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Eurosurveillance, Volume 21, Issue 26, 30 June 2016
Rapid communication
Campos, Cristino, Peixe, and 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

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Citation style for this article: Campos J, Cristino L, Peixe L, Antunes P. 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(26):pii=30270. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.26.30270

Received:17 June 2016; Accepted:30 June 2016


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 food-borne 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´) [1] and Mcr1-Rv2 (5´-CCAGCGTATCCAGCACATTT-3´) [this study].

Table 1

Salmonella isolates from different sources by year and presence of the mcr-1 gene, Portugal, 2002–2015 (n = 1,010)

Source (number of isolates) Years Isolates tested for mcr-1 (serotype/number of isolates)a mcr-1-positive isolates (serotype/number of isolates)a
Human clinical cases (n = 522) 2002–10 258 0
2011–12 155 (S. 1,4,[5],12:i:-/n = 75) 4 (S. 1,4,[5],12:i:-)
2013–15 109 0
Food products (n = 413)
Pork (n = 296) 2002–13 44 0
2014–15 252 (S. 1,4,[5],12:i:-/n = 130; S. Rissen/n = 23) 7 (S. 1,4,[5],12:i:-/n = 5; S. Rissen/n = 2)
Otherb (n = 117) 2002–15 117 0
Food production animals (n = 58)
Pigs/piggeries (n = 54) 2006–08 54 0
Aquacultures (n = 4) 2010–12 4 0
Aquatic environment (n = 17) 2002–11 17 0

a The serotypes of Salmonella isolates are presented only for those among which mcr-1-positive ones were detected.

b Other studied food products comprised: poultry, beef, cow, quail, clam and cooked meals.

The 1,010 isolates comprised the most frequent worldwide Salmonella serotypes (n = 256 S. Typhimurium and n = 34 S. Enteritidis), but also emerging serotypes (n = 436 S. 1,4,[5],12:i:- and n = 93 S. Rissen) or serotypes less frequently detected in European surveillance studies (n = 191 isolates from 54 different serotypes). 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) [2,11-14]. 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).

Table 2

Characterisation of Salmonella isolates recovered from clinical and food samples and carrying the mcr-1 gene, Portugal, 2011–2015 (n = 11)

Serotypea
(number of isolates)
Source-origin
(number of isolates)
Clone designation;
ST(eBG);
PFGE-typeb
(number of isolates, source)
Year/
Regions
Antibiotic resistance phenotype/genotypec
(number of isolates)
Metal tolerance genes
(number of isolates)d
Plasmid-mediated colistin resistance mcr-1
Colistin MIC-mg/L, donor (transconjugant/
number of isolates)e
Plasmid type carrying mcr-1 gene (pMLST, Kb)f
(number of isolates)
ISApl1 upstream of mcr-1
(number of isolates)
1,4,[5],12:i:-
(n = 9)
Clinical
faeces/blood
(n = 4)
European clone;
ST34(eBG1);
C (n = 1, 1 hospital),
E (n = 3, 2 hospitals)
2011–12
North
AMP, (GEN), STR, SUL, TET/
blaTEM, [aac(3)-IV], strA-strB, sul2, tet(B)
(n = 4)
pcoD + silA
+ merA + (terF)
(n = 4)
4–8
(4–8/n = 1)
X4 (35) (n = 1)
HI2 (NT, 120–125) (n = 3)
No (n = 1)
Yes (n = 3)
Pork
carcass
(n = 4)
European clone;
ST34(eBG1);
A (n = 1, 1 slaughterhouse),
B (n = 2, 2 slaughterhouses),
F (n = 1, 1 slaughterhouse)
2014–15
North,
Centre
AMP, (CLO), (CIP, PEF), STR, SUL, TET, (TMP)/
blaTEM, (floR)/(catA-cmlA), (aadA1/aadA2) strA-
strB, sul1-sul3/sul2, tet(A)/tet(B), (dfrA1/dfrA12)
(n = 4)
pcoD + silA
+ (merA) + (terF)
(n = 4)
4–8
(4–8/n = 4)
X4 (35) (n = 2)
HI2 (ST4, 230–300) (n = 2)
No (n = 2)
Yes (n = 2)
Pork
meat
(n = 1)
European clone; STNew/ Single locus variant of ST34;
B (n = 1, 1 meat production unit)
2015
South
AMP, STR, SUL, TET/
blaTEM, strA-strB, sul2, tetB
(n = 1)
pcoD + silA + merA + terF (n = 1) 4
(4/n = 1)
HI2 (ST4, 200)
(n = 1)
No (n = 1)
Rissen
(n = 2)
Pork
carcass
(n = 2)
ST469(eBG66); N
(n = 2, 2 slaughterhouses)
2014–15
North
AMP, CLO, STR, SUL, (TET), TMP/
blaTEM, cmlA, aadA1, sul1-sul3, [tet(A)], dfrA1 (n = 2)
pcoD + silA + merA
(n = 2)
4
(4/n = 1)
X4 (35) (n = 2) No (n = 2)

