissemination between the different settings. Whole genome sequencing (WGS) is nowadays the technology of choice, which permits to rapidly characterize and compare bacterial strains from different settings. In this presentation, the degree of relatedness and genetic characteristics of carbapenemase-producing Enterobacterales (CPE) from companion animals and methicillin-resistant S. aureus (MRSA) from animals was determined and the resulting WGS-based data were compared with those of strains from human origin.

During the summer 2018, a major outbreak of *Escherichia coli* ST410 producing the carbapenemase OXA-181 occurred in a veterinary clinic in Switzerland, where one quarter of the hospitalized companion animals acquired this bacteria during their stay at this clinic [1]. Over the same period, one employee of the clinics was found to be colonized with the same *E. coli* [2]. Nevertheless, CPE were not detected among owners of CPE positive animals [3]. In the following years, OXA-48-producing and multidrug-resistant *Klebsiella pneumoniae*, *Enterobacter cloacae* and *E. coli* also emerged in infection sites of companion animals in the same clinic. The emergence of CPE in veterinary settings raised the question whether they are related to the strains causing infections in humans in Switzerland. Human OXA-181 and OXA-48 producing CPE were made available for comparative genomic analysis from the National Reference Center for Emerging Antibiotic Resistance (NARA), University Fribourg, where human clinical CPE isolates from Switzerland are sent for biochemical and molecular analysis and archived [4].

Cases of human infections with the livestock-associated MRSA (LA-MRSA) ST398 were recorded
in Switzerland in the past years. Comparative genomic analysis with LA-MRSA ST398 from
different animals including pigs, horses, cattle, poultry, companion animals as well as veterinarians
(See Poster of Fernandez et al.) revealed that the human clinical strains were related to strains causing
infections in horses clustering into a specific lineages. They were also detected in veterinary
personnel and in non-hospitalized healthy horses [5]. The LA-MRSA ST398 of this lineage also share
the same resistance and virulence profile.

Acquisition of CPE and MRSA in veterinary clinics poses the evident risk of their further dissemination to other animals, humans and into the environment. However, the direction of transmission between the different settings remains an open question and is likely to occur in both direction. A continuous One-Health and WGS-based surveillance will contribute to rapidly identify new emerging multidrug-resistant bacteria and their potential reservoirs and routes of dissemination in animals, humans and environment.

References

[1] A. Nigg, M. Brilhante, V. Dazio, M. Clément, A. Collaud, et al. (2019). *Euro Surveill* 24:1900071.

[2] A. Endimiani, M. Brilhante, O.J. Bernasconi, V. Perreten, J.S. Schmidt, et al. (2020). *J Antimicrob Chemother* 75:766-768.

[3] V. Dazio, A. Nigg, J.S. Schmidt, M. Brilhante, E.I. Campos-Madueno, et al. (2021). *One Health* 13:100322.

[4] J. Findlay, Perreten V, Poirel L, Nordmann P. (2022). *Eur J Clin Microbiol Infect Dis* 41:1355-1360.

[5] J.I. Hurni, S. Kaiser-Thom, V. Gerber, J.E. Keller, A. Collaud, et al. (2022). *Schweiz Arch Tierheilkd* 164:499-512.
# U.S. FDA's National Center for Toxicological Research AMR Research: Applications across the One Health Continuum

Rajesh NAYAK<fnref n="1" />, Jing HAN<fnref n="2" /> and Steven L. FOLEY<fnref n="2" />

