Salmonella enterica mcr-1 Positive from Food in Brazil: Detection and Characterization

Abstract

The mcr-1 gene has been identified in bacterial isolates obtained from humans, animals, environment, and food, including Salmonella spp., which is one of the major foodborne pathogens worldwide. The aim of this study was to evaluate the presence of mcr-1 gene in Salmonella spp. from food produced in Brazil and to characterize the isolates harboring this gene. A total of 490 Salmonella spp. isolates from the Brazilian National Program for the Control of Foodborne Pathogens were screened for the presence of mcr-1 gene by polymerase chain reaction (PCR). Whole genome sequencing (WGS) was performed in positive isolates to characterize the sequence type (ST), plasmid families and resistance genes. Antimicrobial susceptibility tests were performed by broth microdilution. Selected isolates were submitted to conjugation experiments using the Escherichia coli J53 as a receptor. We detected eight isolates harboring the mcr-1 gene; seven belonged to Salmonella enterica serovar Typhimurium and its monophasic variant 4,[5],12:i:-, and one belonged to serovar Saintpaul. Seven of the mcr-1 positive isolates displayed a high rate of resistance to other antibiotics in addition to colistin. Analysis of the WGS indicated that the ST 19 was the most common ST among the mcr-1 positive isolates. The mcr-1 gene was located in an IncX4 plasmid of ∼33 kb, with no additional resistance genes and with high identity with a plasmid obtained from a clinical isolate of E. coli mcr-1 positive in Brazil. All plasmids harboring the mcr-1 gene were able to conjugate. Our results suggest the spread of a single plasmid type in Brazil harboring the mcr-1 among Salmonella spp. The horizontal transfer of this mobile element has been contributing to the spread of the colistin resistance in the country.

Introduction

The use of polymyxins in human medicine has recently drawn more attention due to the increasing number of infections caused by carbapenemase producing Enterobacteriaceae in the previous years (Poirel et al., 2017). In fact, polymyxins have been used for decades in veterinary medicine and agricultural production (Poirel et al., 2017). Colistin (polymyxin E) is commonly prescribed for treatment and prevention of enteric diseases, mainly in poultry and pigs submitted to intensive husbandry systems. Furthermore, it has been extensively used as growth promoter worldwide (Poirel et al., 2017). In Brazil, the use of colistin as zootechnical additive in animal feed was prohibited only in November 2016 (Brazil, 2016).

Polymyxin resistance is mainly associated with the addition of cationic groups to lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, preventing antibiotic binding (Rhouma et al., 2016). It was believed that this mechanism was restricted to chromosomal mutations, which allowed only vertical transmission of this resistance determinant. However, in 2015, a plasmid transferable polymyxin resistance, mediated by the mcr-1 gene was described for the first time by Liu et al. (2016). The mcr-1 gene encodes a protein that is responsible for the addition of a phosphoethanolamine group to the lipid A, resulting in a more cationic LPS and conferring resistance to colistin (Liu et al., 2016).

The mcr-1 gene has been identified worldwide in bacterial isolates obtained from humans, animals, environment, and food (meat and vegetables), mostly in Escherichia coli but also in other Enterobacteriaceae, such as Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Shigella sonnei, and Salmonella enterica (Schwarz and Johnson, 2016; Poirel et al., 2017). Although there are already several variants of the mcr gene (mcr-1 to mcr-9) described in the literature (Carrol et al., 2019), the vast majority of the reports in Brazil are related to the mcr-1. In fact, in Brazil, mcr-1 was described in E. coli isolated from animals, inpatients, seawater, and food (Fernandes et al., 2016, 2017; Lentz et al., 2016; Monte et al., 2017; Pillonetto et al., 2019), in K. pneumoniae from hospitalized patients (Aires et al., 2017; Dalmolin et al., 2018), and in S. enterica from food (Rau et al., 2018; Moreno et al., 2019).

In view of the extensive use of colistin in the food production chain and the importance of Salmonella spp. as one of the major foodborne pathogens in the world, the aim of this study was to evaluate the presence of the mcr-1 gene in Salmonella spp. obtained from food produced in Brazil, as well as to characterize phenotypically and genotipically the isolates harboring this gene.

