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Article

mcr-1-Mediated Colistin Resistance and Genomic Characterization of Antimicrobial Resistance in ESBL-Producing Salmonella Infantis Strains from a Broiler Meat Production Chain in Italy

by
Patrizia Casagrande Proietti
1,*,†,
Laura Musa
1,†,
Valentina Stefanetti
1,
Massimiliano Orsini
2,
Valeria Toppi
1,
Raffaella Branciari
1,
Francesca Blasi
3,
Chiara Francesca Magistrali
3,
Stefano Capomaccio
1,
Tana Shtylla Kika
4 and
Maria Pia Franciosini
1
1
Department of Veterinary Medicine, University of Perugia, Via S. Costanzo 4, 06126 Perugia, Italy
2
Istituto Zooprofilattico Sperimentale delle Venezie, 35020 Padova, Italy
3
Istituto Zooprofilattico Sperimentale dell’Umbria e Delle Marche ‘Togo Rosati’, 06124 Perugia, Italy
4
Faculty of Veterinary Medicine, Agricultural University of Tirana, 1029 Tirana, Albania
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Antibiotics 2022, 11(6), 728; https://doi.org/10.3390/antibiotics11060728
Submission received: 10 May 2022 / Revised: 25 May 2022 / Accepted: 26 May 2022 / Published: 28 May 2022
(This article belongs to the Special Issue Antimicrobial Compounds and Antimicrobial Resistance: The Big Challenge of the One Health Approach)
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Abstract

:
This work aimed to evaluate phenotypically and genotypically the colistin susceptibility of 85 Salmonella Infantis strains isolated in Italy from the broiler production chain, and to apply a whole-genome approach for the determination of genes conferring antimicrobial resistance (AMR). All isolates were tested by the broth microdilution method to evaluate the colistin minimum inhibitory concentrations (MICs). A multiplex PCR was performed in all isolates for the screening of mcr-1, mcr-2, mcr-3 mcr-4, mcr-5 genes and whole-genome sequencing (WGS) of six S. Infantis was applied. Three out of 85 (3.5%) S. Infantis strains were colistin resistant (MIC values ranged from 4 to 8 mg/L) and mcr-1 positive. The mcr-1.1 and mcr-1.2 variants located on the IncX4 plasmid were detected in three different colistin-resistant isolates. The two allelic variants showed identical sequences. All six isolates harbored blaCTXM-1, aac(6′)-Iaa and gyrA/parC genes, mediating, respectively, beta-lactam, aminoglycoside and quinolone resistance. The pESI-megaplasmid carrying tet(A) (tetracycline resistance), dfrA1, (trimethoprim resistance) sul1, (sulfonamide resistance) and qacE (quaternary ammonium resistance) genes was found in all isolates. To our knowledge, this is the first report of the mcr-1.2 variant described in S. Infantis isolated from broilers chickens. Our results also showed a low prevalence of colistin- resistance, probably due to a reduction in colistin use in poultry. This might suggest an optimization of biosecurity control both on farms and in slaughterhouses.

1. Introduction

Colistin is a polypeptide antibiotic that was initially isolated in 1947 from the soil bacterium Paenibacillus polymyxa subsp. colistinus [1] and belongs to the group of polymyxins. In human medicine, colistin has been used for decades for the treatment of infections caused by Gram-negative bacteria and was later replaced by other antibiotics, such as aminoglycosides, quinolones and β-lactams, due to its toxicity [2]. Nowadays, the use of colistin is reconsidered as a last resort for infections caused by multidrug-resistant (MDR) bacteria [3]. In veterinary medicine, it has been used for a long time as preventive agent against Gram-negative infections [4] but also as a growth promoter in some other species of zootechnical interest [5,6]. The therapeutic usage of colistin is mostly related to enterobacterial infections, particularly in poultry and pigs with gastrointestinal infections, within intensive husbandry systems [7]. The World Health Organization has reclassified colistin in the category of drugs of “critical importance” for human medicine [8], justifying the execution of frequent studies addressed to monitor the resistance displayed by some bacteria, such as Salmonella spp. and Escherichia coli, largely widespread in both the human and veterinary field. Salmonella Infantis is considered an emerging serotype in Europe, widespread in broiler and turkey chain productions, configuring itself as the most common serotype after S. Enteridis and S. Typhimurium [9]. The progressive incidence of S. Infantis infections in humans was related to the spread of MDR S. Infantis strains along the broiler production chain carrying a pSEI-like plasmid with or without extended spectrum β-lactamase (ESBL)-production [10,11,12,13]. Recently, colistin resistance has been attributed to the mcr-1 gene (mobile colistin-resistant gene) located on a transferable plasmid, and first described in China [14] from animals, food and humans.
Nowadays, 32 mcr-1 variants, 8 mcr-2 variants, 40 mcr-3 variants, 6 mcr-4 variants and 4 mcr-5, are described in Enterobacteriaceae according to GenBank records (last accessed 23 December 2021). The predominant replicon types of plasmid-carrying mcr-1 are IncI2, IncX4, IncHI2 and IncP [15]. The (Inc) plasmids groups are classified as incompatibility groups when two plasmids are unable to propagate steadily in the same host [16]. Up to now, 27 different plasmid incompatibility groups have been recognized in the Enterobacteriaceae family [17].
This work aimed to phenotypically and genotypically evaluate the colistin susceptibility of 85 S. Infantis strains isolated in Italy from the broiler production chain, and to determine the genes responsible for antimicrobial resistance (AMR) by whole-genome sequencing (WGS).

