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Mcr-1 and [bla.sub.KPC_3] in Escherichia coli sequence Type 744 after meropenem and colistin therapy, Portugal.

The emergence of the mcr-1 gene (1) and reports on its global dissemination (2) unveiled the danger of plasmid-associated colistin resistance. In July 2016, a 70-yearold woman was admitted to the intensive care unit of Centro Hospitalar do Baixo Vouga-EPE, Aveiro, Portugal, for abdominal pain, ostensibly from an abdominal occlusion. After emergency surgery, the patient received meropenem (20 d), fluconazole, and linezolid (both 10 d) and was transferred to the general medicine ward. After 50 days of antibacterial drug therapy, a urine specimen was positive for Klebsiella pneumoniae (Kp81). Further testing showed a multidrug-resistance profile, including resistance to carbapenems, but susceptibility to colistin and tigecycline (Table). The drug regimen was altered to colistin and tigecycline for 6 days, after which urine cultures were negative for K. pneumoniae.

Urine culture was performed as a standard procedure after 72 days. Escherichia coli (Ec36) was isolated, showing a resistance profile identical to K. pneumoniae Kp81 but expressing colistin resistance (Table). PCR screening and amplicon sequencing confirmed the presence of mcr-1 in Ec36 and [bla.sub.KPC-3] in both isolates (1,3). All treatments were discontinued, and the patient was discharged 72 days after admission.

We sequenced the Ec36 whole genome (GenBank accession no. MUGF00000000) by using the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA); we assembled it de novo by using CLC Genomics (https://www. qiagen.com/us/search/clc-genomics-workbench/) and annotated results by using RAST (http://rast.nmpdr.org/). We used tools available at the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk) to determine the sequence type, resistome, mobilome, serotype, virulence genes, and pathogenicity potential.

Strain Ec36 was assigned to sequence type 744 (ST-744) and predicted as a human pathogen with serotype O89:H10. Testing detected the virulence gene gad, encoding a glutamate decarboxylase involved in acid resistance. Besides mcr-1 and [bla.sub.KPC-3], Ec36 harbored genes encoding resistance to aminoglycosides (strA, strB, aacA4, aadA, aadA5), [beta]-lactams ([bla.sub.TEM-1B], [bla.sub.OXA-9]), macrolides (mph[A]), chloramphenicol (catA1), tetracycline (tet[A], tet[B]), sulfonamides (sul1, sul2), and trimethoprim (dfrA14, dfrA17). We used Plasmidfinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) to identify IncX4 (100%; in the mcr-1-encoding contig), IncFIA, IncFII, IncQl, IncXl, and IncI1. We used pMLST 1.4 (https://cge.cbs.dtu.dk/services/pMLST/) to identify IncFIA and IncFII.

[bla.sub.KPC-3] was in a 16,455-bp contig, 100% identical to plasmid sequences from clinical K. pneumoniae (4). In Portugal, this plasmid was reported in clinical isolates of K. pneumoniae, E. coli, and Enterobacter (5). [bla.sub.KPC-3] was part of Tn4401 isoform d (4), flanked by ISKpn7 and ISKpn6 and located in a cointegrated FIA and FII plasmid (pEc36-KPC3), co-harboring [bla.sub.TEM], [bla.sub.OXA9], aacA4, and aadA1. We analyzed the genetic context of [bla.sub.KpC-3] in Kp81 and Ec36 by using a PCR-based protocol (4), which indicated a similar context in both strains within Tn4401d in a FIA-FII plasmid. As highlighted previously (5), results reinforce the role of Tn4401d on the spread of carbapenemase genes among Enterobacteriaceae in Portugal.

We identified the mcr-1 gene in a 9,085-bp contig, which matched E. coli SHP45 100% (1). Genetic context analysis identified a 2,600-bp mcr-1-containing cassette recognized in different plasmid backbones (6), suggesting its mobilization between different hosts.

The IncX4 plasmid harboring mcr-1 (pEc36_mcr-1) was divided into 2 contigs, which we subsequently cloned by using PCR and sequencing. pEc36_mcr-1 was 33,140 bp and had no other resistance genes, nor ISAp11, found originally associated with mcr-1 and linked to animal reservoirs (7). Plasmid sequence showed high similarity to pESTMCR (GenBank accession no. KU743383), pMCR1IncX4 (accession no. KU761327), and pMCR1-NJ-IncX4 (accession no. KX447768).

We performed mating assays by using Ec36 as donor and E. coliJ53 as recipient. Transconjugants were obtained in Plate-Count-Agar (Merck, Germany) with sodium azide (100 mg/L) and colistin (2 mg/L). The MIC of colistin for the transconjugant (4 mg/L) was 8 times higher than that for E. coli J53. We detected mcr-1 by using PCR for the transconjugant, but not [bla.sub.KPC-3].

mcr-1 was previously detected in carbapenem-susceptible E. coli ST744 in Denmark (8) and in E. coli ST744, co-producing CTX-M-like [beta]-lactamases, in Taiwan (9). Regarding clinical mcr-1-positive E. coli, >10 STs have been reported, including the high-risk ST-131 (8,9). Therefore, the association of a successful clone to the spread of mcr-1 is not evident, but apparently, it is associated with successful plasmids (e.g., IncX4).

