Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle , Spain , September 2015

M Hernández 1 2 3 , MR Iglesias 3 4 , D Rodríguez-Lázaro 3 5 , A Gallardo 4 , NM Quijada 1 , P Miguela-Villoldo 6 , MJ Campos 7 , S Píriz 8 , G López-Orozco 9 , C de Frutos 10 , JL Sáez 9 , M Ugarte-Ruiz 6 , L Domínguez 6 11 , A Quesada 4 12 1. Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico Agrario de Castilla y León, Valladolid, Spain 2. Departamento de Ingeniería Agrícola y Forestal, Tecnología de los Alimentos, Escuela Técnica Superior de Ingenierías Agrarias, Universidad de Valladolid, Palencia, Spain 3. These authors contributed equally to the manuscript 4. Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain 5. Área de Microbiología, Departamento de Biotecnología y Ciencia de los Alimentos, Universidad de Burgos, Burgos, Spain 6. VISAVET Health Surveillance Centre, Universidad Complutense, Madrid, Spain 7. MARE Marine and Environmental Sciences Centre, Instituto Politécnico de Leiria, Peniche, Portugal 8. Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain 9. Subdirección General de Sanidad e Higiene Animal y Trazabilidad, Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente, Madrid, Spain 10. Laboratorio Central de Veterinaria, Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente, Algete, Spain 11. Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain. 12. INBIO G+C, Universidad de Extremadura, Cáceres, Spain

Colistin resistance genes mcr-3 and mcr-1 have been detected in an Escherichia coli isolate from cattle faeces in a Spanish slaughterhouse in 2015.The sequences of both genes hybridised to same plasmid band of ca 250 kb, although colistin resistance was non-mobilisable.The isolate was producing extendedspectrum beta-lactamases and belonged to serotype O9:H10 and sequence type ST533.Here we report an mcr-3 gene detected in Europe following earlier reports from Asia and the United States.
Very recently, in June 2017, Yin et al. detected a third mobile colistin resistance gene mcr-3 on an IncHI2-type plasmid, pWJ1, in a porcine E. coli isolate from Malaysia [1].The authors also identified similar elements in a shotgun genome sequence of a human Klebsiella pneumoniae isolate from Thailand and a human Salmonella enterica serovar Typhimurium isolate from the United States [1].
We found an Escherichia coli isolate carrying the mcr-3 gene among other isolates expressing colistin resistance.It was sampled in cattle faeces at the time of slaughter in Spain in September 2015.The aim of this paper is to describe the presence of mcr-3 in Europe in a strain also carrying the mcr-1 gene.

Screening of bovine samples for colistinresistant bacteria
The VISAVET Health Surveillance Centre in Madrid has been carrying out the national surveillance for detection of extended-spectrum beta-lactamase (ESBL)-producing bacteria in food-producing animals since 2014, commissioned by the Spanish Ministry of Agriculture and Fishing, Food and Environment according to Commission Implementing Decision 2013/652/ EU [2].The screening was performed at slaughterhouses during 2015 on healthy cattle younger than one year from 318 farms (caecal content of 636 animals).The procedure followed the EURL-AR recommendations for detecting ESBL-producing E. coli [3].
A total of 152 samples (47.8%) were suspected to be positive, so antimicrobial susceptibility testing was performed by Sensititre microbroth dilution using EUVSEC and EUVSEC2 plates (Trek Diagnostic Systems, US) to confirm their beta-lactamase production.The antimicrobial drugs to be included in each panel are detailed in the Commission Implementing Decision 2013/652/EU [2].Six E. coli isolates were found resistant to colistin and further characterised.Among them, five were PCR-positive for mcr-1 [4] and one isolate (ZTA15/01169-1EB1) was also PCR-positive for mcr-3 [1].All isolates presented multi-resistant phenotypes (Table 1) and lacked the mcr-2 gene [5].
Both mcr-1 and mcr-3 genes were detected by PCR using previously described primers and conditions [1,4].Minimal inhibitory concentrations were determined by using the two-fold broth microdilution reference method according to ISO 20776-1:2006 [6].The interpretation of the quantitative data was performed as described by the Commission Implementing Decision 2013/652/EU [2], EURL-AR (the EU Reference Laboratory for antimicrobial resistance in the context of animal health and food safety) and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) [7].
mcr-1 was found in a 2,074 bp-length contig, and blastn comparison against the National Center for Biotechnology Information (NCBI) database [14] revealed best match with the IncHI2-type plasmid pECJS-59-244 previously described [13].mcr-3 was found in a 4,098 bp-length contig, and blastn of the gene showed 100% coverage (1-1626/1626) and 99,94% nucleotide identity to mcr-3.A unique polymorphism (C1463T) was found in its coding sequence, giving rise to a T488I variant of the protein encoded by this gene allele, hereafter named mcr-3.2.Moreover, isolate ZTA15/01169-1EB1 contained the mutations S83L and D87N of GyrA, in complete concordance with the phenotypic results (Table 1), in addition to two beta-lactamase-encoding genes (blaCTX-M-55 and blaTEM-1) and several other resistance determinants (Table 2).CTX-M is a widely spread ESBL that could be encoded by IncHI2 and HincI1, among other plasmids [13].
Plasmid location of the mcr-1 and mcr-3.2genes from isolate ZTA15/01169-1EB1 was evidenced by nuclease S1 digestion and pulsed-field gel electrophoresis (PFGE), followed by transfer of DNA to nylon membranes and hybridisation to Dig-labelled probes (Sigma, US).Specific signals obtained by using probes for both mcr-1 and mcr-3, matched a plasmid band of ca 250 kb (Figure).Specificity of the mcr-1 probe was evidenced by using a previously characterised strain carrying mcr-1 on a 30 kb IncX4 plasmid [15].A second ca 75 kb plasmid was identified by PFGE in isolate ZTA15/01169-1EB1 (Figure).However, despite the plasmidic location of the mcr-1 and mcr-3.2genes, colistin resistance was not mobilisable by conjugation in standard conditions (overnight mating at 37 °C) to the receptor strain E. coli J53 after selection in medium with sodium azide (100 mg/L) and colistin (2 mg/L).The previously described E. coli isolate ZTA14/01057 was used as a positive control in parallel, and conjugation to the same recipient was successful with 4.2•10−2 efficiency [16].

