mcr-1 and mcr-2 variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015

Abstract

Objectives

To determine the occurrence of mcr-1 and mcr-2 genes in Gram-negative bacteria isolated from healthy pigs in Great Britain.

Methods

Gram-negative bacteria (n = 657) isolated from pigs between 2014 and 2015 were examined by WGS.

Results

Variants of mcr-1 and mcr-2 were identified in Moraxella spp. isolated from pooled caecal contents of healthy pigs at slaughter collected from six farms in Great Britain. Other bacteria, including Escherichia coli from the same farms, were not detected harbouring mcr-1 or mcr-2. A Moraxella porci-like isolate, MSG13-C03, harboured MCR-1.10 with 98.7% identity to MCR-1, and a Moraxella pluranimalium-like isolate, MSG47-C17, harboured an MCR-2.2 variant with 87.9% identity to MCR-2, from E. coli; the isolates had colistin MICs of 1–2 mg/L. No intact insertion elements were identified in either MSG13-C03 or MSG47-C17, although MSG13-C03 harboured the conserved nucleotides abutting the ISApl1 composite transposon found in E. coli plasmids and the intervening ∼2.6 kb fragment showed 97% identity. Six Moraxella osloensis isolates were positive for phosphoethanolamine transferase (EptA). They shared 62%–64.5% identity to MCR-1 and MCR-2, with colistin MICs from 2 to 4 mg/L. Phylogenetic analysis indicated that MCR and EptA have evolved from a common ancestor. In addition to mcr, the β-lactamase gene, bla_BRO-1, was found in both isolates, whilst the tetracycline resistance gene, tetL, was found in MSG47-C17.

Conclusions

Our results add further evidence for the mobilization of the mcr-pap2 unit from Moraxella via composite transposons leading to its global dissemination. The presence of mcr-pap2 from recent Moraxella isolates indicates they may comprise a reservoir for mcr.

Introduction

The emergence of colistin resistance genes (mcr-1 and mcr-2) on mobile genetic elements in Escherichia coli compromises options for treatment of highly resistant infections.^1,2 We have recently reported the presence of mcr-1 in enteric bacteria from livestock in the UK,³ but not mcr-2.

In this study, we screened the Gram-negative flora of pigs in Great Britain (GB) for the presence of genes related to mcr-1 and mcr-2, using WGS. Moraxella spp. are less common members of the enteric Gram-negative bacteria infrequently causing infection in man, e.g. Moraxella osloensis, or pigs, e.g. Moraxella porci, but also including commensal organisms such as Moraxella pluranimalium.^4–6 The results in this paper extend suggestions that Moraxella spp. could be the source of mcr colistin resistance genes⁷ and describe their occurrence in pig populations from 2014 to 2015.

Materials and methods

Sample collections investigated

A collection of 657 Gram-negative bacteria were isolated from caecal contents of healthy pigs at abattoirs from 57 farms in GB during 2014–15. The bacteria, which included species of Escherichia, Salmonella, Klebsiella and Moraxella, were isolated from pooled pig caecal contents (10 pigs/farm) plated on Brilliance UTI agar (Oxoid Ltd) containing 1 mg/L cefotaxime, 1 mg/L ciprofloxacin or no antibiotic. Type strains M. pluranimalium (248-01^T/DSM-22804) and M. porci (SN9-4M^T/DSM-25326) from the Leibniz Institute DMSZ (Germany), which had previously been isolated from a healthy pig in Spain⁵ and a diseased pig,⁶ were included as reference isolates.

Phenotypic characterization of Moraxella

The MIC of colistin for the Moraxella spp. isolates was determined using the agar dilution method,⁸ because we found that isolates clumped in cation-adjusted Mueller–Hinton broth, and this led to inconclusive results. The MIC of ampicillin and tetracycline for Moraxella isolates was determined using gradient strips (M.I.C.Evaluator, Oxoid Ltd) according to the manufacturer’s instructions. E.coli ATCC 25922 was included as control.