Research scientists at the U.S. FDA's National Center for Toxicological Research (NCTR) have engaged in multiple antimicrobial resistance-related research projects and several of these projects address "One Health" needs within the FDA. This presentation provides an overview of key efforts to generate data which can inform these public health efforts. The projects focused on 1) the characterization of antimicrobial resistance (AMR) genotype and phenotypes in food, feed and companion animals; 2) the development and use of in vitro systems to evaluate factors that potentially contribute to antimicrobial and pathogenicity in enteric bacteria; and 3) the development of database and associated bioinformatics tools to analyze whole genome sequencing (WGS) data to characterize pathogens, including *Salmonella enterica* and *Escherichia coli* that occur throughout the One Health sphere. The exposure of bacterial populations to antimicrobials can contribute to the emergence of AMR in various ecosystems due to selection of resistant mutants and/or the acquisition of AMR genes through horizontal transfer. Research efforts at NCTR have examined plasmids and their role in the dissemination of AMR and virulence genes among different bacteria. The linkage of AMR and virulence is important, since often the more severe manifestations of enteric diseases require antimicrobial therapy and may allow bacteria to adapt in diverse environments across the One Health continuum. Specific efforts have examined *Salmonella*-associated plasmids, including the use of WGS and PCR-based methods to characterize multidrug resistant (MDR) strains of *Salmonella* from a variety of food animal and human clinical sources to understand the distribution of AMR genes in the different bacterial populations <fnref n="1" />⁻³. Research has also centered on the emergence of extended-spectrum β-lactamases (ESBLs) and carbapenem-resistant *E. coli* isolated from companion animals as a potential public health issue⁴,⁵. Many of these resistance and virulence gene determinants in *Salmonella* and *E. coli* are co-located on plasmids that contribute to drug resistance and pathogenicity. Because of the need for better characterization of factors that contribute to the dissemination of AMR, research efforts at NCTR have also addressed extrinsic factors that contribute to resistance transfer, such as the impact of an antimicrobial exposure on plasmid transfer. Our studies showed a diversity of responses to the exposures; however, in several cases, exposure to different concentrations of chloramphenicol and tetracycline increased plasmid transfer among bacterial isolates. To further understand such selective transfers, a plasmid transfer gene database and analyses tools were developed to evaluate the transfer-associated genes that can facilitate the dissemination of plasmids among bacteria (see presentation by Dr. Jing Han at this conference). Overall, research efforts at NCTR have led to a better understanding of mechanisms associated with AMR in various host environments and factors that contribute to the dissemination of AMR across the One Health continuum.

## References

[1] Han, J., David, D., Deck, J., et al. (2011). *J. Clin. Micro.* 49:1130-33.

[2] Han, J., Gokulan, K., Barnette, D., et al. (2013). *Foodborne Path. Dis.* 10:1008-1015.

[3] Aljahdali, N.H., Khajanchi, B. K., Weston, K., et al. (2020). *Genes* 11:1307.

[4] Shaheen, B. W., Nayak, R., Foley, S. L. et al. (2011). *Antimicrob. Agents Chemotherapy* 55:5666-5675.

[5] Shaheen, B. W., Nayak, R., Foley, S. L., Boothe, D. M. (2013). *J. Agents Chemotherapy* 68:1019-1024.

<footnote n="1">¹Regulatory Compliance and Risk Management and</footnote>

<footnote n="2">²Division of Microbiology, FDA National Center for Toxicological Research, Jefferson, Arkansas, USA</footnote>
# Investigating the circulation of AMR between animals, humans and their environment in Cambodia - ARCAHE

Juliette Hayer<sup>1,2&</sup>, Ella Marcy<sup>1,2&</sup>, Mallorie Hidé<sup>1,2</sup>, Savatey Hak<sup>2,3</sup>, Sivhour Chiek<sup>4</sup>, Navin Sreng<sup>3</sup>, Meymey Lem<sup>3,4</sup>, Rina Dork<sup>4</sup>, Chiva Ma<sup>2,3</sup>, Gauthier Delvallez<sup>2,3</sup>, Patrice Piola<sup>5</sup>, Véronique Chevalier<sup>6</sup>, Anne-Laure Bañuls<sup>1,2*</sup> and Sokleaph Cheng<sup>2,3*</sup>

Antimicrobial resistance (AMR) is an increasing public health concern and threatens decades of infectious disease control efforts. The emergence and spread of AMR are mainly attributed to the overuse of antibiotics, whether in humans, animals, or the environment. The development of AMR control and monitoring strategies has become a priority in Low- and Medium-Income Countries (LMICs), particularly in South East Asia, where very high levels of AMR prevalence were reported. Cambodia is also confronted with AMR. We observe the emergence of bacteria resistant to multiple antibiotics, which directly impact human health. Today, we still do not know why and where AMR emerges and how it circulates between humans, animals, and the environment in Cambodia. This information is nevertheless essential for the establishment of effective control strategies. In the framework of the FSPI ARCAHE project (Antibiotic resistance at the Human/Animal/Environment interface in a "One Health" approach in Cambodia), we sought to study, in a "One Health" approach, the resistome present in the environment and animals of patients infected with multi-drug resistant *Enterobacteriaceae*. The research was conducted utilizing genomic and metagenomic sequencing strategies and through the extensive use of bioinformatics workflows.

