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

The mcr-1 gene was found in 11 isolates from a Portuguese Salmonella collection (n = 1,010; 58 serotypes; 2002–15) of clinical samples, foodstuff, foodanimals and water. Mcr-1 has been located on different plasmids (IncX4/IncHI2) in pig-associated multidrugresistant, copper-tolerant S.1,4,[5],12:i:-/ST34 and S. Rissen/ST469 clones from human and pork products since at least 2011. Our data highlight dissemination of mcr-1 by successful resistant clones in Europe and raise questions about the efficacy of copper-based interventions to reduce colistin use.

The mcr-1 gene was found in 11 isolates from a Portuguese Salmonella collection (n = 1,010; 58 serotypes; 2002-15) of clinical samples, foodstuff, foodanimals and water.Mcr-1 has been located on different plasmids (IncX4/IncHI2) in pig-associated multidrugresistant, copper-tolerant S.1,4, [5],12:i:-/ST34 and S. Rissen/ST469 clones from human and pork products since at least 2011.Our data highlight dissemination of mcr-1 by successful resistant clones in Europe and raise questions about the efficacy of copper-based interventions to reduce colistin use.
Since the description of plasmid-mediated colistin resistance encoded by the mcr-1 gene in Enterobacteriaceae from multiple sources in China [1] and its worldwide dissemination mostly in animal sources [2], the use of polymyxins (colistin) in food-producing animals has been questioned in Europe because it may have an impact on human health [3].Nevertheless, data on the transmission of mcr-1-mediated colistin resistance particularly by clonal expansion are lacking [3,4].In fact, the mcr-1 gene has been found in zoonotic foodborne bacteria such as Salmonella [2] but the presence of this gene in particular successful resistant clones has not been demonstrated [3].In this study, we report the presence of the mcr-1 gene in pig-associated clinically relevant Salmonella serotypes and clones recovered from human clinical samples and pork products in Portugal, collected as early as 2011.

Laboratory investigation
We analysed a total of 1,010 Salmonella isolates of 58 serotypes from several sources (human clinical cases, food products, food-animal production settings and aquatic environments) and regions of Portugal, collected between 2002 and 2015 (Table 1).The isolates were screened for the mcr-1 gene by PCR and sequencing, using primers CLR5-F (5´-CGGTCAGTCCGTTTGTTC-3´) They included all isolates previously characterised [5,6] and recent ones from ongoing surveillance studies (data not shown) covering all serotypes, sampling dates, sources, regions, antibiotic susceptibility phenotypes/genotypes and PFGE types.The isolates positive for mcr-1 by PCR were further tested for susceptibility to colistin by the proposed broth microdilution method [7] and interpreted according to the European Committee on Antimicrobial Susceptibility Testing [8].Isolates were also subjected to standard conjugation assays using the recipient strain Escherichia coli HB101 [6].Replicon typing, pMLST, hybridisation experiments (I-CeuI/ S1-PFGE nuclease) [5,9] and detection of the insertion sequence element ISApl1 was performed in Salmonella strains and transconjugants.The presence and location of ISApl1 was determined using primers ISApl1-Fw (5´-GTCGCTTTGGACATTGGGAA-3´) and ISApl1-Rv (5´-GATTGATGTCTTGGTGCTTCGG-3´) designed as part of this study, and CLR5-R (5´-CTTGGTCGGTCTGTAGGG-3´) [1].Clonal relatedness of Salmonella strains was assessed by XbaI PFGE [5,6] and MLST [10].