WGS analysis

DNA was extracted and WGS performed on the 657 Gram-negative isolates, which contained a proportion of non-E. coli, using the Illumina HiSeq platform, as described previously.⁹ The presence of mcr-1 or mcr-2 in the whole-genome sequences of isolates was determined using the APHA SeqFinder pipeline³ by mapping unassembled reads. For mcr-1-/mcr-2-containing isolates, sequences were assembled using SPAdes 3.7.0.¹⁰ They were annotated using Prokka 1.11,¹¹ and BlastN 2.2.25+ for identity, and Jspecies¹² and the Bacterial Pan Genome Analysis pipeline¹³ were used for speciation. WGS data were deposited in the sequence read archive (SRA: PRJEB15347).

Results

Screening for colistin resistance genes

The whole-genome sequences of 657 Gram-negative bacteria recovered from pooled pig caeca from GB farms were screened for the presence of genes with similarity to mcr-1 or mcr-2. Eight isolates of Moraxella spp., with six identified as M. osloensis, one M. porci-like and one M. pluranimalium-like (Figure S1, available as Supplementary data at JAC Online), were found to harbour mcr homologues.

MCR variants and phosphoethanolamine transferase (EptA) in Moraxella spp

A new variant of MCR-1 from E.coli¹ was identified in the M. porci-like isolate, MSG13-C03, hereafter named MCR-1.10 (MF176238). In comparison with nine other MCR-1 variants showing changes at the amino acid level, MCR-1.10 showed seven unique amino acid substitutions with three changes occurring in the N-terminal protein region, i.e. 98.7% amino acid identity (Figure S2A). Analysis of the M. porci-type isolate SN9-4M^T identified a protein with 62.5% identity to MCR-1 and 61.2% identity to EptA from Paenibacillus sophorae. Analysis of the M. pluranimalium type isolate 248-01^T identified an MCR-2 variant (named MCR-2.1; MF176239) that possessed eight amino acid substitutions (98.5% amino acid identity) compared with MCR-2 from E. coli, as reported elsewhere (Figure S2B).⁷ The M. pluranimalium-like MSG47-C17 also harboured a new variant of MCR-2, named MCR-2.2 (MF176240), which showed 65 amino acid substitutions, i.e. 87.9% amino acid identity, compared with MCR-2 from E. coli² and 66 amino acid substitutions, i.e. 87.8% amino acid identity, compared with MCR-2.1 from M. pluranimalium 248-01^T (Figure S2B). The M. osloensis translated eptA genes had 97.8%–98% amino acid identity to EptA from P. sophorae compared with 62%–64% with MCR-1 and MCR-2 (Figure 1). The eight amino acids important for catalytic activity,¹ as well as the cysteine residues that form disulphide bridges,¹⁴ were conserved in all Moraxella MCR variants and EptA sequences, with the exception of EptA from E. coli. Using CLSI guidelines for non-Enterobacteriaceae (>8 mg/L), only the EptA-containing M. porci reference strain was colistin resistant (Table S1). A phylogenetic tree based on amino acid sequences (Figure 1) showed all MCR-1 and MCR-2 homologues to cluster together; these were distinct from the Moraxella EptA cluster. Nevertheless, Moraxella EptA was more closely related to MCR than EptA from the other bacteria examined, which suggested that although these proteins are distinct they had likely evolved from the same common ancestor.

Figure 1.

Phylogenetic tree of EptA, MCR-1-like and MCR-2-like amino acid sequences identified in Moraxella species and those present in other bacteria.

Genomic location of mcr

Chromosomal arrangements of genes flanking mcr and eptA in the Moraxella GB isolates and Moraxella type strains were compared. Comparison of chromosomal genes flanking mcr and eptA indicated gene synteny in the Moraxella mcr region, which was distinct from the eptA region (Figure 2a). In both MSG13-C03 and MSG47-C17 from GB, the mcr genes were downstream of a site-specific recombinase (SSR); whereas in the M. osloensis (including a human isolate) and the M. porci type isolate, the eptA gene was located downstream of a dissimilar hypothetical protein or an RHS type-4 secretion protein. An intervening gene, Carboxylesterase A precursor, was located between SSR and mcr-2.1 in M. pluranimalium 248-01^T. The downstream PAP2 proteins in MSG13-C03 and MSG47-C17 shared 92% identity, but they only showed 42.3%–57.9% identity to PAP2 in the other Moraxella isolates.