In Portugal, mcr-1 has been reported in Salmonella and E. coli from food products and in clinical Salmonella isolates (2,10). Since [bla.sub.KPC-3] is increasingly reported in Portugal, its co-occurrence with mcr-1-harboring plasmids represents a serious concern.

mcr-1 has been found in isolates that produce carbapenemases KPC, NDM, VIM, and OXA-48 (2,7). Carbapenemase genes usually are associated with mobile elements that encode resistance to several antibacterial drugs, and consequently produce multiresistance traits, as in E. coli Ec36. This scenario might predict the emergence of drug-resistant phenotypes, likely jeopardizing treatment.

In summary, we isolated KPC-3-producing and mcr1-harboring E. coli Ec36 from a patient after treatment with meropenem, then colistin. Colistin-resistant Ec36 may have been part of the patient's gut microbiome, acquiring the [bla.sub.KPC-3]-encoding plasmid from the KP81 strain. Although neutropenic, the patient's samples showed an asymptomatic bacteriuria. Thus, prophylactic administration of antibacterial drugs was likely avoidable.

This work was supported by Fundacao para a Ciencia e a Tecnologia (FCT) through CESAM (UID/AMB/50017/2013). I.H. was supported by ESF (EU) and POPH funds (Programa Investigador FCT--IF/00492/2013), and by FCT through SFRH/ BPD/81509/2011 (S.F.) and SFRH/BPD/114855/2016 (M.T.).

Ms. Tacao is a research scientist at the University of Aveiro, Aveiro, Portugal. Her primary interest is microbiology, particularly bacterial genetic determinants of antibiotic resistance and their dissemination.

References

(1.) Liu Y-Y, Wang Y, Walsh TR, Yi L-X, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2015; 3099:1-8.

(2.) Skov RL, Monnet DL. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill. 2016; 21:30155. http://dx.doi.org/10.2807/1560-7917. ES.2016.21.9.30155

(3.) Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010; 65:490-5. http://dx.doi.org/10.1093/ jac/dkp498

(4.) Chen L, Chavda KD, Melano RG, Hong T, Rojtman AD, Jacobs MR, et al. Molecular survey of the dissemination of two blaKPC-harboring IncFIA plasmids in New Jersey and New York hospitals. Antimicrob Agents Chemother. 2014; 58:2289-94. http://dx.doi.org/10.1128/AAC.02749-13

(5.) Rodrigues C, Bavlovic J, Machado E, Amorim J, Peixe L, Novais A. KPC-3-producing Klebsiella pneumoniae in Portugal linked to previously circulating non-CG258 lineages and uncommon genetic platforms (Tn4401d-IncFIA and Tn4401d-IncN). Front Microbiol. 2016; 7:1000. http://dx.doi.org/ 10.3389/fmicb.2016.01000

(6.) Poirel L, Kieffer N, Brink A, Coetze J, Jayol A, Nordmann P Genetic features of MCR-1-producing colistin-resistant Escherichia coli isolates in South Africa. Antimicrob Agents Chemother. 2016; 60:4394-7. http://dx.doi.org/10.1128/ AAC.00444-16

(7.) Nordmann P, Poirel L. Plasmid-mediated colistin resistance: an additional antibiotic resistance menace. Clin Microbiol Infect. 2016; 22:398-400. http://dx.doi.org/10.1016/j.cmi.2016.03.009

(8.) Hasman H, Hammerum AM, Hansen F, Hendriksen RS, Olesen B, Agerso Y, et al. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015. Euro Surveill. 2015; 20:30085. http://dx.doi.org/10.2807/ 1560-7917.ES.2015.20.49.30085

(9.) Kuo SC, Huang WC, Wang HY, Shiau YR, Cheng MF, Lauderdale TL. Colistin resistance gene mcr-1 in Escherichia coli isolates from humans and retail meats, Taiwan. J Antimicrob Chemother. 2016; 71:2327-9. http://dx.doi.org/10.1093/jac/dkw122

(10.) Campos J, Cristino L, Peixe L, Antunes P MCR-1 in multidrugresistant and copper-tolerant clinically relevant Salmonella 1,4,[5],12:i:- and S. Rissen clones in Portugal, 2011 to 2015. Euro Surveill. 2016; 21:30270. http://dx.doi.org/10.2807/ 1560-7917.ES.2016.21.26.30270

Address for correspondence: Marta Tacao, Biology Department, University of Aveiro, Campus Universitario Santiago, 3810-193 Aveiro, Portugal; email: martat@ua.pt

Marta Tacao, Rafael dos Santos Tavares, Pedro Teixeira, Ines Roxo, Elmano Ramalheira, Sonia Ferreira, Isabel Henriques