Materials and Methods

Bacterial isolates and identification procedures

A total of 490 Salmonella spp. isolates were randomly selected from the Brazilian National Program for the Control of Foodborne Pathogens of Ministry of Agriculture, Livestock and Food Supply. All the isolates were obtained according to ISO 6579:2002 procedures, from retail poultry, turkey and pork meat, produced in Brazil between 2014 and 2017, and from pork carcasses, in 2014 and 2015, comprising 154 different slaughterhouses under federal inspection in 140 cities and 13 states. Additional identification was performed by ribotyping, using RiboPrinter^® System (DuPont Qualicon) and PvuII restriction enzyme, to determine the serotype of the isolates.

Identification of mcr-1 gene

Salmonella spp. isolates were evaluated for the presence of the mcr-1 gene by pooling seven isolates together and submitting them to DNA extraction and conventional polymerase chain reaction (PCR), with specific primers CLR5-F (5′-CGGTCAGTCCGTTTGTTC-3′) and CLR5-R (5′-CTTGGTCGGTCTGTAGGG-3′) as previously described (Liu et al., 2016). All isolates from a pool with mcr-1 positive result were retested individually by the same conventional PCR to identify the isolate(s) which presented the gene. For DNA extraction, pure colonies were transferred to TE buffer (Tris HCl 10 mM, EDTA 1 mM, pH 8.0) and submitted to heat shock at 80°C for 20 min, followed by freezing at −20°C. The amplicon of isolates with PCR positive for the mcr-1 gene was sequenced by Sanger method to confirm the mcr-1 gene sequence.

Antimicrobial susceptibility testing

Minimal inhibitory concentration (MIC) of 13 antimicrobials was determined by broth microdilution, according to ISO 20776-1:2006 procedures. The tested antibiotics included ampicillin, azithromycin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, gentamicin, meropenem, nalidixic acid, sulfamethoxazole/trimethoprim, tetracycline, tigecycline, and colistin. Results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints (2019—version 9.0). For those antibiotics which do not have defined breakpoints for Salmonella spp., the results were interpreted with the EUCAST epidemiological cut-off values. E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 were used as quality controls.

Whole-genome sequencing

Total DNA was extracted with the Wizard^® Genomic DNA Purification Kit (Promega Corporation). Libraries were prepared with Nextera XT DNA Sample Preparation Kit and sequenced on an Illumina MiSeq analyzer using a 250 bp paired-end library, according to the manufacturer's instructions. Sequences were assembled on SPAdes Assembly version 3.9 (Nurk et al., 2013). Genome annotation was carried out using the RAST Server (Rapid Annotations using Subsystems Technology) version 2.0 (Aziz et al., 2008). Multilocus sequence typing (MLST) and plasmid incompatibility family were determined using the Center for Genomic Epidemiology tools (MLST 2.0 and PlasmidFinder 2.0), as well as acquired antimicrobial resistance (AMR) genes and chromosomal point mutations (ResFinder 3.1) (Larsen et al., 2012; Zankari et al., 2012; Carattoli et al., 2014). Detailed analyses were performed in Geneious v. 11.0.4 software.

Conjugation experiments

Plasmid transferability was evaluated by conjugation experiments using E. coli J53. Each of mcr-1 positive isolates was incubated with the E. coli J53 on a cellulose membrane, in a Luria-Bertani agar plate. After incubation at 37°C for 24 h, colistin-resistant transconjugants were selected on culture media supplemented with azide (100 mg/mL) and colistin (0.5 μg/mL).

Results

Prevalence and characteristics of mcr-1 positive S. enterica

We detected 8 (1.6%) positive isolates from the 490 Salmonella spp. screened by PCR for the presence of mcr-1 gene. Most (5) isolates mcr-1 positive were obtained from pork carcasses during 2015 (Table 1). All mcr-1 positive isolates presented 100% identity to mcr-1 gene described by Liu et al. (2016). According to ribotyping, the isolates belonged to serovar Typhimurium and its monophasic variant 4,[5],12:i:-, except for the isolate SLRe1401, which belonged to serovar Saintpaul. Phylogeny analysis of mcr-1 positive isolates indicated that there was no correlation between clusters and the epidemiological data (data not shown).

