# ESBL- and mcr-1-producing Escherichia coli in veal calves in Tunisia

Mariem Saidani, Lilia Messadi, Ela Sahmin, Sana Zouaoui, Alya Soudani, Monia Daaloul-Jedidi, Aymen Mamlouk, Faten Ben Chehida, Jean-Yves Madec, Marisa Haenni

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Mariem Saidani, Lilia Messadi, Ela Sahmin, Sana Zouaoui, Alya Soudani, et al., ESBL- and mcr-1-producing Escherichia coli in veal calves in Tunisia. Journal of Global Antimicrobial Resistance, 2019, 19, pp.104-105. 10.1016/j.jgar.2019.08.009. anses-03989543

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Submitted on 14 Feb 2023

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Letter to the Editor

ESBL- and mcr-1-producing Escherichia coli in veal calves in Tunisia

<figure><img src="image_3.png" /></figure>

Sir,

Antimicrobial resistance has become a major public-health concern since resistance to antibiotics such as extended-spectrum cephalosporins (ESCs), carbapenems and colistin have disseminated widely in humans, animals and the environment. In food-producing animals, particular attention has been paid in recent years to plasmid-mediated resistance to ESCs and colistin. Even resistance to carbapenems, which are prohibited antimicrobials in animals, can occur as exemplified by recurrent reports of VIM-1 in the agri-food sector in Germany. Numerous studies have also been performed in chicken and chicken meat, with high rates of ESC-resistant (ESC-R) *Escherichia coli* found worldwide. In contrast, antimicrobial resistance in veal calves is still under-reported, although French and Dutch studies showed a high proportion (20.4–39.0%) of healthy calves carrying ESC-R *E. coli* in slaughterhouses, sometimes associated with the *mcr-1* gene [1,2]. Thus, the aim of the current study was to characterise *E. coli* resistant to ESCs, carbapenems and colistin in faecal samples from veal calves in Tunisia, a sector that has never been explored in this country.

A total of 219 faecal samples were collected between January 2016 and September 2017 from diarrhoeic (n = 160) and healthy (n = 59) calves on 104 different dairy farms. A total of 182 *E. coli* isolates were recovered on MacConkey agar (dominant flora representing major clones), whilst 14 ESC-R *E. coli* (6.4%; 14/219) were recovered on MacConkey agar supplemented with cefotaxime (subdominant flora representing minor clones). In the dominant flora, isolates were frequently resistant to streptomycin (78.0%), tetracycline (72.0%) and amoxicillin (52.7%) but not to carbapenems (Supplementary Table S1). This is in line with the antibiotics most commonly used to treat veal calves in Tunisia and also with a study in France where these resistances were the most frequent [2]. No statistically significant differences were observed in the proportions of resistance in diarrhoeic versus healthy calves (P ≥ 0.05).

All 14 ESC-R *E. coli* were confirmed as extended-spectrum β-lactamase (ESBL)-producers by the double-disk synergy test, among which 6 were also recovered from the dominant flora (Table 1). Only one ESBL-positive isolate was recovered from a healthy animal, highlighting the very low ESBL colonisation rate compared with France (20.4% in 2013) and the Netherlands (39.0% in 2010) [1,2]. Nevertheless, attention should be paid to high-density shedders (i.e. animals carrying ESBL-positive *E. coli* in the dominant flora) since they have been linked to a higher risk of contamination of the food chain after slaughter.

Several CTX-M enzymes were detected, with CTX-M-15 being the most frequent (7/14). Southern blotting on S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) gels using *Xba*I or *I-Ceu*I enzymes revealed that IncF-type plasmids were the most widespread plasmids carrying *bla*<sub>CTX-M</sub> genes (Table 1). Seven different sequence types (STs) were identified, and clonal *E. coli* isolates belonging to ST744, ST162 and ST1722 were identified in unrelated farms located approximately 40 km apart (Supplementary Fig. S1). Only ST1722, which was found in four animals from three different farms, presented a chromosomal insertion of *bla*<sub>CTX-M-15</sub>. Such a chromosomal insertion in the same ST has already been found in migratory birds from Pakistan, suggesting that certain genetic backgrounds may be more prone to chromosomal integration [3]. Five isolates displaying different STs were collected from farm #7, a large state farm that was struggling with numerous cases of diarrhoea at the time of sampling. Of note, the human epidemic clone ST131 was identified in one isolate that carried the *bla*<sub>CTX-M-14</sub> gene on a widespread IncF1:A2:B20 plasmid. To date, ESBL-producing ST131 *E. coli* isolates outside the human sector remain sporadic and are rarely recovered from food-producing animals. However, whether this *E. coli* isolate was transferred from a human to calves is uncertain at this stage of investigation.