Author affiliations: University of Aveiro, Aveiro, Portugal (M. Tacao, R. dos Santos Tavares, P. Teixeira, E. Ramalheira, I. Henriques); Centro Hospitalar do Baixo Vouga-EPE, Aveiro (I. Roxo, E. Ramalheira, S. Ferreira); Instituto de Educacao e Cidadania, Aveiro (I. Roxo, S. Ferreira)

DOI: https://doi.org/ 10.3201/eid2308.170162
Table. MICs of antibacterial drugs for Klebsiella pneumoniae Kp81,
Escherichia coli Ec36, transconjugant E. coli J53::mcr-1, and
recipient strain E. coli J53 *

Drug                             MIC, mg/L (susceptibility)

                                         K. pneumoniae Kp81

Amikacin                          [greater than or equal to] 64 (R)
Aztreonam                         [greater than or equal to] 64 (R)
Cefepime                          [greater than or equal to] 64 (R)
Ceftazidime                       [greater than or equal to] 64 (R)
Ciprofloxacin                     [greater than or equal to] 4 (R)
Colistin                           [less than or equal to] 0.5 (S)
Gentamicin                        [greater than or equal to] 16 (R)
Imipenem                          [greater than or equal to] 16 (R)
Meropenem                         [greater than or equal to] 16 (R)
Piperacillin                     [greater than or equal to] 128 (R)
Piperacillin/tazobactam          [greater than or equal to] 128 (R)
Ticarcillin                      [greater than or equal to] 128 (R)
Ticarcillin/clavulanic acid      [greater than or equal to] 128 (R)
Tigecycline                                    1.5 (S)
Tobramycin                        [greater than or equal to] 16 (R)
Trimethoprim/sulfamethoxazole    [greater than or equal to] 320 (R)

Drug                             MIC, mg/L (susceptibility)

                                      E. coli Ec 36 ([dagger])

Amikacin                                       16 (R)
Aztreonam                         [greater than or equal to] 64 (R)
Cefepime                                        2 (I)
Ceftazidime                       [greater than or equal to] 64 (R)
Ciprofloxacin                     [greater than or equal to] 4 (R)
Colistin                                        8 (R)
Gentamicin                        [greater than or equal to] 16 (R)
Imipenem                          [greater than or equal to] 16 (R)
Meropenem                         [greater than or equal to] 16 (R)
Piperacillin                     [greater than or equal to] 128 (R)
Piperacillin/tazobactam          [greater than or equal to] 128 (R)
Ticarcillin                      [greater than or equal to] 128 (R)
Ticarcillin/clavulanic acid      [greater than or equal to] 128 (R)
Tigecycline                                     1 (S)
Tobramycin                        [greater than or equal to] 16 (R)
Trimethoprim/sulfamethoxazole    [greater than or equal to] 320 (R)

Drug                             MIC, mg/L (susceptibility)

                                        E. coli J53::mcr-1

Amikacin                                        ND
Aztreonam                                       ND
Cefepime                                        ND
Ceftazidime                                     ND
Ciprofloxacin                                   ND
Colistin                                       4 (R)
Gentamicin                                      ND
Imipenem                         [less than or equal to] 0.25 (S)
Meropenem                        [less than or equal to] 0.25 (S)
Piperacillin                                    ND
Piperacillin/tazobactam                         ND
Ticarcillin                                     ND
Ticarcillin/clavulanic acid                     ND
Tigecycline                                  0.25 (S)
Tobramycin                                      ND
Trimethoprim/sulfamethoxazole                   ND

Drug                             MIC, mg/L (susceptibility)

                                            E. coli J53

Amikacin                                        ND
Aztreonam                                       ND
Cefepime                                        ND
Ceftazidime                                     ND
Ciprofloxacin                                   ND
Colistin                                      0.5 (S)
Gentamicin                                      ND
Imipenem                         [less than or equal to] 0.25 (S)
Meropenem                        [less than or equal to] 0.25 (S)
Piperacillin                                    ND
Piperacillin/tazobactam                         ND
Ticarcillin                                     ND
Ticarcillin/clavulanic acid                     ND
Tigecycline                                  0.25 (S)
Tobramycin                                      ND
Trimethoprim/sulfamethoxazole                   ND

* MICs were determined by using VITEK2 system AST.N222 (bioMerieux,
Marcy.l'Etoile, France), except colistin, for which MICRONAUT MIC.
strip (BioConnections, Knypersley, UK) was used, and interpreted
according to the European Committee on Antimicrobial Susceptibility
Testing (http://www.eucast.org).Shaded rows indicate antibacterial
drugs tested for efficacy against each of the 4 organisms.ND, not
determined; R, resistant; S, susceptible.

([dagger]) Strain isolated from the patient in this study.
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Article Details
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Title Annotation:RESEARCH LETTERS
Author:Tacao, Marta; Tavares, Rafael dos Santos; Teixeira, Pedro; Roxo, Ines; Ramalheira, Elmano; Ferreira,
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:4EUPR
Date:Aug 1, 2017
Words:1953
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