Co-occurrence of mcr-1 and bla_KPC-2 in a clinical isolate of Escherichia coli in Brazil

Sir,

Polymyxins are the last resort for the treatment of infections caused by carbapenem-resistant Enterobacteriaceae (CRE). In November 2015, Liu et al.¹ described for the first time a colistin resistance mechanism mediated by a new gene (mcr-1), which was present in a transferable plasmid. Since then, several reports have indicated that mcr-1 has silently spread worldwide since 1980.² Of particular concern is the dissemination of the mcr-1 gene in CRE, potentially leading to pan-drug-resistant isolates. There are only scattered reports of co-occurrence of bla_KPC and mcr-1 genes in the same isolate.^3,4 This study evaluates the characteristics of the first clinical isolate of Escherichia coli harbouring both mcr-1 and bla_KPC-2 genes in Latin America.

Among 1883 CRE clinical isolates screened, 1 KPC-2-producing E. coli was also positive for the mcr-1 gene (isolate 3431F). This isolate was obtained from a rectal swab of a patient hospitalized, in September 2014, at an emergency room of a general hospital in Porto Alegre city, Rio Grande do Sul, the southernmost Brazilian state.

The bla_KPC-2/mcr-1-positive clinical isolate carried at least two distinct plasmids, which were successfully transferred to E. coli J53 by conjugation. Two transconjugants were further evaluated: the transconjugant 3431FT1, which carried only one plasmid and was positive for mcr-1, and the transconjugant 3431FT2, which carried two plasmids and was positive for both mcr-1 and bla_KPC-2 (Table 1). The 3431F clinical isolate presented high MICs (≥32 mg/L) of the carbapenems and low-level resistance to colistin (MIC 4 mg/L); the transconjugants 3431FT1 and 3431FT2 presented significant increases in the MIC of colistin in comparison with E.coli J53 and the transconjugant 3431FT2 also presented increased MICs of the carbapenems (Table 1).

Table 1.

MICs (mg/L) of several antibiotics for E. coli 3431F, transconjugant 3431T1 (IncX4 mcr-1), transconjugant 3431T2 (IncX4 mcr-1 and IncFIB bla_KPC-2) and E. coli J53

Antibiotic	E. coli 3431F	Transconjugant 3431FT1 (IncX4 mcr-1)	Transconjugant 3431FT2 (IncX4 mcr-1 and IncFIB blaKPC-2)	E. coli J53
Ertapenem	32	0.002	0.125	0.004
Meropenem	32	0.012	0.125	0.023
Imipenem	≥32	0.12	0.5	0.12
Ciprofloxacin	4	≤0.125	≤0.125	≤0.125
Amikacin	2	1	1	≤0.5
Gentamicin	1	0.5	0.5	≤0.125
Tigecycline	0.25	0.25	0.25	0.13
Colistin	4	4	2	≤0.125

MICs were determined by broth microdilution, except those of ertapenem, imipenem and meropenem, which were determined by Etest.

The assembled WGS of the clinical isolate 3431F produced 67 scaffolds, which resulted in an estimated draft genome 4 891 834 bp in length, with a G + C content of 50.7% and a total of 4819 genomic features. The in silico analyses of the data indicated that E. coli 3431F belongs to ST744, an E. coli lineage usually associated with resistance genes, including a clinical isolate harbouring mcr-1 and ESBL genes in Denmark.⁵

The WGS data confirmed the presence of the bla_KPC-2 and mcr-1 genes, as well as several other resistance genes, such as genes encoding aminoglycoside-modifying enzymes [strA, strB and aph(3′)-la], a β-lactamase-encoding gene (bla_TEM-1) and genes related to resistance to macrolides [mph(A) and erm(42)], phenicols (floR and catA1), sulphonamides (sul2) and tetracycline [tet(A) and tet(B)].

The in silico analyses also allowed identification of the following plasmid incompatibility types in the 3431F isolate: IncN, IncFIB, IncQ1 and IncX4. Detailed analyses indicated that bla_KPC-2 was located in the IncFIB plasmid and mcr-1 was located in the IncX4 plasmid. bla_KPC-2 was located on a Tn4401 transposon, isoform b. The scaffold bearing bla_KPC-2 was 85 492 bp in length and no other resistance gene was found in this scaffold.

Detailed analysis of the scaffold bearing the mcr-1 gene suggested that it was a complete plasmid (a possible circular molecule 33 511 bp in length). For this reason, primers were designed to close the gaps in this scaffold and PCRs, followed by Sanger sequencing, confirmed the circular plasmid molecule. This plasmid, which was termed pMCR1poa (GenBank MTJV00000000), presented >99% overall identity as compared with pICBEC72H (GenBank CP015977),⁶ pESTMCR (GenBank KU743383),⁷ pMCR1.2-IT (GenBank KX236309)³ and pMCR1-IncX4 (GenBank KU761327),⁸ and 96% identity with pAf48 (GenBank KX032520).⁹ pMCR1poa harbours an ISApl1 insertion and a pap2 gene upstream and downstream of the mcr-1 gene, respectively, as also observed in other mcr-1-bearing plasmids described in other countries.^1,3,6–9

The IncX4-type plasmid carrying the mcr-1 gene, identified in this study, was previously described in several isolates carrying the mcr-1 gene, including a clinical isolate obtained from a hospitalized patient in north-eastern Brazil.⁶ Isolates of E. coli harbouring the IncX4-type plasmid containing the mcr-1 gene have also been identified in poultry from southern Brazil by our group (data not shown).

In summary, we report the occurrence of the mcr-1 gene in a bla_KPC-2-positive E. coli clinical isolate from Brazil. To the best of our knowledge this is the first report of a clinical isolate with both genes in Latin America. In addition, our findings underscore the broad intercontinental distribution of the IncX4 mcr-1-bearing plasmid. Considering the low prevalence of the mcr-1 gene among clinical isolates of CRE observed in our study, it seems that while carbapenem resistance is high, plasmid-mediated colistin resistance is still rare and sporadic among CRE clinical isolates.

Accession number

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MTJV00000000. The version described in this paper is version MTJV01000000.

Funding

This work was supported by Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) (project no. 16-0559). T. V. D. and D. d. L.-M. were supported by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brazil (CNPq) and L. C. was supported by a grant from the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (Fapergs).

Transparency declarations

None to declare.