Table 1.

Epidemiological Features and Phenotypic and Genotypic Characterization of mcr-1 Positive Isolates of Salmonella enterica from Brazil

Isolate	Serotype	Source of isolate	Date of isolation	Antibiotic Phenotypic resistance	Colistin MIC (mg/L)	MLST	mcr-1 Plasmid type	mcr-1 Plasmid size (bp)	Other acquired AMR genes	Chromosomal point mutations associated with AMR	Other plasmid types
SLRe1401	Saintpaul	Turkey meat	May 2014	CST, CHL	4	ST50	IncX4	33,437	aac(6′)-Iaa	—	IncFIB
SLRe1402	Typhimurium	Poultry meat	August 2014	CST, AMP, CIP, GEN, NAL, TET	4	ST19	IncX4	33,578	aph(3″)-Ib, aac(6′)-Iaa, aac(3)-IId, aadA2, aph(6)-Id, bla_TEM-1B, sul2, tet(B)	gyrA p.S83F	IncFIB, IncFIB(S), IncFII(S)
SLRe1501	Typhimurium/4,[5],12:i:-	Pork carcass	February 2015	CST, AMP, CHL, CIP, GEN, NAL, SXT, TET, TGC	4	ST19	IncX4	33,578	aac(6′)-Iaa, aph(3″)-Ib, aph(6)-Id, aadA1, aac(3)-IIa, bla_TEM-1B, qnrE1, floR, sul1, tet(A), dfrA1	gyrA p.S83Y	IncFIA, IncHI2, IncHI2A
SLRe1502	Typhimurium	Pork carcass	February 2015	CST, AMP, CHL, CIP, GEN, NAL, SXT, TET, TGC	4	ST19	IncX4	33,491	aac(3)-IIa, aadA1, strA, strB, bla_TEM-1B, qnrE, sul1, tet(A), dfrA1	gyrA p.S83Y	IncHI2A, IncFIA, IncHI2
SLRe1503	Typhimurium/4,[5],12:i:-	Pork carcass	March 2015	CST, AMP, CIP, NAL	4	ST4556	IncX4	33,376	aac(6′)-Iaa, bla_TEM-1B	gyrAp.D87N	—
SLRe1504	Typhimurium	Pork carcass	September 2015	CST, AMP, CHL, CIP, GEN, NAL, TET, TGC	4	ST19	IncX4	33,473	aadA1, aac(3)-IIa, bla_TEM-1B, qnrE, sul1, tet(A)	gyrA p.S83Y	IncFIA, IncHI2, IncHI2A
SLRe1505	Typhimurium	Pork carcass	September 2015	CST, AMP, AZM, GEN, SXT, TET, TGC	4	ST19	IncX4	32,374	aph(4)-Ia, aph(3′)-Ia, aac(3)-IVa, aadA1, strB, aadA2, strA, bla_TEM-1B, mph(A), sul2, sul1, tet(A), tet(B), dfrA12	—	IncHI2, IncA/C2, IncFIB, ColpVC, IncHI2A
SLRe1601	Typhimurium	Pork meat	June2016	CST, AMP, CHL, TET, TGC	8	ST19	IncX4	33,255	strB, strA, aph(6)-Ic, aph(3′)-IIa, aadA1, bla_TEM-1B, catA1, sul1, tet(B)	—	IncHI1B(R27), IncHI1A, ColpVC, IncFIA(HI1), p0111

AMP, ampicillin; AMR, antimicrobial resistance; AZM, azithromycin; CHL, chloramphenicol; CIP, ciprofloxacin; CST, colistin; GEN, gentamicin; MIC, minimal inhibitory concentration; MLST, multilocus sequence typing; NAL, nalidixic acid; ST, sequence type; SXT, sulfamethoxazole/trimethoprim; TET, tetracycline; TGC, tigecycline.

