New Variant of mcr-3 in an Extensively Drug-Resistant Escherichia coli Clinical Isolate Carrying mcr-1 and bla_NDM-5

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The emergence of multidrug resistance (MDR) in Gram-negative pathogenic bacteria represents one of the greatest global public health threats of the 21st century. In particular, the rise in incidence of MDR Escherichia coli has coincided with an increase in cases of bacteremia infections caused by E. coli (1). The evolution of MDR E. coli has been progressive in nature, with initial emergence due to dissemination of plasmids containing extended-spectrum-β-lactamase (ESBL)-encoding genes such as bla_CTX-M-15 (2). Infection with ESBL-producing E. coli is usually treated with carbapenem antibiotics, but E. coli could acquire carbapenemase genes such as bla_NDM-5 (3) to become carbapenem resistant. The remaining possible treatment for infections caused by carbapenem-resistant isolates is primarily colistin. However, increasing use of this antimicrobial in clinical and veterinary practice has led to the emergence of mobile colistin resistance genes, mcr, which have been reported in various species of the Enterobacteriaceae family (4).

Five plasmid-borne colistin resistance genes, mcr-1 (5), mcr-2 (6), mcr-3 (7), mcr-4 (8), and mcr-5 (9), have now been reported, with mcr-3 having been identified in China in the past few months. The MCR-3 protein has only 32.5%, 31.7%, 49.0%, and 34.7% amino acid identity to MCR-1, MCR-2, MCR-4, and MCR-5, respectively (7–9), suggesting that it is not a recently evolved variant of MCR-1 but rather a distinct class of enzyme. Here we report the first ever clinical isolation of an E. coli strain carrying plasmid-borne bla_NDM-5, mcr-1, and mcr-3 genes.

E. coli strain WCHEC-LL123 was recovered from an abdominal abscess of a 43-year-old male patient in January 2017 in China. The strain was resistant to aztreonam (MIC, 16 μg/ml), ceftazidime (MIC, >512 μg/ml), ceftazidime-avibactam (MIC, >512/4 μg/ml), ciprofloxacin (MIC, 128 μg/ml), colistin (MIC, 8 μg/ml), imipenem (MIC, 32 μg/ml), meropenem (MIC, 64 μg/ml), piperacillin-tazobactam (MIC, >512/4 μg/ml), and trimethoprim-sulfamethoxazole (MIC, 64/1216 μg/ml) but was susceptible to amikacin (MIC, 8 μg/ml), aztreonam-avibactam (MIC, <0.125/4 μg/ml), and tigecycline (MIC, 0.25 μg/ml), as determined using the broth dilution method of the Clinical and Laboratory Standards Institute (10). The patient recovered after repeated surgeries, drainage of the abscess, and antimicrobial treatment with tigecycline.

A draft genome sequence of the strain was generated on the Illumina HiSeq X10 platform, which generated 5,751,013 clean reads assembled into 275 contigs (176 >1,000 bp; N50 88,902 bp) with a 50.4% GC content using SPAdes (11). Strain WCHEC-LL123 belonged to phylogenetic group A, as determined using PCR as described previously (12), and sequence type 206 (ST206), as determined using the genomic sequence to query the E. coli multilocus sequence typing database (http://enterobase.warwick.ac.uk/species/index/ecoli ). Antimicrobial resistance genes were identified from genome sequences using the ABRicate program (https://github.com/tseemann/abricate ). Strain WCHEC-LL123 had both mcr-1 and mcr-3 plasmid-borne colistin resistance genes and the bla_NDM-5 carbapenemase gene. In addition, the strain had 19 antimicrobial resistance genes mediating resistance to aminoglycosides [aac(3)-IVa, aadA1, aadA2, aadA5, aph(3′)-Ia, aph(4)-Ia], β-lactams (bla_CTX-M-14, bla_TEM-106), fosfomycin (fosA), phenicol (catA2, cmlA1, floR), quinolones (oqxA, oqxB), tetracycline [tet(A)], sulfonamides (sul1, sul2, and sul3), and trimethoprim (dfrA17).

