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

Yolanda GONZALEZ-FLORES¹, Margaux GASCHET¹, Mélanie PIMENTA¹, Christophe DAGOT¹, and Marie-Cécile PLOY¹

¹INSERM, CHU Limoges, RESINFIT, UMR1092, University of Limoges, Limoges, France

Mobile genetic elements (MGEs) are essential vehicles for stockpiling and disseminating antibiotic resistance genes (ARGs), allowing their transfer from non-pathogenic (environmental or commensal) to pathogenic bacteria. This strong association of ARGs with MGEs pushes us to explore the diversity of genes associated with mobile elements. This could allow us to recover novel ARGs with no sequence similarity with previously known ARGs. Classes 1-3 mobile integrons are known to host many ARGs as gene cassettes (GCs), being major spreaders and a promising source to search for novel ARGs [1]. Moreover, they display many GCs of unknown function (gcu) that could account for novel ARG activity, as has been previously reported [2].

To further explore the diversity of mobile integron GCs, we developed a novel *cassettomics* strategy combining metagenomics and bioinformatics approaches that allows us to (i) determine the relative proportion of each integron GC within a given sample and (ii) locate to which integron class these GCs are associated, and (iii) identify novel integron GCs. To do this, we took advantage of the 5'/3' conserved regions to specifically amplify each integron class coupled with a specific pipeline, which also allows us to enrich the samples in integrons to access novel GCs which are scarcely represented. This approach has previously allowed us to identify in 20 samples 593 novel GCs, many from class 3 integrons, including some experimentally verified novel ARGs.

Using our *cassettomics* pipeline with a list of 1078 known GCs, we detected more than 150 novel GCs (~4% of them closely related to known ARGs) from 4 water samples with different anthropogenic levels (urban, animal, and littoral settings), and with a threshold of 90% of nucleotide identity. Moreover, 25% of them encode proteins with already known domains with different biochemical activities and 60% proteins of unknown function, which might also be involved in antibiotic resistance. We have developed a filtering and selection workflow based on genetic and epidemiological features to choose the best candidates from these GCs of unknown function in order to perform functional analyses.

Such preemptive antibiotic resistance (AR) detection, using different high throughput sequence-based strategies, allows the detection in the environment of novel ARGs which could be the future superbugs.

## References

[1] S.R. Partridge, S.M. Kwong, N. Firth, S.O. Jensen (2018). *Clin Microbiol Rev*, 31(4):e00088-17.

[2] M.E. Böhm, M. Razavi, N.P. Marathe, C.F. Flach, D.G.J. Larsson (2020). *Microbiome*, 8(1):41.
O55

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

Samuel J. Bloomfield<fnref n="1" />, Aldert L. Zomer<fnref n="2" />, Justin O'Grady<fnref n="1" />, Gemma L. Kay<fnref n="1" />, John Wain<fnref n="1" />,<fnref n="3" />, Nicol Janecko<fnref n="1" />, Raphaëlle Palau<fnref n="1" /> and Alison E. Mather<fnref n="1" />,<fnref n="3" />

Humans are exposed to microorganisms from animals via food, including antimicrobial resistant (AMR) microorganisms. It is difficult to quantify the number of antimicrobial resistant microorganisms on food using traditional culture methods due to the large number of possible microorganisms that can be found on food. Metagenomics may be used to identify AMR genes on food, but directly sequencing food metagenomes is inefficient as DNA from the host (animal or plant) vastly outnumbers the microorganism DNA present. We optimised a host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3-40-fold) compared to undepleted samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.

## References

[1] S.J. Bloomfield, A.L. Zomer, J. O'Grady, G.L. Kay, J. Wain, N. Janecko, R. Palau, A.E. Mather (2023). Food Microbiology 110: 1–12.

<footnote n="1">¹Quadram Institute Bioscience, Norwich, United Kingdom</footnote>

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

<footnote n="3">³University of East Anglia, Norwich, United Kingdom</footnote>
O56

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

Ouléye SIDIBE<fnref n="1" />, Anne-Carmen SANCHEZ<fnref n="2" />, Guillaume KON KAM KING<fnref n="2" />, Fanny CALENGE<fnref n="2" />, Benoît DOUBLET<fnref n="1" />, Sylvie BAUCHERON<fnref n="1" />, Anne-Laure ABRAHAM<fnref n="2" />, Sébastien LECLERCQ<fnref n="1" />

The world is reaching a point where the effectiveness of antibiotics could be completely compromised in the near future, if antimicrobial resistance (AMR) continues to spread globally. The intestinal microbiota of human and domestic animals is suspected to be the main reservoir of AMR bacteria and therefore of antibiotic resistance genes (ARGs) [1]. Advances in DNA sequencing technologies and more specifically metagenomic approaches have shown that several ARGs are prevalent and shared between most gut microbiota [2, 3]. Although nucleotide diversity is documented for well-known ARGs, the specific distribution of each ARG variant in gut resistomes is still completely unknown. This study aims to understand if ARGs described as identical in different microbiomes are actually the same variants.

We adapted DESMAN [4] algorithm previously developed to reconstruct bacterial strain genomes from metagenomic data, to characterize ARG variants from a pool of ARGs. This tool was applied to ARGs detected in metagenomic cecal samples of chickens raised under two different conditions. Among 22 dominant ARGs analyzed, 15 have at least 2 stable variants and 7 (*ant*(6), *bla*<sub>TEM-1</sub>, *erm*(B), *erm*(F), *tet*(Q), *tet*(L) and *tet*(32)) have variants showing different proportions between cecal metagenomes depending on rearing condition and age of animals. Sequence comparison between ARG variants reconstructed by the DESMAN tool and those identified from bacterial isolates revealed a perfect match. These results attest to the reliability of this tool to reconstruct different variants of the same ARG directly from metagenomic data and to infer their relative proportions in different samples. It opens the way for further analyze the relative abundance of ARG variants in metagenomic datasets and finally deciphering their transmissions between microbiomes of different hosts.

References

[1] Anthony et al., 'The Gut Microbiome as a Reservoir for Antimicrobial Resistance'

[2] Wang et al., 'The Shared Resistome of Human and Pig Microbiota Is Mobilized by Distinct Genetic Elements'

[3] Munk et al., 'Abundance and Diversity of the Faecal Resistome in Slaughter Pigs and Broilers in Nine European Countries'

[4] Quince et al., 'DESMAN'.

<footnote n="1">¹ISP UMR 1282, INRAE, Nouzilly, France</footnote>

<footnote n="2">²MaIAGE Unit, INRAE, Jouy-en-Josas, France</footnote>
Development of an *in vitro* biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota

Sarah NAUDIN<fnref n="1" />, Aude FERRAN<fnref n="1" />, Pedro IMAZAKI<fnref n="1" />, Nathalie ARPAILLANGE<fnref n="1" />, Camille MARCUZZO<fnref n="1" />, Maïna VIENNE<fnref n="2" />, Sofia DEMMOU<fnref n="3" />, Felipe RAMON-PORTUGAL<fnref n="1" />, Marlène LACROIX<fnref n="1" />, Claire HOEDE<fnref n="2" />, Maialen BARRET<fnref n="3" />, Véronique DUPOUY<fnref n="1" />, Delphine BIBBAL<fnref n="1" />

**Introduction.** Sewer biofilms are likely to constitute hot spots for selecting and accumulating antibiotic-resistant bacteria and genes. The objective of this study was to optimize culture conditions to obtain a stable and reproducible *in vitro* biofilm, mimicking the biofilm collected in sewers, in order to study the impact of fluoroquinolones on sewer biofilm microbiota.

**Materials and methods.** Biofilms were produced on coupons in CDC Biofilm Reactors®, continuously fed with nutrient (R2A broth) and inoculum (1/100 diluted wastewater), at 21°C. Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs polycarbonate, and (iii) time of culture: 7 days (d7) vs 14 days (d14). Biofilms were collected on d7 and d14. Once the growth conditions have been determined, the impact of fluoroquinolones (ciprofloxacin and norfloxacin) on sewer biofilm microbiota, alone or in combination, was evaluated.

**Results.** 16S rRNA gene and bacteria quantification revealed that the biomass was the highest when *in vitro* biofilm was formed on concrete coupons. 16S rRNA gene sequencing and analysis revealed that the addition of sewer biofilm to the initial inoculum and that the material coupon did not affect the taxonomic diversity. However, the diversity increased on d14 for all tested conditions. The taxonomic composition of environmental samples differed: *Campylobactereles* dominated in wastewater, whereas they were in minority in the sewer biofilm. *In vitro* biofilms were dominated by *Enterobacterales*. Quantification of *qnrA*, B, D and S genes showed that the relative abundance of these genes was higher in *in vitro* biofilms than in sewer biofilm and wastewater. The following growing conditions were chosen: concrete coupons, initial inoculation with sewer biofilm and a time culture of 14 days. Administration of fluoroquinolones had no impact on the abundance of *qnr* genes, except for *qnrA* genes when both fluoroquinolones were administered concomitantly. Sequencing of *gyrA* and *parC* genes was performed to determine the proportion of *E. coli* resistance mutations. Exposure to fluoroquinolones led to the increase in the proportion of mutations in *gyrA* (codons S83L and D87N) and in *parC* (codon S80I).

**Conclusion.** This study allowed determining the culture conditions to develop an *in vitro* model of sewer biofilm. This model should allow for identifying concentrations of antibiotics and antibiotic cocktails in water that promote the selection and dissemination of resistance. Eventually, this work could help define threshold concentrations of antibiotics that exert selection pressure on environmental microbiota for monitoring purposes.

**Funding.** PNREST Anses, 2020/01/142

<footnote n="1">¹INTHERES UMR 1436, Université de Toulouse, INRAE, ENVT, Toulouse, France</footnote>

<footnote n="2">²UR875 MIAT, Bioinfomics, PF GenoToul Bioinfo, INRAE, Université de Toulouse, Castanet-Tolosan, France</footnote>

<footnote n="3">³Laboratoire Ecologie Fonctionnelle et Environnement UMR 5245, Université de Toulouse, CNRS, Toulouse, France</footnote>
# From genotype to antimicrobial resistance (AMR) phenotype prediction: how genomic proficiency tests can facilitate the AMR laboratories' performance evaluation

Athina Andrea, Susanne Karlsmose Pedersen, Lauge Holm Sørensen, Jette Sejer Kjeldgaard, Rene S. Hendriksen

European Union Reference Laboratory for Antimicrobial Resistance, National Food Institute, Technical University of Denmark, Kemitorvet, Building 204, DK-2800 Lyngby, Denmark

**Background:** The transition from traditional microbiology to whole genome sequencing (WGS) based diagnostics has been emerging for the last decade in clinical settings, with the expansion for benchtop WGS platforms. Hence, the need to translate, understand and assess the predicted antimicrobial resistance (AMR) phenotype, based on bioinformatics analysis of WGS data, is gaining ground progressively. Prediction of AMR phenotypes is multifactorial and depends among others on the employed WGS platform, bioinformatic tool as well as scientific expertise for data evaluation. Thus, the European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) recently launched a genomic proficiency test (GPT) with the objective to assess the ability of the network's participating laboratories to detect AMR determinants based on *in silico* data applying own bioinformatic tools as well as the ability to infer an AMR phenotypic profile based on the predicted determinants.

**Methods:** Clinically relevant bacterial species, such as *Salmonella* spp., *Escherichia coli*, *Campylobacter* spp., *Enterococcus* spp. and *Staphylococcus aureus* were included in the 2021 and 2022 GPT iterations; GPT-2021 and GPT-2022. Two strains of three bacterial species were dispatched to the participating laboratories as both live cultures and DNA. Participants were requested to perform WGS followed by bioinformatic analysis to detect AMR determinants (genes and chromosomal mutations) and predict the inferred AMR phenotype, for a selected panel of antimicrobial compounds. Results were submitted to an online reporting and evaluation tool, where proficiency was scored against references results, generated from closed genomes of the same cultures assessed, using ResFinder [1] and PointFinder [2].

**Results:** Twenty-eight and twenty laboratories from Europe participated in GPT-2021 and GPT-2022 iterations respectively. A high concordance (>90% correct results) to the reference data were observed in the laboratories' ability to detect AMR genes from the provided DNA (63% and 70% in GPT-2021 and GPT-2022, respectively) and live cultures (68% and 75% in GPT-2021 and GPT-2022, respectively), despite the differences observed in the applied WGS methodologies and bioinformatics tools. Thus, indicating that the applied WGS methodologies and bioinformatics tools generated reliable results. For chromosomal mutations, >90% correct results were submitted by 86% and 55% of the laboratories (both live cultures and DNA), for GPT-2021 and GPT-2022 respectively. Regarding the laboratories' ability to infer phenotypic AMR profiles, 85% of the participants (live culture) and 90% (DNA) for GPT-2022 as well as 36% of the participants (live culture) and 29% (DNA) in GPT-2021 obtained >90% correct results.

**Conclusion:** The majority of laboratories that participated in GPT-2021 and GPT-2022 (16/18 and 14/14 respectively, for live culture/DNA analysis), obtained a satisfactory overall performance, with >90% correct results. Participation in the DTU GPT, assists the EURL network laboratories to pinpoint flaws in the WGS workflows and/or in the *in silico* AMR identification tools, contributing to provide reliable data for surveillance as well as for control and prevention measurements. A successful *in silico* AMR phenotype prediction based on WGS data is, however, still heavily dependent on the scientific evaluation of the data outputted by the AMR detection tools.

## References

[1] Bortolaia V, Kaas RF, Ruppe E, Roberts MC, Schwarz S, Cattoir V, Philippon A, Allesøe RL, Rebelo AR, Florensa AR, Fagelhauer L, Chakraborty T, Neumann B, Werner G, Bender JK, Stingl K, Nguyen M, Coppens J, Xavier BB, Malhotra-Kumar S, Westh H, Pinholt M, Anjum MF, Duggett NA, Kempf I, Nykäsenoja S, Olkkola S, Wieczorek K, Amaro A, Clemente L, Mossong J, Losch S, Ragimbeau C, Lund O, Aarestrup FM. ResFinder 4.0 for predictions of phenotypes from genotypes. 2020 *Journal of Antimicrobial Chemotherapy* PMID: 32780112, 1:75, 3491-3500, doi: 10.1093/jac/dkaa345.

[2] Zankari, E., Allesøe, R., Joensen, K. G., Cavaco, L. M., Lund, O., & Aarestrup, F. M. PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens. *Journal of Antimicrobial Chemotherapy*. 2017, 72:10-2764-2768, PMID: 29091202. doi: 10.1093/jac/dkx217.
O59

Correlation analysis of bacterial cell morphology and multidrug resistance

Miki IKEBE<fnref n="1" />,<fnref n="3" />, Kota AOKI², Mitsuko HAYASHI-NISHINO<fnref n="1" />,<fnref n="3" />, and Kunihiko NISHINO<fnref n="1" />,<fnref n="3" />,<fnref n="4" />

It has been widely known that cell morphology in Gram-negative rods may change when exposed to stresses such as antimicrobial agents, and that once bacteria acquire antimicrobial resistance (AMR), they no longer undergo significant morphological changes after exposure<fnref n="1" />,². On the other hand, the morphology of drug resistant bacteria in the absence of exposure is still lacks knowledge, even though it is a subject of great interest in understanding the phenomenon of AMR.

We acquired phase-contrast optical microscopy images of multidrug-resistant *Escherichia coli* obtained by evolutionary experiments<fnref n="3" /> under non-exposure condition, and quantified the cell outline information as 10 types of features. The similarity between drug susceptible and resistant bacteria was examined using the histogram crossover method. Then, using Weighted Gene Correlation Network Analysis (WGCNA), we identified a group of genes highly correlated with the morphological characteristics of the resistant bacteria.

We revealed that resistant bacteria have morphological characteristics that are quantitatively different from susceptible bacteria, and that these differences are particularly large in resistant bacteria generated using quinolones and β-lactams. We also identified 56 genes that correlated well with morphological characteristics of resistant bacteria, and GO (Gene Ontology) enrichment analysis revealed that xenobiotic transmembrane transporter activity is significantly enriched for this gene cluster. Furthermore, these analyses revealed that *soxRS* may be one of the key regulators in clarifying the relationship between AMR and morphology.

Our finding provides new insights into the morphological changes of Gram-negative rods during the AMR acquisition process, and indicates the potential for future development of AMR spread prevention methods that utilize morphological information.

References

[1] S. S. Justice, D. A. Hunstad, L. Cegelski, S. J. Hultgren (2008). *Nat Rev Microbiol* 6(2):162-8.

[2] H. Shi, B. P. Bratton, Z. Gitai, K. C. Huang (2018). *Cell* 172(6):1294-1305.

[3] S. Suzuki, T. Horinouchi, C. Furusawa (2014). *Nat Commun* 17:5792.

<footnote n="1">¹Department of Biomolecular Science and Regulation, and ²Department of Intelligent Media, SANKEN (the Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Japan</footnote>

<footnote n="3">³Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan</footnote>

<footnote n="4">⁴Center for Infectious Disease Education and Research, Osaka University, Suita, Japan</footnote>
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**Detection of resistance genes and resistant bacteria in wild birds from the French Antarctic and Austral Territories**

Marisa HAENNI<fnref n="1" />, Laetitia DU FRAYSSEIX<fnref n="1" />, Jeremy TORNOS<fnref n="2" />, Sabine DELANNOY<fnref n="3" />, Amandine GAMBLE<fnref n="4" />, Jean-Yves MADEC<fnref n="1" />, Patrick FACH<fnref n="3" /> and Thierry BOULINIER<fnref n="2" />

Antimicrobial resistance (AMR) in wild birds most likely reflects the contamination of their natural environment, in which the selective pressure of antibiotics is supposed to be very weak, with antimicrobial resistance genes (ARGs) from anthropogenic sources. AMR acquisition in wild birds most probably occurs through foraging (scavenging, predation), but the exact source of AMR in specific wild individuals is often difficult to infer. The aim of this study was to determine the AMR load in fecal samples of wild birds living on islands of the French Antarctic and Austral Territories, which is a highly pristine environment with limited human presence.

Cloacal and fecal samples of wild birds (n=346, mostly form king penguins *Aptenodytes patagonicus* and lesser sheathbill *Chionis minor*) were collected in 2021 from Crozet, Kerguelen and Amsterdam islands using e-swabs™. From each sample, bacteria were isolated on selective ChromID ESBL and non-selective ChromID CPS agar (Biomerieux) and total DNA was extracted (Bacterial DNA kit, Macherey-Nagel). Isolated bacteria were identified by Maldi-TOF and antimicrobial susceptibility was tested by disc-diffusion. High-throughput microfluidic real-time PCR amplification of 23 ARGs associated to gram-negative bacteria was performed using 48.48 dynamic arrays (Biomark™; Fluidigm, USA) on total DNA extracts after a pre-amplification step.

No extended-spectrum beta-lactamase (ESBL) producing isolate was identified on selective plates, while 546 different bacteria were collected on non-selective plates, including 182 *Enterococcus* spp and 73 *Escherichia* spp isolates. Antibiograms revealed very few resistance phenotypes. All tested ARGs, except *mcr-1* and *tetA*, were detected at least in one sample. The most frequent ARGs were those conferring resistance to aminoglycosides (aph(3')-Ia, n=311; *strA* n=169; *strB*, n=190), sulfonamides (*sul1/sul2*, n=144/167) and tetracyclines (*tetB*, n=115). The class 1 and class 2 integrons were respectively found in 147 and 286 samples. ESBL genes *bla*<sub>CTX-M-1-group</sub> and *bla*<sub>CTX-M-9-group</sub> were sporadically identified (n=25 and n=54). Within Possession Island, in Crozet Archipelago, the proportion of resistance genes decreased with distance from the scientific station.

Our results give further evidence that ARGs have now percolated in the most pristine environments. Direct plating of the samples, without any enrichment step, did not allow the isolation of ESBL-producing isolates, even though ESBL genes could be identified using HT-qPCR. This indicates that the AMR burden is still low and restricted to the sub-dominant flora. Interestingly, our results showed that the proportion of ARGs decreased with distance from the scientific station, suggesting the role of anthropization in the spread of ARGs. A new sampling campaign is currently being conducted to provide more evidence for this hypothesis.

<footnote n="1">¹Unité Antibiorésistance et Virulence Bactériennes, ANSES – Université de Lyon, Lyon, France</footnote>

<footnote n="2">²CEFE, University of Montpellier, CNRS, EPHE, IRD, Montpellier, France</footnote>

<footnote n="3">³COLiPATH Unit & Genomics Platform IdentyPath, Laboratory for Food Safety, ANSES, Maisons-Alfort, France;</footnote>

<footnote n="4">⁴School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Glasgow, Scotland</footnote>
# Majority of multi-drug resistant *Aeromonas* isolates from the urban-impacted Akaki river carried enteric toxins

Berhanu Yitayew<fnref n="1" />,<fnref n="2" />, Yimtubezinash Woldeamanuel<fnref n="1" />, Daniel Asrat<fnref n="1" />, Abraham Aseffa<fnref n="3" />, Per-Erik Olsson<fnref n="2" />, Adane Mihret<fnref n="3" />, Jana Jass<fnref n="2" />

The spread of antibiotic-resistant and virulent bacteria through aquatic environments is a global public health challenge (1). *Aeromonas* species are emerging gastrointestinal pathogens that are rapidly becoming antibiotic resistant (2). This study characterized *Aeromonas* isolates from an urban-impacted river that runs through Addis Ababa in Ethiopia. *Aeromonas* spp. were isolated from five sites, chosen based on anthropogenic activities along the Akaki river during dry and wet seasons. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion and carbapenemase production was confirmed by a hydrolysis test. Polymerase chain reaction and whole genome sequencing were employed for molecular characterization. From a total of 163 *Aeromonas* isolates, 144 were implicated in human infections (*Aeromonas caviae*, *Aeromonas hydrophila* and *Aeromonas veronii) and their resistance patterns were assessed. Majority of the isolates were resistant to amoxicillin (144, 100%), ampicillin (142, 98.6%), amoxicillin/clavulanate (117, 81%), imipenem (65, 45.1%), ertapenem (71, 49.3%), and cefotaxime (86, 59.7%). The rate of resistance to the tested antibiotics varied between species, where most of *A. veronii* (37/49, 75.5%) and *A. hydrophila* (28/33, 84.8%) were resistant to imipenem but none of *A. caviae*. The resistance prevalence of *A. veronii*, *A. hydrophila*, and *A. caviae* to meropenem was 63.3%, 57.6%, and 4.8%, respectively. Resistance to carbapenem in most *A. hydrophila* (85.6%) and nearly all *A. veronii* was mediated by carbapenemase production. A significant association of resistance was observed with season of isolation for some antibiotics tested. In these isolates, 6 toxin genes were found. Cytotoxic enterotoxin (alt and ast) and hemolysin (hlyA) were detected in the majority of *Aeromonas* isolates. Cytotoxic enterotoxin (act) gene was found only in *A. hydrophila* and *A. veronii*, whereas none of *Aeromonas* isolates carried Shiga toxins (stx-1 and stx-2). Of the 21 *Aeromonas* isolates that were sequenced, nearly all in the current study were new sequence types. Isolates from farthest upstream and downstream river sites contained fewer antibiotic resistance genes that belonged to the β-lactamases. Presence of bla_cphA gene was associated with the phenotypic carbapenem resistance. These results highlight that river in megacities maybe a reservoir of virulent, multi-drug resistant and carbapenemase-producing *Aeromonas*.