Patient samples were collected at the provincial hospital of Battambang. Environmental samples (e.g., soil, wastewater, drinking water) and oral/rectal swab samples from domestic animals (chickens, pigs, ducks, and dogs) were collected at the patient households. In total, 995 samples were collected. Each sample was collected in duplicate; one for bacterial cultures with antibiotic-selective media and one for metagenomics analysis.

The *Enterobacteriaceae* strains isolated from patients, animal, and environmental samples, have been submitted to Whole Genome Sequencing (WGS) on the Illumina NovaSeq platform (MGX platform, Montpellier, France). For analyzing the WGS datasets, we have implemented a bioinformatics workflow: Bacterial Assembly and Antimicrobial Resistance Genes detection In Nextflow (*baargin*). This tool allows us to assemble the genomes, check for contamination, detect the sequences of plasmid origin, identify the ARGs and mutations conferring resistance, annotate the genomes, and perform a pangenome analysis of all the datasets provided to the workflow. *Baargin* outputs presence/absence matrices that enable comparative study between the different strains based on the resistance profiles.

The comparison between the bacterial resistance profiles of strains isolated from animal and environmental samples collected at the patients' households shows that the animal resistance profiles, such as Extended-Spectrum Beta-Lactamases producing *Enterobacteriaceae* (ESBLE) or Carbapenemase Producing *Enterobacteriaceae* (CPE), were more similar to the patient resistance profiles than the environmental ones. These results suggest similar antibiotic or biological pressure and/or privileged circulation between humans and animals.

<footnote n="1"><sup>1</sup>UMR MIVEGEC, Université de Montpellier, IRD, CNRS, centre IRD, Montpellier, France.</footnote>

<footnote n="2"><sup>2</sup>LMI DRISA "Drug Resistance in South East Asia", Phnom Penh-Montpellier</footnote>

<footnote n="3"><sup>3</sup>Institut Pasteur du Cambodge, Phnom Penh, Cambodia.</footnote>

<footnote n="4"><sup>4</sup>Battambang Provincial Referral Hospital, Cambodia</footnote>

<footnote n="5"><sup>5</sup>CCI France Vietnam</footnote>

<footnote n="6"><sup>6</sup>UMR ASTRE, CIRAD</footnote>

<footnote n="8"><sup>8</sup>*The two authors contributed equally to the work</footnote>
<figure><img src="image_17.png" /></figure>

O47

# The effect of antibiotic usage on resistance in humans and food-producing animals: a longitudinal, one-health study using European data

Sakib RAHMAN¹, and Aidan HOLLIS¹

¹Department of Economics, University of Calgary, Calgary, Canada

This paper estimates the effect of antibiotic usage in humans and food-producing animals on the incidence of resistance in zoonotic bacteria in both humans and animals. We use comprehensive data on usage in humans and food-producing animals of 11 antibiotic classes and occurrence of resistance in three bacterial species common in humans and food-producing animals from European surveillance reports over 11 years in 31 countries. This allows us to make numerous contributions to the growing literature on antibiotic use and resistance.

First, although antibiotic usage occurs simultaneously in humans and other animals, existing studies have almost exclusively considered the effect of antibiotic use in humans *or* other animals. Our study introduces an analysis with usage in both humans and food-producing animals, allowing us to identify the marginal effect of usage in humans and animals. We are also able to estimate their joint effect. Second, our longitudinal data allow us to estimate the effect of usage on resistance in a causal framework, rather than just estimating correlations. We use a methodology pioneered in the economics literature that allows us to bound the causal effect from use to resistance, though not to estimate it precisely.

Third, we show the effect of antibiotic usage in humans on resistance in food-producing animals at an ecological level. To our knowledge, no previous study has attempted to show how antibiotic use in humans is related to resistance in other animals.