2. Materials and Methods

2.1. Collection and Isolate Identification

Eighty-five S. Infantis strains collected in a previous study and selected for ESBL strain presence and for the phenotypic antibiotic resistance profile [18] were investigated. The strains were isolated from 2016 to 2017 in northern, central and southern Italy from cloacal samples (n = 13) on broiler farms and from environmental, skin, liver and meat by product samples (n = 72) from a slaughterhouse, following standard ISO procedures [19]. Salmonella spp. isolates were serotyped by direct slide agglutination with specific antisera (Statens Serum Institute, Copenhagen, Denmark), according to the Kaufmann–White–Le Minor scheme [20].

2.2. Colistin Susceptibility Testing

To assess colistin susceptibility, all S. Infantis isolates were analyzed by the broth microdilution method. Pure cultures were suspended in 4 mL of 0.90% sterile saline solution (final concentration: 5 × 107 CFU/mL), equivalent to a 0.5 McFarland turbidity level (Vitek, bioMérieux Inc., Durham, NC, USA). Ten microlitres of bacterial suspension were transferred to 11 mL of cation-adjusted Müller Hinton broth (Thermo Fisher Scientific, Milan, Italy), and 50 μL of bacterial suspension were dispensed into each well of Euvsec FRCOL microtiter plates (Thermo Fisher Scientific, Milan, Italy) with scalar concentrations of colistin (COL, 0.12–128 mg/L). After inoculation, the plates were incubated for 24 h at 37 °C under aerobic conditions. The results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-offs (MIC > 2 mg/L) [21]. Escherichia coli ATCC 25,922 and ZTA14/0097EC were used as quality and positive control strains, respectively.

2.3. Multiplex PCR Analysis for mcr Genes

Genomic DNA was extracted from individual colonies using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s protocol. Extracted DNA quantity and quality were determined by spectrophotometry (NanoDrop, Thermo Fisher Scientific, Milan, Italy) and electrophoresis on 1% agarose gel, respectively. All isolates were screened by a multiplex PCR for mcr-1, mcr-2, mcr-3 mcr-4, mcr-5 genes, as previously described [22]. One nanogram of DNA was used in a total of 50 µL of reaction mixture contained 10 × buffer, 3 mM MgCl2, 200 µM of each deoxyribonucleotide triphosphate, 1 µM of each primer (Sigma Aldrich, Milan. Italy), 0.5 U Taq DNA polymerase (Microtech, Padova, Italy). In each set of reactions, positive controls [22] and a negative control (negative sample), as well as a negative reaction mix control (containing the reagents and water instead of DNA), were included.

2.4. Whole-Genome Sequencing

Genomic DNA of six S. Infantis (3 mcr-1 positive and 3 mcr-1 negative) isolates was used for library preparation with a commercial kit (Nextera XT, Illumina San Diego, CA, USA). Libraries were sequenced using paired-end Illumina MiSeq, and the quality raw reads was checked using FastQC, (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (accesse on 15 November 2021) removing those showing low quality (Phred Score < 20) and shorter than 70 nucleotides. Processed reads were de novo assembled using SPAdes, version 3.14 [23]. and the generated contigs were passed to CSAR v. 1.1.1 to build the scaffolds with more than 200 nucleotides in length. Subsequently, the genome assembly quality check was performed with QUAST v. 4.3, [24] and the sequences annotated using Prokka v. 1.12 [25]. Genome sequences were analyzed for the presence of genes encoding virulence factors, using databases. Genetic determinants of antibiotic resistance and plasmid replicons were assessed using “The Comprehensive Antibiotic Resistance Database” (CARD, https://card.mcmaster.ca/) (accesse on 15 November 2021) and the STARAMR software (https://github.com/phac-nml/staramr) (accessed on 20 November 2021).

3. Results

3.1. Colistin Susceptibility Test

Overall, three out of 85 (3.5%) S. Infantis strains showed phenotypical resistance to colistin. One strain, isolated from the neck skin of a broiler in 2016 from a slaughterhouse in southern Italy, showed a MIC value = 4 mg/L. The other two strains, isolated from a drumstick in 2017 in a slaughterhouse in northern Italy, presented MIC values = 8 mg/L. The MIC values of the colistin-susceptible isolates ranged from 0.5–1 m/L, and one isolate showed MIC value = 2 mg/L.