## References

[1] Le, T., Truong, T., Tran, LT. et al. Antibiotic resistance in the aquatic environments: the need for an interdisciplinary approach. *Int. J. Environ. Sci. Technol.* 20, 3395–3408 (2023). https://doi.org/10.1007/s13762-022-04194-9

[2] Lamy B, Kodjo A; colBVH Study Group; Laurent F. Prospective nationwide study of Aeromonas infections in France. J Clin Microbiol. 2009 Apr;47(4):1234-7. doi: 10.1128/JCM.00155-09. Epub 2009 Feb 25. PMID: 19244464; PMCID: PMC2668343.

<footnote n="1">¹College of Health Sciences, School of Medicine, Addis Ababa University, Ethiopia,</footnote>

<footnote n="2">²The Life Science Center-Biology, School of Science and Technology, Örebro University, Sweden,</footnote>

<footnote n="3">³Armauer Hansen Research Institute, Ethiopia</footnote>
# Assessment of antimicrobial resistant Enterobacterales from the dairy production environment in low and high zinc containing areas

Elena ANEDDA<fnref n="1" />,<fnref n="2" />, Gillian MADIGAN<fnref n="3" />, Dearbháile MORRIS<fnref n="2" /> and Catherine M. BURGESS<fnref n="1" />

Antimicrobial resistance (AMR) is a critical public health concern and it is acknowledged that a One Health approach is required to address it effectively. Limited information is available on the dissemination AMR in the primary food production environment, where the presence of heavy metals may play a role in promoting AMR gene transmission. In Ireland, where agriculture is of key importance, soils are very well mapped in relation to the levels of heavy metals they contain. This includes zinc, which naturally occurs in low and high concentrations across the country.

Therefore, the objective of this study was to evaluating the presence of AMR Enterobacterales in dairy pasture soil and bovine milk filters on farms in high and low zinc areas across Ireland.

Samples were collected from two distinct areas across Ireland, with varying zinc concentrations. Enterobacterales were enumerated from soil and bovine milk filter samples and the presence of ESBL- producing Enterobacterales (ESBL-PE), carbapenem resistant Enterobacterales (CRE), and ciprofloxacin resistant Enterobacterales (FQR-E) were assessed on selective agars. Suspected colonies were identified by Maldi-TOF and antimicrobial susceptibility testing (AST) was performed on confirmed Enterobacterales. Additionally, chemical analysis was conducted on soil samples.

A variety of AMR Enterobacterales of differing antimicrobial susceptibility were isolated from both sample types. This study demonstrated that the primary food production environment can harbour AMR Enterobacterales, which might enter the food chain and cause a risk for human health.

<footnote n="1">¹Food Safety Department, Teagasc Food Research Centre Ashtown, Dublin, Ireland</footnote>

<footnote n="2">²Antimicrobial Resistance and Microbial Ecology Group, National University of Ireland Galway, Galway, Ireland</footnote>

<footnote n="3">³Bacteriology/Parasitology Division, Department of Agriculture, Food and the Marine, Backweston Complex, Celbridge, Ireland</footnote>
Resistance profiles of viable airborne antibiotic-resistant bacteria from
antibiotic-free broiler operations

Joanie LEMIEUX¹², Juan Manuel DOMINGUEZ<fnref n="3" />, Ève BÉRUBÉ<fnref n="3" />, Meredith Elizabeth GILL<fnref n="4" />, Luc TRUDEL<fnref n="1" />, Nathalie TURGEON<fnref n="2" />, Jacques CORBEIL<fnref n="3" />, Maurice BOISSINOT<fnref n="3" />, Frédéric RAYMOND<fnref n="4" /> and Caroline DUCHAINE¹²⁵

**Introduction.** Animal production represents a reservoir for antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARG). The absence of selective pressure in antibiotic-free productions is thought to reduce the prevalence of ARB in the farm environment. Confined farming activities are also known to produce high concentrations of bioaerosols. These air-suspended biological particles can contain ARB. Bioaerosols can be emitted outside through building ventilation systems and disseminate ARB into the surrounding environment where their impact remains unknown. Our main objective was to characterize the resistome of bacteria present in the indoor air of antibiotic-free broiler operations using selective culture approaches. **Methods.** Two antibiotic-free broilers operations in the province of Quebec, Canada, were sampled once. Airborne culturable bacteria were collected with a high flow rate liquid type air sampler (SASS® 2300: Research International). The liquid air samples were inoculated both on solid media and in broth enrichment with antibiotics in order to recover ARB. According to their colony morphology, randomly selected isolates were identified with MALDI-TOF/MS (Bruker Daltonics) and 16S rRNA sequencing. Whole-genome sequencing (WGS) (NovaSeq6000, Illumina) was performed on twelve isolates and ARGs were predicted using the Comprehensive Antibiotic Resistance Database. **Results.** 36 bacterial species were retrieved from the two farms. Isolates sent to WGS were: *Acinetobacter baumannii*, *Aerococcus viridans*, *Enterobacter cloacae*, *Enterococcus faecium*, *Escherichia coli*, *Globicatella sulfidifaciens*, *Klebsiella pneumoniae*, *Pseudomonas monteilii*, *Rothia nasimurium*, *Staphylococcus saprophyticus*, *Stenotrophomonas* spp. and *Streptococcus alactolyticus*. This latter had resistance genes associated with plasmids, *msrA* (macrolide), *spd* (aminoglycoside), *dfrD* (diaminopyrimidine) and *InuA* (lincomasimde). *R. nasimurium* harbored *IsaE*, a gene previously found in porcine MRSA isolate and *vatE* a transposon-mediated gene found in *E. faecium*. Genes conferring resistance to aminoglycosids (*AcrD*, *AAC(3)-Via*, *ANT(3'')-IIa), beta-lactam (*ompA*), carbapenems (*OXA-317*) and cephalosporins (*ADC-159*, *TEM-135*) were found in *A. baumannii*, *E. coli* and *K. pneumoniae* had *E. cloacae* had a gene conferring resistance to bacitracin. Additionally, antibiotic susceptibility testing will allow comparison of the resistance genotypes and phenotypes. **Conclusion.** Even in the absence of selective pressure during animal production, viable ARB harboring genes conferring resistance to multiple antibiotics are present in the indoor air of two antibiotic-free broiler operations. The same resistome monitoring methodology will be applied to broiler operations administrating antibiotics. This comparison will serve to understand how livestock management approaches can aim to mitigate problems associated with antimicrobial resistance.

<footnote n="1">¹Département de Biochimie, Microbiologie et Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec, Qc</footnote>

<footnote n="2">²Centre de Recherche de l'Institut de Cardiologie et Pneumologie de Québec - Université Laval, Québec, Qc</footnote>

<footnote n="3">³Centre de Recherche en Infectiologie de l'Université Laval, Axe maladies infectieuses et immunitaires, centre de recherche du CHU de Québec - Université Laval, Québec, Qc Canada</footnote>

<footnote n="4">⁴Institut sur la nutrition et les aliments fonctionnels - Université Laval, Québec, Qc</footnote>

<footnote n="5">⁵Research Chair on Bioaerosol</footnote>
# Seasonal Dispersion of Antimicrobial Resistance Genes in Aerosols: A One Year Monitoring at the puy de Dôme summit

Florent Rossi<fnref n="1" />,<fnref n="2" />, Romie Tignat-Perrier<fnref n="4" />,<fnref n="5" />, Muriel Joly<fnref n="3" />, Catherine Larose<fnref n="4" />, Aurélien Dommergue<fnref n="5" />, Nathalie Turgeon<fnref n="1" />,<fnref n="2" />, Marc Veillette<fnref n="1" />,<fnref n="2" />, Pierre Amato<fnref n="3" />, Karine Selegri<fnref n="7" />, Jean-Luc Baray<fnref n="6" />,<fnref n="7" /> and Caroline Duchaine<fnref n="1" />,<fnref n="2" />,<fnref n="8" />

The issue of antimicrobial resistance and its spread in the environment has become a major concern in recent decades. Carried by bacteria, antimicrobial resistance genes (ARGs) are found in many surface ecosystems (soils, rivers and oceans) and the atmosphere could be a major transport route. The atmospheric system is wide and dynamic and the bacteria it contains (~$10^3$ cell.m$^{-3}$ of air) fluctuates in both space and time; season is a critical factor of this variations.

In this work, airborne ARGs and their seasonal fluctuations were assessed between July 2016 and August 2017 at the puy de Dôme meteorological observatory (1465 m a.s.l., France). Sampling consisted in PM10 aerosols collection over one-week periods at high flow rates (60 m<fnref n="3" />.h⁻<fnref n="1" />) on pre-calcinated quartz filters (Ø 150 mm). In total, 48 aerosol samples were retrieved and analyzed for the presence of 34 different subtypes of ARGs from the main existing classes of antibiotic resistance: quinolones, β-lactams, macrolides, tetracyclines, sulfonamides, aminoglycosides, and vancomycins, including one mobile genetic element. These were further related to airborne bacterial concentration (16S qPCR) and diversity (MiSeq Illumina) and to the geographical origins of the air masses from which they originated. Specifically, the ECMWF ERA-5 3D kinematic trajectory model was used to retrace the 72-hours backward trajectories of all the air masses reaching the observatory during each week of sampling.

Our results highlight the presence of 32 different subtypes of airborne ARGs, whose total concentration and related diversity fluctuated seasonally (from 59 to 1.1 x $10^5$ copies.m$^{-3}$), although they were dominated by quinolone resistance overall (> 95%). Such variations were partially related to bacterial abundance and diversity associated with changes in the surrounding landscapes, and with the time spent by the air masses over the continent before reaching the sampling site. Specifically, epiphytic bacteria (i.e. *Sphingomonas*) were particularly abundant during the spring and summer, concomitant with the highest concentrations of ARGs, and could be significantly associated with 9 different resistance genes. Conversely, winter was characterized by a higher contribution of macrolide resistance, which could not be associated to any specific genera. Interestingly, the least abundant genera (<1% of relative abundance) were overall those with the highest number of different resistance genes, exhibiting positive associations with up to 15 different subtypes. Overall, the results of our study indicate that the atmosphere is an important vector of ARGs at short to large spatial scales.

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

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

<footnote n="3">³Université Clermont Auvergne, CNRS, Institut de Chimie de Clermont-Ferrand, Clermont-Ferrand, France</footnote>

<footnote n="4">⁴Laboratoire Ampère, École Centrale de Lyon, CNRS, Université de Lyon, Ecully, France</footnote>

<footnote n="5">⁵Université Grenoble Alpes, CNRS, IRD, INRAE, Grenoble INP, Institut des Géosciences de l'Environnement, Grenoble, France</footnote>

<footnote n="6">⁶Université Clermont Auvergne, CNRS, Observatoire de physique du Globe de Clermont-Ferrand, Clermont-Ferrand, France</footnote>

<footnote n="7">⁷Université Clermont Auvergne, CNRS, Laboratoire de Météorologie physique, Clermont-Ferrand, France</footnote>

<footnote n="8">⁸Canada Research Chair on Bioaerosols</footnote>
<figure><img src="image_33.png" /></figure>

Mechanism reducing carbapenem susceptibility of
environmental *E. coli* isolates

Laëtitia Le Devendec<fnref n="1" />, Mai-Lan Tran<fnref n="2" />, Emeline Larvor<fnref n="1" />, Eric Jouy<fnref n="1" />, Isabelle Kempf<fnref n="1" />, Claire Chauvin<fnref n="3" />, Amandine Wilhelm<fnref n="4" />, Benoit Gassilloud<fnref n="4" />, Patrick Fach<fnref n="2" />, Sandrine Baron<fnref n="1" />, Sabine Delannoy<fnref n="2" />

During a 16-month longitudinal study, bi-monthly water samples were taken upstream and downstream of a watershed. In order to detect carbapenem-resistant *E. coli*, the CHROMID®Carba medium was used. Of the 800 isolates collected from 144 samples, *E. coli* identification was confirmed for only seven of them, isolated on the same day, two upstream and five downstream. For six isolates (five downstream and one upstream), a reduced susceptibility to carbapenems was observed (values for ertapenem: diameter of 19 mm and MIC (in microdilution) between 0.5 and 2 mg.L⁻<fnref n="1" />). The results of the whole genome sequencing indicate that five of the six isolates belong to the same clone (O8:H7, ST196). Furthermore, a mutation of the *ompC* porin coupled with the presence of the *bla*CMY-2 gene, on an *IncI1* plasmid, would be at the origin of the reduced sensitivity of these strains to carbapenems. This type of mechanism has already been described in human clinical cases in the USA, France and Taiwan. To our knowledge, this is the first time that it has been identified in strains from the aquatic environment. The detection of *E. coli* with reduced susceptibility to carbapenems one time in 18 months (one out of 36 sampling dates) could be considered as a one-time event. However, this illustrates the importance of monitoring the aquatic environment but also the methodological difficulties of such surveillance due to the poor performance of the isolation method.

<footnote n="1">¹Mycoplasmosis, Bacteriology and Antimicrobial Resistance Unit, Ploufragan-Plouzané-Niort Laboratory, ANSES, 22440 Ploufragan, France</footnote>

<footnote n="2">²COLiPATH Unit & Genomics Platform IdentyPath, Laboratory for Food Safety, ANSES, 94700 Maisons-Alfort, France</footnote>

<footnote n="3">³Epidemiology, Health and Welfare Unit, Ploufragan-Plouzané-Niort Laboratory, ANSES, 22440 Ploufragan, France</footnote>

<footnote n="4">⁴Water Microbiology Unit, MALDI-TOF platform, Nancy laboratory for hydrology, ANSES, 54000 Nancy, France</footnote>
# Aerosolization behavior of antimicrobial resistance in animal farms: a field study from feces to fine particulate matter

Huibo XIN<fnref n="1" />,<fnref n="2" /> Tianlei QIU<fnref n="1" />, Yajie GUO<fnref n="1" />, Haoze GAO<fnref n="1" />, Liqiu ZHANG<fnref n="2" />, Min GAO<fnref n="1" />

Antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in animal feces can be released into the atmosphere via aerosolization, posing a high health risk to farm workers. Thus far, little attention has been paid to the characterization of the aerosolization process. In this study, fecal and fine particulate matter (PM2.5) samples were collected from 20 animal farms involving swine, cattle, layers, and broilers, and the ARGs, ARB, and human pathogenic bacteria (HPB) were loaded in these two media. The results showed that approximately 70% of ARGs, 60% of ARBs, and 43% of HPBs were found to be preferential aerosolization. The bioaerosolization index (BI) of target 30 ARGs varied from 0.04 to 460.07, and the highest value was detected from *tetW*. The highest BI values of Erythromycin- and Tetracycline-resistant bacteria were for *Kocuria* (13119), and *Staphylococcus* (24746), respectively, and the distribution of BI in the two types of dominant ARB was similar. Regarding the bioaerosolization behavior of HPB, *Clostridium saccharolyticum* WM1 was the most easily aerosolized pathogen in swine and broiler farms, and *Brucella abortus* strain CNM 20040339 had the highest value in cattle and layer farms. Notably, the highest BI values for ARGs, ARB, and HPB were universally detected on chicken farms. Most ARGs, ARB, and HPB positively correlated with animal age, stocking density, and breeding area. Temperature and relative humidity have significant effects on the aerosolization behavior of targets, and the effects of these two parameters on the same target are usually opposite. The results of this study provide a basis for a better understanding of the contribution of animal feces to airborne ARGs and HPBs in farms, as well as for controlling the transport of the fecal microbiome to the environment through the aerosolization pathway.

<figure><img src="image_34.png" /><figcaption>**Figure 1** Bioaerosolization Index (BI) of top 30 bacterial genera of ARGs (A), Erythromycin- (B) and Tetracycline-resistant bacterial genera (C), and top 30 human pathogenic bacteria (D). The BI value was log-transformed.</figcaption></figure>

<footnote n="1">¹Beijing Key Laboratory for Source Control Technology of Water Pollution, Engineering Research Center for Water Pollution Source Control and Eco-remediation, Beijing Forestry University, Beijing, 100083, China.</footnote>

<footnote n="2">²Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China</footnote>
<figure><img src="image_35.png" /></figure>

# Measuring the effects of antimicrobial residues in the environment on AMR transmission in a duck model.

Mike Brouwer, Alieda van Essen-Zandbergen, Yvon Geurts, Frank Harders, Eduardo de Freitas Costa, Kees Veldman, Alex Bossers

Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands

**Background:** In addition to farm animals, wild animals are also often exposed to anthropogenic antibiotic pressure through the contamination of wildlife habitat. Examples include the run-off of antibiotic residuals which are present in manure or the presence antimicrobial residues in effluent water from waste-water treatment plants. As such, various examples of concerning presence of resistant bacteria in wild animals have been described.¹ Transmission of resistant bacteria between aquatic birds through the environment was previously shown in a wild mallard model.²

**Objective:** To determine the effects of antimicrobial residues in pond water on the transmission of resistant *E. coli* in a duck model.

**Methods:** Nine groups of 15 peking ducks were housed for 2 weeks in enclosures containing a pond with various concentrations of either enrofloxacin or ceftiofur, which was refreshed daily. Animals were inoculated with isogenic *E. coli* strains, with or without an IncI1 plasmid, containing either a *qnr*S1 or a *bla*<sub>CTX-M-1</sub> gene. Samples were collected from individual animals and cultured to enumerate colonisation by resistant *E. coli* using selective MacConkey agar plates. The effects on the gut microbiota were determined by 16s sequencing.

**Results/Discussion:** Measurement of the residues of antimicrobials in the pond water and faeces showed that the residues would be present at similar in the gut as in the pond water. A rank-based regression analysis of the semi-quantitative culture results was performed. Both the *qnr*S1 and *bla*<sub>CTX-M-1</sub> genes could be detected in the lowest concentrations of either enrofloxacin or ceftiofur but in the highest concentrations (1000 µg/L) the water in the ponds would cause a selective pressure in the gut for either the *qnr*S1 or *bla*<sub>CTX-M-1</sub> gene to be maintained over time. Despite the levels of antimicrobials in the gut, the microbiome developed similarly over time in all groups of animals.

## References

1. Dolejska et al. Wildlife Is Overlooked in the Epidemiology of Medically Important Antibiotic-Resistant Bacteria. *Antimicrob Agents Chemother*. 2019 Jul 25;63(8):e01167-19.

2. Sandegren et al. Long-term carriage and rapid transmission of extended spectrum beta-lactamase-producing *E. coli* within a flock of Mallards in the absence of antibiotic selection. *Environ. Microbiol. Rep.* 10, 576-582 (2018).
# Environmental characterization of isolated positive patients for carbapenem-resistant Enterobacteriaceae and evaluation of carriage by workers

Annabelle RICHER-FORTIN<fnref n="1" />, Marc VEILLETTE<fnref n="1" />, Yves LONGTIN<fnref n="2" />, Adriana LARROTTA<fnref n="2" />, Nathalie TURGEON<fnref n="1" />, Bianka PAQUET-BOLDUC<fnref n="1" /> et Caroline DUCHAINE<fnref n="1" />,<fnref n="3" />,<fnref n="4" />

The hospital care environment may contain bacteria carrying various antibiotic-resistance genes (ARGs). Surfaces and bioaerosols may have a role in the environmental dissemination of these genes or can allow the monitoring of their presence in the medical environment. More precisely, Carbapenemase-producing Enterobacteriaceae (CPE), enzymes that can hydrolyze almost all beta-lactam antibiotics, are of great concern. Although their dissemination by direct and indirect contact is well documented, the role of air in its dissemination is little investigated.

This work aims to identify and quantify genes associated with CPE in the environment of isolated positive patients (air and surfaces). Some rooms with no positive patient are also sampled to evaluate the ARGs spreading. Also, as part of the project, the healthcare workers' hands and foreheads are tested to assess their occupational exposure and carriage to multi-resistant bacteria.

The sampling takes place in 3 hospitals in the province of Quebec. Air sampling is done in the rooms using the stationary SASS® 3100 Dry Air Sampler with a flow rate of 300L/min. Moisturized swabs and sponges are used to investigate the presence of CPE on surfaces. Multiple surfaces in the rooms are sampled: floors (inside and outside), faucets, drains, doorframes, and no-touch surfaces. No-touch surfaces are sampled to evaluate the deposition of bioaerosols. The genes associated with CPE, namely *bla*-KPC, *bla*-OXA-48, and *bla*-NDM in the various samples, were quantified by quantitative PCR (qPCR), using specific probes and primers. Total bacteria were also quantified using qPCR targeting 16S ribosomal RNA gene.