The estimated effects are both substantial and statistically significant. Strikingly, the lower and upper bounds of the effect of antibiotic use in animals on resistance in humans are not smaller than the effect of antibiotic use in humans. The estimated elasticities are, from the perspective of long-term impact on resistance, disturbingly large. Even at the lower bound, an increase in antibiotic use in animals of only 10% is expected to increase the prevalence of resistance in animals by around 2% and in humans by around 0.3%. Since, as we show in a recent paper, resistance tends to persist over a period of years, increases in usage may lead to long-term increases in resistance.[1]

References

[1] Rahman S, Kesselheim AS, Hollis A. Persistence of resistance: a panel data analysis of the effect of antibiotic usage on the prevalence of resistance. The Journal of Antibiotics. 2023 Feb 28:1-9.
# Comparative genomics of *Escherichia coli* ST131 of human, animal and environmental origin from the Czech Republic

Michaela RUZICKOVA<fnref n="1" />,<fnref n="2" />, Ivana JAMBOROVA<fnref n="1" />, Tomas NOHEJL<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Iva SUKKAR<fnref n="1" />, Jana PALKOVICOVA<fnref n="1" />, Ivo PAPOUSEK<fnref n="2" />, Max CUMMINS<fnref n="3" />, Steven DJORDJEVIC<fnref n="3" />, Monika DOLEJSKA<fnref n="1" />,<fnref n="2" />,<fnref n="4" />,<fnref n="5" />

*Escherichia coli* and its multidrug-resistant sequence types (ST) have been found to be particularly successful at spreading antibiotic resistance. One of the most widespread clones of medical importance is ST131 which is responsible for human nosocomial diseases such as urinary tract or bloodstream infections. This clone is also capable of infecting animals and disseminating through various environments and its sublineages have been also linked with retail meats. The aim of the study was to explore the genetic structure of a large collection of source diverse ST131 in terms of antibiotic resistance genes and plasmids and perform the phylogenetic analysis to evaluate the clonality of isolates coming from different sources.

To this end, we performed a whole-genome sequencing of 898 ST131 isolates originating from humans (n=713), the environment (n=139), wildlife (n=32), food animals (n=13) and companion animals (n=1) in the Czech Republic during years 2009-2021. Isolates were assigned to STs and the content of antibiotic resistance genes, virulence factors and plasmid replicons was assessed. Phylogenetic analysis based on the core-genome of all ST131 isolates, in combination with characterization of *fimH* and *blaCTX-M* genes, enabled categorization of strains into clades A, B, C0, C1 and C2.

Overall, we detected 72.4% of isolates carrying a variant of *blaCTX-M* and 19.8% of isolates carrying a variant of *blaTEM* responsible for resistance to beta-lactam antibiotics regardless of the clade or source. Most isolates belonged to clade C (804/898), characterized by *fimH30* variant, with C1 (29.2%; 262/898) carrying *blaCTX-M-27* and C2 (58.8%; 528/898) linked with *blaCTX-M-15*. This clade frequently contained isolates with point mutations in *parC* and *gyrA* responsible for fluoroquinolone resistance. Forty-nine isolates belonged to clade A carrying *fimH41* and *blaCTX-M-27*, while 45 isolates were assigned to clade B with *fimH22* and *blaCTX-M-14*. Inside clade C1, high clonality was observed among a subset of isolates originating from humans, wild birds, and wastewater (0-50 SNPs difference); a trend also observed in clade C2 within groups of isolates obtained from humans and wastewater. Presence of diverse F-type plasmids was detected throughout all isolates regardless of clade association. Plasmids similar to pUTI89 (F29:A-B10) that carry *cjrABC-senB* cluster contributing to virulence in pathogenic bacteria from urinary tract infections, were associated with clade A isolates from humans and water samples. Clade B isolates frequently carried ColV plasmids of various sequence types and originated mainly from humans, wild birds, and poultry.

Our collection of *E. coli* ST131 comprising isolates from various sources, environments and years was mostly clonal, particularly in phylogenetic groups C1 and C2. Most of the isolates were associated with diverse epidemiologically relevant F plasmids disseminated throughout various niches. These findings highlight the importance of studies focused on previously unmapped parts of the world and support the claim of the global threat these bacteria pose.

Funded by IGA VETUNI 209/2023/FVHE and Czech Health Research Council NU22-09-00645.