3.2. Molecular Characterization

Multiplex PCR-screening revealed that the three colistin-resistant S. Infantis strains were mcr-1-positive.
The raw data of WGS are shown in Supplementary Material (File S1). The results of the AMR genotype of six S. Infantis isolates analyzed by WGS and the phenotypic and PFGE profiles [18] are shown in Table 1. The mcr-1.1 variant was detected in one colistin-resistant isolate and mcr-1.2 in two colistin-resistant isolates. The two allelic variants showed identical sequences and were located on the IncX4 plasmids in the three S. Infantis isolates. (Figure 1 and Figure 2). The isolate harboring the IncX4 plasmid with the mcr 1.1 gene also contained the IncFII plasmid.
Resistance to cefotaxime (ESBL phenotype) was predicted by the presence of the blaCTX-M-1 gene in all cefotaxime-resistant isolates. All isolates were tetracycline-resistant, carrying the tetracycline resistance determinant tet(A). The dfrA1 determinant mediating trimethoprim resistance was identified in all trimethoprim-resistant isolates [18]; two out of six isolates also harbored the dfrA14 gene. The sul1 gene, mediating sulphonamide resistance, was detected in all sulphonamide-resistant strains. All isolates contained qacE determinant, mediating quaternary ammonium resistance. We detected pESI-megaplasmid carrying tet(A), dfrA1, dfrA14, sul1, and qacE genes in all isolates. The aminoglycoside resistance determinant aac(6′)-Iaa was identified in all isolates; one out of six was also gentamicin-resistant, and two out of six aminoglycoside-susceptible isolates harbored aph(3′)-Ia. Two-point mutations in the quinolone resistance, D→G mutation at position 87 of the gyrA gene and T→S mutation at position 57 of the parC gene, were detected in all 6 nalidixic acid-resistant and ciprofloxacin-susceptible isolates.

4. Discussion

Colistin, considered one of the “last resort” treatments against MDR bacterial infections, may be challenged by the mcr genes, which have received widespread attention in different species of Enterobacteriaceae found in animals and humans around the world [26]. In this study, we first evaluated, phenotypically and genotypically, the colistin resistance of 85 S. Infantis strains and determined the genes conferring AMR by WGS of six S. Infantis isolates. Our results demonstrated that three out of 85 (3.5%) strains isolated from slaughtered broilers were colistin-resistant, with MIC values ranging from 4–8 mg/L. In a previous study, Carfora et al. (2018) showed a prevalence of colistin resistance of 1.2% in S. Infantis strains from broiler chickens [27]. These data are in agreement with others obtained in Spain [28] and highlight the efficiency of plans, based on the “One Health” approach, applied at UE level and addressed to control the alarming diffusion of antimicrobial resistance in the animal food chain. In EU countries, the use of colistin as a growth promotor has been prohibited since 2006, although its off-label use is permitted in therapeutic treatments with the exception of Finland, Norway and Iceland, where colistin has never been used [29,30]. Following European countries, China has also banned the use of colistin in 2016 [31]. Other Asian countries, such as India, Vietnam and Bangladesh, using this antimicrobial as growth promoter, showed a remarkable prevalence of resistance, with up to 92% of Salmonella spp. being colistin-resistant strains in Bangladesh [32]. Additionally, a different colistin susceptibility has been described among Salmonella spp. populations. Agerso et al., (2017) demonstrated that S. Dublin and S. Enteritidis were less susceptible than other Salmonella serovars originating from humans [33].
The WGS revealed the presence of the mcr 1.1 and the mcr-1.2 allelic variants located in the IncX4 plasmids in the three colistin-resistant S. Infantis isolates. The two colistin-resistant S. Infantis mcr-1.2 positive displayed the same XbaI 0126 PFGE profile. The one colistin-resistant isolate mcr1.1 positive showed the D PFGE profile, a clonally related group to XbaI 0126 [18]. By WGS, the mcr.1.1 variant was identical to the allelic variant mcr-1.2. The same sequence between these two allelic variants was also found by Simoni et al., (2018) in a colistin-resistant blood isolate of Escherichia coli [34]. In Italy, the mcr-1.2 variant, located in the IncX4 plasmid, was first described in K. pneumonia isolated from humans [35]. Moreover, this variant, associated with the IncX4 plasmid, was found in S. Blockley and in E. coli strains isolated from turkeys [36]. To our knowledge, this is the first report of the mcr1.2 variant described in S. Infantis isolated from broiler chickens. We found the mcr1.1 gene located in the IncX4 plasmid in one colistin-resistant S. Infantis isolate, which is in agreement with Carfora et al. [27]. It is known the ability of the mcr cassette to jump into several types of plasmids, including the IncX4 plasmid, which seems to be one of the predominant replicon types of plasmids, carrying the mcr-1 gene [15]. Moreover, the possibility of the transmission of mcr-positive IncX4 plasmids among different bacterial species, and from animals to humans or vice versa, has been documented, as reported elsewhere [37]. The WGS revealed that all six isolates carried blaCTX-M1, tet(A), dfrA1 and sul1 genes, mediating cefotaxime, tetracycline, trimethoprim and sulfonamide resistance, respectively. These genes were located on the pESI-like megaplasmid, as reported by other authors [11,13]. These data are not surprising as, exploiting data from our previous paper [18], the six S. Infantis isolates sequenced by WGS were ESBL and resistant toward tetracycline, sulfamethoxazole/trimethoprim and nalidixic acid, thus confirming the typical pattern of multi-resistance of the European S. Infantis clone [38]. In Salmonella species, DNA gyrase is the primary target of quinolone action; a single-point mutation in the quinolone resistance-determining region (QRDR) of gyrA can mediate resistance to nalidixic acid and to ciprofloxacin [39]. In our study, six nalidixic acid-resistant and ciprofloxacin-susceptible S. Infantis isolates harbored both gyrA and parC genes, suggesting that the presence of the two genes makes Salmonella strains more susceptible to ciprofloxacin than the isolates harboring a single mutation in gyrA [40]. The mutation parC is considered rare in salmonellae and was only detected in one out of 382 S. Infantis genomes examined in a previous study [13]. Our isolates carried genes, such parc(C) and aph3(III), which have not yet been detected in Italian S. Infantis strains. The aph3(III), mediating aminoglycoside resistance, was detected in association with aac(6′)-Iaa in two aminoglycoside-susceptible S. Infantis isolates. Finally, in our study, pESI-megaplasmid also harbored the qacE gene, involved in resistance to quaternary ammonium compounds (QACs), extensively used in farm buildings, at the end of the production cycle and in food processing due to its cleaning and disinfectant properties [41]. Jaglic and Cervinkova [42] have already reported that bacteria expressing resistance to antiseptics were generally less susceptible to antibiotic and have hypothesized that outer membrane changes could have played a basic role in this non-specific cross-resistance [43]. More recently, qacH-associated non-classic class 1 integrons were seen in conjugative plasmids that could be a tool responsible for co-dissemination of antimicrobial and disinfectant resistance genes [44].