Rooms from *bla*-KPC-positive patients have been sampled. Preliminary data revealed that the *bla*-KPC gene was found on the doorframe in 50% of cases, and it was also found on the patient room floors in 66% of cases and on the floor outside in 83%. The *bla*-KPC gene was also found on no-touch surfaces but less frequently. Surprisingly, *bla*-KPC gene was detected from the sample in one negative patient's room floor. However, no *bla*-KPC has yet been detected in the air of the positive patients' rooms. Analysis and sampling are still ongoing, including workers' sampling. More sampling in hospitals and workers is planned in the next months and another hospital was recruited to the project, to ensure a better statistical power. Bacterial cultures of a few samples will be performed to know if bacteria carrying ARGs are viable. Results achieved in this study will help characterized ARGs in the hospital environment but also provide a better understanding of the role of air in the spread of multi-resistant and thus make the work environment safer.

<footnote n="1">¹Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec/Université Laval, Quebec City, Canada</footnote>

<footnote n="2">²CIUSSS du Centre-Ouest-de-l'Île-de-Montréal, Hôpital général juif/Université McGill, Montréal, Canada</footnote>

<footnote n="3">³Département de biochimie, microbiologie et bio-informatique/Université Laval, Québec, Canada</footnote>

<footnote n="4">⁴Canada Research Chair on Bioaerosols</footnote>
# Characterization of antibiotic resistance genes in the air of the health care environment

Annabelle RICHER-FORTIN<fnref n="1" />, Marc VEILLETTE,<fnref n="1" /> Yves LONGTIN<fnref n="2" />, Adriana LARROTTA<fnref n="2" />, Nathalie TURGEON<fnref n="1" />, Bianka PAQUET-BOLDUC<fnref n="1" /> et Caroline DUCHAINE<fnref n="1" />,<fnref n="3" />,<fnref n="4" />

Various classes of antibiotics are used in human health to treat numerous infectious diseases. The ability to resist antibiotics is conferred to bacteria through antibiotic-resistant genes (ARGs) that can be acquired and/or transferred by bacteria through different mechanisms. The indoor medical environment could represent a potential reservoir for different ARG classes that can potentially be dispersed. The presence of ARGs in the air and the role of the bioaerosols in their propagation have not been extensively studied. This transmission route should be investigated to describe and monitor the antibioresistance evolution in the indoor medical environment.

This work aims to describe the presence of various classes of antibiotic resistance genes (ARGs) and mobile genetic elements in the indoor air of different hospital public areas (cafeteria, corridors, admission, offices). Air sampling was achieved using stationary high-flow air samplers coupled to filters (SASS® 3100 Dry Air Sampler) at different flow rates (200L/min and 300L/min). Each area was sampled three times. Total bacteria concentration in the air was quantified using quantitative PCR (qPCR) targeting the 16S ribosomal RNA gene. The quantification of 34 different subtypes of ARGs from different classes will be achieved by qPCR using specific assays based on a global study focusing on airborne ARG monitoring in various environments (George et al., 2022).

Preliminary data reveal that several beta-lactam resistance genes, such as *blaTEM* and *blaCTXM-1* were detected and quantified in the air of all 4 areas in a hospital. *Bla-KPC*, *bla-CMY2* and *bla-VEB* were also detected in this same hospital. We expected to find similar results as part of a sampling campaign in another hospital in the province of Quebec.

Results will help assess the presence of ARGs in the air of health care environment and provide a better understanding of the role of the aerosol transmission route in the spread of ARGs to general settings. The use of air samples in the environmental investigation could be used to monitor the presence of ARGs.

## References

* George, P. B. L., Rossi, F., St-Germain, M. W., Amato, P., Badard, T., Bergeron, M. G., Boissinot, M., Charette, S. J., Coleman, B. L., Corbeil, J., Culley, A. I., Gaucher, M. L., Girard, M., Godbout, S., Kirychuk, S. P., Marette, A., McGeer, A., O'Shaughnessy, P. T., Parmley, E. J., . . . Duchaine, C. (2022). Antimicrobial Resistance in the Environment: Towards Elucidating the Roles of Bioaerosols in Transmission and Detection of Antibacterial Resistance Genes. *Antibiotics* (Basel, Switzerland), 11(7). https://doi.org/10.3390/antibiotics11070974

<footnote n="1">¹Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec/Université Laval, Quebec City, Canada</footnote>

<footnote n="2">²CIUSSS du Centre-Ouest-de-l'Île-de-Montréal, Hôpital général juif/Université McGill, Montréal, Canada</footnote>

<footnote n="3">³Département de biochimie, microbiologie et bio-informatique/Université Laval, Québec, Canada</footnote>

<footnote n="4">⁴Canada Research Chair on Bioaerosols</footnote>
P11

# Monitoring antibiotic resistance in a water environment with a minor degree of anthropization: The case of the KAMECH watershed

Wejdene MANSOUR<fnref n="1" />, Yosra Mani<fnref n="1" />, Cyrine BRAHAM<fnref n="1" />, Ahlem MAHJOUB<fnref n="1" />, Sana AZAIEZ<fnref n="1" />, Olfa MAHJOUB<fnref n="2" />

Antibiotic resistance remains a major global concern. Indeed, the anthropization of medicines has created a selection pressure favorable to the development of resistant bacteria. The spread of antibiotic resistance in the environment is due not only to the massive and uncontrolled use of antibiotics, but also to their therapeutic and prophylactic use on livestock farms, in agriculture and aquaculture. The aim of this study is to investigate the potential presence of multidrug resistant (MDR) bacteria in a water ecosystem with limited anthropization (deep waters) in the KAMECH catchment area. Our sampling included 9 separate points: 7 wells, a drinking water source and KAMECH lake. Among the one hundred deep-water samples analyzed, 52 bacteria were isolated, of which 37 non-enterobacterial Gram negative bacilli are β-lactam-resistant, 9 are producers of metallo-β-lactamase (MBL) and 2 strains are extended-spectrum- β-lactamase (ESBL) producers. Genotypic analysis revealed the presence of the *bla*~NDM~ gene in 5 strains (*Burkholderia cepacia* and *Pseudomonas oryzihabitans*) and the presence of the *bla*~CTX-M15~ gene in 2 strains (*Stenotrophomonas maltophilia* and *Klebsiella pneumoniae*). The dissemination of these MDR bacteria through water represents a major risk, not only for human health, but also for animal and environmental health.

<figure><img src="image_36.png" /><figcaption>**Fig. 1.** Geographical location of sampling area</figcaption></figure>

<footnote n="1">¹ Laboratoire de Recherche « Biophysique métabolique et Pharmacologie Appliquée » LR12ES02, Faculté de Médecine de Sousse, Université de Sousse, Tunisie</footnote>

<footnote n="2">² Laboratoire de Chimie des Eaux-Sols-Boues, Institut National de Recherche en Génie Rural Eaux et Forêts | INRGREF, Université de Tunis El Manar, Tunisie</footnote>
<figure><img src="image_37.png" /></figure>

# Could *Mytilus galloprovincialis* mussel be a hot-spot of horizontal resistance gene transfer in aquatic environment?

Serena SIMONI<fnref n="1" />, Alessandra DI GREGORIO<fnref n="1" />, Manon AUGUSTE<fnref n="2" />, Elisa TAVIANI<fnref n="2" />, Luigi VEZZULLI<fnref n="2" /> and Carla VIGNAROLI<fnref n="1" />

Antimicrobial resistance (AMR) is one of the most global concerns that recently poses a threat to public health. The continual release of antibiotic resistant bacteria into the environment and their persistence suggests the role that environment may play as an important reservoir and dissemination route of antibiotic resistance genes (ARGs). Horizontal gene transfer (HGT) allows the exchange of genetic elements between different strains and species leading to bacterial genome evolution and contributing to the rapid spread of AMR. Aquatic ecosystems have also become hot-spot for horizontal gene transfer of ARGs representing niches where resistance evolution can occur<fnref n="1" />. Several studies have reported the presence of multidrug-resistant bacteria in coastal waters that could be the result of the exchange of genetic material, such as plasmids, between marine and contaminating bacteria<fnref n="2" />. Bacterial population density and cell proximity differentially impact the various mechanisms of HGT³. For this reason, in this study, we investigated the potential of marine bivalve molluscs to become an optimal hot-spot where conjugation can occurs exploiting their ability to concentrate microorganisms from surrounding water as a result of their filter-feeding activity. To this aim, bivalves were cultured in aquaria in which they were exposed to a mating pair of linezolid resistant enterococci and periodically checked for the development of transconjugants. First, the bioaccumulation capacity of the mussels and the ability of the strains to survive in water were evaluated. Bacterial bioaccumulation experiments showed that the greatest values within the mussels were reached within 72 hours from inoculation. Therefore, conjugation experiments were performed with a maximum duration of 72h, inoculating donor and recipient simultaneously and in a 1:1 ratio. All the transconjugants were obtained within 24h. Conjugation experiments conducted at different temperatures showed that the highest conjugation frequency (~10-7) was obtained at 20°C temperature, while no conjugation occurs at temperature below 20°C. Transconjugants were found in the various body districts of the mussel at similar frequencies (~10-7) both in the digestive gland and in haemolymph. No conjugation seemed to have occurred in the gills. In this study we hypothesized that mussels provide a suitable matrix that might facilitate genetic exchange between pathogenic resistant bacteria reaching coastal waters through sewage. Mussels could contribute both to the spread of ARGs in aquatic environments and to the return of resistant bacteria to human via their consumption.

## References

[1] Larsson DGJ, Flach CF. (2022) *Nat Rev Microbiol* 20(5):257-269.

[2] Taylor NG, Verner-Jeffreys DW, Baker-Austin C. (2011) *Trends Ecol Evol* 26(6):278-84.

[3] Andersson DI, Hughes D. (2016) *Microbiol Spectr* 5(4).

<footnote n="1">¹Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy</footnote>

<footnote n="2">²DISTAV, Department of Earth, Environment, and Life Sciences, University of Genoa, Genoa, Italy</footnote>
# How *Salmonella enterica* acquires resistance to Basil's essential oils, and how this affects the bacteria's characteristics

Gany Beer-Davidsonª, Assaf Rokneyᵇ and Sima Yaronª

Essential oils (EOs) are complex mixtures of volatile compounds produced in various parts of plants, and are widely used as natural preservatives in food, beverages, fragrances, sanitary, cosmetics, and pharmaceutical markets. Linalool, one of the main components of the basil's essential oil (BEO), has antibacterial activity. It perforates the bacteria's outer membrane, inhibits cell motility and forms bacterial aggregates. In 2007 a foodborne outbreak originating from contamination of prepacked fresh basil spread across Europe. It was caused by Linalool resistant *Salmonella* Senftenberg (*S. Senftenberg*). It was also demonstrated that the development of resistance to linalool is associated with cross resistance to several antibiotics¹. In the current study, we hypothesize that extensive commercial use of EOs can lead to the development of resistant pathogens, similar to the acquisition of multiple antibiotic resistance caused by the overuse of antibiotics. In order to understand the specific evolutionary path of acquiring resistance to basil oil and its main components we sequentially exposed *S. Senftenberg* and *S. Typhimurium* to increasing concentrations of linalool under different conditions. In total, we developed 16 different lineages of *S. Senftenberg*. Isolates from these lineages could grow with up to 16% linalool (MIC increased by up to 128 folds in comparison with the WT strain). For *S. Typhimurium* we developed 8 different lineages, but the bacteria were not able to grow with more than 2% linalool (MIC increased by up to 32 folds in comparison with the WT strain). Furthermore, we were interested to study a potential link between basil oil resistance and multiple antibiotic resistance in food borne pathogens. To do so we selected linalool resistant variants from the above described lineages and tested their susceptibility to antibiotics (according to CLSI guidelines). We observed various phenotypic differences out of 15 selected variants, 8 were resistant to trimethoprim, and 1 to kanamycin. On the other hand, 7 variants were found to be susceptible to nalidixic acid. Whole genome sequencing of 50 *S. Senftenberg* variants revealed a total of 66 Single nucleated polymorphisms (SNPs), 52 of them resulted in a single amino acid change. In the *S. Typhimurium* lineages (total of 17 variants were sequenced), 5 SNPs were found, from which 1 resulted in a single amino acid change and 4 resulted in a gap, leading to a truncated protein. The SNPs found were located in different genes, some are responsible for multidrug resistance (*ramA*), heat shock response (*hsUI*), oxidative stress response (*rcsC*) and virulence (*lon*). Moreover, stable linalool resistant variants were developed with no genetic variations. These results indicate that the genetic changes occurring after sequential exposure to increasing concentrations of linalool are random. Further research is required to fully understand how the resistance to linalool accompanied by the change in antibiotic susceptibility occurs, and what precautions should be recommended in order to prevent or control the development of EOs resistant pathogens.

## Reference

[1] Kalily E, Hollander A, Korin B, Cymerman I, Yaron S. Mechanisms of resistance to linalool in *Salmonella* Senftenberg and their role in survival on basil. Environ Microbiol. 2016

<footnote>ªDepartment of Biotechnology and Food Engineering, Technion–Israel Institute of Technology</footnote>

<footnote>ᵇPublic Health Laboratories –Jerusalem (PHL-J), Public Health Services (PHS), Ministry of Health (MOH), Israel</footnote>
<figure><img src="image_38.png" /></figure>

# Uncovering the Hidden World of Caves Reveals the Key Role of Multidrug Efflux Pumps in Formation of Resistomes

Yuping Cao¹, ²*, Huihong Li¹, ², Sima Yaron¹, ², and Ka Yin Leung², ¹

¹Biotechnology & Food Engineering, Technion, Israel

²Biotechnology & Food Engineering, Guangdong Technion, China

Antibiotic resistance, a natural phenomenon, has become a global concern due to the misuse of antibiotics. Human activities have a substantial impact on the environment, which serves as a reservoir for antibiotic resistance genes (ARGs). Exploring pristine environments free from human interferences provides unique insights into the natural development of microbiomes and resistomes, the collection of all ARGs within a community. The current studies examined 47 publicly available metagenomes from 14 caves, classified into five microhabitats: sediments, microbial mats, water, biofilms, and minerals. Our findings revealed a core microbiome across all samples, with specific taxa displaying differential abundance and discrimination in various microhabitats due to their distinct environmental conditions. Moreover, microhabitats led to significantly different bacterial communities and resistomes. Notably, multidrug resistance (MDR) genes, primarily multidrug efflux pumps, were prevalent in all samples. The resistome differences mainly originated from variations in multidrug efflux pumps, such as *mex*-, *emr*-, and *mdt*-type efflux pumps. Interestingly, the mobile colistin resistance gene *mcr* was pervasive in cave environments. Furthermore, bacterial hosts carrying MDR genes, particularly multidrug efflux pumps, maintained a high relative abundance in ARG-host profiles. These results align with our previous studies using a germfree soil model, which demonstrated that the hosts for MDR genes, such as Burkholderiaceae, are crucial for the establishment of soil resistomes, irrespective of antibiotic selections¹. This research provides a comprehensive overview of microbiomes and resistomes in pristine cave habitats. The findings highlight the importance of MDR genes, especially multidrug efflux pumps, in the formation of resistomes and the ancient origin of clinically significant ARG *mcr* and possibly other functions of ARGs that have no clinical values.

<figure><img src="image_39.png" /><figcaption>**Fig. 1.** The locations and types of pristine cave samples</figcaption></figure>

**Reference:**

1. Cao, Yuping, et al. "Burkholderiaceae and multidrug resistance genes are key players in resistome development in a germfree soil model." *mSystems* 6.6 (2021): e00988-21.
# ICEs in *Vibrio cholerae* from water samples from France: Occurrence, diversity and impact for human health

Sandrine BARON<fnref n="1" />, Laëtitia LE DEVENDEC<fnref n="1" />, Jean LESNE, Xavier BELLANGER<fnref n="2" />, Claudia JÄCKEL<fnref n="3" />, Jens A. HAMMERL<fnref n="3" />

*Vibrio cholerae* is found in aquatic ecosystems throughout the world. While isolates of the O1/O139 serogroup are capable of causing cholera due to the presence of cholera toxin (CTX) and toxin coregulated pilus (TCP) as primary virulence factors, isolates of other serogroups (non-O1/O139) are usually considered less harmful. However, some of them have been shown to be involved in intestinal and extra-intestinal infections (i.e., wound, soft tissue and ear infections or bacteraemia) due to a wide range of virulence factors (i.e., mannose-sensitive haemagglutinin pilus (MSHA), various hemolysins, repeat toxin clusters (RTX), outer membrane proteins and the type III secretion system (TTSS)) that usually act synergistically during the infection process. Besides pathogenicity, self-transferring integrative conjugative elements (ICEs) gain further importance as they represent large genomic segments carrying several bacterial adaptive functions including antimicrobial resistance (AMR). In this study the occurrence and diversity of ICE-carrying *V. cholerae* from France and their impact for human health was studied.

*V. cholerae* isolation was conducted from water/sediment samples (total: n=586) collected along a salinity gradient under a temperate climate (France): freshwater (2 sites: n=49 and n=91), brackish water (n=207) and sediments (n=41) from brackish areas, seawater (n=59) and wastewater (n=139) between June 2000 and September 2001. In addition, 49 samples of cockles were also investigated. Isolates from different sources were subjected to a molecular survey on the presence of ICE. ICE-carrying isolates (n=64) were further subjected to paired-end, short read whole-genome sequencing (WGS) for in depth characterization.

Non-O1/O139 isolates were successfully recovered by cultivation from all sources investigated in this study. Molecular detection on ICE revealed substantial differences between the different water types, while samples from brackish and seawater exhibited the highest number of ICE-carrying *V. cholerae* in comparison to freshwater samples. Bioinformatic analysis revealed 21 different MLST allele compositions among 64 *V. cholerae* isolates, of which 11 belong to unassigned sequence types (ST). The AMR genes detected were linked to chromosomal contigs and conferred resistance to carbapenems (*varG*), colistin (*almG*) and phenicol (*catB9*). The number of virulence factors among the isolates ranged from 151 to 166 and were involved in adherence, biofilm formation, effector delivery systems, exoenzymes, exotoxins, immune modulation, motility, and nutritional/metabolic factors.

The detection of ICE-carrying isolates in about 10% of the analyzed *V. cholerae* provides a first insight into the presence of self-transmissible sequence regions in naturally occurring isolates. The genomes of the isolates found so far are not heavily loaded with AMR, but may represent a hot spot for the acquisition of insertional elements carrying medically important resistance genes. Selective pressures on the bacteria, such as exposure to antimicrobials or residues in water (wastewater), may force their spread and adaptation, but will also hamper medical treatment purposes against *V. cholerae* infections.

Further surveillance and characterization data is needed and will provide information about diversity of ICE in *V. cholerae* and their specific mechanisms for acquiring and spreading AMR.

<footnote n="1">¹Mycoplasmosis-Bacteriology and Antimicrobial Resistance Unit, Anses, France</footnote>

<footnote n="2">²LCPME, CNRS, Université de Lorraine, Nancy, France</footnote>

<footnote n="3">³Department Biological Safety, German Federal Institute for Risk Assessment, Germany</footnote>
<figure><img src="image_40.png" /></figure>

# Microplastics as cargo for pathogenic bacteria and hotspot of antimicrobial resistance genes transfer? Insights into microbial communities and the emergence of antibiotic resistance

Méril Massot, Stéphanie Bedhomme

CEFE, CNRS, 1919 route de Mende, 34293 Montpellier

Microplastics (MP) have emerged as a significant source of pollution in marine ecosystems¹. Microbial biofilms rapidly establish on the surface of these MPs and studying these microcosms is crucial to characterize the role of MP as vector for pathogens, and their role as reservoir of antimicrobial resistance (AMR) genes.

To tackle these questions, we used the fish-pathogen *Vibrio anguillarum*, as model pathogenic bacteria and evaluated by flow cytometry and microscopy the intraspecific variability in the efficiency of colonization of different types of MP: polystyrene spheres (30 µm), and polypropylene and polyethylene terephthalate fragments (38-50 µm). The set of sixteen strains tested revealed a broad spectrum of attachment phenotypes. We therefore looked for an association between colonization phenotype and gene content and phylogenetic relatedness.

Furthermore, we investigated the potential role of MP in structuring microbial community composition in marine environments. MP samples were incubated at three distinct locations in the Mediterranean Sea, representing varying levels of human activities: an aquaculture area (étang de Thau), a harbor (Carnon city), and a less polluted area (national reserve of Cerbères-Banyuls). Variation in composition of the MP-associated biofilms among locations and MP polymers were analyzed by shotgun metagenomics, using MetaSpades². Obtained sequences were screened against the CARD database³ for AMR gene identification.

To understand the influence of marine MP-associated biofilms on the emergence of antibiotic-resistant pathogenic bacteria, we investigated the impact of natural marine microbial communities on the attachment of three Vibrio strains covering the range of attachment phenotype. Attachment efficiency was measured by comparing attachment to sterile MP versus colonized MP. Additionally, we are planning to study whether the spatial structure provided by MP in the water column promotes horizontal transfer of resistance genes detected in these communities.

It is crucial to decode the potential consequences of MP-associated biofilms on the emergence of antibiotic-resistant pathogenic bacteria. The colonization of microplastics by bacteria in marine environment enables their long-distance transport, facilitating the spread to susceptible new host populations.

## References

1. Bowley, J., Baker-Austin, C., Porter, A., Hartnell, R. & Lewis, C. Oceanic Hitchhikers – Assessing Pathogen Risks from Marine Microplastic. *Trends Microbiol.* **29**, 107–116 (2021).

2. Nurk, S., Meleshko, D., Korobeynikov, A. & Pevzner, P. A. metaSPAdes: a new versatile metagenomic assembler. *Genome Res.* **27**, 824–834 (2017).

3. Alcock, B. P. et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. *Nucleic Acids Res.* **48**, D517–D525 (2020).
<figure><img src="image_41.png" /></figure>

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

P17

# Fluoroquinolone resistance in *E. coli* in the broiler production chain: the role of parent stock and the hatchery

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

## 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 *Escherichia coli* (*E. coli*) in broilers. The purpose of this study was to describe the spread of FQ resistant *E. coli* throughout the broiler production chain from parent stock farms up to day-old broiler chicks and to investigate their role in the dynamics of the spread.

