# Analysis of ESBL genes-carrying plasmids in Escherichia coli from livestock, wildlife and food sources

Sandra Martínez-Álvarez, Pierre Châtre, Susana Sanz, Carmen Olarte, Myriam Zarazaga, Jean-Yves Madec, Carmen Torres, Marisa Haenni

► To cite this version:

Sandra Martínez-Álvarez, Pierre Châtre, Susana Sanz, Carmen Olarte, Myriam Zarazaga, et al. Analysis of ESBL genes-carrying plasmids in Escherichia coli from livestock, wildlife and food sources. 33rd European Congress of Clinical Microbiology & Infectious Diseases (ECCMID), Apr 2023, Copenhagen, Denmark. 10.13039/501100011033. anses-04276203

HAL Id: anses-04276203

https://anses.hal.science/anses-04276203

Submitted on 8 Nov 2023

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INTRODUCTION AND OBJECTIVES

In *E. coli*, resistance to 3th-4th generation cephalosporins associated with the production of extended-spectrum β-lactamases (ESBLs) has become an increasing health problem. In contrast to ESBL-producing *E. coli* of clinical origin, less information is available from isolates of environmental, food, livestock or wildlife origin. The goal of this study was to unravel some of the transmission routes of ESBL in non-human settings.

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MATERIALS AND METHODS

**Sampling:** Thirty *E. coli* isolates from different sources: a) swine farms: slurry (n=7) and nasal cavities (n=5); b) broiler farm (n=8): air (1) and manure (7); c) migratory birds (n=5) and d) meat (n=5)) from Spain were selected.

**Clonal characterization of bla<sub>ESBL</sub> carrying isolates:** The genetic diversity of ESBL-positive *E. coli* isolates was previously analyzed using the triplex PCR-based phylogenetic typing (chuA, yjaA, TSPE4.C2) and multilocus-sequence typing (MLST) to select the ESBL *E. coli* for the fully characterization by whole genome sequencing.

**Plasmids characterization:** bla<sub>ESBL</sub>-carrying plasmids were examined by PCR-based replicon typing, S1-PFGE, conjugation experiments and were fully characterized by long-read-sequencing (Oxford Nanopore) as well as by short-read sequencing NovaSeq600 Technology (Illumina).

**Antimicrobial resistance genes, integrons and bla<sub>ESBL</sub> genetic environment:** Co-located resistance genes, integrons and bla<sub>ESBL</sub> genetic environments were investigated by WGS and using bioinformatic tools.

<figure><img src="image_5.png" /><figcaption>**Figure 2.** PFGE-S1 and hybridizations by Southern Blot for IncI1 and IncFIB</figcaption></figure>

RESULTS

* The genetic backgrounds of the 30 ESBL-producing *E. coli* isolates were diverse (Figure 1), with 17 STs identified. Only ST117/B2 and ST10/A were found in different contexts but did not always present the same ESBL gene: ST117/B2 was identified in four samples from broiler farms and chicken meat sharing a closely related MLVA pattern and in one sample originating from swine, while ST10/A was found in one chicken meat and one swine sample.

* The ESBL phenotype was due to the presence of the *bla*<sub>SHV-12</sub> (n=15), *bla*<sub>CTX-M-14</sub> (n=8), *bla*<sub>CTX-M-1</sub> (n=3), *bla*<sub>CTX-M-32</sub> (n=2), *bla*<sub>CTX-M-65</sub> (n=1) and *bla*<sub>TEM-52</sub> (n=1) genes, among which 28 were horizontally transferable by conjugation (Table 1).

* Six ESBL genes from three different STs were carried by 220-230 kb IncHI2 plasmids belonging to pST5 (in 4/5 *bla*<sub>SHV-12</sub> from broiler farms) and pST5 (in one *bla*<sub>CTX-M-65</sub> from livestock and in one *bla*<sub>SHV-12</sub> from chicken meat). IncHI2 plasmids carrying *bla*<sub>SHV-12</sub> were found in the same clones from different sources (broiler and meat) and showed identical sizes (220 kb) (Figure 2).