The *mcr-1* gene was detected in 3 ESBL-producing and 8 non-ESBL-producing *E. coli* isolates, meaning that 5.0% (11/219) of the samples harboured a colistin-resistant *E. coli* (Table 1). No other gene of the *mcr* family was found. The *mcr-1* gene was systematically carried on an IncHI2 plasmid and was co-localised with the *bla*<sub>CTX-M</sub> gene in ESBL-producing isolates. This *bla*<sub>CTX-M/mcr-1</sub> IncHI2 plasmid has frequently been reported in animals, including in Tunisia where it has been described in poultry production and in dromedaries [4,5]. This plasmid has thus disseminated in the animal sector in Tunisia, although not in high proportions (4.6% in veal calves, 0.4% in dromedaries, 10.2% of ESBL-producing *E. coli* in chicken). Nine *mcr-1*-positive isolates originated again from farm #7 where colistin was used to treat diarrhoea, likely explaining the spread of the *mcr-1* gene in five genetic backgrounds (ST162, ST88, ST533, ST162 and ST744). Co-selection through other antibiotic treatments is also plausible given the number of associated antimicrobial resistances (Table 1).

In this study, 6.4% (14/219) of the samples presented an ESBL-producing *E. coli* and 5.0% (11/219) presented a colistin-resistant *E. coli*. Epidemic plasmids and *E. coli* clones were found, highlighting the importance both of prudent use of antibiotics and appropriate hygiene and biosecurity measures to control the spread of antimicrobial resistance in the agri-food sector in Tunisia.

Funding

This study was supported by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) and the
<table><caption>**Table 1**

Characteristics of extended-spectrum β-lactamase (ESBL)- and MCR-1-producing *Escherichia coli* isolates collected from veal calves in Tunisia.</caption><thead><tr><th>Strain<sup>a</sup></th><th>Health status</th><th>Farm</th><th>Phylogroup</th><th>ST</th><th>ESBL gene</th><th>Associated resistances</th><th>Colistin resistance gene (MIC in mg/L)</th><th>ESBL-carrying plasmid</th><th>FAB formula</th><th>Plasmid size (kb)</th></tr></thead><tbody><tr><td>BV47</td><td>DC</td><td>1</td><td>B2</td><td>131</td><td>bla<sub>CTX-M-14</sub></td><td>NAL, ENR, TET, SXT</td><td>–</td><td>IncF</td><td>F1:A2:B20</td><td>150</td></tr><tr><td>BV82</td><td>HC</td><td>2</td><td>A</td><td>167</td><td>bla<sub>CTX-M-15</sub></td><td>CHL, GEN, NAL, ENR, TET, SXT</td><td>–</td><td>IncF</td><td>F1:A1:B49</td><td>130</td></tr><tr><td>V55</td><td>DC</td><td>3</td><td>A</td><td>744</td><td>bla<sub>CTX-M-55</sub></td><td>STR, TET, CHL, FFC, SXT, NAL, ENR</td><td>–</td><td>IncF</td><td>F18:A:-B1</td><td>170</td></tr><tr><td>V56</td><td>DC</td><td>4</td><td>A</td><td>744</td><td>bla<sub>CTX-M-55</sub></td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR</td><td>–</td><td>IncF</td><td>F18:A:-B1</td><td>170</td></tr><tr><td>V61</td><td>DC</td><td>5</td><td>B1</td><td>162</td><td>bla<sub>CTX-M-1</sub></td><td>STR, TET, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (6)</td><td>IncH12</td><td>–</td><td>260</td></tr><tr><td>V62</td><td>DC</td><td>6</td><td>B1</td><td>162</td><td>bla<sub>CTX-M-1</sub></td><td>STR, TET, CHL, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>260</td></tr><tr><td>V66</td><td>DC</td><td>7</td><td>A</td><td>744</td><td>bla<sub>CTX-M-55</sub></td><td>STR, TET, CHL, FFC, SXT, NAL, ENR</td><td>–</td><td>IncF</td><td>F18:A:-B1</td><td>170</td></tr><tr><td>V73</td><td>DC</td><td>7</td><td>B1</td><td>162</td><td>bla<sub>CTX-M-1</sub></td><td>STR, TET, CHL, FFC, SXT, NAL, COL</td><td>mcr-1 (8)</td><td>IncH12</td><td>–</td><td>260</td></tr><tr><td>V75</td><td>DC</td><td>7</td><td>A</td><td>10</td><td>bla<sub>CTX-M-15</sub></td><td>STR, TET, CHL, FFC, SXT, NAL</td><td>–</td><td>IncX1</td><td>–</td><td>70</td></tr><tr><td>V76</td><td>DC</td><td>7</td><td>D</td><td>8149</td><td>bla<sub>CTX-M-15</sub></td><td>GEN, STR, TET, SXT, NAL, ENR</td><td>–</td><td>IncF</td><td>F18:A:-B30</td><td>170</td></tr><tr><td>V85</td><td>DC</td><td>7</td><td>D</td><td>1722</td><td>bla<sub>CTX-M-15</sub></td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR</td><td>–</td><td>Chromosome</td><td>–</td><td>–</td></tr><tr><td>V89</td><td>DC</td><td>8</td><td>D</td><td>1722</td><td>bla<sub>CTX-M-15</sub></td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR</td><td>–</td><td>Chromosome</td><td>–</td><td>–</td></tr><tr><td>V90</td><td>DC</td><td>8</td><td>D</td><td>1722</td><td>bla<sub>CTX-M-15</sub></td><td>STR, TET, CHL, FFC, SXT, NAL, ENR</td><td>–</td><td>Chromosome</td><td>–</td><td>–</td></tr><tr><td>V92</td><td>DC</td><td>9</td><td>D</td><td>1722</td><td>bla<sub>CTX-M-15</sub></td><td>STR, TET, CHL, SXT, NAL, ENR</td><td>–</td><td>Chromosome</td><td>–</td><td>–</td></tr><tr><td>BV6</td><td>DC</td><td>7</td><td>B1</td><td>88</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>BV15</td><td>DC</td><td>7</td><td>B1</td><td>533</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>BV87</td><td>HC</td><td>7</td><td>B1</td><td>533</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>BV95</td><td>DC</td><td>7</td><td>B1</td><td>533</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>BV96</td><td>DC</td><td>7</td><td>B1</td><td>533</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>BV118</td><td>HC</td><td>7</td><td>A</td><td>744</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>V47</td><td>DC</td><td>7</td><td>A</td><td>88</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr><tr><td>V52</td><td>DC</td><td>7</td><td>A</td><td>88</td><td>None</td><td>GEN, STR, TET, CHL, FFC, SXT, NAL, ENR, COL</td><td>mcr-1 (4)</td><td>IncH12</td><td>–</td><td>280</td></tr></tbody></table>