## Materials and methods

In this study, 4 broiler parent stock farms were included (which were supplying eggs to 3 different hatcheries). On these parent stock farms, 30 faeces samples, 30 eggs, swabs of the egg trays and information on antimicrobial usage, were collected. The batch of on-farm sampled eggs of each parent stock farm was traced to the hatchery and again 30 eggs of the same batch were sampled on day 18 of hatching. On the day of hatching, 30 day-old chicks were collected from the same batch as well. Environmental swabs were collected in the hatchery from several crucial points where the eggs or day-old chickens came into contact with the environment, being the hatching crates, suction cups, collection belt, and the transport crates of the day-old chicks. Both the outside of the eggs, the crushed eggshell, and the contents of the eggs were analysed following the method of Mehzoud *et al*. (2016) [1]. In all samples, the presence of *E. coli* and FQ resistant *E. coli* isolates was detected by plating on plain MacConkey agar or supplemented with 0.25 µg/ml enrofloxacin (ECOFF). All isolates were identified using MALDI-TOF MS. For a selection of isolates whole genome sequencing was applied to investigate their phylogenetic relatedness using cgMLST analysis, and to detect resistance determinants.

## Results

On all 4 parent stock farms, FQ resistant *E. coli* were detected [56.7-93.3%]. A low number of eggs was positive for *E. coli*, both just after laying [0-13.3%] and after 18 days of incubation [0-3.3%]. Except for one egg after laying, no FQ resistant *E. coli* could be found in or on the eggs. In the day-old chicks sampled at the hatchery, 3 out of 4 batches were positive for FQ resistant *E. coli* [0-90%]. On the day of hatching all environmental swabs from the collection belts were positive for FQ resistant *E. coli* and in 3 out of 4 batches, FQ resistant *E. coli* was discovered on the transport crates of the 1-day-old chicks. The results of the whole genome sequencing will be presented during the conference.

## References

[1] H. Mezhoud, I. Chantziaras, M. Iguer-Ouada, N. Moula, A. Garmyn, et al. (2016). Avian Pathology, 45:4, 493-500

<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>
P18

Multi-drug resistant Klebsiella spp. in hospitals, wastewaters and surface waters

Jarmila LAUSOVA<fnref n="1" />,<fnref n="2" />, Iva SUKKAR<fnref n="2" />, Lenka DAVIDOVA GERZOVA<fnref n="2" />, Monika DOLEJSKA<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="4" />

Multi-drug resistant bacteria pose a significant challenge to the treatment of infectious diseases. Of particular concern are bacteria belonging to the genus *Klebsiella*, especially *K. pneumoniae* (*Klpn*) and *K. oxytoca* (*Klox*), which are frequently associated with hospital-acquired infections and have potential to spread outside hospitals via wastewaters. The municipal wastewater treatment plants (mWWTP) have been identified as a reservoir of resistant bacteria and are considered a hotspot that facilitates the spread of antibiotic resistance into the environment. The aim of this study was to compare clinical isolates of *Klebsiella* spp. with resistant strains obtained from wastewaters and surface water.

A total of 397 *Klebsiella* spp. (383 *Klpn* and 14 *Klox*) were obtained from patients (n=138) and five types of water: hospital sewage (n=97), inflow (n=58) and outflow from the mWWTP (n=73), river upstream (n=22) and downstream mWWTP (n=19) from three cities in Czech Republicusing selective cultivation (cefotaxime, 2 mg/l). All strains were tested for minimum inhibitory concentrations of 24 antimicrobials, extended-spectrum beta-lactamase (ESBL) & AmpC production and subjected to whole-genome sequencing (Illumina). Analysis of antibioticresistance genes, sequence types (STs) and plasmid replicons was performed by BLAST algorithm and CGE tools (http://www.genomicepidemiology.org/). The phylogenetic tree was constructed using Prokka, Pirate and RAxML.

Most isolates (95%) showed multi-drug resistance profile, resistance to cefazolin (100%), aminoglycosides (92%), trimethoprim (87%), ciprofloxacin (76%), tetracyclines (64%) and some of them were resistant to last line drugs carbapenems (14%) and colistin (8%). Resistance to cephalosporins was associated with ESBL phenotype and encoded mainly by *bla*<sub>CTX-M-15</sub> gene (n=379/397). Carbapenemase-encoding genes including *bla*<sub>GES-5</sub> (n=15), *bla*<sub>OXA-48</sub> (n=4), *bla*<sub>NDM-1</sub> (n=4) and *bla*<sub>KPC-3</sub> (n=1) were found in isolates originating from patients, hospital sewage, outflow/inflow of mWWTPs and river. Isolates were assigned to 81 different STs with predominance of *Klpn* ST307 (n=53) that was found in all water types and clinical samples. Among *Klox*, ST27 (n=7) was the most prevalent, found in outflow from mWWTP and river upstream mWWTP and showed high phylogenetic relatedness (SNPs<50).

This study pointed out the importance of the surveillance of pathogenic resistant bacteria in the
hospital and community wastewaters and their spread through the WWTPs to surface water and the
environment.

<footnote n="1">¹Department of Biology and Wildlife Diseases, VETUNI Brno, Brno, Czech Republic</footnote>

<footnote n="2">²Central European Institute of Technology, VETUNI Brno, Brno, Czech Republic</footnote>

<footnote n="3">³Department of Microbiology, Faculty of Medicine and University Hospital Pilsen, Charles University, Pilsen, Czech Republic</footnote>

<footnote n="4">⁴Department of Clinical Microbiology and Immunology, Institute of Laboratory Medicine, The University Hospital Brno, Brno, Czech Republic</footnote>
# Characterisation of extended spectrum cephalosporin resistance in *Escherichia coli* on Irish pig farms using whole genome sequencing

Lorcan O'NEILL<sup>1,2</sup>, Maria RODRIGUES da COSTA<sup>1,2,3</sup>, Edgar GARCIA MANZANILLA<sup>1,2</sup>, Fiona CRISPIE<sup>4</sup>, Finola C. LEONARD<sup>1</sup>

Extended spectrum cephalosporin (ESC) resistance in *Escherichia coli* is a major public health issue. Although not the primary source of human infections, animals represent an important reservoir of extended spectrum (ESBL) and AmpC beta lactamase producing *E. coli*. As one of the highest users of veterinary antimicrobials, the pig sector is a particular focus of concern and there is a need for further studies investigating the epidemiology and genetic context of ESC-resistant *E. coli* during the pig lifecycle. This study sought to investigate the prevalence and molecular epidemiology of ESBL- and AmpC producing *E. coli* in batches of pigs on Irish farms during their lifecycle.

Twelve farrow-to-finish farms participated in a longitudinal study. Ten litters of piglets were selected from each farm; pooled faecal samples were collected during each production stage (piglet, weaner, grower and finisher), at slaughter, from the piglets' dams and also from unrelated batches in different houses. ESC-resistant *E. coli* were selected using media supplemented with 1 mg/L cefotaxime and plate counts were used to calculate the proportions of resistance. Up to five isolates from each sampling point were selected for antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS).

All farms were positive for ESC resistant *E. coli* at at least one sampling point during the study. The highest proportions of resistance were observed in piglets (median = 0.00005, range = 0 - 0.15) and were lowest in finishers (median = 0, range = 0 - 0.01). In total, 198 isolates were selected for AST and WGS analysis. In addition to beta lactam and cephalosporin resistance, 95% of isolates were resistant to at least one other antimicrobial from different classes such as: doxycycline (82.3%); trimethoprim and sulfamethoxazole (64.6%); gentamicin (21.7%) and ciprofloxacin (67.2%). Eighty-seven isolates were positive for an ESBL gene, mainly *bla*<sub>CTX-M-15</sub> (n = 57), *bla*<sub>CTX-M-27</sub> (n = 18), *bla*<sub>CTX-M-65</sub> (n = 10). The other isolates were AmpC producers and possessed either *bla*<sub>CMY-2</sub> (n = 46) or a mutation of the chromosomal *ampC* promoter gene (n = 64). Twenty-six sequence types (ST) and 26 serotypes were identified with 40 unique combinations. Each unique combination of ST and serotype was associated with one ESC resistance mechanism. One ESC resistance mechanism dominated on each farm throughout the production cycle although six farms were positive for more than one ESBL/AmpC producing gene. In addition, *bla*<sub>OXA-1</sub> was found in 33 isolates on three farms and in all cases co-occurred with the dual aminoglycoside and fluoroquinolone resistance gene, *aac*(6')-Ib-cr.

This study gives important insights into the epidemiology and diversity of ESC resistant *E. coli* on Irish pig farms. More in depth bioinformatical analysis of this data is to follow and will allow for determination of phylogeny and the zoonotic or pathogenic potential of the isolates and for improved genetic context of the detected resistance genes.

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

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

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

<footnote n="4"><sup>4</sup>Food Research Centre, Teagasc, Fermoy, Ireland</footnote>
# Hospital and community wastewaters as a source of multidrug-resistant ESBL-producing *Escherichia coli*

Lenka DAVIDOVA GERZOVA<fnref n="1" />, Jarmila LAUSOVA<fnref n="1" />,<fnref n="2" />, Iva SUKKAR<fnref n="1" />, Kristina NESPOROVA<fnref n="1" />, Lucie NECHUTNA<fnref n="3" />,<fnref n="4" />, Katerina VLKOVA<fnref n="3" />,<fnref n="4" />, Katerina CHUDEJOVA<fnref n="3" />,<fnref n="4" />, Marcela KRUTOVA<fnref n="5" />, Jana PALKOVICOVA<fnref n="1" />, Jakub KASPAR<fnref n="6" />, Monika DOLEJSKA<fnref n="1" />,<fnref n="2" />,<fnref n="4" />,<fnref n="7" />

Hospitals and wastewaters represent a recognized hot spot for the selection and dissemination of antibiotic-resistant bacteria to the environment, but the total participation of hospitals in the spread of nosocomial pathogens to the municipal wastewater treatment plants (WWTPs) and adjacent rivers were to be revealed. Therefore, we used a combination of culturing and whole-genome sequencing to explore the transmission routes of *Escherichia coli* from hospitalized patients suffering from urinary tract infections (UTI) via wastewaters to the environment. Samples were collected in two periods in three locations (A, B, C) and cultured on selective antibiotic-enhanced plates. In total, 409 *E. coli* isolates were obtained from patients with UTI (n=80), raw hospital sewage (n=74), WWTPs inflow (n=95)/outflow (n=106), and river upstream (n=23)/downstream (n=31) of WWTPs. The majority (96%) of the isolates produced extended-spectrum beta-lactamase, mainly CTX-M-15, and showed multidrug resistance (MDR) profiles. Seven carbapenemase-producing isolates with GES-5 or OXA-244 were obtained in two locations from wastewaters and river samples. Isolates were assigned to 76 different sequence types (ST) with the predominance of ST131 (n=80) that was found in all sources including rivers. Extraintestinal pathogenic lineages frequently found in hospital sewage (ST10, ST38, ST69) were also found in river water. Despite generally high genetic diversity, phylogenetic analysis of ST10, ST295 and ST744 showed highly related isolates (SNP 0-18) from different sources, providing the evidence for the transmission of resistant strains through WWTPs to surface waters. Results of this study suggest that 1/ UTI share a minor participation in hospitals wastewaters; 2/ high diversity of STs and phylogenetic groups in municipal wastewaters stress the urban influence rather than hospitals; 3/ pathogenic lineages and bacteria with emerging resistance genotypes associated with hospitals spread into surface waters. Our study highlights the contribution of wastewaters to the transmission of ESBL- and carbapenemase-producing *E. coli* with MDR profiles to the environment.

Funded by NU20J-09-00040

<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 Microbiology, Faculty of Medicine and University Hospital Pilsen, Charles University, Pilsen, Czech Republic</footnote>

<footnote n="4">⁴Biomedical Center, Faculty of Medicine, Charles University, Pilsen, Czech Republic</footnote>

<footnote n="5">⁵Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic</footnote>

<footnote n="6">⁶Center of cardiovascular and transplant surgery, St. Anne's University Hospital Brno, Czech Republic</footnote>

<footnote n="7">⁷Department of Clinical Microbiology and Immunology, Institute of Laboratory Medicine, The University Hospital Brno, Czech Republic</footnote>
# Genotypic characterization of ESBL/AmpC-producing *E. coli* from the German National Zoonoses Monitoring from 2016-2021 within the food production chain

Carolina Plaza-Rodriguez, Silke Jahn, Tanja Skladnikiewicz-Ziemer, Silvia Schmoger, Janina Malekzadah, Mirjam Grobbel, Annemarie Käsbohrer, Bernd-Alois Tenhagen, Alexandra Irrgang,

German Federal Institute for Risk Assessment

Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases are able to hydrolyze 3rd generation cephalosporins, which are widely used in human medicine. Resistances mediated by ESBL/AmpCs are of great concern and closely monitored in human as well as in veterinary medicine. Here, we present data on the ESBL/AmpC genotypes of 4601 *E. coli* isolates obtained from the regular monitoring on antimicrobial resistance (AMR) in zoonotic and commensal bacteria from 2016-2021.

The AMR monitoring was conducted within the framework of the Zoonoses Directive 2003/99/EC, following technical specifications given by Commission Implementing Decision (CID) 2013/652/EU and CID 2020/1729/EU complemented by additional national zoonoses programs. Isolates were obtained from different matrices from broilers (N = 1297), fattening turkeys (N = 1005), fattening pigs (N = 992) and cattle (N = 1206), and at different levels of the food production chain including primary production, slaughterhouse and retail. In addition, isolates from wild animals, small ruminants, freshwater fish and plant-based food were included (N = 101). In the years 2016-2020 phenotypic presumed ESBL/AmpC isolates were analyzed by real-time PCR and genotype was determined by PCR sequencing. TEM variant was only determined if no other possible ESBL gene was detected, as high number of isolates harbor additional *bla*TEM-1 (non-ESBL). In 2021, presumed ESBL/AmpC-producing isolates were characterized by Illumina short read sequencing.

The variability of ESBLs genes is fairly low with 23±3, different detected genes and gene combinations each year. Although PCR primers used do not allow to detect all known gene variants, variability of ESBL genes found did not increase by NGS analysis. However, NGS supports detection of chromosomal AmpC gene alterations. In general, the majority of isolates harbored one ESBL/AmpC gene, only few carried two or more genes. In cattle and pigs (uneven years), the majority of isolates (75.8%) produce CTX-M-1 or CTX-M-15, whereas in poultry (even years), a more even distribution of CTX-M-1, CTX-M-15, CMY-2, TEM-52 and SHV-12 was observed. ESBL/AmpC-beta lactamases CTX-M-2, -3,-8, -9, DHA-1 or EC-5 were found rarely. Comparing the genes found at slaughter (caecum content) over the years, there were no major variations in isolates from pigs and calves. However, in broilers a significant decrease in CMY-2 and CTX-M-1 beta-lactamases and a significant increase in SHV-12 and TEM-52 were observed. Significant changes in several genes were also observed in turkeys over time (decrease in CTX-M-1 and TEM-52, and increase in CTX-M-15, -27 and -55). Distribution of ESBL/AmpC genes in isolates from wild animals, small ruminants, freshwater fish and plant-based food was similar to the combined frequency in pigs and cattle.

In conclusion, close observation to detect trends on the specific ESBL genes in different livestock production chains is important to assess the potential sources of human exposure. In addition, further research is needed to understand what caused the shift in poultry, as the use of cephalosporins is not allowed in poultry production
<figure><img src="image_42.png" /></figure>

P22

# Extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing *Escherichia coli* among bivalve samples of the Portuguese coast

Samanta Freire, Teresa Grilo, Bruna Rodrigues, Rui Oliveira, António Marques, Laurent Poirel, Marta Aires-de-Sousa

**Background.** Bivalves are filter-feeding organisms and biomarkers of bacterial pollution. Our study aimed to analyze the occurrence and characteristics of extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing *Escherichia coli* among bivalve samples from Portuguese coastal waters.

**Methods.** As part of the national monitoring program for shellfish production area management, 551 bivalve samples were collected between december 2021 and september 2022, by the Instituto Português do Mar e da Atmosfera (IPMA), from 20 shellfish production areas along the Portuguese coastal waters. Homogenized samples were screened for *E. coli* contamination by plating onto tryptone bile X-glucuronide (TBX) selective plates. A total of 206 TBX plates showing *E. coli* bacterial growth (37%) were provided by IPMA for this study. After overnight broth enrichment, the bacterial growth of each TBX plate was inoculated onto selective media for ESBL producers (CHROMagar ESBL) and for carbapenem-resistant isolates (ChromID Carba Smart). Antimicrobial susceptibility testing was performed on all isolates recovered from the two selective media using the disc diffusion method. Screening for ESBL and carbapenemases was performed by PCR amplification and sequencing. Clonality was evaluated by multilocus sequence typing.

**Results.** Nine bivalve samples were contaminated with an ESBL producer (4.4%) and one carried a carbapenemase-producing isolate (0.005%). The 10 isolates belonged to two species: *E. coli* (n=7; 70%) and *K. pneumoniae* (n=3). All ESBLs were from the CTX-M-type: CTX-M-32 (n=4), CTX-M-15 (n=4) and CTX-M-14 (n=1). The single carbapenemase producer harbored *bla*GES-5. The seven ESBL-producing *E. coli* isolates belonged to five distinct sequence types (ST10, ST23, ST540, ST617, ST746, SLV206, SLV2325), while the two ESBL-producing *K. pneumoniae* isolates belonged to ST834 and ST15. The carbapenemase producer (*K. pneumoniae*) belonged to a new ST. Mating-out assay followed by PCR-based replicon typing revealed that the *bla*GES-5 gene was located on a ColE plasmid.

**Conclusions.** Bivalves constitute a reservoir of ESBL- and carbapenemase-producing Enterobacteriaceae. We identified the first enterobacterial isolate producing a carbapenemase of GES-type recovered from bivalve samples. This carbapenemase, which has been previously detected in Portugal among humans, animals and now in the marine environment, seems to be widely spread in the country, despite the prevalence in bivalves is still reduced.
Phylogenetic analysis of *Acinetobacter* spp. isolates from raw meat

Leila HAMZE<fnref n="1" />, Raquel GARCIA FIERRO<fnref n="1" />, Pauline FRANCOIS<fnref n="1" />, Debora VOGT<fnref n="2" />, Antoine DRAPEAU<fnref n="1" />, Andrea ENDIMIANI<fnref n="3" />, Marisa HAENNI<fnref n="1" />, Vincent PERRETEN<fnref n="2" />, Jean-Yves MADEC<fnref n="1" />, and Agnese LUPO<fnref n="1" />

The *Acinetobacter* genus includes 76 species. Among those, *Acinetobacter baumannii* is the most threatening for public health, because of its ability to develop multidrug resistance and to cause opportunistic and severe infections. Multidrug-resistant *A. baumannii*, especially those belonging to international clonal complexes (ICs) I-III, are able to spread epidemically. More recently, the medical importance of *Acinetobacter nosocomialis* has been demonstrated [1]. Furthermore, extra-hospital reservoirs of *Acinetobacter* species have been unveiled with increasing interest for food sources. The aim of this study was to characterize 76 *Acinetobacter* spp. isolates collected in the frame of two screening campaigns (November 2012-May 2013, December 2018-July 2019) in raw meat samples [2,3]. Susceptibility to 14 antibiotics was evaluated by disc diffusion (EUCAST, 2022). Total DNA was extracted and sequenced generating short-reads (Illumina). After reads' quality check (FastQC v. 0.11.9), assembly was obtained using Shovill v.1.0.4. Identification was confirmed by rMLST (https://pubmlst.org/species-id). Presence of antibiotic resistance genes and determination of the sequence type was achieved using the Comprehensive Antibiotic Resistance Database and the Pasteur Institute scheme, respectively. Genomic relationships among isolates was analyzed by parsnp v.1.5.2. Two out of 76 isolates were identified as *A. nosocomialis*, the remaining were *A. baumannii* harboring *bla*OXA-51-*type* and *bla*ADC-25 genes. Thirteen *A. baumannii* isolates were resistant to tetracycline and harbored a *tet*(39) gene. Two possessed the *aph*(3'')-*Ib* and *aph*(6)-*Id* genes conferring resistance to streptomycin. The phylogenetic tree representation evidenced heterogeneity among genomes of the study, reflected by the diversity of their sequence types (n=51). Four sequence types (ST42, ST109, ST351, ST364) were present in both screening campaigns and several non-epidemic (n=20) were shared with isolates associated to infections in humans and from diverse regions of the globe. Raw meat could serve as important reservoir of different clones of *A. baumannii* with potential for causing infections. Through food, such clones could propagate in the community and under selective pressure develop multidrug resistance. Food products need careful surveillance for understanding their role in the global picture of the antibiotic resistance burden.

References

[1] D. Wong, T.B. Nielsen, R.A. Bonomo, P. Pantapalangkoor, B. Luna, B. Spellberg (2016). *Clin Microbiol Rev.* 30:409-447.

[2] A. Lupo, D. Vogt, S.N. Seiffert, A. Endimiani, V. Perreten. (2014). *J Food Prod* 77:1976-1981.

[3] C. Rodrigues, K. Hauser, N. Cahill, M. Ligowska-Marzęta, G. Centorotola, A. Cornacchia, R. Garcia Fierro, M. Haenni, et al. (2022). *Microbiol Spectr* 10:e0237621.

<footnote n="1">¹Unité Antibiorésistance et virulence bactérienne, AVB, ANSES, Lyon, France</footnote>

<footnote n="2">²Division of Molecular Bacterial Epidemiology and Infectious Diseases, Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland</footnote>

<footnote n="3">³Institute for Infectious Diseases, University of Bern, Bern, Switzerland.</footnote>
<figure><img src="image_43.png" /></figure>

# Time-calibrated WGS-based phylogeny revealed successful spread of two major Staphylococcus aureus CC398 phylogroups around Europe and novel CC398 MRSA lineages in Switzerland.