5. Conclusions

The WGS revealed the presence of the mcr1.1 and mcr-1.2 allelic variants located in the IncX4-type plasmid. The mcr-1.2 allelic variant has not yet been described in S. Infantis isolated from broiler meat production in Italy. Our results showed a low prevalence of colistin-resistant strains of S. Infantis, carrying the mcr-1 gene, probably due to a reduction in colistin use in poultry [45]. Moreover, the pESI-megaplasmid, detected in our study, in addition to other genes, carried the qacE gene involved in resistance to quaternary ammonium salts, one of the most common disinfectants used in the poultry industry. In this work, we confirmed the diffusion and persistence of the ESBL multiresistant S. Infantis strains in broiler meat production chain highlighting the need to improve biosecurity measures both on farms and in slaughterhouses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antibiotics11060728/s1, File S1. WGS data.

Author Contributions

Conceptualization, P.C.P. and M.P.F.; methodology, P.C.P., L.M., V.S., F.B. and C.F.M. software, S.C.; investigation, L.M., V.S., V.T., R.B. and T.S.K.; data curation, M.O. and P.C.P.; writing—original draft preparation, P.C.P., M.P.F., V.S. and L.M.; writing—review and editing, P.C.P., M.P.F., V.S. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This project has been funded by Ricerca di Base 2020, University of Perugia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank Hendrixen (National Food Institute, European Union Reference Laboratory for Antimicrobial Resistance (EURL-AMR), Alessia Franco and Antonio Battisti (National Reference Laboratory for Antimicrobial Resistance, IZSLT) for kindly providing the positive strains used as controls in PCR assay. We would also like to thank Lucas Dominguez Rodriguez, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain for kindly provided the positive strain for MIC assay.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Benedict, R.G.; Langlykke, A.F. Antibiotic Activity of Bacillus Polymyxa. J. Bacteriol. 1947, 54, 24. [Google Scholar]
  2. Poirel, L.; Jayol, A.; Nordmann, P. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clin. Microbiol. Rev. 2017, 30, 557–596. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Kempf, I.; Jouy, E.; Chauvin, C. Colistin Use and Colistin Resistance in Bacteria from Animals. Int. J. Antimicrob. Agents 2016, 48, 598–606. [Google Scholar] [CrossRef] [PubMed]
  4. Kieffer, N.; Aires-de-Sousa, M.; Nordmann, P.; Poirel, L. High Rate of MCR-1-Producing Escherichia coli and Klebsiella pneumoniae among Pigs, Portugal. Emerg. Infect. Dis. 2017, 23, 2023–2029. [Google Scholar] [CrossRef] [Green Version]
  5. Rhouma, M.; Beaudry, F.; Thériault, W.; Letellier, A. Colistin in Pig Production: Chemistry, Mechanism of Antibacterial Action, Microbial Resistance Emergence, and One Health Perspectives. Front. Microbiol. 2016, 7, 1789. [Google Scholar] [CrossRef]
  6. Kumar, H.; Chen, B.-H.; Kuca, K.; Nepovimova, E.; Kaushal, A.; Nagraik, R.; Bhatia, S.K.; Dhanjal, D.S.; Kumar, V.; Kumar, A.; et al. Understanding of Colistin Usage in Food Animals and Available Detection Techniques: A Review. Animals 2020, 10, 1892. [Google Scholar] [CrossRef]
  7. Catry, B.; Cavaleri, M.; Baptiste, K.; Grave, K.; Grein, K.; Holm, A.; Jukes, H.; Liebana, E.; Lopez Navas, A.; Mackay, D.; et al. Use of Colistin-Containing Products within the European Union and European Economic Area (EU/EEA): Development of Resistance in Animals and Possible Impact on Human and Animal Health. Int. J. Antimicrob. Agents 2015, 46, 297–306. [Google Scholar] [CrossRef]