* Eleven ESBL genes from seven different genetic backgrounds (Figure 1) were located on 80-110 kb IncI1 plasmids: three *bla*<sub>SHV-12</sub>, two *bla*<sub>CTX-M-1</sub> and six *bla*<sub>CTX-M-14</sub>. Isolates from chicken meat or storks presented the pST3, while isolates from livestock presented the pST80.

* All the IncHI2 *bla*<sub>SHV-12</sub> carrying plasmids from the broiler farm carried the same unusual class 1 integron (*sat*-psp-aadA2-cmlA-aadA1) and another unusual class 1 integron was detected in one isolate from chicken meat (*bla*<sub>SHV-12</sub>-positive; *estX*-psp-aadA2-cmlA-aadA1). This integron was also detected in the IncF plasmid carrying *bla*<sub>SHV-12</sub>. Other unusual gene-cassette arrangements were detected associated with *bla*<sub>CTX-M-14/32/65</sub> (*estX*-IS1294-aadA2-cmlA1-aadA1) (Table 1).

<figure><img src="image_6.png" /><figcaption>**Figure 1.** Representation *bla*<sub>SHV-12</sub>, *bla*<sub>CTX-M-1</sub>, *bla*<sub>CTX-M-14</sub>, *bla*<sub>CTX-M-32</sub> and *bla*<sub>CTX-M-65</sub> genetic environment. IR: Inverted Repeats.</figcaption></figure>