Javier Eduardo Fernandez<fnref n="1" />, Adrian Egli<fnref n="2" />, Gudrun Overesch<fnref n="1" />, Vincent Perreten<fnref n="1" />.

A time-calibrated whole genome-based phylogeny of 130 Swiss methicillin-resistant *Staphylococcus aureus* (MRSA) of clonal complex (CC) 398 originating from animal, food samples, and humans as well as of an international CC398 collection of 638 strains from all 5 continents revealed the presence of distinct phylogroups in Asia, North and South America, and Europe. The majority of Swiss MRSA CC398 clustered within two major European phylogroups (EP1 and EP2) and formed three major novel lineages (LE), LEd in EP1 and LEa, LEb in EP2. The Bayesian inferred phylogeny estimated that the European phylogroups EP1 and EP2 started to diverge from each other in the 1970s. LEa (n=57) is characterized by MRSA CC398 carriage of the staphylococcal cassette chromosome *mec* (SCC*mec*) elements IVa and *spa* type t011 (t011-SCC*mec*IVa), LEb (n=25) by t011-SCC*mec*Vc, and LEd (n=32) by t011/t034/t571-SCC*mec*Vc.

Antibiotic resistance gene patterns of Swiss lineages were conserved within strains of LEa [*aac(6')-Ie-aph(2'')-Ia*, tet(M), *dfrK*] (horses, human carriage and infections) and LEd [*ant(9)-Ia, tet(M), tet(K), erm(A), vga(E), dfrG*] (pigs and cattle carriage), and diverse in LEb strains (carriage and infection in different animals and humans). Although spillover was frequent between human and animals based on phylogenetic relatedness, virulence profiling revealed that there were no human or animal specific virulence factors and human adaptation (i.e. acquisition of the immune evasion cluster) was scarcely detected in human and horse strains.

By placing international CC398 into a temporal, geographical, and phylogenetic context, we showed that some Swiss MRSA CC398 (n=89) from animals and human origin clustered into novel lineages, which evolved, from two globally spreading European phylogroups and that bacterial exchange occurs between host species, with some of them developing into infections especially in horses and humans. This study provides a baseline for future global WGS-based One-Health studies of the evolution of MRSA CC398 as well as for local outbreak investigations.

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

<footnote n="2">²Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.</footnote>
Antimicrobial and biocide susceptibility among *Staphylococcus aureus* and *Staphylococcus pseudintermedius* from dogs and cats

Angela R. SCHUG<sup>1,2</sup>, Anissa D. SCHOLTZEK<sup>1,2</sup>, Barbara KOHN<sup>2,3</sup>, Christiane WEINGART<sup>2,3</sup>,
Anne-Kathrin SCHINK<sup>1,2</sup>, Astrid BETHE<sup>1,2</sup>, Antina LÜBKE-BECKER<sup>1,2</sup>, Andrea T. FEßLER<sup>1,2</sup>
and Stefan SCHWARZ<sup>1,2</sup>

**Introduction:** Since *Staphylococcus aureus* and *Staphylococcus pseudintermedius* have a zoonotic potential, monitoring of their antimicrobial and biocide resistance is an important issue. The aim of the present study was to investigate the resistance situation of canine and feline *S. aureus* and *S. pseudintermedius* isolates from diagnostic submissions.

**Materials and Methods:** A total of 114 isolates, including 62 *S. aureus*, 52 *S. pseudintermedius* were investigated for (i) their susceptibility to 27 antimicrobial agents by broth microdilution according to CLSI [1] and the detection of the respective resistance genes. Moreover, the isolates were tested for their susceptibility to four biocides, namely benzalkonium chloride, chlorhexidine, polyhexanide and octenidine by a recently developed biocide susceptibility testing protocol [2].

**Results:** Penicillin resistance mediated by the *blaZ* gene was the dominant resistance property with 80.65% among *S. aureus* and 86.54% *S. pseudintermedius*. About one quarter of the isolates (*S. aureus* 33.87%; *S. pseudintermedius* 15.38%) proved to be methicillin-resistant and carried the genes *mecA* or *mecC*. Macrolide resistance with 27.2% was second most prevalent and all isolates harbored the resistance genes *erm*(A), *erm*(B), *erm*(C), *erm*(T) or *msr*(A), alone or in combinations. Among all isolates tested fluoroquinolone resistance was detected in 21.1% and tetracycline resistance mediated by *tet*(K) and/or *tet*(M) occurred in 19.3% of the isolates. Resistance to last resort antimicrobial agents for human medicine was only detected in single isolates, if at all. The biocide susceptibility testing showed unimodal minimal inhibitory concentration (MIC) distributions of the four biocides and were very similar for *S. aureus* and *S. pseudintermedius*.

**Conclusions:** While various antimicrobial resistance properties were determined and a considerable number of the isolates displayed a multiresistance phenotype, the biocide susceptibility testing revealed unimodal distributions not pointing towards resistance development. Further monitoring is necessary to early detect changes in the resistance situation.

**References**

[1] CLSI Supplement VET01S5th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2020

[2] Schug AR, Bartel A, Scholtzek AD, Meurer M, Brombach J, Hensel V, Fanning S, Schwarz S, Feßler AT. (2020). *Vet Microbiol.* 248:108791.

[3] Feßler AT, Scholtzek AD, Schug AR, Kohn B, Weingart C, Schink AK, Bethe A, Lübke-Becker A, Schwarz S. (2022). Antibiotics (Basel). 11:127.

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

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

<footnote n="3"><sup>3</sup>Small Animal Clinic, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany</footnote>
<figure><img src="image_44.png" /></figure>

Highly conserved IncK/bla<sub>CMY-2</sub> plasmids in <i>E. coli</i> ST38 from broiler production

Solveig Sølverød MO<sup>1,2</sup>, Jannice Schau SLETTEMEÅS<sup>1</sup>, Håkon KASPERSEN<sup>1</sup> and Marianne SUNDE<sup>1</sup>

*Escherichia coli* belonging to multilocus sequence type (MLST) 38 have been predominant among extended-spectrum cephalosporin (ESC)-resistant *E. coli* detected in Norwegian broiler production. Previously we have shown that IncK/bla<sub>CMY-2</sub> plasmids from broiler production in different European countries are highly similar, but with some differences with regard to size. We were interested in looking into the IncK/bla<sub>CMY-2</sub> plasmid epidemiology and evolution in ESC-resistant *E. coli* ST38 over a six-year period (2011-2016). Isolates for long-read sequencing were selected based on an ST38 phylogenetic tree [1], picking representatives from different parts of the tree, different levels of the production pyramid and different years to get a representative selection. A total of 10 isolate were sequenced using Oxford Nanopore Technology (ONT, minION). Plasmids were assembled with Unicycler, annotated with Bakta and compared using the gggenomes package in R (Figure 1). We will include complete IncK/bla<sub>CMY-2</sub> plasmids originating from *E. coli* ST38 from broiler production in other European countries, and thus evaluate diversity and, if possible, evolution of the plasmids in a European perspective.

<figure><img src="image_45.png" /><figcaption>Figure 1. Alignment of IncK/bla<sub>CMY-2</sub> plasmids originating from *E. coli* ST38 isolated from Norwegian broiler production during a six-year period (2011-2016).</figcaption></figure>

**References**

[1] S.S. Mo, E.Z. Fiskebeck, J.S. Slettemeås, K. Lagesen, O. Nilsson et al. (2023). Under review in *Front Microbiol*.

<footnote n="1"><sup>1</sup>Section for Food Safety and Animal Health Research, Department of Animal Health and Welfare and Food Safety, Norwegian Veterinary Institute, Ås, Norway</footnote>

<footnote n="2"><sup>2</sup>Section for Bacteriology, Department of Analysis and Diagnostics, Norwegian Veterinary Institute, Ås, Norway</footnote>
## AMR monitoring on rabbit farms

Alieda van ESSEN-ZANDBERGEN, Yvon GEURTS, Michal BROUWER and Kees T. VELDMAN

Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands

**Background:** In 2014 antimicrobial resistance (AMR) monitoring of livestock and meat became mandatory within the European Union (EU). The animal sectors covered by the mandatory monitoring are: veal calves, fattening pigs, turkeys and broilers. However, other animal sectors, such as meat rabbits and dairy goats, are not included. Therefore, no recent AMR data are available from these animal sectors. The latest report of the Netherlands Veterinary Medicines Institute (SDa) shows that antibiotic use in the rabbit sector has decreased but is still relatively high compared to other animal sectors. Therefore, a baseline study on antibiotic resistance in meat rabbit farms in the Netherlands is performed.

**Objectives:** Monitoring of antimicrobial resistance in the rabbit sector in the Netherlands.

**Methods:** At each of 15 rabbit farms, five fresh feacal samples were taken from the floor of the pens close to slaughter date. In line with European legislation, indicator *Escherichia coli*, ESBL/AmpC/carbapenemase-suspected *E. coli*, *Campylobacter coli* and *C. jejuni* were isolated from the collected samples. In addition, MRSA was isolated from five dust samples, collected with wipes and up to 5 nose-swabs of dead animals per farm. Antimicrobial susceptibility was tested with broth microdilution method with standard European antibiotic panels.

**Results:** Bacterial culture was conducted on a total of 75 feacal samples. No ESBL/AmpC/carbapenemase-suspected *E. coli*, *Campylobacter coli* and *jejuni* were isolated from these fecal samples. Also, no MRSA was isolated from dust samples and nose-swabs. Indicator *E. coli* was present in all feacal samples, MIC testing of these isolates showed high percentage of resistance to tetracycline (56.0%), trimethoprim (54.7%) and sulfamethoxazole (52.0%). Five *E. coli* isolates originating from 3 different farms showed a colistin MIC of 4-16 mg/L.

<table><caption>**Table 1:** MIC-value and resistance percentage (%) of *E. coli* isolated from rabbits</caption><thead><tr><th rowspan="2">MIC (g/L)</th><th rowspan="2">AMP</th><th rowspan="2">FOT</th><th rowspan="2">TAZ</th><th rowspan="2">GEN</th><th rowspan="2">TET</th><th rowspan="2">SMX</th><th rowspan="2">TMP</th><th colspan="8">Antibiotic</th></tr><tr><th>CIP</th><th>NAL</th><th>CHL</th><th>AZI</th><th>COL</th><th>MERO</th><th>TGC</th><th>AMI</th></tr></thead><tbody><tr><td>0.015</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>36</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>0.03</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>27</td><td>0</td><td>0</td><td>0</td><td>0</td><td>73</td><td>0</td></tr><tr><td>0.06</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>9</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>0</td></tr><tr><td>0.125</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>0.25</td><td>0</td><td>71</td><td>48</td><td>0</td><td>0</td><td>0</td><td>9</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>50</td><td>0</td></tr><tr><td>0.5</td><td>0</td><td>4</td><td>23</td><td>46</td><td>0</td><td>0</td><td>14</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>25</td><td>0</td></tr><tr><td>1</td><td>0</td><td>0</td><td>4</td><td>25</td><td>0</td><td>0</td><td>10</td><td>1</td><td>0</td><td>0</td><td>0</td><td>70</td><td>0</td><td>0</td></tr><tr><td>2</td><td>2</td><td>0</td><td>0</td><td>4</td><td>22</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td></tr><tr><td>4</td><td>33</td><td>0</td><td>0</td><td>10</td><td>0</td><td>0</td><td>1</td><td>0</td><td>71</td><td>0</td><td>22</td><td>0</td><td>0</td><td>72</td></tr><tr><td>8</td><td>27</td><td>0</td><td>0</td><td>1</td><td>36</td><td>0</td><td>0</td><td>2</td><td>54</td><td>45</td><td>2</td><td>0</td><td>0</td><td>3</td></tr><tr><td>16</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>17</td><td>7</td><td>3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>32</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>40</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>64</td><td>13</td><td>0</td><td>0</td><td>0</td><td>42</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>128</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2</td><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>256</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>512</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>1024</td><td>0</td><td>0</td><td>0</td><td>0</td><td>39</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>2048</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>N tot</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td><td>75</td></tr><tr><td>R%</td><td>17.3%</td><td>5.3%</td><td>0.0%</td><td>0.0%</td><td>56.0%</td><td>52.0%</td><td>54.7%</td><td>4.0%</td><td>2.7%</td><td>5.3%</td><td>0.0%</td><td>6.7%</td><td>0.0%</td><td>0.0%</td></tr><tr><td>Nres</td><td>13</td><td>4</td><td>0</td><td>0</td><td>42</td><td>39</td><td>41</td><td>3</td><td>2</td><td>4</td><td>0</td><td>5</td><td>0</td><td>0</td></tr></tbody></table>

**Conclusion and discussion:** In indicator *E. coli* from meat rabbits resistance levels are high for tetracycline, trimethoprim and sulfamethoxazole compared to livestock. This might reflect the relatively high antibiotic use in the rabbit sector. In contrast, no ESBL/AmpC/carbapenemase-suspected *E. coli*, *C. coli* and *C. jejuni* were isolated from feacal samples and no MRSA was isolated from dust or nasal swabs.

**References**

[1] Het Gebruik van antibiotica bij landbouwhuisdieren in 2021, november 2022

https://www.autoriteitdiergeneesmiddelen.nl/nl/publicaties/sda-rapporten-antibioticumgebruik

[2] MARAN, Monitoring of Antimicrobial Resistance and Antibiotic usage in animals in the Netherlands in 2021, June 2022
<figure><img src="image_46.png" /></figure>

# Unexpected high resistance rates for ertapenem in *Campylobacter* isolates obtained from livestock

Kees VELDMAN, Alieda VAN ESSEN-ZANDBERGEN and Michael BROUWER

Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands

**Background:** A new European legislation on antimicrobial resistance monitoring in livestock and meat came into force in 2021 based on a scientific EFSA report [1]. Therefore, susceptibility testing of *Campylobacter jejuni* and *Campylobacter coli* isolates obtained from broilers and cattle (<1 year) and from *C. coli* isolates from fattening pigs were included in the mandatory monitoring program. In addition, the harmonised antimicrobial panel for susceptibility testing of *Campylobacter* was modified: nalidixic acid and streptomycin were substituted by chloramphenicol and ertapenem.

**Methods:** Isolation of *Campylobacter* was performed according to a standardised protocol designed by the EURL for *Campylobacter* [2]. Susceptibility testing was performed with broth microdilution in standardised European antibiotic panels (Sensititre, panel EUCAMP3) according to ISO 20776-1. Results were interpreted using epidemiological cut-off values (ECOFFs) published by EUCAST. For ertapenem, tentative ECOFFs of 0.5 mg/L (advised by EFSA) were applied for both *C. jejuni* and *C. coli*. As a follow-up, a set of 30 *Campylobacter* isolates with variable ertapenem MIC-values were selected for further analysis conducting Whole Genome Sequencing (Illumina) to reveal the presence of potential carbapenem resistance mechanisms.

**Results:** In 2021 AMR monitoring of *C. jejuni* and *C. coli* from caecal samples of broilers was mandatory. In addition, AMR monitoring of *C. coli* from fattening pigs and *C. jejuni* and *C. coli* from cattle were included in the Dutch AMR monitoring program on a voluntary base. Applying the tentative ECOFF for ertapenem resulted in high resistance rates for ertapenem, especially in *C. coli* from poultry (76.2%) and to a lesser extent in broilers (39.4%) compared to low resistance in *C. coli* from fattening pigs (2.8%) and *C. jejuni* from broilers and veal calves. WGS data analysis showed the presence of *bla*OXA-184 (n=2), *bla*OXA-193 (n=15) and *bla*OXA-489 (n = 9), without association with elevated ertapenem MIC-values.

<table><caption>Table 1. Ertapenem resistance percentages of *C. jejuni* and *C. coli* isolated from faecal samples of livestock in 2021</caption><thead><tr><th colspan="2">C. jejuni (R%)</th><th colspan="3">C. coli (R%)</th></tr><tr><th>Broilers<br>(n = 131)</th><th>Veal calves<br>(n = 222)</th><th>Broilers<br>(n = 84)</th><th>Veal calves<br>(n = 137)</th><th>Pigs<br>(n = 287)</th></tr></thead><tbody><tr><td>6.1</td><td>0.5</td><td>76.2</td><td>39.4</td><td>2.8</td></tr></tbody></table>

**Discussion and conclusions:** The tentative ECOFFs for ertapenem seems to overestimate the proportion of resistance for ertapenem, especially for *C. coli* by cutting through the wild-type population. Nonetheless, the observed animals-specific differences in ertapenem resistance remains unexplained after analysis of the WGS data. Additional research is required to uncover intrinsic or acquired carbapenem resistance mechanisms in both *C. jejuni* and *C. coli*.

## References

[1] EFSA (European Food Safety Authority), (2019). *EFSA Journal 2019;17(6):5709, 122 pp*

[2] EURL Campylobacter (2021), Protocol for isolation, identification and storage of *Campylobacter jejuni* and/or *Campylobacter coli* for AMR monitoring, www.sva.se/en/about-us/eurl-campylobacter/laboratory-procedures/
# Resistances to Cephalosporins and Fluoroquinolones in Veterinary medicine in Livestock
## Results of the German National Antibiotic Resistance Monitoring

Heike KASPAR, Antje ROEMER, Maria KLUGE, Stefan FIEDLER, Anne-Kathrin KARAALP, Britta BALLHAUSEN, Fabian HORN, Ulrike STEINACKER

Federal Office of Consumer Protection and Food Safety (BVL), Berlin, Germany

### Objectives

Cephalosporins (3rd and 4th generation) and Fluoroquinolones are classified from WHO as “highest priority critically important antimicrobials” for human use and from WOAH as “critically important antimicrobials”. They should be used careful and prudent and only then where it is necessary. Nevertheless, they are important substances to treat bacterial infections in human and veterinary medicine. Since 2001, an annual representative German-wide monitoring study (GERM-Vet) on bacterial isolates from diseased animals generates resistance data amongst others against a set of five different cephalosporins and a set of three different fluoroquinolones.

### Methods

Based on a statistically valid sampling plan the bacterial isolates were investigated by using the broth microdilution method according to CLSI document VET01 5th ed. The MIC values were assessed with their corresponding clinical veterinary breakpoints). If no breakpoints were available, MIC90 values were used for classification.

### Results

The resistance data were evaluated according the bacterial species, animal species and indications that were investigated.

#### Mastitis (dairy cow)

S. aureus isolates showed low resistance rates against cephalosporins (0-1.5%) and fluoroquinolones (MIC90 0.25 mg/L), E. coli: MIC90 values were low over a period of five years (Cefquinom MIC90 0.12 mg/L, Ceftiofur about 3% in 2021).

#### Calves

MIC90 values for cephalosporins of the 3rd and 4th generation and for fluoroquinolones are high for bacterial strains isolated from calves (for all tested cephalosporins >32 mg/L, fluoroquinolones >16mg/L). The rate for ESBL positive E. coli isolates from calves is decreasing since 2017 from 32% to 13% in 2021. ESBLs of the class blaCTX-M-1 and blaCTX-M-15 were identified most frequently

#### Pigs

MIC90 values for cephalosporins of the 3rd and 4th generation and fluoroquinolones were much lower for bacterial strains isolated from pigs than for those isolated from calves (for all tested cephalosporins 0.12 – 0.5 mg/L, fluoroquinolones 1 mg/L). The rate for ESBL positive E. coli was still low about 3% in 2021.

#### Poultry

Cephalosporins are not approved for veterinary use in poultry. Nevertheless, we see high MIC90 values for broilers although the ESBL rates for E. coli are still at 2%. Resistance rates for isolates from turkeys to fluoroquinolones are low (about 7%) for isolates derived from broilers the values are slightly higher (16%).

### Conclusions

An intelligent and rational application of antimicrobial agents is needed to minimise the development and the spread of antimicrobial resistant bacteria and their resistance genes as far as possible. Depending on the affiliation to animal and bacterial species we see large differences in resistance data and a very different impact on resistance situation in veterinary medicine. With these representative and quantitative data, we are able to monitor and to estimate the development of antimicrobial resistance in veterinary pathogens to 3rd and 4th generation cephalosporins.
<figure><img src="image_47.png" /></figure>

Linezolid-resistant *Enterococcus faecalis* of chicken origin harbored chromosome-borne *optrA* and plasmid-borne *cfr*, *cfr(D)* and *poxtA2* genes

Xiang-Dang Du<fnref n="1" />*, Hong Yao<fnref n="1" />, Chunyan Xu<fnref n="1" />, Chenglong Li<fnref n="1" />, Tang Biao<fnref n="2" />, Jinfeng Zhao<fnref n="1" />, Xinxin Shan<fnref n="1" /> and Stefan Schwarz<fnref n="3" />,<fnref n="4" />

The aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in an *Enterococcus faecalis* isolate QZ076, co-carrying *optrA*, *cfr*, *cfr(D)* and *poxtA2* genes. MICs were determined by broth microdilution. WGS was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, using *E. faecalis* JH2-2 and clinical MRSA 109 as recipients. *E. faecalis* QZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, with *optrA* located in the chromosomal DNA. The gene *cfr* was located on a novel pseudo-compound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515 generated 8-bp direct target duplications (5'-GATACGTA-3'). The genes *cfr(D)* and *poxtA2* were co-located on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. The *cfr*-carrying plasmid pQZ076-1 could transfer from *E. faecalis* QZ076 to *E. faecalis* JH2-2, along with the *cfr(D)*- and *poxtA2*-co-carrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [*optrA*, *cfr*, *cfr(D)*, *poxtA2*] being simultaneously present in the same *E. faecalis* isolate. The location of the *cfr* gene on a pseudo-compound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, the *cfr*-carrying pheromone-responsive conjugative plasmid in *E. faecalis* was also able to mobilize the inter-species transfer of the *cfr(D)*- and *poxtA2*-co-carrying plasmid between enterococci and staphylococci.

References

[1] Schwarz S, Zhang W, Du XD, Krüger H, Feßler AT, Ma S, Zhu Y, Wu C, Shen J, Wang Y (2021). *Clin Microbiol Rev* 34:e0018820.

