Colistin Resistance in Monophasic Isolates of Salmonella enterica ST34 Collected From Meat-Derived Products in Spain, With or Without CMY-2 Co-production

Abstract

Colistin is a last-resort antibiotic in fighting severe infections caused by multidrug resistant Gram negative pathogens in hospitals. Zoonotic bacteria acquire colistin resistance in animal reservoirs and mediate its spread along the food chain. This is the case of non-typhoid serovars of Salmonella enterica. Colistin-resistant S. enterica in foods represents a threat to human health. Here, we assessed the prevalence of colistin-resistance in food-borne isolates of S. enterica (2014-2019; Asturias, Spain), and established the genetic basis and transferability of this resistance. Five out of 231 isolates tested (2.2%) were resistant to colistin. Four of them, belonging to the European monophasic ST34 clone of S. Typhimurium, were characterized in the present study. They were collected from pork or pork and beef meat-derived products, either in 2015 (three isolates) or 2019 (one isolate). Molecular typing with XbaI-PFGE and plasmid profiling revealed distinct patterns for each isolate, even though two of the 2015 isolates derived from the same sample. The MICs of colistin ranged from 8 to 16 mg/L. All isolates carried the mcr-1.1 gene located on conjugative plasmids of the incompatibility groups IncX4 (2015 isolates) or IncHI2 (2019 isolate). Apart from colistin resistance, the four isolates carried chromosomal genes conferring resistance to ampicillin, streptomycin, sulfonamides and tetracycline [bla _TEM-1, strA-strB, sul2, and tet(B)] and heavy metals, including copper and silver (silESRCFBAGP and pcoGE1ABCDRSE2), arsenic (arsRSD2A2BCA1D1) ± mercury (merEDACPTR), which are characteristically associated with the European ST34 monophasic clone. The 2019 isolate was also resistant to other antibiotics, comprising third generation cephalosporins and cephamycins. The latter phenotype was conferred by the bla _CMY-2 gene located on an IncI1-I(α)-ST2 plasmid. Results in the present study identified meat-derived products as a reservoir of a highly successful clone harboring transferable plasmids which confer resistance to colistin and other clinically important antibiotics. An important reduction in the number of food-borne S. enterica detected during the period of the study, together with the low frequency of colistin resistance, underlines the success of One Health initiatives, such as those implemented at the UE, to control zoonotic bacteria along the food chain and to halt the spread of antimicrobial resistance.

Keywords: European ST34 monophasic clone; IncH12; IncI2; IncX4; blaCMY–2; colistin resistance; food – borne pathogens; mcr-1.

FIGURE 1

Typing of food-borne isolates belonging to the European monophasic clone, and plasmid analysis. (A) XbaI-PFGE and dendrogram showing the similarity between the generated profiles. Lane 1, LSP 295/15; lane 2, LSP 298/15; lane 3, LSP 38/19; lane 4, LSP 237/15. (B) Plasmid profiles generated with the Kado-Liu method. Lanes C1 and C2, plasmids obtained from Escherichia coli 39R861 (NCTC 50192) and V517 (NCTC 50193) used as size standards for undigested DNA; lane 1, LSP 237/15; lane 2, LSP 295/15; lane 3, LSP 298/15; lane 4, LSP 38/19. (C) S1-PFGE. Lane B, XbaI-digested DNA of Salmonella enterica serovar Braenderup H9812 used as size standard; lane 1, LSP 38/19. Resistance plasmids are indicated in (B,C).

FIGURE 2

Genetic environment of the mcr-1.1 and bla_CMY–2 genes detected in food-borne isolates of the European monophasic clone. (A) Genetic context of mcr-1.1 carried by IncX4 plasmids in the LSP 237/15, LSP 295/15, and LSP 298/15 isolates. (B) Genetic organization of a ca. 83 kb contig carrying the mcr-1.1 gene in the IncHI2 plasmid of LSP 38/19. The alignment of this contig with homologous regions in two related plasmids either lacking mcr-1.1 (pKUSR18; accession number KM396298) or having the gene outside this region (pSE08-00436-1; accession number NZ_CP020493) was created with EasyFig BLASTn. The gray shading between the regions reflects nucleotide sequence similarity ranging from 67 to 100%, according to the scale shown at the lower right part of the figure. (C) Genetic environment of the bla_CMY–2 gene in a 35.6 kb contig of the IncI1-I(α) plasmid of LSP 38/19. The open reading frames (ORFs) are represented by arrows pointing to the direction of transcription and having different colors according to their function: red, resistance; yellow, plasmid replication, stability and maintenance; brown, conjugation; blue, DNA mobility, with the tnpA gene of IS26 shown in dark blue; gray, other roles; white, pap2, adjacent to mcr-1.1 in the IncX4 and IncHI2 plasmids, and bcl-sugE, adjacent to bla_CMY–2 in the IncI1-I(α) plasmid. The scale is different for (A–C).

FIGURE 3

Genetic organization of the tetra-resistance regions found in food-borne isolates of the European monophasic clone. The alignment was created with EasyFig BLASTn including the tetra-resistance regions of strains 105/7/03 (accession number HQ331538, shown above) and 07-2006 (accession number KR856283; shown below) for comparison. The open reading frames (ORFs) are represented by arrows pointing to the direction of transcription and having different colors according to their origin or roles: white, chromosomal ORFs of the S. Typhimurium LT2 chromosome (named according to accession number NC_003197); red, resistance; blue, DNA mobility, with the tnpA gene of IS26 shown in dark blue; yellow, plasmid replication genes; pale orange, other roles. The gray shading between the structures connects regions of nucleotide sequence similarity ranging from 97 to 100%, according to the scale shown at the lower right part of the figure.