<table><caption>**Table 1.** Characteristics of *bla*<sub>SHV-12</sub>-carrying plasmids in studied *E. coli* collection.</caption><thead><tr><th rowspan="2">E. coli strain (source)</th><th rowspan="2">Clones (ST/phylogroup)</th><th rowspan="2">Replicons</th><th colspan="5">bla<sub>ESBL</sub> carrying plasmids</th><th rowspan="2">Other co-located resistance genes</th></tr><tr><th>ESBL gene</th><th>Replicon type</th><th>pMLST/ FAB formula</th><th>Size (kb)</th><th>Conjugation frequency</th><th>Genetic environment of bla<sub>ESBL</sub></th></tr></thead><tbody><tr><td>X2583 (BF, A)</td><td>770/E</td><td>FII, FIB</td><td>bla<sub>SHV-12</sub></td><td>FIB</td><td>F64:A:-B27</td><td>70</td><td>5 x 10<sup>-6</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>tet(A)</td></tr><tr><td>X2630 (BF, M)</td><td>770/E</td><td>FII, FIB</td><td>bla<sub>SHV-12</sub></td><td>FIB</td><td>F64:A:-B27</td><td>70</td><td>1,4 x 10<sup>-5</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>tet(A)</td></tr><tr><td>X2639 (BF, M)</td><td>770/E</td><td>FII, FIB</td><td>bla<sub>SHV-12</sub></td><td>FIB</td><td>F64:A:-B27</td><td>70</td><td>2 x 10<sup>-6</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>tet(A)</td></tr><tr><td>X2632 (BF, M)</td><td>68/E</td><td>HI2</td><td>bla<sub>SHV-12</sub></td><td>HI2</td><td>NHF</td><td>110</td><td>4,4 x 10<sup>-5</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>-</td></tr><tr><td>X2631 (BF, M)</td><td>117/B2</td><td>B/O/K/Z, FIB, HI2</td><td>bla<sub>SHV-12</sub></td><td>HI2</td><td>ST5</td><td>230</td><td>1,1 x 10<sup>-6</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>tet(A)</td></tr><tr><td>X2641 (BF, M)</td><td>117/B2</td><td>B/O/K/Z, FIB, HI2</td><td>bla<sub>SHV-12</sub></td><td>HI2</td><td>ST5</td><td>230</td><td>1,3 x 10<sup>-6</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>-</td></tr><tr><td>X2643 (BF, M)</td><td>10992/A</td><td>FIB, HI2</td><td>bla<sub>SHV-12</sub></td><td>HI2</td><td>ST5</td><td>230</td><td>5,4 x 10<sup>-5</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>tet(A)</td></tr><tr><td>X2645 (BF, M)</td><td>10992/A</td><td>HI2, I1</td><td>bla<sub>SHV-12</sub></td><td>HI2</td><td>ST5</td><td>230</td><td>NC</td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>sat-psp-aadA2-cmlA-aadA1</td><td>tet(A)</td></tr><tr><td>X3078 (M)</td><td>117/B2</td><td>FIB, I1, X1</td><td>bla<sub>CTX-M-1</sub></td><td>I1</td><td>ST3 (CC3)</td><td>110</td><td>2,3 x 10<sup>-6</sup></td><td>wbuC-bla<sub>CTX-M-1</sub>-IS1380-like</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X3561 (M)</td><td>752/A</td><td>-</td><td>bla<sub>TEM-52C</sub></td><td>CHR</td><td>-</td><td>-</td><td>9,7 x 10<sup>-7</sup></td><td>Tn2-bla<sub>TEM-52</sub></td><td>-</td><td>-</td><td>-</td></tr><tr><td>X3556 (M)</td><td>117/B2</td><td>B/O/K/Z, FIB, HI2</td><td>bla<sub>SHV-12</sub></td><td>HI2</td><td>ST5</td><td>220</td><td>5 x 10<sup>-3</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>estX-psp-aadA2-cmlA-aadA1</td><td>-</td></tr><tr><td>X3081 (M)</td><td>223/B1</td><td>I1</td><td>bla<sub>SHV-12</sub></td><td>I1</td><td>ST3 (CC3)</td><td>100</td><td>1,7 x 10<sup>-2</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X3274 (M)</td><td>10/A</td><td>X3</td><td>bla<sub>SHV-12</sub></td><td>X3</td><td>-</td><td>45</td><td>1,1 x 10<sup>-3</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C10533 (SF, S)</td><td>542/A</td><td>FII, I1, X1, X4, Y</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>2,5 x 10<sup>-7</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C10524 (SF, S)</td><td>453/B1</td><td>FII, FIA, FIB</td><td>bla<sub>CTX-M-14</sub></td><td>FII, FIA-FIB</td><td>F31:A4:B58</td><td>180</td><td>NC</td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>+/+</td><td>dfrA17-aadA5</td><td>-</td></tr><tr><td>C10534 (SF, S)</td><td>4038/B1</td><td>I1, R, X1, X4</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>1,6 x 10<sup>-3</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C10535 (SF, S)</td><td>4038/B1</td><td>I1, R, X1, X4</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>1,3 x 