# Genomic epidemiology, resistome, virulome and plasmidome analysis of extended spectrum beta-lactamases-producing Escherichia coli and Klebsiella pneumoniae from migratory birds

Sandra Martínez Álvarez, Ursula Höfle, Pierre Châtre, Teresa

Cardona-Cabrera, Pauline François, Myriam Zarazaga, Jean-Yves Madec,

Marisa Haenni, Carmen Torres

► To cite this version:

Sandra Martínez Álvarez, Ursula Höfle, Pierre Châtre, Teresa Cardona-Cabrera, Pauline François, et al.. Genomic epidemiology, resistome, virulome and plasmidome analysis of extended spectrum beta-lactamases-producing Escherichia coli and Klebsiella pneumoniae from migratory birds. 33rd European Congress of Clinical Microbiology & Infectious Diseases (ECCMID), Apr 2023, Copenhagen, Denmark. 10.13039/501100011033 . anses-04276206

HAL Id: anses-04276206

https://anses.hal.science/anses-04276206

Submitted on 8 Nov 2023

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## INTRODUCTION AND OBJECTIVES

The presence of ESBL-producing Enterobacterales is actively monitored in humans and animals, but the role of migratory birds as sentinels or reservoirs of resistance genes is much less understood. This study aimed at detecting and deeply characterizing ESBL-producing Enterobacterales isolated from white storks feeding on specific landfills in Spain.

## MATERIALS AND METHODS

Faecal samples (n=211) of adult white storks were sampled at two landfills in Spain (Figure 1). A total of 72 ESBL-producing *E. coli* (n=60; 28.4%) and *K. pneumoniae* (n=12; 5.2%,) isolates were recovered on selective media and included in this study. The ESBL-positive isolates were sequenced using NovaSeq6000 technology (Illumina). Resistance and virulence genes, sequence types (ST) and replicon patterns were determined using tools from the Centre for Genomic Epidemiology (CGE). Localization of ESBL genes was performed using southern blots on S1-PFGE gels.

<figure><img src="image_3.png" /><figcaption>**Figure 4.** Accessory genome profiling obtained by S1-PFGE for ESBL and/or pAmpC-producing *E. coli*/*K. pneumoniae*.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Location of the two sampling points (2 landfills) carried out during the summer of 2021 in South-central Spain</figcaption></figure>

## RESULTS

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* A large diversity of genetic lineages (*E. coli*, n=26; *K. pneumoniae*, n=4) were identified, including human-associated high-risk (*E. coli* ST131, ST58 or ST69; *K. pneumoniae* ST307) (Figure 2, 3; respectively) and more ubiquitous clones (*E. coli* ST10, ST155, ST117, ST617) (Figure 2).
* WGS revealed the presence of a wide variety of ESBL-genes (bla<sub>CTX-M-1</sub>, bla<sub>CTX-M-15</sub>, bla<sub>CTX-M-55</sub>, bla<sub>CTX-M-32</sub>, bla<sub>CTX-M-14</sub>, bla<sub>CTX-M-65</sub>, bla<sub>CTX-M-27</sub>, bla<sub>SHV-12</sub>), and the sporadic occurrence of the pAmpCs bla<sub>CMY-2</sub>, bla<sub>DHA-1</sub> genes (Figure 2, 3). All ESBL *K. pneumoniae* harboured bla<sub>CTX-M-15</sub> (Figure 3).
* Plasmid analysis revealed that 71.8% of the isolates carried IncF (IncFA, IncFB, IncFII) replicons, while IncY, IncI1, IncX1 and ColV replicons were also frequently identified (Figure 2, 3, 4).
* ESBL-producers also displayed virulence determinants, since two atypical enteropathogenic *E. coli* carrying *eae* and *tir* genes, as well as 28 extra-intestinal pathogenic *E. coli* carrying *pap*, *afa*, *iuc* and/or *iutA* genes, were observed (Figure 2).
* Moreover, in eight ESBL-positive isolates, three of four genes (*chuA*, *fyuA*, *vat* and *yfcV*) associated with uropathogenic *E. coli* were detected simultaneously (Figure 2).

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<figure><img src="image_8.png" /><figcaption>**Figure 2.** Maximum-likelihood phylogeny of ESBL/pAmpC *E. coli* producing isolates from storks. Branch lengths represent the number of nucleotide substitutions per site (scale, 100000 substitutions per site). Metadata columns include sequence types shown with coloured bars (each colour correspond to one ST), phylogroups and geographical origin of isolates (landfill 1/2). ESBL/AmpC genes as well as plasmid content, antimicrobial resistance genes and virulome are indicated with rectangles, triangles or circles denoting the presence (filled shapes) or absence (empty shapes) of the gene.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 3.** Maximum-likelihood phylogeny of ESBL *K. pneumoniae* producing isolates from white storks. The phylogenetic tree was constructed based on nucleotide sequence alignments of 4214 core genes. Branch lengths represent the number of nucleotide substitutions per site (scale, 100000 substitutions per site). Metadata columns include sequence types shown with coloured bars (each colour correspond to one ST) and geographical origin of isolates (landfill 1/2). ESBL genes, other AMR genes as well as plasmid content are indicated with rectangles denoting the presence (filled shapes) or absence (empty shapes) of the gene.</figcaption></figure>

## CONCLUSIONS

This study demonstrates that storks are an important reservoir of resistance and virulence genes with high clinical impact. The wide variety of STs and resistance genes identified suggests that these birds can acquire and accumulate resistant bacteria, including human high-risk clones, along their migratory routes. These results highlight the importance of unravelling the transmission routes of antibiotic resistance, and the resulting risks, in a One Health perspective.

## ACKNOWLEDGEMENTS

S. M-A has a predoctoral fellowship FPI from Gobierno de La Rioja. This work was financed in part by MCIN/AEI/10.13039/501100011033 of Spain and also by project SBPLY/19/180501/000325 of the regional government of Castilla - La Mancha by the European Union's funds for regional development (FEDER).