<footnote n="1">¹Central European Institute of Technology, University of Veterinary Sciences Brno, Brno, Czech Republic,</footnote>

<footnote n="2">²Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Brno, Czech Republic,</footnote>

<footnote n="3">³Australian Institute for Microbiology and Infection, University of Technology Sydney, Sydney, Australia,</footnote>

<footnote n="4">⁴Department of Clinical Microbiology and Immunology, Institute of Laboratory Medicine, The University Hospital Brno, Brno, Czech Republic,</footnote>

<footnote n="5">⁵Biomedical Centre, Charles University, Pilsen, Czech Republic</footnote>
# Understanding the risks associated with recirculating aquaculture systems: persistence of antimicrobial-resistant *Enterococcus* spp. and comparison with isolates from human infections

Joyshri SARKER<fnref n="1" />, Aylar SABA SHIRVAN<fnref n="1" />, Céline DAVID<fnref n="1" />, Stephane CORVEC<fnref n="2" />, Eric BATARD<fnref n="3" />, Maria UGARTE-RUIZ<fnref n="4" />, Ségolène CALVEZ<fnref n="1" />, Nora NAVARRO-GONZALEZ<fnref n="1" />

Antimicrobial resistance (AMR) is recently becoming a concern in the aquaculture sector. In rainbow trout farms from Portugal, Novais et al. (2018) found that inflow water and commercial feed were vehicles of AMR-*Enterococcus*. Enterococci are considered indicators of fecal contamination from birds and mammals, and indicators of AMR from the environment.

With this longitudinal observational study, we aim at monitoring the presence and persistence of *Enterococcus* spp. in a recirculating aquaculture system (RAS) raising rainbow trout at Oniris (Nantes, France). We also aim at determining AMR phenotypes and at comparing with isolates from human infections. Over five monthly sampling events (February-July 2022), 95 samples were collected from all inputs and compartments of the RAS: water (n=33), biofilm (n=23), sediment (n=5), fish feces (n=23) and feed administered to fish (n=4). Water was sampled at each treatment step: arrival of city water, sand filtration, biological filtration, UV treatment, arrival at fish tanks and reservoir tank. Suspected colonies isolated on Slanetz and Barley agar (37°C, 48h) were confirmed with the use of MALDI-TOF. Ten samples out of 95 were positive for *Enterococcus* spp. In fish feed, *Enterococcus faecium* was found (1 sample out of 4), as well as *E. gallinarum* (2/4) and *E. casseliflavus* (1/4). Oppositely, uncommon species such as *E. moraviensis*, *E. silesiacus* or *E. termitis* were found in RAS samples: 3 samples out of 33 (9%) in water, 3/23 (13%) in fish feces, 1/23 (4%) in biofilm and 2/5 (40%) in sediment. The highest prevalence was observed in May (50%) followed by July (20%), whereas *Enterococcus* spp. were not detected in February. It is remarkable that the RAS water temperature increased over time (14°C to 18°C). These results suggest that *Enterococcus* introduced by feed do not persist in a RAS raising rainbow trout. All *Enterococcus* isolates, in addition to 20 *E. faecium* from human infections retrieved from the University Hospital (CHU), Nantes, will be tested with the broth microdilution method against Vancomycin, Teicoplanin, Quinupristin-Dalfopristin, Tetracycline, Daptomycin, Ciprofloxacin, Erythromycin, Tigecycline, Linezolid, Gentamicin, Ampicillin and Chloramphenicol. These results will shed light on the similarity between AMR phenotypes in *Enterococcus* spp. from the RAS environment and human infections.

## Reference

Novais C., Campos J., Freitas, A.R., Barros M., Silveira E., Coque T.M., Antunes P., Peixe L. (2018). Water supply and feed as sources of antimicrobial-resistant *Enterococcus* spp. in aquacultures of rainbow trout (*Oncorhynchus mykiss*), *Portugal. Science of the Total Environment* 625:1102–1112.