At least 8 variants of mcr-1 have been found up to now, and the mcr-1 gene in our strain is identical to the original mcr-1 variant. However, the mcr-3 in our strain is a novel variant; compared with the original mcr-3 variant on plasmid pWJ1 (GenBank accession no. KY924928 ) it has 3 nucleotide differences (99.82% identity), which lead to 3 amino acid substitutions (M23V, A456E, and T488I). In GenBank, there are three additional mcr-3 variants in species other than Aeromonas spp., which are believed to be the origin of mcr-3. Compared to the original MCR-3, the three additional mcr-3 variants encode MCR-3 proteins with a single amino acid substitution, D295E in Klebsiella pneumoniae strain PB395 (GenBank accession no. NZ_FLWO01000034 ) from humans in Thailand, G373V in K. pneumoniae strain PB517 (GenBank accession no. NZ_FLXA01000011 ) from humans in Thailand, and M23V in Citrobacter freundii strain D36-1 (GenBank accession no. NZ_NEFW01000046 ) from swine in China. The mcr-3 variant in our strain was therefore assigned the designation mcr-3.5 here. The MIC of colistin for the E. coli transconjugant containing mcr-3.5 was 4 μg/ml (see Table 1), which was identical to that for E. coli containing the original mcr-3 variant (7). This suggests that the amino acid substitutions of MCR-3.5 have not altered its activity against colistin.

Plasmids carrying mcr-1, mcr-3.5, and bla_NDM-5 were circularized using PCR and Sanger sequencing to fill in gaps between contigs. Plasmid replicon types were determined by using the PlasmidFinder tool at http://genomicepidemiology.org/ . Conjugation experiments were carried out in broth and on filters with the azide-resistant E. coli strain J53 as the recipient. The mcr-3.5 gene was carried by a self-transmissible 52.2-kb IncP plasmid, designated pMCR3_LL123. pMCR3_LL123 is almost identical to two mcr-1-carrying IncP plasmids, both of which were recovered from sewage of the same hospital in 2015, pMCR_1511 (GenBank accession no. KX377410 ) found in a K. pneumoniae isolate (13) and pMCR_1622 (GenBank accession no. KY463452 ) found in an E. coli isolate (ST7068), but there are two differences. First, there is no mcr-3 on pMCR_1622, while a truncated mcr-3 (a remnant with the last 645 bp of the 1,626-bp mcr-3) is present on pMCR_1511 (Fig. 1), which is identical to the corresponding region of the original mcr-3 variant and does not have the mutations seen in mcr-3.5. The truncated mcr-3 on pMCR_1511 had a genetic context similar to that of mcr-3.5 on pMCR3_LL123 (Fig. 1). The mechanism for the truncation of mcr-3 on pMCR_1511 was not clear. Second, the composite transposon formed by ISApl1 carrying mcr-1 on pMCR_1511 (13) and the ISApl1-mcr-1-orf element on pMCR_1622 with the open reading frame (ORF) referring to a gene encoding a putative phosphoesterase were absent from pMCR3_LL123 (Fig. 1). Nonetheless, the identical backbone suggests that pMCR3_LL123, pMCR_1511, and pMCR_1622 were derived from a common ancestor. IncP is a broad-host-range type, and the association of mcr-3 with an IncP plasmid is worrisome because it may have the potential to mediate the dissemination of mcr-3 beyond the Enterobacteriaceae family and Aeromonas spp.