Antimicrobial susceptibility

Antimicrobial susceptibility testing indicated that most of the mcr-1 positive isolates displayed resistance to other antibiotics in addition to colistin; most isolates were resistant to ampicillin (seven isolates), tetracycline (six isolates), nalidixic acid (five isolates), chloramphenicol (five isolates), ciprofloxacin (five isolates), and gentamicin (five isolates) (Table 1). In contrast, none of the isolates was resistant to cefotaxime, ceftazidime, or meropenem. MIC for colistin varied from 4 to 8 mg/L, which were just above the breakpoint to be classified as resistant by the EUCAST (Table 1).

Genetic characterization and plasmid analysis

Genome analysis using Center for Genomic Epidemiology tools revealed that six isolates belonged to the sequence type (ST)19 and two isolates (SLRe1401 and SLRe1503) belonged to ST50 and ST4556, respectively. Different types of plasmids (Table 1) were identified among the mcr-1 positive isolates by the in silico analysis of whole-genome sequencing (WGS) data. However, all eight isolates carried an IncX4 plasmid harboring the mcr-1 gene. No additional resistance genes were found in IncX4 plasmids. Nevertheless, several other acquired antibiotic resistance-encoding genes, as well as chromosomal point mutations, were identified according to ResFinder 3.1; these genetic determinants were associated to resistance to aminoglycoside, β-lactam, fluoroquinolone, sulfonamide, and tetracycline classes. None of the isolates had mutations in the pmrAB, phoPQ, or mgrB genes associated with colistin resistance.

Detailed WGS analysis, performed in Geneious software, indicated that the IncX4 plasmids presented ∼33 kb. The insertion sequence ISApl1 was not found neither upstream nor downstream the mcr-1 gene. However, the mcr-1-pap2 sequence and the transposase IS26 were present in all IncX4 plasmids analyzed. Alignment with pICBEC72Hmcr, a plasmid obtained from a clinical isolate of E. coli mcr-1 positive from Brazil (GenBank CP015977.1), indicated 99% of identity (Fig. 1).

FIG. 1.

Genomic context of IncX4 mcr-1 carrying plasmid from isolate SLRe1504 compared to pICBEC72Hmcr (GenBank CP015977). Plasmid region represented by arrows shows sequence details close to mcr-1 gene insertion. Plasmid region represented by rectangle shows sequences related to plasmid functionality. Gray arrows represent hypothetical proteins. * The plasmid from isolate SLRe1504 was used as reference. The other mcr-1 carrying plasmids described in this study presented the same gene organization.

Transfer of mcr-1 gene

To investigate the transference potential of the plasmids harboring the mcr-1 gene, four of the S. enterica isolates were submitted to conjugation experiments using the E. coli J53 as a recipient strain. Results demonstrated that all plasmids were able to conjugate. The presence of the mcr-1 gene in the transconjugants was confirmed by PCR.

Discussion

In this study, we have detected and confirmed the presence of the mcr-1 gene in eight Salmonella isolates from meat (turkey, poultry, and pork origin), belonging to two different serotypes. Our data showed that most of the isolates (seven isolates) which harbored the mcr-1 gene were Salmonella Typhimurium or its monophasic variant 4,[5],12:i:-. Although mcr-1 bearing plasmids have been identified in different Salmonella serovars, Typhimurium seems to be the most often serotype associated with this resistance gene. This association still needs further investigation; however, it may indicate the requirement of a specific genetic background for acquisition and maintenance of mcr-1 harboring plasmids (Doumith et al., 2016; Li et al., 2016b; Cui et al., 2017a).

The antimicrobial susceptibility profile varied among different Salmonella spp. mcr-1 positive isolates, although the vast majority of isolates (except isolate SLRe1401) displayed a multidrug resistance profile, being resistant to three or more classes of antibiotics (Magiorakos et al., 2012). In fact, multiple resistance genes were detected by in silico analysis of WGS in most isolates. Although the frequency of multiresistance varies in different Salmonella serotypes, Salmonella Typhimurium is commonly associated to this pattern worldwide (Leekitcharoenphon et al., 2016; Mąka and Popowska, 2016; Wang et al., 2017). It is important to highlight that none of the isolates was resistant to third-generation cephalosporins or to carbapenems, which are considered critically important antimicrobials for human medicine, according to the World Health Organization (WHO, 2016).

