Identification of a novel plasmid-mediated colistin-resistance gene , mcr-2 , in Escherichia coli , Belgium , June 2016

BB Xavier 1 2 3 , C Lammens 1 2 3 , R Ruhal 1 2 3 , S Kumar-Singh 1 3 4 , P Butaye 5 6 7 , H Goossens 1 2 3 , S Malhotra-Kumar 1 2 3 1. Laboratory of Medical Microbiology, Wilrijk, Belgium 2. Vaccine & Infectious Disease Institute, Wilrijk, Belgium 3. University of Antwerp, Wilrijk, Belgium 4. Molecular Pathology group, Cell Biology and Histology, Wilrijk, Belgium 5. Ghent University, Faculty of Veterinary Medicine, Ghent, Belgium 6. CODA-CERVA, Brussels, Belgium 7. Ross University School of Veterinary Medicine, Basseterre, Saint Kitts and Nevis


mcr-2 is harboured on IS1595 with likely origins in Moraxella spp.
mcr-2-harbouring plasmids from all three E. coli isolates were analysed.The mobile element harbouring mcr-2 was identified as an IS element of the IS1595 superfamily, which are distinguished by the presence of an ISXO2-like transposase domain [10].
We also identified a 297 bp open reading frame downstream of mcr-2 on this element, which encodes a PAP2 membrane-associated lipid phosphatase with 41% identity to Moraxella osloensis phosphatidic acid phosphatase (71% query coverage).Interestingly, a blastn search of the IS1595 backbone, after removal of the mcr-2 and pap2 phosphatase gene sequences, identified a single hit to Moraxella bovoculi strain 58069 (GenBank accession number CP011374) genomic region

Figure 1
Genetic organisation and structure of the mcr-2-harbouring plasmid pKP37-BE from a colistin-resistant Escherichia coli isolate not harbouring mcr-1, Belgium, June 2016 The plasmid map was generated using GenomeVx [23].
Plasmid pKP37-BE isolated from one of the porcine ST10 E. coli isolates was found to have a size of 35,104 bp, 41.3% GC content and 56 protein-encoding gene sequences (RAST) (Figure 1); European Nucleotide Archive accession numbers PRJEB14596 (study) and LT598652 (plasmid sequence).
Apart from IS1595, pKP37-BE did not carry any other resistance genes and the plasmid backbone was highly similar to Salmonella enterica subsp.enterica serovar Heidelberg plasmid pSH146_32 (GenBank accession number JX258655), with 98% identity and 90% query coverage.Several Salmonella-associated virulence genes were found on pKP37-BE, including virB/D4 that encodes a type 4 secretion system [11].

Structure predictions and phylogenetic analyses of the MCR-2 protein
MCR-2 protein was predicted to have two domains, with domain 1 (1 to 229 residues) as a transporter and domain 2 (230 to 538 residues) as a transferase domain (Figure 2).

Discussion
Identification of plasmid-mediated colistin resistance represents a paradigm shift in colistin-resistance mechanisms, which until recently were restricted to chromosomal mutations and vertical transmission.Since mcr-1 conferring plasmid-mediated colistin resistance was first detected in China, mcr-1 has been identified in 30 countries across five continents [14][15][16][17] (Figure 4).

Figure 2 MCR-2 and MCR-1 predicted tertiary structures
RaptorX [24] was used to generate the structures.For both MCR-2 and MCR-1, domain 1 was predicted to be a transporter and domain 2 a phosphoethanolamine transferase (sulfatase).
Our analysis identified a novel plasmid-mediated phosphoethanolamine transferase-encoding gene, mcr-2, which was detected at an even higher prevalence than that of mcr-1 among colistin-resistant porcine E. coli in our study.We were, however, limited by small sample numbers.It should also be noted that the calves and piglets were from different regions of the country (calves from Wallonia and piglets from Flanders).
Phylogenetic analysis of MCR-2 provided strong evidence that this protein was distinct from MCR-1, and that it might have originated from Moraxella catarrhalis.The latter set of data are further strengthened by the fact that mcr-2 is co-harboured with a lipid phosphatase gene that shows highest homology to a phosphatase from Moraxella spp., and that the genetic element IS1595 harbouring these two genes might itself have originated from Moraxella spp.While Moraxella spp.are not polymyxin producers, this bacterial genus is known to be intrinsically resistant to polymyxins [18] and potential intergeneric transfer of mcr-2 from co-habiting Moraxella spp. of animal, human or environmental origin is therefore highly likely.Phosphoethanolamine transferases are housekeeping enzymes that catalyse the addition of the phosphoethanolamine moiety to the outer 3-deoxy-Dmanno-octulosonic acid (Kdo) residue of a Kdo(2)-lipid A [19].The fact that we did not identify any chromosomal mutations in the known colistin resistance-conferring genes in our E. coli isolates (by whole genome sequencing, data not shown) additionally supports the role of the acquired phosphoethanolamine transferase in conferring colistin resistance.
Finally, the high transfer frequency of the mcr-2-harbouring IncX4 plasmid might underlie the higher prevalence of mcr-2 in our porcine isolates.In the three mcr-2 harbouring isolates analysed, IS1595 showed presence of direct repeats and a complete tnpA gene, while inverted repeats were not found (data not shown).However, the carrier plasmid IncX4 is itself highly transmissible, showing 10 2 -10⁵-fold higher transfer frequencies than, for instance, epidemic IncFII plasmids, as shown previously [20] as well as in our own transconjugation experiments.Importantly, a lack of fitness-burden of IncX4 carriage on bacterial hosts [20] Figure  Maximum likelihood tree generated by bootstrap analysis using 1,000 replicates.The analysis was carried out using CLC Genomics workbench v9.0.1 (clcbio, Qiagen) in-built tool for this evolutionary relationship with other related sequences.Branch length is proportional to the number of substitutions per site.Bootstrap values are indicated in the nodes.
makes this plasmid replicon a highly effective vehicle for dissemination of mcr-2.IncX4 plasmids have also been previously shown to harbour mcr-1 [21] as well as extended spectrum beta-lactamase genes, bla CTX- M [20].Interestingly, the pKP37-BE backbone, which likely originated from Salmonella spp., harboured a battery of virulence genes including the virB4/D4 genes encoding a type-IV secretion system that has been shown to mediate downregulation of host innate immune response genes and an increased bacterial uptake and survival within macrophages and epithelial cells [11].Outer membrane modifications leading to colistin resistance have been shown to attenuate virulence [22]: whether these co-harboured virulence genes are able to compensate the pathogenic abilities of colistin-resistant E. coli remains to be explored.
Taken together, these data call for immediate inclusion of mcr-2 screening in ongoing molecular epidemiological surveillance to gauge the worldwide dissemination of mcr-2 in both human and animal colistin-resistant Gram-negative bacteria of medical importance.

* Authors' correction
The number of countries in which mcr-1 has been identified was updated to 32 and supporting references were added on 11 July 2016.The references in the article were renumbered accordingly.

3
Phylogenetic analysis of the entire MCR-2 protein sequence