[2] Antonelli A, D'Andrea MM, Brenciani A, Galeotti CL, Morroni G, Pollini S, Varaldo PE, Rossolini GM (2018). *J Antimicrob Chemother* 73:1763-1769.

[3] Baccani I, Antonelli A, Di Pilato V, Coppi M, Di Maggio T, Spinicci M, Villagran AL, Revollo C, Bartoloni A, Rossolini GM (2021). *Antimicrob Agents Chemother* 65:e0069521.

<footnote n="1">¹International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China.</footnote>

<footnote n="2">²State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products & Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China</footnote>

<footnote n="3">³Institute of Microbiology and Epizootics, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.</footnote>

<footnote n="4">⁴Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, Berlin, Germany</footnote>

<footnote>*Corresponding author. E-mail: xddu@henau.edu.cn</footnote>
# Antimicrobial resistance of *Escherichia coli* isolated from slaughterhouse effluents in France: monitoring 15 years apart

V. Dupouy<fnref n="1" />, M.M. Um<fnref n="2" />, N. Arpaillange<fnref n="1" />, F. Auvray<fnref n="2" />, A. Bousquet-Mélou.<fnref n="1" />, H. Brugère<fnref n="2" />, D. Bibbal<fnref n="1" />

**Introduction.** Livestock is a reservoir of antibiotic resistant *E. coli* strains, and such strains are present in slaughterhouse effluents. As the presence of these strains is poorly documented, the aim of this study was to identify factors having an impact on the prevalence of antibiotic-resistant *E. coli* in slaughterhouse effluents: species slaughtered, season, step of sewage treatment. Moreover, some effluents were sampled after an interval of 10/15 years in order to estimate the impact of the French national plans Ecoantibio, implemented in 2012, which aimed to reduce the use of veterinary antibiotics.

**Materials and methods.** *E. coli* strains were isolated from effluents of various types of domestic ungulate slaughterhouses, at different sampling campaigns:

(i) slaughterhouses with large tons carcass weight equivalent per year, possessing a Wastewater Treatment Plant (WWTP), allowing them to discharge the treated effluents into the environment: 6 slaughterhouses sampled in 2002/2003, 1 in 2011 and 4 in 2017,

(ii) smaller slaughterhouses, performing a pre-treatment of their effluents before they were sent to a municipal WWTP: 6 slaughterhouses sampled in 2002/2003.

Antimicrobial resistance was investigated by disc diffusion method. Resistance determinants and integrase genes were amplified by PCR. During the 2017 sampling campaign, resistance to colistin and Critically Important Antibiotics (CIA) was specifically screened on antibiotic-supplemented plates.

**Results.** The percentage of resistant *E. coli* isolates among the total *E. coli* strains isolated was not different between wastewater, pre-treated and treated effluents. The season (autumn, winter, summer), as well as the year of sampling (before *versus* after Ecoantibio plans), also had no impact on this percentage. A variation was only observed according to the type of slaughterhouse. It was between 63% and 96% for the smallest slaughterhouses which were multispecies slaughterhouses. It was also high for plants where swine and veal calves were mainly slaughtered (from 80 to 96%). In contrast, it was lowest in plants where adult cattle was mainly slaughtered (from 7% to 46%). Resistance was mainly detected for tetracycline, ampicillin, streptomycin, sulfonamides and trimethoprim. During the 2017 sampling campaign conducted in cattle slaughterhouses, no strain resistant to colistin was isolated, and only a few CIA-resistant strains was detected: one *E. coli* isolate harbouring the *bla*<sub>CTX-M-15</sub> gene and *E. coli* isolates resistant to ciprofloxacin belonging to 5 distinct ERIC2 PCR (Enterobacterial Repetitive Intergenic Consensus) profiles.

**Conclusion.** The percentage of resistant *E. coli* isolates in slaughterhouse effluents was related to the category of domestic ungulates slaughtered in the plants.

<footnote n="1">¹INTHERES, Université de Toulouse, INRAE, ENVT, Toulouse, France</footnote>

<footnote n="2">²IRSD, Université de Toulouse, INSERM, INRAE, ENVT, UPS, Toulouse, France</footnote>
# Plasmids carrying carbapenemase gene in Carbapenemase-producing *Enterobacteriaceae* isolated from companion animals in Korea

Jong-Chan CHAE<fnref n="1" />, Jae Hong JEONG<fnref n="1" />, Sumin KWAK<fnref n="1" />, Kwang-Won SEO<fnref n="2" /> and Jae-Young OH<fnref n="1" />

Carbapenem-producing *Enterobacteriaceae* (CPE) are resistant to the carbapenem class of antibiotics which are considered as the drugs of last resort for human infections. It is known that most of the treatment of companion animals with clinical sign depends on human antibiotics, and even carbapenems are used in severe cases. Since the first detection of carbapenem-resistant *Escherichia coli* from domestic companion animals in Korea, 2017, the seven CPEs among carbapenem-resistant *Enterobacteriaceae* (CRE) were collected from fecal samples of companion animals between 2019 and 2022. Whole genome sequencing analysis was performed using Nanopore and Illumina platforms in order to characterize them under genomic level. Plasmids harboring the carbapenemase genes were identified from the IncX3 plasmids in 5 strains and the IncFIA plasmids in 2 strains, respectively. A *bla*~NDM-5~ gene was detected in 6 CPEs and an IncX3 plasmid (54,805 bp) carrying *bla*~KPC-2~ was found in CPE isolated from asymptomatic companion dog in 2022. IncX3 plasmids (46,161 bp) carrying *bla*~NDM-5~ in 4 CREs were identical to the plasmids reported in 2017. Because of concerns about close contact between humans and companion animals and the possibility of cross-species transmission, a multifaceted surveillance of antibiotic resistant microorganisms in companion animals is required.

<figure><img src="image_48.png" /><figcaption>**Fig. 1.** Comparison of IncX3 plasmids carrying carbapenemase genes in companion animals</figcaption></figure>

## References

[1] J.S. Hong, W. Song, H.M. Park, J.Y. Oh, J.C. Chae, et al. (2019). *Microb Drug Resist* 25:344–349.

[2] S.F. Mouftah, T. Pál, D. Darwish, A. Ghazawi, et al. (2019). *Infect Drug Resist* 12:1719-1742.

<footnote n="1">¹Division of Biotechnology / Jeonbuk National University, Iksan, Republic of Korea</footnote>

<footnote n="2">²College of Veterinary Medicine / Chungbuk National University, Cheongju, Republic of Korea</footnote>
# Comparison of geno- and phenotypic characteristics of *E. faecalis* and *E. faecium* with whole genome sequencing results

Marlena GOŁAŚ-PRĄDZYŃSKA, Jolanta G. ROLA

Department of Hygiene of Food of Animal Origin, National Veterinary Research Institute, Pulawy, Poland

**Background,** Enterococci are a group of bacteria of significant importance as they cause bacterial infections in humans. It is important to study this bacteria derived from dairy products, and enrich the presently insufficient data on the antimicrobial resistance and virulence factors.

**Objectives,** The aim of the study was genotypic characterization of archival strains obtained from milk and dairy products and comparison of the results with those obtained by phenotypic methods and classical PCR.

**Methods and Results,** A total of 36 strains from milk and dairy products were analyzed. The identification of *Enterococcus faecalis* and *Enterococcus faecium* species selected antibiotic resistance genes were detected using polymerase chain reaction. Panel of 18 antibiotics was performed using the broth dilution method. Deoxyribonucleic acid (DNA) isolation for sequencing was performed using the Maxwell Rapid Sample Concentrator kit. Quantitative and qualitative DNA assessments were performed and libraries created using the KAPA Hyper Plus kit. Sequencing was performed on the Nextseq instrument using a 2 × 150-bp paired-end protocol. Phenotypic resistance to tetracycline, kanamycin, streptomycin, erythromycin, chloramphenicol, tylosin and single strains resistant to vancomycin, gentamicin, teicoplanin and ciprofloxacin was determined. The tested virulence genes in most cases overlapped with the results of phenotypic resistance, with the exception of isolates sensitive to vancomycin (with *VanA* gene), erythromycin, tetracycline. Resistant strains lacked the *vgaA* and *vatD* gene. *E. faecalis* and *E. faecium* tetracycline-, erythromycin- and chloramphenicol-resistant genes found in the ResFinder 4.1 database were associated with resistance phenotypes.
# Raw milk and cheeses made from raw milk as a potential source of microbiological hazards

Jolanta G. ROLA, Katarzyna KAMIŃSKA, Dominika DĘBCZAK

Department of Hygiene of Food of Animal Origin, National Veterinary Research Institute, Pulawy, Poland

**Background,** Raw milk and cheeses can be transmitters of many microorganisms that are dangerous to humans. Food borne pathogens can come from animal and the food production environment.

**Objectives,**

The aim of the study is to assess the occurrence and hazard characterization associated with the presence of *Salmonella* spp., *Listeria monocytogenes*, *Campylobacter* spp. and verotoxic *Escherichia coli* in raw milk (cow, sheep, goat milk). In addition, the research included the occurrence of enterotoxic positive staphylococci (CPS) and methicillin-resistant staphylococci (MRSA) in raw milk and cheese.

**Methods and Results,**

The study was carried out on the basis of the screening immunoenzymatic method with the use of mini Vidas analyser, and positive samples were confirmed with the reference methods. Pheno- and genotypic characterization of isolates were performed. Culture, biochemical, minimal inhibitory concentration and polimerase chain reaction methods were used.

In 2019-2022, a total of 1,075 bulk milk samples and cheeses from dairy farms from all over Poland were analyzed. The presence of *Listeria monocytogenes*, *Campylobacter* spp., *Salmonella* spp. and verotoxic *Escherichia coli* was found. The percentage of the positive samples was 8% for *Listeria monocytogenes*, 3% for *Campylobacter* spp., 0,1% for *Salmonella* spp. and 0,9% for *E. coli* O157. More than half of the samples were contaminated with CPS. Among the CPS, the presence of enterotoxigenic strains and MRSA was confirmed. Some strains of CPS showed the presence of staphylococcal enterotoxin genes encoding classic staphylococcal enterotoxins SEA, SEB, SEC and SED. The *seg*, *ser*, *sei*, *sej* and *sep* genes were also detected.
# ESBL gene dynamics of *Enterobacteriaceae* populations in the human-animal-environment interface

Javier Fernandez-Favieres, Jose F. Delgado-Blas, Carlos Serna, Mario Pulido-Vadillo, Natalia Montero and Bruno Gonzalez-Zorn

Antimicrobial Resistance Unit (ARU), Animal Health Department and VISAVET Health Surveillance Centre, Complutense University of Madrid, Madrid, Spain

Antimicrobial resistance (AMR) is a growing concern for both human and animal public health. The interaction between these two compartments and the environment has long been considered crucial in epidemiological and ecological studies. Extended-spectrum β-lactamases (ESBLs) are clinically relevant AMR mechanisms, given their capacity to degrade cephalosporins, a β-lactam class considered essential for the treatment of complicated infections caused by multi-drug resistant (MDR) pathogens, including *Enterobacteriaceae* species. Hence, this work aims to investigate the ESBL gene dynamics from a One Health perspective within a small municipality in the South of Spain. A total of 250 samples were taken from: pig farms (10 farms, 110 samples), hospital patients (35 samples) and water sources, both waste 2 WWTPs, 18 of WWTP samples; 11 community wastewater canalsizations, 33 wastewater samples) and natural (3 rivers, 36 river samples, 3 underground wells, 18 samples). Total *Enterobacteriaceae* isolates were counted on MacConkey agar plates. Likewise, ESBL-producing *Escherichia coli* and *Klebsiella pneumoniae* isolates were counted and selected in ESBL Brilliance plates, and identification was confirmed by MALDI-TOF. Minimal Inhibitory Concentration (MIC) to multiple antibiotic classes were assessed by broth microdilution with EUVSEC3 Sensititre plates. Whole Genome Sequencing (WGS) data from Illumina and Nanopore technologies were analysed by Unicycler, ABRicate and Roary, among others, in order to study genome structure, resistome/plasmidome content and pangenome structure, respectively. Total *Enterobacteriaceae* counts in pig faeces remained stable among farms, while the amount of ESBL-producing *E. coli* varied greatly. The same applied for extensive and intensive farms, with the latter presenting significantly higher levels of resistant bacteria (Mann-Whitney-Wilcoxon test, p-value = 5.329e-06). In water samples, a direct correlation was found between the presence of *Enterobacteriaceae* isolates and the amount of ESBL-producing bacteria, suggesting an effect of geographical distribution and faecal contamination on AMR levels. When selecting ESBL-producing isolates (evidenced by the high MICs against ampicillin, cefotaxime and ceftazidime,) resistance to chloramphenicol, trimethoprim and sulfamethoxazole were found as well. Preliminary WGS analyses revealed that ESBL-producing *E. coli* isolated from human patients belonged predominantly to ST131, a highly virulent, highly successful clone. These isolates carried mainly *blaCTX-M-15* and *blaCTX-M-27*, highly prevalent and relevant ESBL genetic variants. Such AMR genes were associated to IncFII and IncFIB plasmids, respectively, pointing out their transmission potential through plasmid dissemination. Moreover, a CTX-M-15-producing ST131 isolate was found in the municipal WWTP, which suggests the community spread of this clinically relevant clone. Subsequent genomic analyses will uncover the resistome-plasmidome associations and the pangenome structure of the whole ESBL-producing *E. coli* and *K. pneumoniae* populations, as well as their relationship with the phenotypic profiles and epidemiological context. These will lead to an indepth understanding of the ecological forces governing intra- and inter-compartment AMR dynamics and the establishment of regulatory measures to preserve regional public health.
# NDM-producing Enterobacterales in Environment of Broiler Farm Rapidly Colonized in chicks

Zhongpeng CAI<fnref n="1" />, Wenxian PU<fnref n="1" />, Yanxiang JIAO<fnref n="1" />, Luchao LV<fnref n="1" />, Guolong GAO<fnref n="1" />, Yuman BAI<fnref n="1" />, Wanyun HE<fnref n="1" />, Jun YANG<fnref n="1" />, Sheng CHEN<fnref n="2" />, Jian-Hua LIU<fnref n="1" />,<fnref n="3" />

Carbapenems play an important role in clinical medicine by serving as “last resort” antibiotics in treatment of serious infections caused by multidrug-resistant Gram-negative bacteria. Therefore, the emergence and global spread of New Delhi metallo-β-lactamases-producing carbapenem-resistant *Enterobacterales* (NDM-CRE) has become one of the most urgent global health threats. Usage of carbapenems in food-producing animals is prohibited. However, detection of NDM-CRE in farm animals is increasingly common. Our previous study revealed the long-term persistence of NDM-CRE in three floor-rearing chicken farms in Anhui province, China. To identify the risk factors associated with the persistence of NDM-CRE in these farms, we investigated the epidemiological characteristics of NDM-CRE in one broiler farm by testing samples collected within two complete breeding periods of two batches of broilers during April to August 2021. We collected a total of 3244 samples during the vacancy and raising periods in the farm, including 1030 chicken faeces (CF), 1841 environmental samples collected inside the chicken house (EI) and 373 outside the chicken house (EO). After enriching in LB broth, the samples were spread onto MAC agar plates containing 0.5 mg/L meropenem. One to three colonies (one colony per morphological type) from each plate were selected and PCR was performed to screen for the presence of the *bla*<sub>NDM</sub> genes. The results showed that, during the vacancy period, 15.92% environmental samples were positive for NDM-CRE, and that such strains were rapidly acquired by chicks, for which the detection rate of NDM-CRE in faecal samples was up to 60% within 6 hours of being transferred to the farm; the rate continued to rise in the following 72 hours, reaching a peak in around 2 weeks (100%). The rate of carriage of NDM-CRE in the chicken fell afterwards but remained stable and relatively high prior to being sold in markets (65%). Three NDM variants were identified, namely NDM-5(n=178), NDM-1(n=22), and NDM-13(n=16). The predominant NDM-CRE species among the samples were *Escherichia coli* (CR-EC, 145/216) and *Klebsiella pneumoniae* (CR-KP, 36/216). Both clonal and horizontal transmission of *bla*<sub>NDM</sub> were observed in both CR-EC and CR-KP strains isolated from chicken and environmental samples during the vacancy and raising periods. Several common sequence types (STs), such as CR-EC ST9388 and CR-KP ST1758 which carried plasmids of the same *bla*<sub>NDM</sub>-IncHI2 type and exhibited a high degree of sequence similarity, were identified. In summary, our findings show that chicks can be rapidly colonized by environmental NDM-CRE in the farm; such strains can rapidly spread and persist among the animals and in the farm environment during raising period. It is important to implement “all-in/all-out” management policy and carry out thorough cleaning and disinfection in broiler farms to prevent NDM-producing bacteria from being able to persist and disseminate in chicken farms.

<footnote n="1">¹College of Veterinary Medicine, South China Agricultural University, Guangzhou, China</footnote>

<footnote n="2">²Department of Food Science and Nutrition, The Hong Kong polytechnic University, Hung Hom, Hong Kong</footnote>

<footnote n="3">³Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China</footnote>
# Genomic analysis of antimicrobial-resistant *Escherichia coli* in a gull colony in the Czech Republic

Marketa RYSAVA<fnref n="1" />,<fnref n="2" />, Kristina NESPOROVA<fnref n="1" />, Michaela RUZICKOVA<fnref n="1" />,<fnref n="2" />, Hassan TARABAI<fnref n="1" />,<fnref n="6" />, Simon KREJCI<fnref n="2" />, Martina MASARIKOVA<fnref n="1" />,<fnref n="3" />, Jarmila LAUSOVA<fnref n="1" />,<fnref n="2" />, Ivan LITERAK<fnref n="1" />,<fnref n="2" />, Monika DOLEJSKA<fnref n="1" />,<fnref n="2" />,<fnref n="4" />,<fnref n="5" />

Wildlife plays an important role in the spread of antibiotic resistance and is one of the main reservoirs for the emergence and dissemination of antibiotic-resistant bacteria in the environment. Migrating animals are of great importance as they may spread antibiotic resistance in different geographical areas. This study aimed to investigate the presence and dynamics of antibiotic-resistant *Escherichia coli* in Caspian gulls (*Larus cachinnans*) from the Czech Republic and their potential role in the spread of antibiotic resistance.

Cloacal samples from birds breeding in the Nove Młyny water reservoir were collected in June 2018 (n=72) and May 2019 (n=45) and 6 water samples were taken from various locations the reservoir in 2019. Isolates were selected on media with ciprofloxacin (0,05 mg/ml), cefotaxime (0,2 mg/ml) or colistin (3,5 mg/ml). Resistant *E. coli* isolates were whole genome sequenced using Illumina. In addition, 1393 isolates from EnteroBase were analysed phylogenetically since they belonged to the same sequence type (ST) as our gull isolates. The search on EnteroBase was restricted to the Czech Republic and neighbouring countries, where the gulls were observed to migrate according to our telemetry data. The phylogenetic tree, which comprised 1534 isolates, underwent assessment and recalculation, with only isolates that differed by up to 100 SNPs from ours being included.

A total of 176 *E. coli* isolates were obtained, out of which 141 were sequenced. In 2018, a high occurrence (79%, 57/72) of ESBL/AmpC-producing *E. coli* was observed compared to 38% (17/45) in 2019. The predominant clonal lineage in 2018 was ST11983 (37/57 isolates), which carried a F34:A:-B- plasmid with *bla*CMY-2 gene. In year 2019, ST2325 (12/17 isolates) carrying F2:A:-B- plasmid and *bla*TEM gene was the most common. This ST was also found in water samples obtained this year. High proportions of several STs commonly recognized as internationally high-risk lineages, such as ST10, ST58, ST88, ST117, ST648, or ST74415, were found in the samples from both years as well as in the water sample. According to the results of the phylogenetic analysis, certain STs were detected in multiple sources, including humans, and a subset of these STs were found to be closely related to isolates recovered from gulls.

Our findings showed that the presence of *E. coli* strains resistant to antibiotics can fluctuate significantly over time in synanthropic birds, highlighting the importance of longitudinal studies. We identified both a temporary dominant lineage and persistent sequence types. The similarity between gull isolates and those in EnteroBase underscores the importance of assessing the potential risks associated with migratory birds as well as with anthropogenic influence on nature.

Funded by IGA VETUNI 208/2021/FVHE from University of Veterinary Sciences Brno.

<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 Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Brno, Czech Republic,</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>

<footnote n="6">⁶University of South Bohemia, Faculty of Science, Ceske Budejovice, Czech Republic</footnote>
# Impact of biosecurity and hygiene measures on different routes of introduction and persistence of antimicrobial resistance in pig and broiler farms.