Regarding the genetic context, MLST analysis revealed that most isolates belonged to ST19. Salmonella spp. mcr-1 positive of this ST has been described only in Denmark and in Spain (Torpdahl et al., 2017; Lozano-Leal et al., 2019). Of note, most reports of mcr-1 Salmonella Typhimurium are associated to ST34, including reports from Asia, Europe, and the only reported case in Latin America, in Colombia (Doumith et al., 2016; Li et al., 2016b; Saavedra et al., 2017). ST19 differs from ST34 by a single nucleotide change in the dnaN locus (Li et al., 2017).

To the best of our knowledge, this is the first report of ST50 in Salmonella Saintpaul and the ST4556 in Salmonella Typhimurium harboring the mcr-1 gene. ST4556 has a single nucleotide change in the sucA locus when compared to ST19.

Several plasmid incompatibility groups have already been associated with the mcr-1 gene. However, the global dissemination seems to be associated with three major groups: IncX4, IncI2, and IncHI2 (Matamoros et al., 2017). Among all of our isolates, the mcr-1 gene was located in IncX4 plasmids, with ∼33 kb. IncX4 plasmids have been reported in different Salmonella serotypes, mainly in Europe (Campos et al., 2016; Veldman et al., 2016; Torpdahl et al., 2017; Garcia-Graells et al., 2018) and Asia (Chiou et al., 2017; Cui et al., 2017b). There are no reports of IncX4 plasmids harboring mcr-1 gene in Salmonella isolates in Latin America, except in Brazil (Moreno et al., 2019). Indeed, all mcr-1 reports in Brazil are associated with IncX4 plasmids, including E. coli from humans, foods, and environment (Fernandes et al., 2016, 2017; Monte et al., 2017; Pillonetto et al., 2019), and K. pneumoniae from inpatients (Aires et al., 2017; Dalmolin et al., 2018).

The high identity between the IncX4 plasmids harboring mcr-1 gene from our study and pICBEC72Hmcr (GenBank CP015977.1) suggests the spread of a single plasmid type in Brazil, probably of the same origin, which can be found in different bacteria from distinct sources. This fact underlines the potential for horizontal transfer of the mcr-1 through the IncX4 plasmids. However, in silico analysis showed the absence of the mobile element ISApl1 in all of our plasmids (the same was found in pICBEC72Hmcr). This insertion sequence is associated with the mobilization and dissemination of mcr-1 gene among different bacterial strains and species, but is not present in IncX4 plasmids (Luo et al., 2017). The exact mechanism related to the dissemination of polymyxin resistance by IncX4 plasmids requires more investigation, but it seems to be associated with the mcr-1-pap2 sequence (Li et al., 2016a), which was present in all eight isolates that we have sequenced, near the transposase IS26.

Conjugation experiments demonstrated that all the four Salmonella isolates tested were able to transfer their mcr-1 bearing plasmids to E. coli J53, which is in concordance with earlier findings about the conjugative nature of IncX4 plasmids (Campos et al., 2016; Cui et al., 2017a, b).

Conclusions

We reported eight mcr-1 positive Salmonella isolates from food in Brazil. Our results suggest the spread of a single plasmid type in Brazil harboring the mcr-1 gene, indicating that horizontal transfer of this mobile genetic element has been contributing to the spread of the colistin resistance in the country. The presence of mcr-1 gene in a relevant foodborne pathogen emphasizes the importance of monitoring the AMR in the food-production chain and the need of promoting the responsible use of antibiotics in agriculture and livestock.

Sequence Data

Sequences were deposited to GenBank under accession numbers RXNI00000000, RXNJ00000000, RXNK00000000, RXNL00000000, RXNM00000000, RXNN00000000, RXNO00000000, and RXNP00000000.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by “Instituto Nacional de Pesquisa em Resistência a Antimicrobianos” (INPRA), by Ministry of Agriculture, Livestock and Food Supply from Brazil (MAPA) and by “Fundo de Incentivo à Pesquisa” (FIPE/HCPA 17-0541).