ST, sequence type; MIC, minimum inhibitory concentration; DC, diseased calf; HC, healthy calf; NAL, nalidixic acid; ENR, enrofloxacin; TET, tetracycline; SXT, sulfonamides/trimethoprim; CHL, chloramphenicol; GEN, gentamicin; STR, streptomycin; FFC, florfenicol; COL, colistin.

This research project 'ResAntibioVet 06.680' funded by the Ministry of Agriculture of Tunisia. MS received a fellowship from the Tunisian Ministry of Higher Education and Scientific Research.

## Competing interests

None declared.

## Ethical approval

Not required.

## Acknowledgments

The authors especially thank all of the veterinarians for their help during the sampling process. The authors also thank Estelle Saras, Pierre Châtre, Antoine Drapeau, Véronique Métayer and Sihem Chebil for their helpful assistance.

## Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jgar.2019.08.009.

## References

[1] Hordijk J, Wagenaar JA, van de Giessen A, Dierikx C, van Essen-Zandbergen A, Veldman K, et al. Increasing prevalence and diversity of ESBL/AmpC-type β-lactamase genes in *Escherichia coli* isolated from veal calves from 1997 to 2010. *J Antimicrob Chemother* 2013;68:1970–3.

[2] Gay E, Bour M, Cazeau G, Jarrige N, Martineau C, Madec J-Y, et al. Antimicrobial usages and antimicrobial resistance in commensal *Escherichia coli* from veal calves in France: evolution during the fattening process. *Front Microbiol* 2019;10:792.

[3] Mohsin M, Raza S, Schaufler K, Roschanski N, Sarwar F, Semmler T, et al. High prevalence of CTX-M-15-type ESBL-producing *E. coli* from migratory avian species in Pakistan. *Front Microbiol* 2017;8:2476.

[4] Saidani M, Messadi L, Mefteh J, Chaouechi A, Soudani A, Selmi R, et al. Various Inc-types plasmids and lineages of *Escherichia coli* and *Klebsiella pneumoniae* spreading *bla*<sub>CTX-M-15</sub>, *bla*<sub>CTX-M-1</sub> and *mcr-1* genes in camels in Tunisia. *J Glob Antimicrob Resist* 2019(May), doi:http://dx.doi.org/10.1016/j.jgar.2019.05.007 [Epub ahead of print].

[5] Saidani M, Messadi L, Chaouechi A, Tabib I, Saras E, Soudani A, et al. High genetic diversity of Enterobacteriaceae clones and plasmids disseminating resistance to extended-spectrum cephalosporins and colistin in healthy chicken in Tunisia. *Microb Drug Resist* 2019(July), doi:http://dx.doi.org/10.1089/mdr.2019.0138 [Epub ahead of print].

Mariem Saidani<sup>a,b</sup>

<sup>a</sup>Service de Microbiologie et d'Immunologie, Ecole Nationale de Médecine Vétérinaire, Université de La Manouba, Sidi Thabet, Tunisia

<sup>b</sup>Institut National Agronomique de Tunisie, Université de Carthage, Tunis, Tunisia

Lilia Messadi
Ela Sahmin
Sana Zouaoui
Alya Soudani

Monia Daaloul-Jedidi

Aymen Mamlouk

Faten Ben Chehida

Service de Microbiologie et d'Immunologie, Ecole Nationale de Médecine Vétérinaire, Université de La Manouba, Sidi Thabet, Tunisia

Jean-Yves Madec

Marisa Haenni*

Unité Antibiorésistance et Virulence Bactériennes, Université de Lyon
– ANSES Site de Lyon, 31 avenue Tony Garnier, 69364 Lyon, France

* Corresponding author.

E-mail address: marisa.haenni@anses.fr (M. Haenni).

Received 3 June 2019

Available online 19 August 2019

<footnote>a *Escherichia coli* isolates found both in the dominant and subdominant flora are underlined.</footnote>