10<sup>-4</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C10538 (SF, S)</td><td>4038/B2</td><td>I1, R, X1, X4</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>9 x 10<sup>-7</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C10540 (SF, S)</td><td>4038/B3</td><td>I1, R, X1, X4</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>2,4 x 10<sup>-3</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X60 (SF, S)</td><td>4038/B4</td><td>I1, R, X1, X4</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>4,6 x 10<sup>-3</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X5084 (SF, N)</td><td>48/A</td><td>FII, I1, X1</td><td>bla<sub>CTX-M-32</sub></td><td>I1</td><td>ST36 (CC3)</td><td>80</td><td>6,1 x 10<sup>-2</sup></td><td>wbuC-bla<sub>CTX-M-32</sub>-ISKpn26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X5093 (SF, N)</td><td>117/F</td><td>FIB, I1, N, R</td><td>bla<sub>CTX-M-14</sub></td><td>I1</td><td>ST80 (CC31)</td><td>100</td><td>2,2 x 10<sup>-6</sup></td><td>IS930-bla<sub>CTX-M-14</sub>-IS26</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X5159 (SF, N)</td><td>847/B1</td><td>FIB, I1, Y</td><td>bla<sub>SHV-12</sub></td><td>I1</td><td>ST3 (CC3)</td><td>100</td><td>2,3 x 10<sup>-6</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X5388 (SF, N)</td><td>5440/B1</td><td>X1, FIC, FIB</td><td>bla<sub>CTX-M-1</sub></td><td>X1</td><td>-</td><td>45</td><td>2,7 x 10<sup>-6</sup></td><td>IS26-bla<sub>CTX-M-1</sub>-mrx</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X5726 (SF, N)</td><td>10/B1</td><td>FIB, FIC, HI2, I1, Y</td><td>bla<sub>CTX-M-65</sub></td><td>HI2</td><td>ST3</td><td>230</td><td>3,6 x 10<sup>-4</sup></td><td>IS930-bla<sub>CTX-M-65</sub>-ISEcp1</td><td>+/-</td><td>-</td><td>-</td></tr><tr><td>X5229 (MB, F)</td><td>58/B1</td><td>FIB, FII, I1, Q1, Y</td><td>bla<sub>SHV-12</sub></td><td>I1</td><td>ST3 (CC3)</td><td>100</td><td>2,8 x 10<sup>-5</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>-</td><td>-</td><td>-</td></tr><tr><td>X5239 (MB, F)</td><td>155/B1</td><td>FIB, X1</td><td>bla<sub>CTX-M-32</sub></td><td>FIB</td><td>F4:A:-B-</td><td>90</td><td>3,3 x 10<sup>-5</sup></td><td>wbuC-bla<sub>CTX-M-32</sub>-ISKpn26</td><td>+/-</td><td>aadA22</td><td>bla<sub>TEM-1A</sub></td></tr><tr><td>X5279 (MB, F)</td><td>38/D</td><td>FIB, FII, X1, Y</td><td>bla<sub>SHV-12</sub></td><td>FIB-FII</td><td>F24:A:-B58</td><td>90</td><td>5,1 x 10<sup>-3</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>estX-IS1294-aadA2-cmlA1-aadA1</td><td>-</td></tr><tr><td>X5282 (MB, F)</td><td>38/D</td><td>FIB, FII, X1, Y</td><td>bla<sub>SHV-12</sub></td><td>FIB-FII</td><td>F24:A:-B58</td><td>90</td><td>1,8 x 10<sup>-6</sup></td><td>IS26-bla<sub>SHV-12</sub>-deoR</td><td>+/-</td><td>estX-IS1294-aadA2-cmlA1-aadA1</td><td>-</td></tr><tr><td>X5854 (MB, F)</td><td>162/B1</td><td>FIB, I1</td><td>bla<sub>CTX-M-1</sub></td><td>I1</td><td>ST3 (CC3)</td><td>110</td><td>4,2 x 10<sup>-4</sup></td><td>wbuC-bla<sub>CTX-M-1</sub>-ISEcp1</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

BF, A: broiler farm air; BF, M: broiler farm manure; M: chicken meat; SF, S: swine farm, slurry; SF, N: swine farm, nasal; MB, F: migratory birds faeces NHF, No hit found; CHR, Chromosomic location; NC, Not conjugative

CONCLUSIONS

* ✓ Incl1 plasmids were predominant carrying *bla*<sub>CTX-M-14</sub> and *bla*<sub>CTX-M-1</sub>, while IncHI2 were predominant for *bla*<sub>CTX-M-65</sub> and also detected carrying *bla*<sub>SHV-12</sub>.

* ✓ Some isolates from different sources and geographical origin were clonally related or showed similar or even indistinguishable plasmids, which suggests that both clonal and horizontal plasmid transfer facilitates the spread of *bla*<sub>ESBL</sub> genes.

* ✓ The high prevalence of class 1 integrons linked to the *sul*3 gene and carrying large cassette arrays in IncHI2 and IncF plasmids may play an important role in the persistence of these ESBL isolates.

* ✓ This study demonstrate that the only plasmid for which we evidenced cross-sector transmission is Incl1/pST3 mainly carrying *bla*<sub>SHV-12</sub>.

ACKNOWLEDGEMENTS