References

Aires

CAM

, Conceição-Neto

, Oliveira

TRT

, Dias

, Montezzi

, Picão

, Albano

, Asensi

, Carvalho-Assef

APD

. Emergence of the plasmid-mediated mcr-1 gene in clinical KPC-2-producing Klebsiella pneumonia sequence type 392 in Brazil. Antimicrob Agents Chemother, 2017; 61:e00317-17.

Aziz

, Bartels

, Best

, DeJonghM, Disz

, Edwards

, Formsma

, Gerdes

, Glass

, Kubal

, Meyer

, Olsen

, Olson

, Osterman

, Overbeek

, McNeil

, Paarmann

, Paczian

, Parrello

, Pusch

, Reich

, Stevens

, Vassieva

, Vonstein

, Wilke

, Zagnitko

. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics, 2008; 9:75.

Brazil. Ministry of Agriculture, Livestock and Food Supply. Normative Instruction n° 45, of November 22, 2016. Available at: www.agricultura.gov.br/assuntos/insumos-agropecuarios/insumos-pecuarios/alimentacao-animal/arquivos-alimentacao-animal/legislacao/instrucao-normativa-no-45-de-22-de-novembro-de-2016.pdf/view, accessed August 25, 2019 .

Campos

, Cristino

, Peixe

, Antunes

. MCR-1 in multidrug-resistant and copper-tolerant clinically relevant Salmonella 1,4,[5],12:i:- and S. Rissen clones in Portugal, 2011 to 2015. Euro Surveill, 2016; 21:30270.

Carattoli

, Zankari

, García-Fernández

, Larsen

, Lund

, Villa

, Aarestrup

, Hasman

. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother, 2014; 58:3895–3903.

Carrol

, Gaballa

, Guldimann

, Sullivan

, Henderson

, Wiedmann

. Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype Typhimurium isolate. MBio, 2019; 10:e00853-19.

Chiou

C-S

, Chen

Y-T

, Wang

Y-W

, Liu

Y-Y

, Kuo

H-C

, Tu

Y-H

, Lin

A-C

, Liao

Y-S

, Hong

Y-P

. Dissemination of mcr-1-carrying plasmids among colistin-resistant Salmonella strains from humans and food-producing animals in Taiwan. Antimicrob Agents Chemother, 2017; 61:e00338-17.

Cui

, Zhan

, Gu

, Li

, Chan

EW-C

, Yan

, Wu

, Xu

, Chen

Prevalence and molecular characterization of mcr-1 positive Salmonella strains recovered from clinical specimens in China. Antimicrob Agents Chemother, 2017a;61:e02471-16.

Cui

, Zhang

, Li

, Chan

EW-C

, Wu

, Chen

. Distinct mechanisms of acquisition of mcr-1-bearing plasmid by Salmonella strains recovered from animals and food samples. Sci Rep, 2017b;7:13199.

10.

Dalmolin

, Martins

, Zavaski

, Lima-Morales

, Barth

. Acquisition of the mcr-1 gene by a high-risk clone of KPC-2-producing Klebsiella pneumoniae ST437/CC258, Brazil. Diagn Microbiol Infect Dis, 2018; 90:132–133.

11.

Doumith

, Godbole

, Aston

, Larkin

, Dallman

, Day

, Muller-Pebody

, Ellington

, de Pinna

, Johnson

, Hopkins

, Woodford

. Detection of the plasmid-mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales. J Antimicrob Chemother, 2016; 71:2300–2305.

12.

Fernandes

, McCulloch

, Vianello

, Moura

, Pérez-Chaparro

, Esposito

, Sartori

, Dropa

, Matté

, Lira

DPA

, Mamizuka

, Lincopan

. First report of globally disseminated IncX4 plasmid carrying the mcr-1 gene in a colistin-resistant Escherichia coli sequence type 101 isolate from a human infection in Brazil. Antimicrob Agents Chemother, 2016; 60:6415–6417.

13.

Fernandes

, Sellera

, Esposito

, Sabino

, Cerdeira

, Lincopan

. Colistin-resistant mcr-1-positive Escherichia coli on public beaches, and infectious threat emerging in recreational waters. Antimicrob Agents Chemother, 2017; 61:e00234-17.