<footnote n="1">¹ BIOEPAR UMR 1300, Oniris - INRAE, Nantes, France</footnote>

<footnote n="2">² Université de Nantes CHU Nantes Service de Bactériologie et des contrôles microbiologiques, INSERM, INCIT UMR 1302, Nantes, France</footnote>

<footnote n="3">³ Nantes Université, Cibles et médicaments des infections et de l'immunité, IICiMed, UR 1155, Nantes, France</footnote>

<footnote n="4">⁴ VISAVET Health Surveillance Centre. Universidad Complutense Madrid, Madrid, Spain</footnote>
Successful host adaptation of IncK2 plasmids.

Marta ROZWANDOWICZ¹, Henrik HASMAN², Mike SM BROUWER³, Muna F ANJUM⁴,
Stefan BÖRJESSON⁵,⁶, Manuela CANIÇA⁷, Jens A HAMMERL⁸, Virginia CARFORA⁹, Benoît
DOUBLET¹⁰, Antoni PA HENDRICKX¹, Emma ÖSTLUND¹¹, Jannice S SLETTEMEÅS¹²,
Magdalena ZAJĄC¹³, Pieter-Jan CEYSSENS¹⁴, Joost HORDIJK¹, Full Force consortium

¹National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands

2 Statens Serum Institute (SSI), Denmark

3 Wageningen Bioveterinary Research (WBVR), the Netherlands

4 Animal and Plant Health Agency (APHA), UK

5 Public Health Agency of Sweden (PHAS), Sweden

6 Örebro University, Sweden

7 National Institute of Health Doctor Ricardo Jorge (INSA), Portugal

8 German Federal Institute for Risk Assessment (BfR), Germany

9 Istituto Zooprofilattico Sperimentale del Lazio e della Toscana "M. Aleandri" (IZSLT), Italy

10 National Research Institute for Agriculture, Food and the Environment (INRAE), France

11 National Veterinary Institute (SVA), Sweden

12 Norwegian Veterinary Institute (NVI), Norway

13 National Veterinary Research Institute (PIWET), Poland

14 Sciensano, Belgium

Antimicrobial resistance is a global health threat and because it is often encoded on plasmids, it is
crucial to understand the dynamics of plasmid spread and host adaptation. It was shown that IncK
plasmids can be divided into two separate lineages named IncK1 and IncK2. IncK2 plasmids are
found predominantly in poultry. The relatively high body temperature of chicken influences IncK2
plasmids fitness cost, copy number and stress response in the *Escherichia coli* host. These data shed
a light on IncK2 plasmid's success and persistence in *E. coli* of chicken origin.

This study analyzed 50 IncK2 carrying isolates of human, poultry, cattle, pig and environmental
origin from 10 European countries and Lebanon, as well as 14 publicly available IncK2 plasmid
sequences. IncK2 carrying isolates analyzed in this study were sequenced using both Illumina and
Nanopore technology. A phylogenetic analysis of all plasmids was performed in order to determine
the genetic relatedness of IncK2 plasmids isolated in different countries and from different sources.
Additionally, a genome wide association study (GWAS) was performed on annotated sequences to
assess if an association exists between specific genetic features and various sources, that could
explain the suspected IncK2 plasmid adaptation to the chicken host. The obtained results show that
an antitoxin for the Hok/Gef family protein and YdeA protein were predominantly found on plasmids
isolated from chicken and therefore are significantly associated with IncK2 from the chicken host.
Moreover, protein YffA is significantly associated with IncK2 from human.

In combination with prior findings, this study shows that adaptation of plasmids to a chicken host is
a complex process that involves both physiological and genetic determinants. Understanding the basis
of plasmid adaptation may lead to the development of intervention strategies that reduce the spread
of AMR plasmids between different sources.
O51

# Indications of transmission of *mcr*-1.26 IncX4 plasmids along the poultry food chain to humans

Ulrike BINSKER<fnref n="1" />,*, Kathrin OELGESCHLÄGER<fnref n="1" />, Annemarie KÄSBOHRER<fnref n="1" />,<fnref n="2" /> and
Jens A. HAMMERL<fnref n="1" />