  8. World Health Organization. GLASS: The Detection and Reporting of Colistin Resistance; World Health Organization: Geneva, Switzerland, 2021; ISBN 978-92-4-001904-1. [Google Scholar]
  9. European Food Safety Authority; European Centre for Disease Prevention and Control. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-Borne Outbreaks in 2017. EFSA J. 2018, 16, e05500. [CrossRef]
  10. Aviv, G.; Tsyba, K.; Steck, N.; Salmon-Divon, M.; Cornelius, A.; Rahav, G.; Grassl, G.A.; Gal-Mor, O. A Unique Megaplasmid Contributes to Stress Tolerance and Pathogenicity of an Emergent Salmonella Enterica Serovar Infantis Strain. Environ. Microbiol. 2014, 16, 977–994. [Google Scholar] [CrossRef]
  11. Franco, A.; Leekitcharoenphon, P.; Feltrin, F.; Alba, P.; Cordaro, G.; Iurescia, M.; Tolli, R.; D’Incau, M.; Staffolani, M.; Di Giannatale, E.; et al. Emergence of a Clonal Lineage of Multidrug-Resistant ESBL-Producing Salmonella Infantis Transmitted from Broilers and Broiler Meat to Humans in Italy between 2011 and 2014. PLoS ONE 2015, 10, e0144802. [Google Scholar] [CrossRef] [Green Version]
  12. Szmolka, A.; Szabó, M.; Kiss, J.; Pászti, J.; Adrián, E.; Olasz, F.; Nagy, B. Molecular Epidemiology of the Endemic Multiresistance Plasmid PSI54/04 of Salmonella Infantis in Broiler and Human Population in Hungary. Food Microbiol. 2018, 71, 25–31. [Google Scholar] [CrossRef] [Green Version]
  13. Alba, P.; Leekitcharoenphon, P.; Carfora, V.; Amoruso, R.; Cordaro, G.; Di Matteo, P.; Ianzano, A.; Iurescia, M.; Diaconu, E.L.; Study Group, E.-E.-A.N.; et al. Molecular Epidemiology of Salmonella Infantis in Europe: Insights into the Success of the Bacterial Host and Its Parasitic PESI-like Megaplasmid. Microb. Genom. 2020, 6. [Google Scholar] [CrossRef]
  14. Liu, Y.-Y.; Wang, Y.; Walsh, T.R.; Yi, L.-X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; et al. Emergence of Plasmid-Mediated Colistin Resistance Mechanism MCR-1 in Animals and Human Beings in China: A Microbiological and Molecular Biological Study. Lancet Infect. Dis. 2016, 16, 161–168. [Google Scholar] [CrossRef]
  15. Sun, J.; Fang, L.-X.; Wu, Z.; Deng, H.; Yang, R.-S.; Li, X.-P.; Li, S.-M.; Liao, X.-P.; Feng, Y.; Liu, Y.-H. Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the Mcr-1 Acquisition. Sci. Rep. 2017, 7, 424. [Google Scholar] [CrossRef]
  16. Carattoli, A. Plasmid-Mediated Antimicrobial Resistance in Salmonella Enterica. Curr. Issues Mol. Biol. 2003, 5, 113–122. [Google Scholar]
  17. Yang, Q.-E.; Sun, J.; Li, L.; Deng, H.; Liu, B.-T.; Fang, L.-X.; Liao, X.-P.; Liu, Y.-H. IncF Plasmid Diversity in Multi-Drug Resistant Escherichia coli Strains from Animals in China. Front. Microbiol. 2015, 6, 964. [Google Scholar] [CrossRef]
  18. Casagrande Proietti, P.; Stefanetti, V.; Musa, L.; Zicavo, A.; Dionisi, A.M.; Bellucci, S.; Mensa, A.L.; Menchetti, L.; Branciari, R.; Ortenzi, R.; et al. Genetic Profiles and Antimicrobial Resistance Patterns of Salmonella Infantis Strains Isolated in Italy in the Food Chain of Broiler Meat Production. Antibiotics 2020, 9, 814. [Google Scholar] [CrossRef]
  19. ISO 6579-1:2017/AMD 1:2020 ISO 6579-1:2017/Amd 1:2020. Available online: https://www.iso.org/cms/render/live/en/sites/isoorg/contents/data/standard/07/66/76671.html (accessed on 19 March 2022).