Figure 2.

(a) Chromosomal arrangement of eptA, mcr-1-like and mcr-2-like genes in Moraxella species. mcr and other flanking genes have been annotated; hypothetical genes have been left blank. The shadow parallelograms between each sequence denote sequence identity. (b) Alignment of the mcr-1.10-pap2 unit in M. porci-like MSG13-C03 with E. coli plasmids harbouring mcr-1-pap2. Alignment of the mcr-1-pap2 region in E. coli plasmid pECJS-59-244 (KX084394), M. porci-like MSG13-C03 and E. coli KX528699. The upstream and downstream flanking sequences are shown, with the underlined bases indicating 100% identity, and the conserved trinucleotide and dinucleotide sequences are shown in red.

Comparison of the Moraxella mcr region with E. coli plasmids harbouring the mcr-1-pap2 composite transposon showed a 2618 bp fragment was shared with 96.5% identity. No intact copies of ISApl1 were detected in MSG13-C03, but the ATA trinucleotide abutting the upstream ISApl1¹⁵ and the CG dinucleotide abutting the downstream ISApl1¹⁵ were conserved (Figure 2b). The region encompassing mcr-2.2 (1637 bp) in MSG47-C17 had the highest (85.6%) identity to the mcr-2-harbouring E. coli pKP37-BE plasmid, with only a small portion (86 bp) of the PAP2 gene conserved in the latter (Figure S3). However, the insertion element ISEc69 belonging to the IS1595 family, and associated with mcr-2 in pKP37-BE,² was not found in MSG47-C17. The homologous sequence (CAAGTTTAAT) downstream of the PAP2 gene in MSG13-C03 and MSG47-C17 was identical to pECJS-59-244, which harbours the mcr-1 composite transposon.

Both MSG13-C03 and MSG47-C17 harboured the bla_BRO-1 β-lactamase gene reported in Moraxella catarrhalis¹⁶ and the tetracycline resistance gene, tetL, from Gram-positive bacteria¹⁷ was identified in MSG47-C17 (Figure S4). The ampicillin MIC was 32 mg/L for MSG13-C03 and 1 mg/L for MSG47-C17. The tetracycline MIC for M. pluranimalium-like MSG47-C17 was 1 mg/L.

Discussion

We have previously reported on the presence of mcr-1 in E. coli (n = 1050) from healthy and diseased pigs in GB,^3,18 where four mcr-1-positive E. coli were characterized. This collection was further found to be negative for the presence of mcr-2 (data not shown). In this study, we extended on previous work by examining whole-genome sequences of 657 Gram-negative bacteria isolated from healthy pigs from 57 different farms, between 2014 and 2015, which were screened for the presence of mcr-1 and mcr-2. Eight Moraxella from six farms, which did not harbour mcr-positive E. coli, were identified harbouring eptA and homologues of mcr. Six were EptA-harbouring M. osloensis, whilst an M. porci-like isolate harboured the variant MCR-1.10 and an M. pluranimalium-like isolate harboured the MCR-2.2 variant. It has been previously suggested that Moraxella spp. could have been the source of mcr-like genes owing to the high amino acid identity to EptA in this species.^2,14 The data presented here and recent data from others⁷ substantiate this speculation, with our data also showing conservation of the mcr-pap2 unit in different Moraxella species. This phenomenon is not new and would be similar to mobilization of the chromosomal bla_CTX-M β-lactamase genes from Kluyvera to other Enterobacteriaceae,¹⁹ where it is now widely disseminated. Gene synteny within the mcr-pap2 region indicated this unit to be intrinsic to the Moraxella spp. genome. In addition, conservation within recent Moraxella pig isolates of the ∼2.6 kb mcr-pap2 unit and the dinucleotide/trinucleotide sequences that abut the mcr composite transposon in E. coli suggests Moraxella may comprise a natural reservoir of mcr, which may be mobilized via IS elements.

In conclusion, our results add further evidence that Moraxella was the likely progenitor, and the mcr-pap2 unit was mobilized from Moraxella,^7,14 being disseminated widely via a composite transposon. It also indicates that mcr-harbouring Moraxella occur in pig populations and may be a recurrent source of mcr.