Silvia STRONATI<fnref n="1" />,<fnref n="2" />, Christopher TEALE<fnref n="1" />, Francesca MARTELLI<fnref n="1" />, Benjamin SWIFT<fnref n="2" />, Lucy BRUNTON<fnref n="2" />

Evidence suggests that antimicrobial resistant (AMR) bacteria and genes can persist and spread within and between livestock premises even in settings where antibiotic usage is low or absent. To allow for effective surveillance and control strategies of resistant organisms in the livestock sector, it is key to identify main entry points and test current routine cleaning protocols. This study aimed to assess the presence of AMR *E. coli*, as an indicator organism, in livestock housing and in main farm inputs. To evaluate the efficacy of cleaning and disinfection protocols applied in different sectors, environmental swabs were collected from pig and broiler farms. On each site, one building that was used to house livestock was sampled three times – while populated by one group of animals, after being vacated and cleaned, and finally after introduction of a new production group. Swabs were collected from indoor surfaces in different sections of the houses, as well as from outside areas surrounding the entrance to the buildings. Water, bedding, feed, and replacement animals were sampled to evaluate their potential role in introducing contamination. To date, 299 confirmed *E. coli* isolates have been isolated from 50 broiler house samples and 303 isolates from 54 finisher pig pens samples. These were tested for susceptibility to 10 antibiotics through breakpoint sensitivity testing: ampicillin, cefotaxime, ceftazidime, ciprofloxacin, tetracycline, sulfamethoxazole, trimethoprim, streptomycin, apramycin, florfenicol. Overall, 61% of isolates from the broiler house and 24% of the finisher pig pens were resistant to three or more antibiotic classes and identified as multidrug resistant (MDR). Most of the *E. coli* isolates from the broiler farm were resistant to ampicillin (77%), sulfamethoxazole (71%) and tetracycline (57%). A similar resistance profile - but at lower levels - was found in the pig housing isolates: ampicillin (26%), tetracycline (15%) and sulfamethoxazole (11%). The percentage of *E. coli* isolates that were MDR at each of the three visits was 69%, 45% and 70% respectively for the broiler house, and 32%, 16% and 23% respectively for the pig house. After cleaning, all indoor samples from the broiler house - except for one floor swab - tested negative for *E. coli*, while isolation was possible from all samples of the pig housing throughout the sampling period. No statistically significant difference was observed in the proportion of MDR isolates between the three broiler farm visits. However, there was a significant reduction in the percentage of MDR *E. coli* after depopulation and cleaning of the pig pens (P = 0.009). These results suggest that the cleaning and disinfection strategy applied in the broiler sector, may help to reduce carryover of *E. coli* contamination between production groups in comparison to the pig sector. However, MDR *E. coli* appear to play a role in residual AMR contamination in livestock housing also in the context of relatively successful cleaning and disinfection. Genetic analysis on the collected MDR isolates will provide more information on their potential persistence and eventual genetic features preventing their elimination. Pilot data identified replacement animals as a key source of MDR *E. coli*, suggesting the need for surveillance in hatcheries and monitoring of pigs - whether reared on farm or externally sourced - before introducing them in finishing units.

<footnote n="1">¹Animal and Plant Health Agency, Weybridge, UK</footnote>

<footnote n="2">²Royal Veterinary College, Hatfield, UK</footnote>
# Interventions to control the dynamics of antimicrobial resistance from chickens through the environment: ENVIRE

Wejdene MANSOUR<fnref n="1" />, Lucie COLLINEAU<fnref n="2" />, Mindaugas MALAKAUSKAS<fnref n="3" />, Marta KUZMINSKA-BAJOR<fnref n="4" />, Tina KABELITZ<fnref n="5" /> and Roswitha MERLE<fnref n="6" />

The overall objective of the project ENVIRE is to contribute to the reduction of the selection and the spread of antimicrobial resistance in broiler chickens and from chicken farms to the environment, and ultimately to humans. Different intervention studies will investigate the potential of various on-farm measures: i) Antibiotic-free chicken raising, ii) Phytotherapy as alternative for antibiotics, iii) *E. coli* vaccination, iv) Application of bacteriophages, v) Treatment or storage of manure, vi) Depollution of farm effluents to remove antibiotics and their residues. Experimental studies will be complemented by field studies whenever possible. Focus will be laid on ESBL *Escherichia coli* and *Enterobacteriaceae* and on resistance against fluoroquinolones as well as colistin. A quantitative risk assessment model will be developed and used to assess the effectiveness as well as potential synergistic effects of the interventions, to reduce human exposure via the foodborne, occupational and environmental pathways. Data already available for the participating countries will be included in the model, and new, essential data will be generated within the studies. As a result, specific as well as general interventions will be identified that have the potential to reduce AMR in chicken and in the environment of chicken farms for Europe and Tunisia. To achieve this, six working groups from Germany, France, Lithuania, Poland, and Tunisia, bundle their leading expertise for the respective issue.

<footnote n="1">¹ Laboratoire de Recherche « Biophysique métabolique et Pharmacologie Appliquée » LR12ES02, Faculté de Médecine de Sousse, Université de Sousse, Tunisie</footnote>

<footnote n="2">² French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Laboratory of Lyon, Epidemiology and support to Surveillance Unit, Lyon, France</footnote>

<footnote n="3">³ Veterinary Academy of Lithuanian University of Health Sciences (LSMU), Department of Food Safety and Quality, Kaunas, Lithuania</footnote>

<footnote n="4">⁴ Wrocław University of Environmental and Life Sciences
The Department of Biotechnology and Food Microbiology (UPWR), Poland</footnote>

<footnote n="5">⁵ Leibniz Institute for Agricultural Engineering and Bioeconomy e.V. (ATB), Department Engineering for livestock management, Germany</footnote>

<footnote n="6">⁶ Department Veterinary Medicine
Institute for Veterinary Epidemiology and Biostatistics, Freie Universität Berlin (FUB), Germany</footnote>
Persistence of multidrug resistant *Escherichia coli* in chicken breeding environment

Sébastien Leclercq<fnref n="1" />, Philippe Bochereau<fnref n="1" />, Isabelle Foubert<fnref n="1" />, Christophe Souchet<fnref n="2" />, Angélique Travel<fnref n="2" />, Yannick Baumard<fnref n="3" />, Maria-Céleste Le Bourhis<fnref n="3" />, Benoît Doublet<fnref n="1" />, and Sylvie Baucheron<fnref n="1" />

Antimicrobial resistance (AMR) has decreased in French broiler production for the last 10 years, linked to lower antibiotics use. Nonetheless, animal gut and the breeding environment are still considered as reservoirs of resistant zoonotic bacteria. The aim of this study was to characterize the dynamic of AMR transmission and persistence in an experimental facility simulating a broiler production system with no antibiotic exposition.

Chickens from five families were followed over three generations and two breeding environments (meat production and lineage reproduction), and commensal AMR *E. coli* strains were isolated from feces at different weeks of age, as well as from building surfaces before flock entry. Among the ca. 600 non-redundant isolates collected, a subset of 128 multidrug resistant isolates (at least 4 antibiotic classes) were subjected to sequencing with Illumina and/or Oxford Nanopore technologies.

The vast majority of isolates (91%) belonged to six unrelated sequence types from phylogroups A (ST206, ST2701, ST93) and B1 (ST162, ST1611 and ST453), none being reported as major causes of human or animal infection. These ST were shared between chicken families, generations and sample type (feces or surfaces), but segregated by breeding environment: isolates of phylogroup A were associated with reproductive animals while isolates of phylogroup B1 were associated with production animals. The AMR gene repertoire of sequenced isolates showed a weak diversity, with several genes (i.e. *tet(A)*, *bla*TEM-1, *sul2*, *dfrA1*, *aadA1*) being shared by most ST. However, they were carried by independent plasmids, i.e. pIncF and pIncHI1 in ST2701 isolates, pIncZ in ST93 isolates, or pIncI1 in ST453 isolates.

Our results indicate that the transmission of AMR in animals of successive generations is mainly driven by the persistence of specific clones in our environment facility, rather than direct vertical transmission (from mother to offspring) or horizontal transmission of resistance plasmids.

This study is supported by the grant (2017-448) from the french national plan for the reduction of the risks of antimicrobial resistance in veterinary medicine (Ecoantibio2) of the Ministère de l'agriculture et de l'alimentation.

<footnote n="1">¹INRAE, Université de Tours, ISP, F-37380, Nouzilly, France</footnote>

<footnote n="2">²ITAVI, French Poultry Institute, France</footnote>

<footnote n="3">³INRAE, Pôle d'Expérimentation Avicole de Tours, Nouzilly, France</footnote>
# Antimicrobial resistance in commensal *Escherichia coli* on Irish pig farms: a longitudinal study

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" />, Rachel GRIMES<fnref n="4" />, Finola C. LEONARD<fnref n="1" />

New European Union regulations restricting antimicrobial use (AMU) in livestock aim to minimise potential human exposure to antimicrobial resistant bacteria of animal origin. Nevertheless, a better understanding is required of how different levels of AMU during the production cycle affect the development of antimicrobial resistance (AMR) in indicator bacteria such as *Escherichia coli*. This is especially relevant to pig farming since it is typically the livestock sector with the highest AMU. This study sought to evaluate how AMR in *E. coli* evolves during the lifecycle of pigs on Irish farrow-to-finish farms with different levels of AMU.

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). Twenty *E. coli* isolates from each pooled sample were tested against a panel of 16 antimicrobials using EUCAST defined epidemiologic cut off values (ECOFF). For each of the six most prevalent resistance phenotypes, as well as for the multidrug resistant (resistance to ≥ 3 antimicrobial classes) and pan suseptible phenotypes, mixed effects logistic regression models were constructed using AMU practice and stage of production as explanatory variables.

The highest frequencies of resistance were to doxycycline, ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, gentamicin and the fluoroquinolones. Resistance to these agents was highest in weaners except for fluoroquinolones where it was highest in piglets. Similarly, multidrug resistance was highest, and pan suseptibilty was lowest, in isolates from the weaner stage. Resistance was lower on farms not using medicated feed (low AMU) compared to moderate or high AMU farms with the exception of fluoroquinolone and trimethoprim/sulfamethoxazole resistance. Resistance to amikacin or imipenem was not observed and resistance to the cephalosporins was low.

Stage of production and in-feed AMU practices influence the occurrence of AMR in *E. coli* in pig production. These findings have implications for strategies to control AMR at farm level.

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

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

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

<footnote n="4">⁴Department of Clinical Microbiology, Trinity College Dublin, Dublin 2, Ireland</footnote>
<figure><img src="image_49.png" /></figure>

# Investigation into the occurrence of AMR in *Escherichia coli* in wild birds

Catherine FEARNLEY<fnref n="1" />, Angela Lopez GARCIA<fnref n="1" />, Beaulieu PUDDICOMBE<fnref n="1" />, Jaspreet TIWANA<fnref n="1" />, Catherine MAN<fnref n="2" />, Manal Abu OUN<fnref n="1" />, Chris TEALE<fnref n="3" />, Muna ANJUM<fnref n="2" />

Antimicrobial resistance (AMR) is a critical and growing problem worldwide. *Escherichia coli* is a commensal bacterium commonly present in the intestine of animals and people, and a useful indicator to monitor AMR circulating between hosts and the environment. There is evidence to suggest that wild birds can carry AMR-harbouring bacteria which they can spread through migration. Livestock feed, surface waters and pastures are all susceptible to contamination from wildlife excreta.

AMR harbouring *E. coli* have been isolated from many bird species, occupying diverse ecological niches, including ducks and geese, cormorants, birds of prey, gulls, doves, and passerines [1]. Gulls especially scavenge at wastewater treatment plants and landfill sites, and therefore can act as sentinels, mirroring AMR in people and possible transmission into the environment. Wild birds are also potential spreaders of AMR through their ability to migrate long distances in short periods of time, presence on pastures with livestock or access to livestock accommodation, and potential contamination of surface waters / environment.

In this study we have investigated the prevalence of AMR *E. coli* in over 100 wild birds across UK using selective media for the detection of ESBL/AmpC- and carbapenemase-producing *E. coli* [2]. We have also investigated the prevalence of *E. coli* resistant to two antimicrobials used in animal production in the UK and Europe, tetracycline and apramycin. Phenotypic and genotypic characterization to determine AMR profiles of *E. coli* that are circulating in the wild bird population across UK has been undertaken, with comparisons made to isolates recovered from other sources.

## References

[1] J. Bonnedahl and J. D Järhul (2014). *Upsala J. Med. Sci.* 119: 113-116.

[2] Isolation of ESBL, AmpC and carbapenemase-producing *E. coli* from caecal samples-December 2019, DTU Food, National Food Institute; https://www.eurl-ar.eu/protocols.aspx

<footnote n="1">¹Animal and Plant Health Agency, Addlestone, UK</footnote>

<footnote n="2">²Animal and Plant Health Agency, Starcross, UK</footnote>

<footnote n="3">³Animal and Plant Health Agency, Shrewsbury, UK</footnote>
# Influence of management factors on Austrian dairy farms and the prevalence of antibiotic resistance among ESBL/AmpC-producing *E. coli*

Thomas WERNER<fnref n="1" />, Annemarie KÄSBOHRER<fnref n="1" />,<fnref n="2" /> Barbara WASNER<fnref n="3" />, Sandra KOEBERL-JELOVCAN<fnref n="4" />, Sebastian VETTER<fnref n="1" /> and Clair FIRTH<fnref n="1" />

A total of 51 dairy farms in four federal states in Austria were included in the project. Farmers were recruited by their local veterinarian. Antibiotic use data were provided from the veterinary practice software. Farmers were requested to complete an online survey of management factors. Faecal samples were collected directly from pre-weaned calves (< 6 weeks of age), weaned calves (> 6 weeks of age) and the freestall/tie-stall alleys of dairy cows. Faecal samples were collected from calves using rectal swabs, which were then pooled in the laboratory. Cow samples were collected using boot swabs.

For the detection of ESBL/AmpC-producing *E. coli*, incubated sample material was spread on a selective MacConkey agar containing 1 mg/l cefotaxime and incubated. A subculture was spread on selective culture medium (MacConkey agar containing 1 mg/l CTX) and incubated again. The pure culture was confirmed by MALDI-TOF. The presence of resistance in all pure cultures was checked using the Sensititre™ EU Surveillance (EUVSEC3) plate. Isolates that showed resistance to cefotaxime, ceftazidime or meropenem were re-screened for resistance using the Sensititre™ EU Surveillance ESBL (EUVSEC2) plate.

Overall, 50 farmers completed the online survey. Cephalosporins and penicillins were the most frequently used antibiotics on these farms. Waste milk was routinely fed to calves on 88% of farms. Blanket dry cow therapy was used on 26.9% of farms, while over 40% of farmers stated that they used antibiotic dry cow therapy only on symptomatic cows. From 190 faecal samples, 56 isolates (29.5%) were confirmed as ESBL/AmpC-producing *E. coli*. ESBL/AmpC-producing *E. coli* were found on 27/51 (52.9%) of dairy farms tested. The lowest prevalence of ESBL/AmpC-producing *E. coli* were found among samples from weaned calves (22.5%), while dairy cows (31.9%) and pre-weaned calves (31.9%) were equally high.

Statistical analyses demonstrated links between the presence of ESBL/AmpC-producing *E. coli* and the feeding of waste milk to calves in these herds. Farms where calf pens/hutches were washed and disinfected between restocking with new calves were more likely to have ESBL/AmpC-producing *E. coli* present than farms where pens/hutches were simply mucked out and straw replaced. This is likely to be indicative of a higher level of infection and general health issues leading to a need for disinfection, rather than a causal relationship of disinfection causing resistance.

<footnote n="1">¹Unit of Veterinary Public Health & Epidemiology, University of Veterinary Medicine, Vienna Austria</footnote>

<footnote n="2">²Department for Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany</footnote>

<footnote n="3">³Upper Austrian Animal Health Organization Laboratory, Clinical Microbiology, Ried im Innkreis, Austria</footnote>

<footnote n="4">⁴Institute for Medical Microbiology and Hygiene, Centre for Foodborne Infectious Diseases, Division for Public Health, Austrian Agency for Health and Food Safety (AGES GmbH), Graz, Austria</footnote>
# Human carriage of ESBL/pAmpC-producing *Escherichia coli* and *Klebsiella pneumoniae* in relation to the consumption of raw or undercooked vegetables, fruits and fresh herbs

Anouk P. MEIJS<fnref n="1" />, Marta ROZWANDOWICZ<fnref n="1" />, Paul D. HENGEVELD, Cindy M. DIERIKX<fnref n="1" />, Joost HORDIJK<fnref n="1" />, Sabine C. DE GREEFF<fnref n="1" />, Engeline VAN DUIJKEREN<fnref n="1" /> and Jaap T. VAN DISSEL<fnref n="1" />

**Background and objectives:** Vegetables, fruits and herbs can be contaminated with ESBL-producing Enterobacterales. Although fresh produce appears to be less frequently colonized with antimicrobial resistant bacteria compared to meat products, their role as a reservoir might be important since many of these products are consumed without preparation, such as washing or heating. In this study, we investigated to what extend the consumption of raw vegetables, fruits and fresh herbs influences carriage rates of ESBL-producing *Escherichia coli* and *Klebsiella pneumoniae* (ESBL-E/K) in the general population. Secondary, we assessed long-term carriage as well as changes in ESBL-E/K prevalence during the COVID-19 pandemic, by comparing the results to earlier findings in the same population.

**Materials and methods:** Between July and December 2021 participants sent in two faecal samples, three months apart, accompanied by a digital questionnaire. During the study period food frequency questionnaires were sent on a monthly basis. Faeces was cultured using selective enrichment and subsequent culture on selective agar. Phenotypically positive ESBL-E/K isolates were sequenced using Illumina short read sequencing. Long read sequencing (Oxford Nanopore) was performed in participants that carried the same ESBL gene and bacterial ST in both samples. Multivariate logistic regression models were established to assess the association between the average weekly consumption of specified vegetables, fruits and ESBL-E/K carriage.

**Results:** The ESBL-E/K prevalence based on the first faecal sample was 7.6% (41/537; 95% CI 5.7-10.2) and 7.0% based on the second sample (34/489; 95% CI 5.0-9.6). The multivariate models did not result in statistical significance for any of the selected fruit and vegetable types. Trends for increased carriage rates were observed for the consumption of bell pepper, celery and several types of berries (raspberry, blueberry, blackberry). *bla*<sub>CTX-M-15</sub> was the predominant ESBL/pAmpC gene among the participants, followed by *bla*<sub>DHA-1</sub>. The most frequently found sequence types were *E. coli* ST 69, 10 and 131. The ESBL-E/K prevalence was comparable to a previous cross-sectional study of the same cohort performed in 2015-2017 (7.5%; 95% CI 5.6-10.1%). A comparison of the core genome showed that in six persons the ESBL-E/K was genetically highly similar to the bacteria found approximately five years earlier.

**Conclusion:** This population study showed that the contribution of the consumption of raw fruits and vegetables to ESBL-E/K carriage in humans in the Netherlands is probably low. In addition, we found indications for long-term carriage over a period of 5 years and demonstrated that the effect of the COVID-19 pandemic and its containment measures on ESBL-E/K carriage rates in the general population is small.

<footnote n="1">¹Centre for Infectious Disease Control, RIVM, Bilthoven, the Netherlands</footnote>
Mobile tigecycline resistance gene found in live bird markets in Dhaka,
Bangladesh

Alistair R. Davies<fnref n="1" />, Thomas Chisnall<fnref n="1" />, Shamima Akter<fnref n="2" />, Mohammad Golam Azam Chowdhury<fnref n="2" />, Md. Mohibul Hassan Afrad<fnref n="3" />, Zakiul Hasan<fnref n="3" />, Mohammad Habibur Rahman<fnref n="3" />, Eric Brum<fnref n="3" />, Roderick M. Card<fnref n="1" />

Multidrug-resistant pathogens present a significant risk to public health. Indicator species such as *Escherichia coli* can be used to assess the risk to consumers of the spread of AMR around a population. There is currently a limited understanding of AMR characteristics in Bangladesh, as well as in live bird markets. Caecal contents were collected from Sonali and Broiler chickens from a live bird market in Dhaka, Bangladesh. Samples were collected in 2018 (108) and 2020 (109) and isolated on MAC. A selection of 83 isolates were sent to the UK for broth microdilution assays to determine the MIC using commercially available broth microdilution plates. WGS was performed on all isolates to identify AMR genes as well as evaluate phylogenetic similarity based on core genome SNPs. 77/83 isolates were MDR, with the most common pattern of resistance to tetracycline, trimethoprim, sulfamethoxazole, ampicillin, and ciprofloxacin. All ciprofloxacin resistant isolates contained *qnrS* genes or *gyrA* and *parC* chromosomal mutations. 39/83 of isolates were resistant to azithromycin associated with *mphA* genes. Four isolates were ESBLs, two with *bla*<sub>CTX-M-55</sub>, two with *bla*<sub>CTX-M-65</sub>. Two isolates were AmpC phenotypes with *bla*<sub>DHA-1</sub> genes. Seven isolates were colistin resistant, confirmed with the presence of *mcr1.1* genes. Two isolates were resistant to tigecycline, one with an acquired *tetX4* gene. The presence of MDR E. coli in live bird markets presents concern to public health. The discovery of *tetX4* has not yet been characterised in Bangladesh and is therefore significant. Finding that 8% of isolates were colistin resistant with *mcr1* genes is also noteworthy given colistin is prohibited in animal use. An increase in food safety awareness and improved AMU stewardship in farms and in hospitals may contribute to a risk reduction. Continued surveillance at slaughter can allow monitoring of trends and impact of efforts to reduce AMR.

<footnote n="1">¹FAO Reference Centre for AMR, Department of Bacteriology, Animal and Plant Health Agency, Addlestone, United Kingdom</footnote>

<footnote n="2">²Central Disease Investigation Laboratory (CDIL), Dhaka, Bangladesh</footnote>

<footnote n="3">³Emergency Centre for Transboundary Animal Diseases (ECTAD), Food and Agriculture Organization (FAO) of the United Nations, Dhaka, Bangladesh</footnote>
One Health approach applied to the following up of antimicrobial resistance dissemination in freshwater fishfarms at the watershed level

Sandrine BARON<fnref n="1" />, Laëtitia LE DEVENDEC<fnref n="1" />, Emeline LARVOR<fnref n="1" />, Eric JOUY<fnref n="1" />, Aurélien TOCQUEVILLE<fnref n="2" />, Matthieu GAUME<fnref n="2" />, Pascal GALLOT<fnref n="2" />, Claudia JÄCKEL<fnref n="3" />, *Jens A. HAMMERL*<fnref n="3" /> Rodolphe THOMAS<fnref n="4" />, Sophie LE BOUQUIN<fnref n="4" />, Claire CHAUVIN<fnref n="4" />

A research project (Resist3A) aimed to investigate the AMR dissemination upstream and downstream of two trout fish farms, located respectively at the source (FF1) and at the mouth (FF2) of a river impacted by agricultural, terrestrial farming and wastewater treatment plant.

During 17 months, every two weeks, water samples were collected upstream and downstream two fish farms and biofilm samples were collected in a same fishpond per farm. Abiotic environmental parameters (pH, conductivity...) were measured too. Enumeration of *E. coli*, *Aeromonas* spp. and *Pseudomonas aeruginosa* was performed using culture methods on the 144 water samples and 72 biofilm samples collected.