  20. Grimont, P.A.D.; Weill, F.-X. Antigenic Formulae of the Salmonella Serovars; WHO: Geneva, Switzerland, 2007. [Google Scholar]
  21. EUCAST. European Committee on Antimicrobial Susceptibility Testing. Available online: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_10.0_Breakpoint (accessed on 19 March 2022).
  22. Rebelo, A.R.; Bortolaia, V.; Kjeldgaard, J.S.; Pedersen, S.K.; Leekitcharoenphon, P.; Hansen, I.M.; Guerra, B.; Malorny, B.; Borowiak, M.; Hammerl, J.A.; et al. Multiplex PCR for Detection of Plasmid-Mediated Colistin Resistance Determinants, Mcr-1, Mcr-2, Mcr-3, Mcr-4 and Mcr-5 for Surveillance Purposes. Eurosurveillance 2018, 23, 17-00672. [Google Scholar] [CrossRef]
  23. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [Green Version]
  24. Gurevich, A.; Saveliev, V.; Vyahhi, N.; Tesler, G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 2013, 29, 1072–1075. [Google Scholar] [CrossRef]
  25. Seemann, T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef]
  26. Luo, Q.; Wang, Y.; Xiao, Y. Prevalence and Transmission of Mobilized Colistin Resistance (Mcr) Gene in Bacteria Common to Animals and Humans. Biosaf. Health 2020, 2, 71–78. [Google Scholar] [CrossRef]
  27. Carfora, V.; Alba, P.; Leekitcharoenphon, P.; Ballarò, D.; Cordaro, G.; Di Matteo, P.; Donati, V.; Ianzano, A.; Iurescia, M.; Stravino, F.; et al. Corrigendum: Colistin Resistance Mediated by Mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016–2017). Front. Microbiol. 2018, 9, 2395. [Google Scholar] [CrossRef] [Green Version]
  28. Vázquez, X.; García, V.; Fernández, J.; Bances, M.; de Toro, M.; Ladero, V.; Rodicio, R.; Rodicio, M.R. Colistin Resistance in Monophasic Isolates of Salmonella Enterica ST34 Collected from Meat-Derived Products in Spain, with or without CMY-2 Co-Production. Front. Microbiol. 2021, 12, 735364. [Google Scholar] [CrossRef]
  29. Maron, D.F.; Smith, T.J.S.; Nachman, K.E. Restrictions on Antimicrobial Use in Food Animal Production: An International Regulatory and Economic Survey. Glob. Health 2013, 9, 48. [Google Scholar] [CrossRef] [Green Version]
  30. Moulin, G.; Catry, B.; Baptiste, K.; Cavaco, L.; Jukes, H.; Kluytmans, J. Updated Advice on the Use of Colistin Products in Animals within the European Union: Development of Resistance and Possible Impact on Human and Animal Health. Eur. Med. Agency 2016, 44, 56. [Google Scholar]
  31. Walsh, T.R.; Wu, Y. China Bans Colistin as a Feed Additive for Animals. Lancet Infect. Dis. 2016, 16, 1102–1103. [Google Scholar] [CrossRef]
  32. Uddin, M.B.; Hossain, S.M.B.; Hasan, M.; Alam, M.N.; Debnath, M.; Begum, R.; Roy, S.; Harun-Al-Rashid, A.; Chowdhury, M.S.R.; Rahman, M.M.; et al. Multidrug Antimicrobial Resistance and Molecular Detection of Mcr-1 Gene in Salmonella Species Isolated from Chicken. Animals 2021, 11, 206. [Google Scholar] [CrossRef] [PubMed]
  33. Agersø, Y.; Torpdahl, M.; Zachariasen, C.; Seyfarth, A.; Hammerum, A.M.; Nielsen, E.M. Tentative Colistin Epidemiological Cut-off Value for Salmonella spp. Foodborne Pathog. Dis. 2012, 9, 367–369. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Simoni, S.; Morroni, G.; Brenciani, A.; Vincenzi, C.; Cirioni, O.; Castelletti, S.; Varaldo, P.E.; Giovanetti, E.; Mingoia, M. Spread of Colistin Resistance Gene Mcr-1 in Italy: Characterization of the Mcr-1.2 Allelic Variant in a Colistin-Resistant Blood Isolate of Escherichia Coli. Diagn. Microbiol. Infect. Dis. 2018, 91, 66–68. [Google Scholar] [CrossRef]