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FIG 1

The genetic context of mcr-3 on pMCR3_LL123 (second from top). The genetic context of mcr-3 on pWJ1 (top), that of the truncated mcr-3 on pMCR_1511 (third from top), and the corresponding region on non-mcr-3-carrying plasmid pMCR_1622 (bottom) are shown for comparison. The 5-bp direct repeats (DRs) characteristic of the transposition of Tn3 family transposons, including TnAs3 in this case, are indicated. dgkA refers to a gene encoding diacylglycerol kinase. The numbers represent genes or mobile genetic elements, including: 1, an open reading frame (ORF) of unknown function, which is broken into two parts (two 1Δ); 2, ISEnca1 truncated; 3, a gene encoding an NimA/NimC family protein, which is truncated with a 219-bp remnant on pWJ1 and a 24-bp remnant on pMCR3_LL123; 4, a gene encoding a DUF4942 domain-containing protein; 5, an ORF of unknown function; 6, a truncated (41-bp remnant) ISKpn40; 7, an ORF of unknown function; 8, an ORF of unknown function, which is interrupted by the composite transposon formed by ISApl1 carrying mcr-1 on pMCR_1511 and the ISApl1-mcr-1-orf element on pMCR_1622 with the 2-bp direct repeats (AC) characteristic of the insertion of ISApl1.

Analysis of the complete sequence of pMCR3_LL123 identified that mcr-3 was located on the transposon TnAs3, which belongs to the Tn3 family and was originally identified in Aeromonas salmonicida. Both boundaries (inverted repeats [IRs]) of TnAs3 were present on pMCR3_LL123 and were flanked by a 5-bp repeat (TACAT) (Fig. 1), which is characteristic of the transposition of TnAs3. TnAs3 is inserted into a gene which has no known function and is located between the parA gene encoding plasmid partition and the traX gene encoding plasmid conjugation (Fig. 1). However, the transposase gene tnpA is interrupted with only 861 bp of 2,967 bp remaining. It is therefore unlikely that the transposition of TnAs3 is due to its own transposase. It is known that two IRs of the Tn3 family of transposons can be transposed together with their intervening genetic components in the presence of transposases encoded by other Tn3 family transposons (14). However, the mechanism mediating the introduction of mcr-3 into TnAs3 warrants further investigation.

The mcr-1 gene was carried by a separate 223-kb IncHI2 plasmid in strain WCHEC-LL123, which was also self-transmissible and was highly similar (99% identity) to the corresponding region of pMCR_1613 (GenBank accession no. CP019214 ), an IncHI2 plasmid with an additional IncN replicon in E. coli strain WCHEC1613 (ST48) isolated from the sewage of the same hospital in 2015. In strain WCHEC-LL123, mcr-1 was located in a composite transposon formed by two copies of ISApl1, which was inserted into a gene encoding a putative signal peptidase I/pili assembly chaperon protein belonging to the IncHI2 backbone with the characteristic 2-bp direct target repeats.

The coexistence of two colistin resistance plasmids in a single bacterial isolate is intriguing. The MICs of colistin against E. coli transconjugants containing mcr-3 or mcr-1 were 4 μg/ml, while that against WCHEC-LL123 (containing both mcr-1 and mcr-3) was 8 μg/ml (Table 1). This suggests that the coexistence of mcr-1 and mcr-3 may not provide a significant additive effect. Astonishingly, the carbapenemase-encoding bla_NDM-5 gene was found on a third resistance plasmid, a self-transmissible 47-kb IncX3 plasmid. This is identical to pNDM5_WCHEC0215 (GenBank accession no. KY435936 ), a bla_NDM-5-carrying IncX3 plasmid in an ST167 E. coli clinical isolate from the same hospital in 2013 (15), except for several single nucleotide variations.

In conclusion we report the clinical isolation from a patient in China of an E. coli strain which carries three distinct resistance plasmids, one carrying mcr-1, one carrying mcr-3, and one carrying bla_NDM-5. The isolation of this strain is highly significant not only from a clinical treatment perspective but also from the perspective of the evolution of antimicrobial resistance. It also suggests that clinical strains of E. coli are capable of acquiring and maintaining multiple large MDR plasmids with no apparent loss of fitness with respect to ability to successfully colonize and cause clinical infections in patients.