The co-transfer of plasmid-borne colistin-resistant genes mcr-1 and mcr-3.5, the carbapenemase gene blaNDM-5 and the 16S methylase gene rmtB from Escherichia coli

We found an unusual Escherichia coli strain with resistance to colistin, carbapenem and amikacin from sewage. We therefore characterized the strain and determined the co-transfer of the resistance determinants. Whole genome sequencing was performed using both Illumina HiSeq X10 and MinION sequencers. Short and long reads were subjected to de novo hybrid assembly. Sequence type, antimicrobial resistance genes and plasmid replicons were identified from the genome sequences. Phylogenetic analysis of all IncHI2 plasmids carrying mcr-1 available in GenBank was performed based on core genes. Conjugation experiments were performed. mcr-3.5 was cloned into E. coli DH5α. The strain belonged to ST410, a type with a global distribution. Two colistin-resistant genes, mcr-1.1 and mcr-3.5, a carbapenemase gene blaNDM-5, and a 16S methylase gene rmtB were identified on different plasmids of IncHI2(ST3)/IncN, IncP, IncX3 and IncFII, respectively. All of the four plasmids were self-transmissible and mcr-1.1, mcr-3.5, blaNDM-5 and rmtB were transferred together. mcr-1-carrying IncHI2 plasmids belonged to several sequence types with ST3 and ST4 being predominant. MIC of colistin (4 μg/ml) for DH5α containing mcr-3.5 was identical to that containing the original mcr-3 variant. In conclusion, carbapenem resistance, colistin resistance and high-level aminoglycoside resistance can be transferred together even when their encoding genes are not located on the same plasmid. The co-transfer of multiple clinically-important antimicrobial resistance represents a particular challenge for clinical treatment and infection control in healthcare settings. Isolates with resistance to both carbapenem and colistin are not restricted to a given sequence type but rather are diverse in clonal background, which warrants further surveillance. The amino acid substitutions of MCR-3.5 have not altered its activity against colistin.


Materials and Methods
Recovery of the strain and in vitro antimicrobial susceptibility testing. E. coli strain WCHEC025943 was recovered from the influx mainstream of hospital sewage at West China Hospital, Chengdu, western China, in April 2017. The sewage sample was mixed with 100 ml brain heart infusion broth (Oxoid, Hampshire, UK) in a 500 ml flask. After overnight incubation at 37 °C, the culture suspension was diluted to 0. 5 McFarland standard and an 100 μl aliquot was plated onto a CHROMAgar Orientation agar plate (CHROMAgar, Paris, France) containing 4 μg/ml colistin and 16 μg/ml meropenem. The plate was then incubated at 37 °C overnight. The pink colony that represents E. coli was screened for mcr-1 as described previously 3 . Species identification was established by Vitek II (bioMérieux, Marcy-l′Étoile, France) and by MALDI-TOF MS (Bruker, Billerica, MA, USA). MICs of amikacin, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, ciprofloxacin, colistin, imipenem, meropenem, tigecycline and trimethoprim-sulfamethoxazole were determined using the broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI) 12 . For ceftazidime-avibactam, colistin and tigecycline, the breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/) were used, while the breakpoints of aztreonam were applied for aztreonam-avibactam.
Whole genome sequencing and analysis. Genomic DNA of strain WCHEC025943 was prepared using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and was subjected to whole genome sequencing using both the HiSeq X10 platform (Illumina, San Diego, CA, USA) and the long-read MinION Sequencer (Nanopore, Oxford, UK). The de novo hybrid assembly of both short Illumina reads and long MinION reads was performed using Unicycler 13 under conservative mode for increased accuracy. Complete circular contigs generated were then corrected using Plion 14 with Illumina reads for several rounds until no change was detected.
Nucleotide sequence accession numbers. Complete sequences of the chromosome and plasmids of strain WCHEC025943 have been deposited into GenBank under the accession no. CP027199 to CP027205.
Phylogenetic group typing. E. coli phylogenetic group of strain WCHEC025943 was determined using PCR as described previously 17 . Cloning of mcr-3.5. The complete coding sequence of mcr-3.5 was amplified with primers mcr3.5-up (CTGGTCGGAGATATGGGTGT) and mcr3.5-dw (GGCATTCAACATCAGAGCAA) using PrimeSTAR Max DNA Polymerase (Takara, Dalian, China). The primers were designed to amplify the gene with 222-bp upstream and 540-bp downstream sequences of mcr-3.5. Amplicons were ligated to the pMD20-T vector using the Mighty TA-cloning kit (Takara). The ligated fragments were transformed into E. coli DH5α. mcr-3.5-containing transformants were selected on LB agar plates containing 2 μg/mL colistin. The presence of mcr-3.5 in transformants was confirmed by PCR. MIC of colistin was determined for transformants carrying mcr-3.5 using the broth microdilution method 12 .
Phylogenetic analysis of IncHI2 plasmids. Complete sequences of all IncHI2 plasmids carrying mcr-1 (n = 25 in addition to pMCR1_025943 and pMCR1_020123) were retrieved from GenBank. Plasmid replicon types and sequence types of these plasmids were determined using PlasmidFinder and pMLST. Annotation was performed using Prokka 18 and antimicrobial resistance genes were identified using ResFinder. Orthologues of these plasmids were identified using OrthoFinder 19 with default settings, resulting in a sum of 56 genes representing the core genome of these 27 plasmids. The alleles of orthologous genes were aligned using MAFFT 20 and concatenated into a single sequence containing 56 aligned genes for each plasmid. The maximum-likelihood phylogenetic tree was inferred based on the core genome using RAxML 21 with a 1000-bootstrap test.