  35. Di Pilato, V.; Arena, F.; Tascini, C.; Cannatelli, A.; Henrici De Angelis, L.; Fortunato, S.; Giani, T.; Menichetti, F.; Rossolini, G.M. Mcr-1.2, a New Mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella Pneumoniae Strain of Sequence Type 512. Antimicrob. Agents Chemother. 2016, 60, 5612–5615. [Google Scholar] [CrossRef] [Green Version]
  36. Alba, P.; Leekitcharoenphon, P.; Franco, A.; Feltrin, F.; Ianzano, A.; Caprioli, A.; Stravino, F.; Hendriksen, R.S.; Bortolaia, V.; Battisti, A. Molecular Epidemiology of Mcr-Encoded Colistin Resistance in Enterobacteriaceae from Food-Producing Animals in Italy Revealed through the EU Harmonized Antimicrobial Resistance Monitoring. Front. Microbiol. 2018, 9, 1217. [Google Scholar] [CrossRef]
  37. Viñes, J.; Cuscó, A.; Napp, S.; Alvarez, J.; Saez-Llorente, J.L.; Rosàs-Rodoreda, M.; Francino, O.; Migura-Garcia, L. Transmission of Similar Mcr-1 Carrying Plasmids among Different Escherichia coli Lineages Isolated from Livestock and the Farmer. Antibiotics 2021, 10, 313. [Google Scholar] [CrossRef]
  38. EFSA Panel on Biological Hazards (EFSA BIOHAZ Panel); Koutsoumanis, K.; Allende, A.; Alvarez-Ordóñez, A.; Bolton, D.; Bover-Cid, S.; Chemaly, M.; De Cesare, A.; Herman, L.; Hilbert, F.; et al. Salmonella Control in Poultry Flocks and Its Public Health Impact. EFSA J. 2019, 17, e05596. [Google Scholar] [CrossRef]
  39. Hopkins, K.L.; Davies, R.H.; Threlfall, E.J. Mechanisms of Quinolone Resistance in Escherichia coli and Salmonella: Recent Developments. Int. J. Antimicrob. Agents 2005, 25, 358–373. [Google Scholar] [CrossRef]
  40. Eaves, D.J.; Randall, L.; Gray, D.T.; Buckley, A.; Woodward, M.J.; White, A.P.; Piddock, L.J.V. Prevalence of Mutations within the Quinolone Resistance-Determining Region of GyrA, GyrB, ParC, and ParE and Association with Antibiotic Resistance in Quinolone-Resistant Salmonella Enterica. Antimicrob. Agents Chemother. 2004, 48, 4012–4015. [Google Scholar] [CrossRef] [Green Version]
  41. Kawai, R.; Yada, S.; Yoshimura, T. Characterization and Solution Properties of Quaternary-Ammonium-Salt-Type Amphiphilic Gemini Ionic Liquids. ACS Omega 2019, 4, 14242–14250. [Google Scholar] [CrossRef]
  42. Jaglic, Z.; Cervinkova, D. Genetic Basis of Resistance to Quaternary Ammonium Compounds—The Qac Genes and Their Role: A Review. Vet. Med. 2012, 57, 275–281. [Google Scholar] [CrossRef] [Green Version]
  43. Russell, A.D. Do Biocides Select for Antibiotic Resistance? J. Pharm. Pharmacol. 2000, 52, 227–233. [Google Scholar] [CrossRef]
  44. Jiang, X.; Xu, Y.; Li, Y.; Zhang, K.; Liu, L.; Wang, H.; Tian, J.; Ying, H.; Shi, L.; Yu, T. Characterization and Horizontal Transfer of QacH-Associated Class 1 Integrons in Escherichia coli Isolated from Retail Meats. Int. J. Food Microbiol. 2017, 258, 12–17. [Google Scholar] [CrossRef]
  45. Aconiti Mandolini, N.; Perugini, G.; Filippini, G.; Pierucci, P.; Baiguini, A.; Vaccaro, A.; Pelagalli, G.; Marinelli, F.; Capuccella, M. Evaluation of Colistin Consumption in Swine and Poultry of Marche Region during the 2017–2019 Period. Eur. J. Public Health 2020, 30, ckaa166.1305. [Google Scholar] [CrossRef]
Antibiotics 11 00728 g001 550
Figure 1. IncX4 plasmid content of the S. Infantis isolate, carrying mcr1.1.
Figure 1. IncX4 plasmid content of the S. Infantis isolate, carrying mcr1.1.