14.

Garcia-Graells

, De Keersmaecker

SCJ

, Vanneste

, Pochet

, Vermeersch

, Roosens

, Dierick

, Botteldoorn

. Detection of plasmid-mediated colistin resistance, mcr-1 and mcr-2 genes, in Salmonella spp. isolated from food at retail in Belgium from 2012 to 2015. Foodborne Pathog Dis, 2018; 15:114–117.

15.

Larsen

, Cosentino

, Rasmussen

, Friis

, Hasman

, Marvig

, Jelsbak

, Sicheritz-Pontén

, Ussery

, Aarestrup

, Lund

. Multilocus Sequence Typing of total-genome-sequenced bacteria. J Clin Microbiol, 2012; 50:1355–1361.

16.

Leekitcharoenphon

, Hendriksen

, Hello

, Weill

F-X

, Baggesen

, Jun

S-R

, Ussery

, Lund

, Crook

, Wilson

, Aarestrup

. Global genomic epidemiology of Salmonella enterica Serovar Typhimurium DT104. Appl Environ Microbiol, 2016; 82:2516–2526.

17.

Lentz

, Lima-Morales

, Cuppertino

, Nunes

, Motta

, Zavascki

, Barth

, Martins

. Letter to the editor: Escherichia coli harbouring mcr-1 gene isolated from poultry not exposed to polymyxins in Brazil. Euro Surveill, 2016; 21:30267.

18.

, Yang

, Miao

, Chavda

, Mediavilla

, Xie

, Feng

, Tang

Y-W

, Kreiswirth

, Chen

, Du

Complete sequences of mcr-1-harboring plasmids from extended-spectrum-β-lactamase and carbapenemase-producing Enterobacteriaceae.

Antimicrob Agents Chemother, 2016a;60:4351–4354.

19.

, Li

, Liu

, Shi

, Jiang

, Lin

, Qiu

, Zhang

, Chen

, Zhou

, Sun

, Hu

. Clonal expansion of biofilm-forming Salmonella Typhimurium ST34 with multidrug-resistance phenotype in the southern coastal region of China. Front Microbiol, 2017; 8:2090.

20.

X-P

, Fang

L-X

, Song

J-Q

, Xia

, Huo

, Fang

J-T

, Liao

X-P

, Liu

Y-H

, Feng

, Sun

. Clonal spread of mcr-1 in PMQR-carrying ST34 Salmonella isolates from animals in China. Sci Rep, 2016b;6:38511.

21.

Liu

Y-Y

, Wang

, Walsh

, Yi

L-X

, Zhang

, Spencer

, Doi

, Tian

, Dong

, Huang

, Yu

L-F

, Gu

, Ren

, Chen

, Lv

, He

, Zhou

, Liang

, Liu

J-H

, Shen

. 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.

22.

Lozano-Leal

, Garcia-Omil

, Dalama

, Rodriguez-Souto

, Martinez-Urtaza

, Gonzalez-Esacalona

. Detection of colistin resistance mcr-1 gene in Salmonella enterica serovar Rissen isolated from mussels, Spain, 2012 to 2016. Euro Surveill, 2019; 24:1900200.

23.

Luo

, Yu

, Zhou

, Guo

, Shen

, Lu

, Huang

, Xu

, Xiao

, Li

. Molecular epidemiology and colistin resistant mechanism of mcr-positive and mcr-negative clinical isolated Escherichia coli . Front Microbiol, 2017; 8:2262.

24.

Magiorakos

A-P

, Srinivasan

, Carey

, Carmeli

, Falagas

, Giske

, Harbarth

, Hindler

, Kahlmeter

, Olsson-Liljequist

, Paterson

, Rice

, Stelling

, Struelens

, Vatopoulos

, Weber

, Monnet

. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect, 2012; 18:268–281.

25.

Mąka Ł, Popowska

Antimicrobial resistance of Salmonella spp. isolated from food. Rocz Panstw Zakl Hig, 2016; 67:343–358.

26.

Matamoros

, van Hattem

, Arcilla

, Willemse

, Melles

, Penders

, Vinh

, Hoa NT

MBAT consortium, de Jong

, Schultsz

. Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction. Sci Rep, 2017; 7:15364.

