Emergence of the Plasmid-Mediated mcr-1 Gene in Clinical KPC-2-Producing Klebsiella pneumoniae Sequence Type 392 in Brazil

LETTER

Since the first report of the plasmid-mediated colistin resistance mcr-1 gene in Escherichia coli and Klebsiella pneumoniae isolates from China (1), mcr-1 has already spread to most continents, being detected in different species from several sources, including carbapenemase-producing clinical isolates (2). In Brazil, mcr-1 has been identified in E. coli isolates from food-producing animals (3), migratory birds (4), and a human clinical sample (5). In this study, we report the detection of mcr-1 in KPC-2-producing K. pneumoniae from a human clinical specimen in Brazil.

In September 2016, a 61-year-old man diagnosed with thrombotic thrombocytopenic purpura was admitted to the intensive care unit of a hospital in Vitória, Espírito Santo (southern Brazil), with ischemic stroke, bicytopenia, and pulmonary focus sepsis. After mechanical ventilation, bladder catheterization, and multiple catheter punctures, he developed a urinary tract infection caused by a K. pneumoniae strain (CCBH24080) resistant to polymyxin B and imipenem by Etest (bioMérieux, France). Interestingly, the patient responded well to therapy with polymyxin B (500,000 IU every 12 h) and meropenem (1 g every 8 h). The patient remained in isolation and died in November due to a severe hemorrhage.

Bacterial identification was confirmed by matrix-assisted laser desorption ionization (Bruker Daltonics, Germany). The MICs of colistin (16 μg/ml) and imipenem (>64 μg/ml) were confirmed by microdilution with cation-adjusted Mueller-Hinton broth (6), while for the other drugs, testing was performed by Vitek 2 (bioMérieux). Antimicrobial susceptibility was interpreted according to CLSI guidelines (7), except for tigecycline and colistin, for which the EUCAST criteria were used (8) (Table 1).

TABLE 1.

Molecular and phenotypic characterization of MCR-1-producing K. pneumoniae strain CCBH24080 and its transconjugants

Isolate	Resistance determinantsa	Virulence genesb	MIC (μg/ml)c
			TZP	FOX	CXM	CRO	CAZ	FEP	AMK	GEN	CIP	ETP	MEM	IPM	TGC	CST
CCBH24080	mcr-1, blaKPC-2, aac(6′)Ib-cr, aac(3)-IIa, strA, strB, blaTEM-1, blaOXA-1, blaSHV-11, blaCTX-M-15, oqxAB, qnrB66, gyrA mutation N87D, parC mutation S80I, fosA, catB3, sul2, tet(A), dfrA14	entB, fimH, iutA, kpn, mrkD, traT, uge, ureA, wabG, ycfM	≥128	≥64	≥64	≥64	≥64	≥64	16	≥16	≥4	≥8	≥16	>64	2	16
TC-mcrd	mcr-1	NDe	≤4	≤4	4	≤1	≤1	≤1	≤2	≤1	≤0.25	≤0.5	≤0.25	0.25	≤0.5	8
TC-mcr/blaKPCd	mcr-1, blaKPC-2	ND	≥128	≥4	≥64	2	2	≤1	≤2	≤1	≤0.25	0.5	1	2	≤0.5	8
J53	NAf	NA	≤4	≤4	4	≤1	≤1	≤1	≤2	≤1	≤0.25	≤0.5	≤0.25	0.25	≤0.5	<0.125

^aResistance determinants were detected by whole-genome sequencing for CCBH24080 and by PCR for the transconjugants.

^bVirulence determinants were associated with the production of adhesins (fimH, mrkD, kpn), lipopolysaccharides (wabG, uge, ycfM), siderophores (iutA, entB), urease (ureA), and serum resistance (traT).

^cAbbreviations: TZP, piperacillin-tazobactam; FOX, cefoxitin; CXM, cefuroxime; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; ETP, ertapenem; MEM, meropenem; IPM, imipenem; TGC, tigecycline; CST, colistin.

^dTC, transconjugant.

^eND, not determined.

^fNA, not applicable.

Whole-genome sequencing of CCBH24080 (GenBank accession no. NBOS00000000) was performed on the MiSeq platform (Illumina, USA). Genome assembly was carried out with the A5 assembly pipeline (9), and annotation was performed on RAST v.2.0 (http://rast.nmpdr.org). Multilocus sequence typing and searching for resistance genes were done with the Center for Genomic Epidemiology platform (www.genomicepidemiology.org). Virulence genes and plasmids were searched for by manual curation with Geneious v.6.1.8 (Biomatters Ltd., New Zealand) and the BLAST tool (https://www.ncbi.nlm.nih.gov). CCBH24080 belongs to sequence type 392, a member of internationally successful clonal group 147 (10). In addition, the isolate presented a wide variety of resistance and virulence genes (Table 1). In Brazil, polymyxin B resistance in K. pneumoniae has been associated with mgrB mutations (11), but no mutations in the mgrB, pmrAB, phoPQ, and crrAB sequences were detected in CCBH24080.

By reference mapping, we were able to identify an IncX4 plasmid of 33.3 kb carrying the mcr-1 gene that is identical to an E. coli plasmid from Brazil (GenBank accession no. CP015977.1) (5) with 100% coverage. The bla_KPC-2-bearing plasmid was very similar to an IncN plasmid detected in São Paulo (GenBank accession no. CP004367.2), except for a 2.250-bp deletion in the CCBH24080 plasmid. Analysis by S1 pulsed-field gel electrophoresis, followed by Southern blotting, confirmed that mcr-1 and bla_KPC-2 were located on plasmids of ∼33 and ∼44 kb, respectively. Mobilization of both plasmids was successfully assayed by mating donor cells with E. coli J53, and transconjugants were selected in 300 μg/ml sodium azide Mueller-Hinton agar containing colistin (2 μg/ml) or imipenem (1 μg/ml). Colistin was found to select transconjugants carrying either both plasmids or the mcr-1-carrying plasmid only. Nevertheless, imipenem selected only transconjugants carrying both plasmids (Table 1). The presence of the HicBA toxin/antitoxin system encoded by the IncX4 plasmid may explain this phenomenon (12).

Isolates of K. pneumoniae harboring mcr variants have been reported in Asia and Europe (1, 13–17), including carbapenemase (NDM-5 and KPC-3)-producing ones (15, 17). To our knowledge, this is the first description of mcr-1 in a human KPC-2-producing K. pneumoniae isolate. This finding raises a major concern, since KPC-producing K. pneumoniae is disseminated worldwide, and highlights the potential for the dissemination of mcr-1 associated with multidrug-resistant international clones. Furthermore, the possibility of the simultaneous transfer of these genes poses a threat to infection control strategies and clinical therapy.