Conjugation.
Conjugation experiments were carried out in brain heart infusion broth at 30 °C using azide-resistant E. coli strain J53 as the recipient. Transconjugants were selected on LB agar plates containing 150 μg/ml sodium azide plus 2 μg/ml colistin for mcr-1.1 and mcr-3.5, plus 1 μg/ml meropenem for bla NDM-5 or plus 64 μg/ml amikacin for rmtB. Transconjugants were also selected on LB agar plates containing 150 μg/ml sodium azide plus 2 μg/ml colistin, 1 μg/ml meropenem and 64 μg/ml amikacin to examine whether mcr, bla  and rmtB could be transferred together. The presence of mcr-1.1, mcr-3.5, bla NDM-5 and/or rmtB in transconjugants was screened using PCR and Sanger sequencing. Conjugation frequency was calculated as the number of transconjugants per recipient cell.
Of note, mcr-3.5 encodes three amino acid substitutions (M23V, A456E and T488I) compared with the original mcr-3 variant on plasmid pWJ1 (GenBank accession no. KY924928). MCR-3 has been predicted to have two domains, i.e. Domain 1 (residues 1 to 172) containing 5 transmembrane α-helices and Domain 2 (residues 173 to 541), a periplasmic domain containing the putative catalytic center 5 . The amino acid substitutions of MCR-3.5 occurred in the first α-helix (M23V) of Domain 1 and in Domain 2 (A456E and T488I). However, the MIC of colistin for E. coli DH5α containing mcr-3.5 was 4 μg/ml, which was identical to that for E. coli containing the original mcr-3 variant 5 . This confirms that the amino acid substitutions of MCR-3.5 have not altered its activity against colistin as described previously 11 .
The rmtB gene in strain WCHEC025943 was carried on a 75.8-kb IncF plasmid containing an IncFII replicon (FII_47 allele) and an IncFIB replicon (a new allele). By contrast, strain WCHEC020123 did not have IncF plasmids. Like strain WCHEC020123, in strain WCHEC025943, mcr-1.1, mcr-3.5 and bla NDM-5 were carried by three plasmids belonging to different replicon types (Table 1). bla NDM-5 was carried by an IncX3 plasmid, which was almost identical to the bla NDM-5 -carrying IncX3 plasmid in strain WCHEC020123. mcr-3.5 was carried on a 50.5-kb IncP plasmid, designated pMCR3_025943, in strain WCHEC025943. pMCR3_025943 is identical to pMCR3_020123, the mcr-3.5-carrying IncP plasmid in strain WCHEC020123 11 , except that an insertion sequence, IS1294, is absent from pMCR3_025943 but is inserted in a spacer region in pMCR3_020123. mcr-1 was carried on a 265.5-kb plasmid (designated pMCR1_025943) containing both IncHI2 (ST3) and IncN replicons in strain WCHEC025943, which was larger than the 223.7-kb mcr-1-carrying IncHI2 (ST3) plasmid (pMCR1_020123) in strain WCHEC020123. The major differences between the two ST3-IncHI2 plasmids, pMCR1_025943 and pMCR1_020123, are the presence of IncN replicon and a 30-kb region containing several genes (traN, traU, traW) encoding conjugation in the former but absent from the latter. ST3-IncHI2 plasmids have been found increasingly as the vector of mcr-1 and are particularly large and complex in structure with the ability to acquire multiple antimicrobial resistance genes and additional plasmid replicons [27][28][29] . mcr-1-carrying IncHI2 plasmids were mostly found in E. coli and were also present in several other species of the Enterobacteriaceae (Fig. 1). A few IncHI2 plasmids also contain additional replicons, among which IncN replicon was the most common (Fig. 1). These plasmids were large in size (125,572 to 256,620 bp for plasmids  containing IncHI2 replicons alone and 238,539 to 369,298 bp for those containing additional replicons) and commonly carried multiple antimicrobial resistance genes (Fig. 1). Most of these plasmids belong to ST3 (n = 15) or ST4 (n = 8), while one belongs to ST14 and the sequence type is not assigned to three plasmids due to the absence of an allele for IncHI2 pMLST. This suggests that several types of IncHI2 plasmids could mediate the transfer of mcr-1 and ST3-IncHI2 is the most common type (Fig. 1). These plasmids were also aligned against pSLK172-1 (GenBank accession no. CP017632), the largest (369,298 bp) mcr-1-carrying IncHI2 plasmid, using BRIG 30 . This revealed that mcr-1-carrying IncHI2 plasmids are complex and highly variable in structure (Fig. 2). In strain WCHEC025943, the four plasmids carrying mcr-1.1, mcr-3.5, bla NDM-5 or rmtB were all self-transmissible at a 10 −3 , 10 −4 , 10 −3 and 10 −4 frequency, respectively. Alarmingly, the four plasmids could be transferred together to a single transconjugant at a 10 −6 frequency. This suggests that carbapenem resistance, colistin resistance and aminoglycoside resistance can be transferred together even when their encoding genes are located on separate plasmids.

Conclusion
The above findings suggest that carbapenem resistance, colistin resistance and high-level aminoglycoside resistance can be transferred together even when their encoding genes are not located on the same plasmid. The co-transfer of multiple clinically-important antimicrobial resistance represents a particular challenge for clinical treatment and infection control in healthcare settings, which warrant more surveillance and further studies to explore counter measures. Isolates with resistance to both carbapenem and colistin are not restricted to a given sequence type but rather are diverse in clonal background. mcr-1-carrying IncHI2 plasmids belonged to several sequence types with ST3 and ST4 being predominant.