27.

Monte

, Mem

, Fernandes

, Cerdeira

, Esposito

, Galvão

, Franco

BDGM

, Lincopan

, Landgraf

. Chicken meat as a reservoir of colistin-resistant Escherichia coli strains carrying mcr-1 genes in South America. Antimicrob Agents Chemother, 2017; 61:e02718-16.

28.

Moreno

, Gomes

VTM

, Moreira

, de Oliveira

, Peres

, Silva

APS

, Thakur

, La Ragione

, Moreno

. First report of mcr-1-harboring Salmonella enterica serovar Schwarzengrund isolated from poultry meat in Brazil. Diagn Microbiol Infect Dis, 2019; 93:376–379.

29.

Nurk

, Bankevich

, Antipov

, Gurevich

, Korobeynikov

, Lapidus

, Prjibelsky

, Pyshkin

, Sirotkin

, Stepanauskas

, McLean

, Lasken

, Clingenpeel

, Woyke

, Tesler

, Alekseyev

, Pevzner

. Assembling genomes and mini-metagenomes from highly chimeric reads. J Comput Biol, 2013; 7821:158–170.

30.

Pillonetto

, Mazzetti

, Becker

, Siebra

, Arend

LNVS

, Barth

. Low level of polymyxin resistance among nonclonal mcr-1-positive Escherichia coli from human sources in Brazil. Diagn Microbiol Infect Dis, 2019; 93:140–142.

31.

Poirel

, Jayol

, Nordmann

. Polymyxins: Antibacterial activity, susceptibility testing and resistance mechanisms encoded by plasmid or chromosomes. Clin Microbiol Rev, 2017; 30:557–596.

32.

Rau

, de Lima-Morales

, Wink

, Ribeiro

, Martins

, Barth

. Emergence of mcr-1 producing Salmonella enterica serovar Typhimurium from retail meat: First detection in Brazil. Foodborne Pathog Dis, 2018; 15:58–59.

33.

Rhouma

, Beaudry

, Thériault

, Letellier

. Colistin in pig production: Chemistry, mechanism of antibacterial action, microbial resistance emergence, and One Health perspectives. Front Microbiol, 2016; 7:1789.

34.

Saavedra

, Diaz

, Wiesner

, Correa

, Arévalo

, Reyes

, Hidalgo

, de la Cadena

, Perenguez

, Montaño

, Ardila

, Ríos

, Ovalle

, Díaz

, Porras

, Villegas

, Arias

, Beltrán

, Duarte

. Genomic and molecular characterization of clinical isolates of Enterobacteriaceae harboring mcr-1 in Colombia, 2002 to 2016. Antimicrob Agents Chemother, 2017; 61:e00841-17.

35.

Schwarz

, Johnson

. Transferable resistance to colistin: A new but old threat. J Antimicrob Chemother, 2016; 71:2066–2070.

36.

Torpdahl

, Hasman

, Litrup

, Skov

, Nielsen

, Hammerum

. Detection of mcr-1-encoding plasmid-mediated colistin-resistant Salmonella isolates from human infection in Denmark. Int J Antimicrob Agents, 2017; 49:261–262.

37.

Veldman

, van Essen-Zandbergen

, Rapallini

, Wit

, Heymans

, van Pelt

, Mevius

. Location of colistin resistance gene mcr-1 in Enterobacteriaceae from livestock and meat. J Antimicrob Chemother, 2016; 71:2340–2342.

38.

Wang

, Cao

, Alali

, Cui

, Li

, Zhu

, Qang

, Meng

, Yang

. Distribution and antimicrobial susceptibility of foodborne Salmonella serovars in eight provinces in China from 2007 to 2012 (except 2009). Foodborne Pathog Dis, 2017; 14:393–399.

39.

World Health Organization. Critically Important Antimicrobials for Human Medicine, 5th rev. Geneva: World Health Organization, 2016.

40.

Zankari

, Hasman

, Cosentino

, Vestergaard

, Rasmussen

, Lund

, Aarestrup

, Larsen

. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother, 2012; 67:2640–2644.