Antimicrobial resistance is one of the major global health challenges. The drivers of emergence and evolution of antimicrobial resistances include antimicrobial use and abuse in human, animal and environmental sectors. Their interconnection can contribute to the spread of resistant bacteria and resistance determinants between sectors, inevitably affecting the health of these contiguous habitats. The association of resistance determinants with mobile genetic elements facilitates transmission across habitat boundaries further aggravating the problem. One such example is the transmissible plasmid-mediated colistin resistance (*mcr*) first discovered in 2016. Since then, the main determinant *mcr*-1 has been found in a vast variety of plasmid backbones in diverse bacterial species across all sectors. To date, 34 variants of *mcr*-1 have been described with varying prevalence rates. Whereas common variants, such as *mcr*-1.1 can be used for quantification of transmission events, rare variants allow for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. *mcr*-1.26 is rare and was detected in 2018 in an *E. coli* isolated from a hospitalized patient in Germany. We report on the presence of *mcr*-1.26 in 16 *E. coli* and one *K. pneumoniae* originating from poultry, such as feces and retail meat, already found in 2014. The *mcr*-1.26 was located on transmissible IncX4 plasmids. Comparative bioinformatics analysis suggest horizontal transfer of a conserved *mcr*-1.26 IncX4 plasmid type within poultry husbandry and demonstrate a high similarity to the plasmid reported for the human sample. We further identified two novel *mcr*-1.26-IncX4 types. Three *E. coli* carried an *mcr*-1.26-IncX4 plasmid that acquired an additional resistance gene (*bla*<sub>TEM</sub>). Notably, in addition to the *bla*<sub>TEM</sub> gene, the *mcr*-1.26-IncX4 plasmid of the *K. pneumoniae* isolate encoded a further transposase.

Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of *mcr*-1.26 and its transmission along the poultry food chain to humans. We highlight the transmission of *mcr*-1.26-IncX4 between Enterobacterales to acquire resistance to colistin, a last-line antimicrobial in human medicine. Finally, our study indicates ongoing plasmid evolution of *mcr*-1.26-IncX4 in different bacterial hosts by the acquisition of an additional beta-lactam resistance gene and a transposase.

<footnote n="1">¹Department Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany</footnote>

<footnote n="2">²Department for Farm Animals and Veterinary Public Health, Institute of Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria</footnote>
## The present and future in antibiotic resistance surveillance

Etienne Ruppé

INSERM, Université de Paris, IAME, F-75018 Paris, France

Antibiotic resistance is a major, global health issue which is now recognized as a major cause of deaths worldwide. Especially, resistance to beta-lactam in Gram-negative bacilli such as Enterobacterales, *Pseudomonas aeruginosa* and *Acinetobacter baumannii* is driving a significant part of this burden. Surveillance of antibiotic resistance is crucial to undertake preventive and mitigation measures. Surveillance be applied to different levels of the microorganisms (gene, mobile genetic element, clone), in different sectors (human, animal and environment), in different settings (community, healthcare structures, farms, wildlife, polluted or pristine environments), times and places. Hence, surveillance shall be driven by the needs and the actions taken beyond. In the last decade, major advancements have been achieved in the field of sequencing. Especially, we have now the possibility to finely reconstruct genomes and mobile genetic elements and to track them with a high precision. Sequencing technologies together with bioinformatic tools and knowledge databases also enable the analysis of the antibiotic resistance gene contents (the resistome) from a large diversity of samples and thereby provide knowledge about the circulation of antibiotic resistance gene between pathogens and their environment. Here, we will present how the application of sequencing technologies can be applied to the surveillance of multidrug resistant bacteria in healthcare structures and in a One Health perspective. Then, we will also see how metagenomic sequencing improved our knowledge about the gut resistome and how it can be used to clinical samples.
<figure><img src="image_18.png" /></figure>

O53

# An in-house 45-plex array for the detection of antimicrobial resistance genes in Gram-positive bacteria

Carole KOWALEWICZ, Michael TIMMERMANS, David FRETIN, Pierre WATTIAU and Cécile BOLAND