Antimicrobial susceptibility testing of a selection of *E. coli* (n=142), *Aeromonas* (n=355), and *Pseudomonas aeruginosa* (n=123) isolates was performed using agar diffusion method.

The proportion of *E. coli* isolates, which were susceptible to all tested antimicrobials, was higher in water than in biofilm (92.7% vs 89%). No isolate was resistant to carbapenems or extended-spectrum beta-lactams. No multidrug-resistant isolate was detected. Only 13 isolates were resistant to one or two classes of antibiotics.

The *Aeromonas* isolates collected were collected from water samples (n=172) and from biofilm (n=183). From the source to the mouth in water sample, no decrease of susceptibility was observed for oxolinic acid, florfenicol, oxytetracycline, ceftazidime and meropenem. At the opposite, a slight reduction of susceptibility was observed for enrofloxacin. Isolates collected in the biofilm of the FF1, showed a reduced susceptibility to oxytetracycline and oxolinic acid. No reduction of susceptibility was observed among isolates collected in the biofilm of the FF2.

On the contrary, of the results observed for *E. coli*, the proportion of *Pseudomonas aeruginosa* susceptible to all the 13 antibiotics tested was higher in biofilm than in water (100% vs 74.5%).

These results confirm the importance of an approach including several compartments of the environment (water, biofilm), and three bacteria models allowed us to have a broader view of the AMR dissemination.

<footnote n="1">¹Mycoplasmosis-Bacteriology and Antimicrobial Resistance Unit, Anses, France</footnote>

<footnote n="2">²Aquaculture Service, Technical Institute for Poultry, Fish and Rabbit Farming (ITAVI), France</footnote>

<footnote n="3">³Department Biological Safety, German Federal Institute for Risk Assessment, Germany</footnote>

<footnote n="4">⁴Epidemiology, Health and Welfare Unit - Anses, France</footnote>
# Assessment of the diffusion of AMR in aquatic environments: specificity of river fish farming

Sandrine BARON<fnref n="1" />, Laëtitia LE DEVENDEC<fnref n="1" />, Claudia JÄCKEL<fnref n="2" />, Jens A. HAMMERL<fnref n="2" />, Claire CHAUVIN<fnref n="3" />, LE BOUQUIN Sophie<fnref n="3" />

Antimicrobial resistance (AMR) is now recognized as among the top 10 threats to global health, with current trends in resistant infections in humans and livestock pointing toward a potential postantibiotic era (Liguori, et al., 2022). The aquatic environment is considered as a hot spot for the dissemination of AMR, due to its direct link with the aquatic environment, the issue of monitoring AMR in aquaculture arises.

According to Liguori and collaborators (2022), a fundamental stumbling block to the advancement of AMR monitoring of water environments is a lack of agreed upon targets and standardized methods, including a lack of benchmarking and threshold data to inform evolutionary, epidemiological, and other risk modeling efforts.

AMR monitoring in aquaculture is concerned by the same problematics. Using trout fish farming as a case study, we will provide some answers to the following questions:

* Where? Fish farming depends on the quality of the water received. Sampling strategy should then include data collected upstream and downstream of the fish farming.

* What? What kind of samples water or/and biofilm or/and sediment? A sample should be easy collected and always available.

* Who? *E. coli* is a well-characterized bacterium used for the monitoring of AMR in humans and livestock (in national surveys, at the European level and elsewhere), as well as for the monitoring of water quality (as a fecal contamination indicator). Could *E. coli* be used? What alternatives can be offered?

* How? Currently AMR monitoring plans are based on culture depend methods and phenotypic criteria. Nevertheless, the use of genetic indicator or genomic approach are under progress.

## Reference

Liguori K, Keenum I, Davis BC, Calarco J, Milligan E, Harwood VJ, Pruden A. Antimicrobial Resistance Monitoring of Water Environments: A Framework for Standardized Methods and Quality Control. Environ Sci Technol. 2022 Jul 5;56(13):9149-9160. doi: 10.1021/acs.est.1c08918. Epub 2022 Jun 22. PMID: 35732277; PMCID: PMC9261269.

<footnote n="1">¹Mycoplasmosis-Bacteriology and Antimicrobial Resistance Unit, Anses, France</footnote>

<footnote n="2">²Department Biological Safety, German Federal Institute for Risk Assessment, Germany</footnote>

<footnote n="3">³Epidemiology, Health and Welfare Unit – Anses, France</footnote>
# Session 3 - Mechanisms and dissemination of antimicrobial resistance in animal and zoonotic pathogens

P48

## Orf121 carried by the IS91 insertion sequence negatively influences its mobility

Aurélien Fauconnier, Margaux Gaschet, Marie-Cécile Ploy, Cécile Pasternak

INSERM, University of Limoges, CHU Limoges, RESINFIT, UMR1092, Limoges, France

Prokaryotic insertion sequences (IS) contribute to bacterial multidrug resistance. IS91 is usually associated with virulence and antibiotic-resistance genes, but its contribution to their dissemination is unclear. IS91 belongs to an atypical IS family with a HUH transposase mediating transposition events using a rolling circle transposition mechanism1. This IS family displays two functionally distinct ends: transposition initiates at the so-called oriIS end and terminates at the terIS end. Nevertheless, terIS could not be recognized, leading to the mobilization of an adjacent DNA fragment (“one-ended transposition”)2. Unlike other members of the family, IS91 carries upstream the tnpA transposase gene, a small ORF potentially encoding a 121 amino acids polypeptide: Orf1212. The termination codon of orf121 overlaps the initiation codon of tnpA, suggesting that the expression of both genes may be translationally coupled via Termination-Reinitiation mechanism.

We investigated the role of Orf121 in the *in vivo* transposition of IS91 using a genetic system based on the mating-out procedure in *Escherichia coli*. Furthermore, we have detected two potentially functional promoters (*Porf121* and *PtnpA*) that could drive the expression of the two ORFs.

We showed that co-expressing Orf121 (*in cis* or *in trans*), in addition to the transposase gene, strongly decreased both the IS91 transposition frequency and the one-ended transposition rate; these results indicate the Orf121 polypeptide may exert a negative effect on the *in vivo* transposition of IS91 and that it may be required for accurate recognition and cleaving of the *terIS* end. Then, we mapped more than 1000 *in vivo* IS91 insertion sequences indicating that the target site specificity of IS91 does not depend on the presence or absence of Orf121. Furthermore, Orf121 does not tetranucleotide the *terIS* cleavage site. Lastly, we showed that the activity of the *Porf121* promoter is significantly higher than that of the *PtnpA* promoter.

## References:

[1] Garcillan-Barcia, M. P., and al. "Mobile DNA II." (2002): 891-904.

[2] Bernales, and al. "Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions." Molecular microbiology 33.2 (1999): 223-234.
# Resistance of *Salmonella* to heavy metals, a key element for dominance in the agro-food industry in Europe?

GRANDJEAN Hugo<fnref n="1" />, DOUARRE Pierre-Emmanuel<fnref n="1" />, ROMERO Karol<fnref n="1" />, TRAN Mai-Lan<fnref n="2" />,
MAZURAIS Clément<fnref n="3" />, JITARU Petru<fnref n="3" />, LEBLANC Jean-Charles<fnref n="1" /> and CADEL-SIX Sabrina<fnref n="1" />

For the last 50 years, the success of the epidemic clones of the *Salmonella* Typhimurium and its monophasic variant (S. 4,[5],12:i:-) (DT9, DT204, DT104 and DT193) was mostly attributed to their antimicrobial resistance profile. The recent discovery of a genetic island SGI-4 carrying resistance genes to copper, arsenic and silver and its role in the emergence of the European clone (DT193 or ST34) suggested that other factors, such as heavy metal resistance, can favorise the selection of epidemic clones enabling them to survive along the agro-food production chain until humans [1, 2]. In this study, the heavy metal resistance genes from the BacMet database were screened in 168 genomes belonging to the 11 *Salmonella* serovars prevalent in agro-food sectors in France. The minimal inhibitory concentration (MIC) and growth kinetics were performed in concentrations of copper relevant to farm environments for four strains carrying (or not) operons *pco* and *sil*, identified as coding for copper resistance in the SGI-4. The intracellular copper concentration of these strains was quantified by mass spectrometry and the expression of 18 genes coding for copper resistance was explored by reverse transcription PCR.

Within the 52 genes identified, the ones involved in copper metabolism and resitance were prevalent within the 168 genomes analyzed. Interestingly, S. Senftenberg carry 25 of the 89 SGI-4 genes with both *pco* and *sil* operons and some S. Agona strains (n=3/10) encoded *sil* cluster, confirming the mobile nature of these elements. The presence of *pco* and *sil* clusters did not affect the MIC, growth kinetics and mass spectrometry analyses in aerobiosis. However, in anaerobiosis, strains carrying *pco* and/or *sil* clusters were advantaged with both higher growth resistance rate and greater biomass production suggesting that copper resistance likely plays a role during intracellular life of *Salmonella* in the host. The use of copper as feed complement could modify the intestinal concentration of this compound in the hosts and exert selective pressure on microorganisms able of resisting its toxicity. The transcriptomic analysis confirmed the involvement of genes described and predicted as involved in copper resistance, especially *sil* operon. Finally, our results underlined that the copper resistance appears to be a key factor in the survival of *Salmonella* in the host and suggest that relationship between heavy metal resistance and antibiotics for *Salmonella* would deserves further study.

## References

[1] Branchu, P.; Charity, O. J.; Bawn, M.; Thilliez, G.; Dallman, T. J.; Petrovska, L.; Kingsley, R. (2019) *Front Microbiol*, *10*, 1118.

[2] Cadel-Six, S.; Cherchame, E.; Douarre, P. E.; Tang, Y.; Felten, A.; Barbet, P.; Litrup, E.; Banerji, S.; Simon, S.; Pasquali, F.; Gourmelon, M.; Mensah, N.; Borowiak, M.; Mistou, M. Y.; Petrovska, L. (2021). *Front Microbiol*, *12*, 651124.

<footnote n="1">¹ANSES, Laboratory for Food Safety, Salmonella and Listeria Unit, Maisons-Alfort, France</footnote>

<footnote n="2">²ANSES, Laboratory for Food Safety, IdentyPath Genomics Platform, Maisons-Alfort, France</footnote>

<footnote n="3">³ANSES, Laboratory for Food Safety, Trace Metals and Minerals Unit, Maisons-Alfort, France</footnote>
# Effects of subinhibitory vancomycin and gentamycin concentrations on biofilm formation by *Staphylococcus aureus*

Aniba RAFIK<fnref n="1" />,<fnref n="2" />*, Barguigua ABOUDDIHAJ<fnref n="1" />, Dihmane ASMAA<fnref n="1" />, Raqrqa HABIBA<fnref n="1" />, Ressmi AMINA<fnref n="1" />, Nayme KAOTAR<fnref n="2" />, Timinouni MOHAMMED<fnref n="3" />

**Objectives:** To examine the effect of sub-inhibitory concentrations (sub-MIC) of antibiotics on biofilm formation by *Staphylococcus aureus* (S. aureus) clinical isolates.

**Methodology:** Twenty S. aureus isolates were investigated using conventional microbiological methods. The susceptibility of uropathogenic staphylococci to vancomycin was performed by broth microdilution following the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The biofilm-forming ability of S. aureus isolates treated and untreated with vancomycin and gentamycin sub-MICs was assessed in vitro using the tissue culture plate (TCP) method combined with a crystal violet staining assay.

**Results:** According to the tissue culture plate approach, all strains of S. aureus have shown the capacity of biofilm formation, 29% of the isolates were strong biofilm producers, 57% of the isolates were moderate biofilm producers, and 10% of the isolates were weak biofilm producers.

Different changes in the ability to form biofilms were detected in S. aureus isolates treated with vancomycin and gentamicin sub-MIC concentrations. MIC/4 concentration of vancomycin had an inhibitory effect on biofilm formation in 92.96% of isolates, while biofilm formation increased in 31.3% of isolates at the MIC-16 concentration. MIC/4 and MIC/8 concentrations of gentamycin had inducing effects on the rate of biofilm formation in 54.55% and 83.33% of isolates, respectively.

**Conclusion:** The findings of this research demonstrated that the reduction and induction effects on biofilm formation ability are antibiotic-dependent and even vary from strain to strain.

## References

1. Chen J, Zhou H, Huang J, Zhang R, Rao X. Virulence alterations in staphylococcus aureus upon treatment with the sub-inhibitory concentrations of antibiotics. Journal of Advanced Research. 2021;31:165-175.

2. Rasigade JP, Moulay A, Lhoste Y, Tristan A, Bes M, Vandenesch F, et al. Impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by Staphylococcus aureus. BMC Microbiology. 2011;11(1):263.

3. Ferrer MD, Rodriguez JC, Álvarez L, Artacho A, Royo G, Mira A. Effect of antibiotics on biofilm inhibition and induction measured by real-time cell analysis. J Appl Microbiol. 2017;122(3):640-650.

4. Peng Q, Tang X, Dong W, Sun N, Yuan W. A Review of Biofilm Formation of Staphylococcus aureus and Its Regulation Mechanism. Antibiotics. 2023;12(1):12.

5. Belbase A, Pant ND, Nepal K, Neupane B, Baidhya R, Baidya R, et al. Antibiotic resistance and biofilm production among the strains of Staphylococcus aureus isolated from pus/wound swab samples in a tertiary care hospital in Nepal. Ann Clin Microbiol Antimicrob. 2017;16:15.

<footnote n="1">¹Team of Biotechnology and Sustainable Development of Natural Resources, Polydisciplinary Faculty, Sultan Moulay Slimane University, Beni Mellal, Morocco.</footnote>

<footnote n="2">²Molecular Bacteriology Laboratory, Pasteur Institute of Morocco, Casablanca, Morocco.</footnote>

<footnote n="3">³Biotechnology and bioinformatic laboratory, School of Advanced Studies in Biotechnology and Health, Casablanca, Morocco.</footnote>
# KSA-1, a new Ambler class A extended-spectrum β-lactamase from a plant bacterial enterobacterial species

Claudine Fournier<fnref n="1" />,²³, Patrice Nordmann<fnref n="1" />,<fnref n="3" />,<fnref n="4" />, Jose-Manuel Ortiz de la Rosa<fnref n="1" />, Ayda Kusaksizoglu<fnref n="1" />, Laurent Poirel<fnref n="1" />,<fnref n="3" />

Several bacterial species belonging to the *Gammaproteobacteria* possess intrinsic class A β-lactamase genes in their chromosome that may represent sources of further dissemination and acquisition in other Gram-negative species.

Here we characterized the KSA-1 class A β-lactamase which gene was identified from the chromosome of an environmental *Enterobacterales*, namely *Kosakonia sacchari* (formerly *Enterobacter sacchari*), recently identified as a progenitor of an MCR-like colistin resistance determinant.

## Methods

In-silico analysis using the GenBank database identified a class A β-lactamase gene in the chromosome of *Kosakonia sacchari* SP1 (GenBank accession no. WP_065368351). The corresponding protein KSA-1 shared 63% amino-acid identity with intrinsic CKO-1 from *Citrobacter koseri* and 53% with TEM-1. The *bla*<sub>KSA-1</sub> was amplified, cloned into plasmid pUCp24 and expressed in *E. coli* TOP10. MICs and kinetic parameters were obtained from the purified enzyme.

## Results

*K. sacchari* SP1 was isolated from a stem of sugar cane cultivar grown in China. Once produced in *E. coli*, KSA-1 showed a typical ESBL resistance pattern with high level resistance to penicillins and temocillin, with a significative reduction of the MICs value in presence of class A β-lactamase inhibitors (clavulanic acid, tazobactam and avibactam). The strain was resistant to cefoxitin, ceftazidime, aztreonam, but remained susceptible to cefotaxime.

Kinetic assays were performed using a purified extract of KSA-1, showed a high hydrolysis rate for benzylpenicillin and piperacillin. Determination of inhibitory constants showed IC₅₀ values of 2.2, 3 and 1.8 nM for clavulanic acid, tazobactam and avibactam, respectively. Analysis of sequences surrounding the *bla*<sub>KSA-1</sub> gene did not reveal any mobile element that could have been involved in the acquisition of this β-lactamase gene.

## Conclusion

KSA-1 was characterized as a class A extended spectrum β-lactamase (ESBL), distantly related to known extended-spectrum β-lactamases, with a high activity against temocillin. The *bla*<sub>KSA-1</sub> gene was considered as intrinsic to the species, that latter being therefore a putative progenitor of an ESBL encoding gene.

<footnote n="1">¹Medical and Molecular Microbiology Unit, University of Fribourg, Fribourg, Switzerland,</footnote>

<footnote n="2">²Laboratory of Clinical Microbiology, Hôpital Cantonal fribourgeois, Fribourg, Switzerland,</footnote>

<footnote n="3">³Swiss National Center for Emerging Antibiotic Resistance, University of Fribourg, Fribourg, Switzerland,</footnote>

<footnote n="4">⁴University of Lausanne and University Hospital Centre, Lausanne, Switzerland</footnote>
<figure><img src="image_50.png" /></figure>

# Hidden antimicrobial resistances in *Vibrio parahaemolyticus*: Environmental bacteria as sources or vehicles for the spread of plasmid encoded clinically important antimicrobial resistances

Jens Andre HAMMERL<fnref n="1" />,<fnref n="2" />

Antimicrobial resistance (AMR) is on a rise and challenges global One Health increasingly. The emergence of different antimicrobial-/biocide resistances in the individual One Health compartments (environment, animals and humans) is usually associated with an adaption of the bacteria against prevailing selection pressures (antimicrobials, residues or biocides). Mobile genetic elements (MGEs) carrying transmissible resistance determinants are common and are widely spread among bacteria. Because of their localization on plasmids, bacteriophages and insertion sequences, they can also be transmitted between bacteria by different mechanisms during horizontal gene transfer. Investigation and monitoring on the emergence of transmissible resistances is important, but sometimes their phenotypic development is masked by their hosts. Recently, a *V. parahaemolyticus* isolate was notified to carry a carbapenemase-producing plasmid, which only lead to slightly increases MIC values for carbapenems in *Vibrio* spp. Nevertheless, the location of the resistances on a self-transmissible plasmids results to a high resistance phenotype against different carbapenems after natural transmission into a broad range of *Enterobacteriaceae* isolates. The properties and the genome of the plasmid associated with this hidden resistance phenotype as well as the genotypic and phenotypic features of the *V. parahaemolyticus* isolate will be presented and discussed. The data clearly showed that some bacteria can acquire and mask resistance plasmids, which were further spread to clinically relevant genera associated with severe nosocomial infection in human.

<footnote n="1">¹Department Biological Safety, Unit Diagnostics, Pathogen Characterisation and Food Borne Parasites, Department Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany</footnote>

<footnote n="2">²Landesuntersuchungsamt Bremen, Referat 20 – Mikrobiologie, Bremen, Germany</footnote>
*Staphylococcus aureus* plasmids of animal origin, a vector of antibiotic resistance

Rachel CONTARIN¹,², Emilie DORDET FRISONI¹ and Marisa HAENNI²

¹INTHERES, Université de Toulouse, INRAE, ENVT, Toulouse, France

²Unité Antibiorésistance et Virulence Bactériennes, Agence Nationale de Sécurité Sanitaire (ANSES) - Université de Lyon, Lyon, France

*Staphylococcus aureus* is a human and animal opportunistic pathogen that has demonstrated the capacity to develop resistance to a wide range of antibiotics. Its ability to acquire antibiotic resistance genes (ARGs) is closely associated with the large proportion of mobile genetic elements (MGEs) present in its genome (between 15 to 20%) and its capacity to exchange genetic material with its environment1. We recently conducted an *in silico* analysis of MGEs and their linked ARGs on a dataset of 1257 S. aureus genomes isolated from animals, obtained from the NCBI database. As this analysis suggested the central role of plasmids in the dissemination of antibiotic resistance, our aim here was to provide further evidence from the field by characterizing 340 S. aureus isolates collected from different animal species by the Resapath network2 in France from 2010 to 2021. Their antibiotic resistance genes were detected by PCR, their plasmid content was identified by PFGE and finally the plasmid/resistance gene associations were established with Southern Blot.

Data showed that almost all isolates (304/340, 89.4%) carried at least one resistance gene, of which 60% contained *blaZ* gene (beta-lactam resistance), 55% *tet(M)* (tetracycline resistance) and 40% *aac(6')-aph(2'')* (aminoglycoside resistance). The diversity of ARGs in these S. aureus field isolates was similar to that found in our *in silico* analyses. A large proportion of isolates (55%) contained at least one plasmid, with an average of three plasmids per isolate. The plasmid sizes varied between 2kb and 160kb, but isolates mostly carried plasmids whose size was either 25kb-35kb (27%) or 4.3kb (21%). Southern Blot analyses showed that 40% of S. aureus plasmids carried at least one of the four ARGs *aadD*, *erm(C)*, *str*, or *tet(K)*. Contrarily to *in silico* data, *tet(M)* was only found localized on the chromosome. A large diversity of ARG/plasmid associations (42 different profiles) was observed. Each gene was found to be more commonly associated with a specific size of plasmid: *str*, *tet(K)* and *erm(C)* were located on small (<5kb) plasmids while *aadD* was found either associated with medium-size plasmids (25kb) or associated with *erm(C)* on a small 9kb plasmid.

This study confirmed that plasmids play an important role in antimicrobial resistance in S. aureus, as suggested by *in silico* analyses. Short- and long-read sequencing are ongoing on a subset of isolates carrying representative patterns of plasmids in order to refine their characterization. Further research is needed to uncover the mechanisms behind the wide dissemination of these plasmids and to explain their epidemic success.

References

[1] Alibayov, B., Baba-Moussa, L., Sina, H., Zdeňková, K., & Demnerová, K. (2014). Molecular Biology Reports, 41(8), 5005-5018

[2] Resapath: Surveillance network of antimicrobial resistance in pathogenic bacteria from animal origin.
https://resapath.anses.fr/