AMP: ampicillin; CIP: ciprofloxacin; CLO: chloramphenicol; GEN: gentamicin; MIC: minimum inhibitory concentration; PEF: pefloxacin; pMLST: plasmid multilocus sequence type; STR: streptomycin; SUL: sulfamethoxazole; TET: tetracycline; TMP: trimethoprim.

a The serotypes of Salmonella isolates were determined by classical serotyping, performed at the National Centre of Salmonella (INSA, Lisbon, Portugal) and/or PCR assay for determination of S. 4,[5],12:i:- [5].

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.

c Antimicrobial susceptibility was evaluated by disc diffusion assay. Variable antibiotic resistance phenotypes and genotypes are presented between brackets; Antibiotic resistance patterns and genes transferred by conjugation are underlined; In two S. 1,4,[5],12:i:- isolates, transfer of genes strA-strB and/or blaTEM was observed; Some genes were included on class 1 integrons (1,700bp (dfrA1-aadA1) or 2,000bp (dfrA12-orfF-aadA2)); Integrons were located on the chromosome in S. Rissen (n = 2) and on the IncHI2/ST4 plasmid in S. 1,4,[5],12:i:- isolates (n = 2).

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.

e Recipient strain used in conjugation assays: Escherichia coli HB101 (azide sodium, resistant to streptomycin and kanamycin); colistin MIC = 0.125 mg/L).

f Plasmid types carrying the mcr-1 gene transferred by conjugation are underlined.

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 food-producing 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].

The 11 mcr-1-positive Salmonella isolates belonged to the serotypes S. 1,4,[5],12:i:- and S. Rissen (Table 2), which have been strongly associated with pig production and caused human infections in Europe [5,6,20-22] including in Portugal [23]. In both cases, we found them associated with particular successful multidrug-resistant (MDR) clonal lineages, either of the S. Rissen/ST469 clone or the S. 1,4,[5],12:i:-/ST34 European clone that is currently spreading epidemically in European countries [5,20,22] and has been dominant in our Salmonella collection for the last years [5,6,20; unpublished data]. A previous report on mcr-1 in the clinically relevant Salmonella serotype S. Typhimurium/ST34 was associated with travel to South-East Asia [13].

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 non-transferable) 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 [2,12-14]. 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 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-1-carrying 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].


Acknowledgements

This work was supported by FCT/MEC (Fundação para a Ciência e a Tecnologia / Ministério da Educação e Ciência) through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 [grant number UID/MULTI/04378/2013 – POCI/01/0145/FERDER/007728]. JC is supported by a Ph.D. fellowship from Fundação para a Ciência e a Tecnologia (grant number SFRH/BD/93091/2013).

The authors would like to thank to Linda Cavaco for providing the mcr-1-positive control DNA. We are also very grateful to Jorge Machado (INSA, Lisbon, Portugal) for the Salmonella serotyping.

Conflict of interest

None declared.

Authors’ contributions

JC, LP and PA designed the study and analysed epidemiological, microbiological and molecular data, JC and LC performed the phenotypic and molecular assays, JC and PA wrote the first draft of the manuscript, PA and LP participated in the coordination and concept of the manuscript and revised the final version.


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