Veterinary Bacteriology, Sciensano, Ixelles, Belgium

Identifying antimicrobial resistance (AMR) genes and determining their occurrence in Gram-positive bacteria provide useful data to understand how resistance can be acquired and maintained in these bacteria. We describe an in-house bead array targeting AMR genes of Gram-positive bacteria and allowing their rapid detection all at once at a reduced cost. A total of 41 AMR probes were designed to target genes frequently associated with resistance to tetracycline, macrolides, lincosamides, streptogramins, pleuromutilins, phenicols, glycopeptides, aminoglycosides, diaminopyrimidines, oxazolidinones and particularly shared among *Enterococcus* and *Staphylococcus* spp. A collection of 124 enterococci and 62 staphylococci isolated from healthy livestock animals through the official Belgian AMR monitoring (2018–2020) was studied with this array from which a subsample was further investigated by whole-genome sequencing. The array detected AMR genes associated with phenotypic resistance for 93.0% and 89.2% of the individual resistant phenotypes in enterococci and staphylococci, respectively. Although linezolid is not used in veterinary medicine, linezolid-resistant isolates were detected. These were characterized by the presence of *optrA* and *poxtA*, providing cross-resistance to other antibiotics. Rarer, vancomycin resistance was conferred by the *vanA* or by the *vanL* cluster. Numerous resistance genes circulating among *Enterococcus* and *Staphylococcus* spp. were detected by this array allowing rapid screening of a large strain collection at an affordable cost. Our data stress the importance of interpreting AMR with caution and the complementarity of both phenotyping and genotyping methods. This array is now available to assess other One-Health AMR reservoirs.

<table><caption>**Fig. 1.** List of the AMR genes targeted by the array and their related antimicrobials and antimicrobial classes monitored by broth microdilution in *Enterococcus* spp. and/or *Staphylococcus* spp. Antimicrobials monitored by broth microdilution in *Staphylococcus* spp. only. [1]</caption><thead><tr><th>Classes</th><th>Antimicrobials</th><th>Targeted genes</th></tr></thead><tbody><tr><td rowspan="3">Aminoglycosides</td><td>Kanamycin<sup>a,1</sup> (KAN)</td><td rowspan="3">aadD<sup>a</sup>, aadE<sup>c</sup>, aacA-aphD<sup>a,b</sup>, aphA3<sup>a</sup>, aph2-Id<sup>a,b</sup>, aph2-1e<sup>b,c</sup></td></tr><tr><td>Gentamicin<sup>b</sup> (GEN)</td></tr><tr><td>Streptomycin<sup>c</sup> (STR)</td></tr><tr><td>Glycopeptides</td><td>Vancomycin (VAN)</td><td>vanA, vanB, vanC<sub>1</sub>, vanC<sub>2-3</sub></td></tr><tr><td>Lincosamides</td><td>Clindamycin<sup>CL</sup></td><td>ermA, ermB, ermC, IsaA, IsaE, InuA, InuB</td></tr><tr><td>Macrolides</td><td>Erythromycin (ERY)</td><td>ermA, ermB, ermC, mefA/E, mphC</td></tr><tr><td>Oxazolidinones</td><td>Linezolid (LZD)</td><td>cfr, optrA, poxtA</td></tr><tr><td>Phenicol</td><td>Chloramphenicol (CHL)</td><td>catp<sub>C194</sub>, catp<sub>C221-223</sub>, cfr, fexA, optrA, poxtA</td></tr><tr><td>Pleuromutilins</td><td>Tiamulin<sup>TIA</sup></td><td>cfr, IsaA, IsaE, vgaA, vgaB, vgaD</td></tr><tr><td rowspan="2">Streptogramins</td><td rowspan="2">Quinupristin (group B-streptogramin) / dalfopristin (group-A-streptogramin) (Synercid, SYN)</td><td>Streptogramin A (dalfopristin): IsaA, IsaE, vatA, vatB, vatC, vatD, vatE, vgaA, vgaB, vgaD, cfr</td></tr><tr><td>Streptogramin B (quinupristin): ermA, ermB, ermC, IsaA, IsaE, vgbB</td></tr><tr><td>Diaminopyrimidines</td><td>Trimethoprim<sup>TMP</sup></td><td>dfrA/C, dfrD, dfrK, dfrG</td></tr><tr><td>Tetracyclines</td><td>Tetracycline (TET)</td><td>poxtA, letO, letK, tetL, tetM</td></tr></tbody></table>

## Reference

[1] Kowalewicz C., Timmermans M., Fretin D., Wattiau P., & Boland C. (2022). An in-house 45-plex array for the detection of antimicrobial resistance genes in Gram-positive bacteria. MicrobiologyOpen, e1341.