Discussion and conclusions
The first plasmid-mediated polymyxin resistance mechanism, mcr- pneumoniae and S. enterica serovar Typhimurium from Thailand and the United States, respectively [1].We also demonstrated in 2016 the presence of mcr-1 in E. coli and S. enterica isolates from poultry and swine in Spain [15].In addition to these findings, this work describes the results of screening for multidrug-resistant E. coli (including polymyxin resistance) of bovine origin in Spain.Among the colistin-resistant isolates found, three genotypes were identified: strains carrying mcr-1 alone, strains carrying mcr-1 and mcr-3.2, and strains without any plasmidic determinants.This study shows the appearance of the colistin-resistant mcr-3 gene in Europe as early as in 2015, as well as the coexistence of two plasmid-mediated colistin resistance genes, mcr-1 and mcr-3.2 in the same cells of isolate ZTA15/01169-1EB1.Resistance is indicated by (R).mcr-1 and mcr-3 genes were detected by PCR using previously described primers and conditions [1,4].Minimal inhibitory concentrations were determined by using the two-fold broth microdilution reference method according to ISO 20776-1:2006 [6].The interpretation of the quantitative data was performed as described by the Commission Implementing Decision 2013/652/EU [2], EURL-AR (EU Reference Laboratory for antimicrobial resistance in the context of animal health and food safety) and EUCAST (The European Committee on Antimicrobial Susceptibility Testing (EUCAST) [7].
Most mcr-1, mcr-2 and mcr-3 genes are plasmidic sequences [1,4,5].Although the PFGE and further hybridisation with mcr-1-and mcr-3-specific probes did not exclude the possibility of independent carriage by two different similar-sized plasmids, genome sequencing of isolate ZTA15/01169-1EB1 only identified the IncHI2 replicon as an appropriate candidate to harbour colistin resistance genes.We therefore assume that both genes were located on the same plasmid in our isolate.
Further efforts are focused on investigating the structure of plasmids, transmission potential, gene expression and stability of the mcr-1 and mcr-3.2genes.Furthermore, the reason why co-occurrence of mcr-1 and mcr-3 genes confers low colistin resistance needs to be elucidated.

Table 1
Colistin resistance genes and antimicrobial resistance of Escherichia coli isolates of bovine origin, Spain, September 2015 (n = 6)

Table 2
Resistome and plasmid profiles of Escherichia coli ZTA15/01169-1EB1, Spain, September 2015 Resistance and plasmid determinants were identified against the ResFinder and PlasmidFinder databases, respectively [9,12].a Number of query nucleotides found in the obtained draft genome compared to the total length of each reference gene sequence deposited in the ResFinder and/or PlasmidFinder databases.
b GenBank accession number.