Detection of mcr-1 gene in pig-associated clinically-relevant clones
The mcr-1 gene was detected in 11 (1.1%) of the 1,010 Portuguese Salmonella isolates, recovered from human clinical sources and pork food products from across the country (Table 1, Table 2).This gene had 100% homology with the first published mcr-1 sequence in an Escherichia coli strain from China (GenBank accession number: KP347127) [1], which was further described in diverse other Enterobacteriaceae including sporadic Salmonella isolates from European countries (France, the Netherlands, Spain, the United Kingdom) [2,[11][12][13][14].In most of these studies, detection of mcr-1 gene was only performed in colistin-resistant isolates.This impairs the determination of its real prevalence because the gene may be silent, as described in one E. coli strain [15].All our isolates carrying the mcr-1 gene presented a minimum inhibitory concentration (MIC) of 4-8 mg/L for resistance to colistin (Table 2).
During the study period (2002 to 2015), Salmonella isolates harbouring the mcr-1 gene were only recovered between 2011 and 2015 and originated from human clinical sources (0.8%, n = 4/522) and pork products, mostly from slaughterhouses, (2.4%, n = 7/296) (Table 1).Colistin has been widely used in veterinary medicine, particularly in food-producing animals, primarily in pigs [16,17].The available data from 2004 to 2006 had already shown high use of colistin for foodproducing animals in Portugal [18], which is one of the European countries with highest consumption of polymyxins that has been increasing in the last years (2011-13) [3,19].Taking into account the current picture of colistin use in Portugal, the detection of mcr-1 in the most recent collections and in pork products is of concern.Nevertheless, data on chronology, current prevalence of the mcr-1 gene and its evolution in bacteria from animals, food and humans are lacking [3].
Of note, all S. 1,4, [5],12:i:-and S. Rissen mcr-1-carrying isolates were co-resistant to antibiotics used in a human and/or veterinary context and carried diverse metal tolerance genes, remarkably those conferring tolerance to copper (all carrying pcoD + silA on the chromosome) (Table 2), a feed additive mostly used for pigs or piglets in Europe.The fact that these successful clones presented higher tolerance to copper, as previously demonstrated [6,20], can contribute to their selection and wider expansion with potential repercussions for mcr-1 transmission.

Location of mcr-1 gene in diverse plasmid backbones
The mcr-1 gene was located on two plasmid types, IncX4 (n = 5; 35 kb; 4 transferable) and IncHI2 (n = 6), either of ST4 subtype (n = 3; 200-300 kb; all transferable) or non-typeable (n = 3; 120-125 kb; all nontransferable) and mostly associated with the ISApl1 transposable element (Table 2).IncHI2/ST4 and IncX4 plasmids have been widely implicated in the spread of mcr-1 gene in diverse Salmonella serotypes and other Enterobacteriaceae in European and non-European countries, both from human and animal sources [2,[12][13][14].Transferability of the mcr-1 gene was achieved from S. Rissen (n = 1) and S. 1,4, [5],12:i:-(n = 6) isolates and was associated with a 32-64-fold increase in the colistin MIC and, in some isolates, with acquisition of   b PFGE types are designated by capital letters and include previously described types [5,6] and types described for the first time in this study.The human clinical isolates (n = 4 from four patients) were recovered from three hospitals, and pork products (n = 7) were recovered from six slaughterhouses and one meat production unit.
d Screening for genes encoding tolerance to metals were done by PCR [20].Metal tolerance genes that were not observed in all the isolates are presented between brackets; Metal tolerance genes transferred by conjugation are underlined.All pcoD + silA genes were chromosomally located.
f Plasmid types carrying the mcr-1 gene transferred by conjugation are underlined.
resistance to other antibiotics and metals tolerance genes (Table 2).The fact that successful MDR S. 1,4, [5],12:i:-and S. Rissen clones have the ability to acquire plasmids carrying the mcr-1 gene is of concern because colistin resistance may contribute to their further expansion, particularly in the pig reservoir.In addition, those strains could act as reservoir of mcr-1-carrying plasmids with a broad host range enhancing colistin resistance transmission for other clinically relevant bacteria sharing the same ecological niche.

Conclusions
This study has evidenced the acquisition of mcr-

Table 1
Salmonella isolates from different sources by year and presence of the mcr-1 gene, Portugal, 2002-2015 (n = 1,010) The serotypes of Salmonella isolates are presented only for those among which mcr-1-positive ones were detected.
a b Other studied food products comprised: poultry, beef, cow, quail, clam and cooked meals.

Table 2
Characterisation of Salmonella isolates recovered from clinical and food samples and carrying the mcr-1 gene, Portugal, 2011-2015 (n = 11) [3]arrying plasmids by two clinically relevant MDR and copper-tolerant clones of S. 1,4,[5],12:i:-and S. Rissen, strongly associated with pork food products and which were dominant in the collection studied.The detection of S. 1,4,[5],12:i:-from human infections, already in 2011, is also of note, suggesting long-term dissemination of this resistance gene in humans in Portugal.Finally, the detection of mcr-1 in copper-tolerant clones raises questions about the efficacy of recently suggested metal-based interventions (e.g.copper) to reduce the use of colistin and contain mcr-1 dissemination[3].