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Antibiotics 11 00728 g002 550
Figure 2. IncX4 plasmid content of the S. Infantis isolate, carrying mcr1.2.
Figure 2. IncX4 plasmid content of the S. Infantis isolate, carrying mcr1.2.
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Table
Table 1. Genotypic and phenotypic profiles of the six S. Infantis isolates.
Table 1. Genotypic and phenotypic profiles of the six S. Infantis isolates.
IsolateOriginSourceYearTopologyAMR GenotypePhenotypic Profile [18]PFGE [18]
S1Northern ItalyNeck Skin2019ChromosomicparC (T57S), blaCTX-M-1, aac(6′)-Iaa, gyrA(D87G)A, Ams, Cl, Ctx, Gm, Na, Sxt, TeNA
Plasmidic pESI-likesul1, qacE, dfrA1, tet(A)
S2Northern ItalyNeck Skin2016ChromosomicparC (T57S), blaCTX-M-1, aac(6′)-Iaa, gyrA(D87G)Ams, Cl, Col, Ctx, Caz Na, Sxt, TeD
Plasmidic pESI-likesul1, qacE, dfrA1, tet(A)
Pasmidic IncX4 mcr-1.1
Plasmidic IncFII
S3Northern ItalyDrumstick2017ChromosomicparC (T57S), blaCTX-M-1, aac(6′)-Iaa, aph(3′)-Ia, gyrA(D87G)A, Cl, Col, Ctx, Na, Sxt, TeXbaI 0126
Plasmidic pESI-likesul1, qacE, dfrA1, drfA14, tet(A)
Plasmidic IncX4mcr-1.2
S4Northern ItalyNeck Skin2017ChromosomicparC (T57S), blaCTX-M-1, aac(6′)-Iaa, gyrA(D87G)A, Ams, Cl, Ctx, Gm, Na, TeNA
Plasmidic pESI-likesul1, qacE, dfrA1, tet(A)
S5Northern ItalyDrumstick2017ChromosomicparC (T57S), blaCTX-M-1, aac(6′)-Iaa, aph(3′)-Ia gyrA(D87G)A, Cl, Col, Ctx, Na, Sxt, TeXbaI 0126
Plasmidic pESI-likesul1, qacE, dfrA1, drfA14, tet(A)
Plasmidic IncX4mcr-1.2
S6Southern Italy 2016ChromosomicparC (T57S), blaCTX-M-1, aac(6′)-Iaa, gyrA(D87G)A, Ctx, Cz, Na, Sxt, TeXbaI 0126
Plasmidic pESI-likesul1, qacE, dfrA1drfA14, tet(A)
Legend: A (ampicillin), Ams (ampicillin/sulbactam), Cl (cefalexin), Col (colistin), Ctx, (cefotaxime) Caz (ceftazidime), Gm (gentamicin), Na, (nalidixic acid) Sxt (trimethoprim/sulphametoxazole), Te (tetracycline); PFGE (Pulsed field gel electophoresis); NA (profile cluster not assigned); D, (profile cluster not yet labeled by ECDS nomenclature).
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Casagrande Proietti, P.; Musa, L.; Stefanetti, V.; Orsini, M.; Toppi, V.; Branciari, R.; Blasi, F.; Magistrali, C.F.; Capomaccio, S.; Kika, T.S.; et al. mcr-1-Mediated Colistin Resistance and Genomic Characterization of Antimicrobial Resistance in ESBL-Producing Salmonella Infantis Strains from a Broiler Meat Production Chain in Italy. Antibiotics 2022, 11, 728. https://doi.org/10.3390/antibiotics11060728

AMA Style

Casagrande Proietti P, Musa L, Stefanetti V, Orsini M, Toppi V, Branciari R, Blasi F, Magistrali CF, Capomaccio S, Kika TS, et al. mcr-1-Mediated Colistin Resistance and Genomic Characterization of Antimicrobial Resistance in ESBL-Producing Salmonella Infantis Strains from a Broiler Meat Production Chain in Italy. Antibiotics. 2022; 11(6):728. https://doi.org/10.3390/antibiotics11060728

Chicago/Turabian Style

Casagrande Proietti, Patrizia, Laura Musa, Valentina Stefanetti, Massimiliano Orsini, Valeria Toppi, Raffaella Branciari, Francesca Blasi, Chiara Francesca Magistrali, Stefano Capomaccio, Tana Shtylla Kika, and et al. 2022. "mcr-1-Mediated Colistin Resistance and Genomic Characterization of Antimicrobial Resistance in ESBL-Producing Salmonella Infantis Strains from a Broiler Meat Production Chain in Italy" Antibiotics 11, no. 6: 728. https://doi.org/10.3390/antibiotics11060728

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