Colistin- and Carbapenem-Resistant Escherichia coli Harboring mcr-1 and blaNDM-5, Causing a Complicated Urinary Tract Infection in a Patient from the United States

ABSTRACT Colistin is increasingly used as an antibiotic of last resort for the treatment of carbapenem-resistant Gram-negative infections. The plasmid-borne colistin resistance gene mcr-1 was initially identified in animal and clinical samples from China and subsequently reported worldwide, including in the United States. Of particular concern is the spread of mcr-1 into carbapenem-resistant bacteria, thereby creating strains that approach pan-resistance. While several reports of mcr-1 have involved carbapenem-resistant strains, no such isolates have been described in the United States. Here, we report the isolation and identification of an Escherichia coli strain harboring both mcr-1 and carbapenemase gene blaNDM-5 from a urine sample in a patient without recent travel outside the United States. The isolate exhibited resistance to both colistin and carbapenems, but was susceptible to amikacin, aztreonam, gentamicin, nitrofurantoin, tigecycline, and trimethoprim-sulfamethoxazole. The mcr-1- and blaNDM-5-harboring plasmids were completely sequenced and shown to be highly similar to plasmids previously reported from China. The strain in this report was first isolated in August 2014, highlighting an earlier presence of mcr-1 within the United States than previously recognized.

clinical infections caused by carbapenem-resistant Gramnegative bacteria, especially carbapenem-resistant Enterobacteriaceae (CRE) (1). The recent discovery of transmissible colistin resistance in China (2) has led to numerous reports of the plasmid-borne gene mcr-1 in animal, food, and clinical samples worldwide (3). Of particular concern is the spread of mcr-1 into CRE, thereby creating highly drug-resistant strains that are potentially untreatable. Several reports outside the United States have already described mcr-1 in bacterial strains harboring carbapenemases, including NDM-1 (4), NDM-5 (5-7), NDM-9 (8), KPC-2 (9), OXA-48 (10), and VIM-1 (11). Most recently, mcr-1 was detected in two clinical carbapenem-susceptible Escherichia coli strains from the United States, isolated from urine cultures ob-tained in April 2016 (12) and May 2015 (13), respectively. Here, we report the identification from urine of an E. coli strain harboring both mcr-1 and carbapenemase gene bla NDM-5 , originally isolated in August 2014 from a U.S. patient with recurrent urinary tract infections and no recent travel history.
A 76-year-old man presented to a tertiary-care hospital in New Jersey with subjective fever and flank pain in August 2014. The patient had emigrated from India and resided continuously in the United States for 1 year prior to this presentation. He had a history of prostate cancer treated with radiation therapy and subsequently developed recurrent urinary tract infections. He had recently undergone cystoscopy to evaluate the possibility of colovesicular fistula formation. The procedure was complicated by bladder perforation requiring bilateral nephrostomy tube placement. The OBSERVATION crossmark nephrostomy tubes were clamped 5 days prior to presentation, and he then developed subjective fever, chills, and generalized weakness. Laboratory testing on presentation indicated a leukocyte count of 14.1 ϫ 10 3 cells/l, associated with pyuria. The initial antimicrobial regimen included intravenous piperacillintazobactam and vancomycin. No fistula was seen on imaging studies, and the nephrostomy tubes were unclamped.
The susceptibility results of urine cultures obtained prior to the initiation of antimicrobial therapy are shown in Table 1. A cleancatch urine culture grew greater than 100,000 CFU per ml of Pseudomonas aeruginosa, Citrobacter koseri, and Enterococcus faecium. A urine culture obtained from the nephrostomy tube grew Ͼ100,000 CFU/ml of P. aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterococcus spp., and Staphylococcus aureus (methicillin resistant). Susceptibility testing results showed that the E. coli isolate was resistant to colistin and all ␤-lactams (including carbapenems) except aztreonam, but remained susceptible to amikacin, gentamicin, nitrofurantoin, tigecycline, and trimethoprim-sulfamethoxazole ( Table 1). The P. aeruginosa isolate was susceptible to piperacillin-tazobactam and colistin but exhibited resistance or intermediate resistance to other agents tested, including carbapenems. In contrast, the C. koseri and K. pneumoniae isolates were resistant to only ampicillin, with the latter also exhibiting intermediate resistance to nitrofurantoin. Based on these culture results, oral trimethoprim-sulfamethoxazole was added to the antimicrobial regimen, although by this point the patient had remained afebrile and the leukocyte count had normalized. A sterile clean-catch urine culture was obtained after 6 days of antimicrobial therapy. The patient returned home after undergoing a urinary diversion with ileal conduit.
The mcr-1-and bla NDM-5 -harboring plasmids from E. coli strain MCR1_NJ were transferred to E. coli DH10B by electroporation, thereby confirming the mutually exclusive presence of mcr-1 and bla NDM-5 in the resulting transformants. The conjugability of the mcr-1 plasmid was further confirmed by experiments using E. coli J53 Azi r as the recipient strain. Plasmid DNA was isolated using a Qiagen Plasmid Midi kit (Hilden, Germany) and subjected to complete plasmid sequencing using Illumina NextSeq as described previously (6). Sequencing reads were assembled de novo using SPAdes software (15), and gaps were closed by Sanger sequencing as described previously (6,16). The mcr-1harboring plasmid from E. coli MCR1_NJ (subsequently named pMCR1-NJ-IncX4) was 33,395 bp in length and had 100% BLAST query coverage and 99.6% nucleotide identity to conjugative plasmid pMCR1-IncX4 (GenBank accession no. KU761327), which we previously described in CTX-M-55-producing E. coli and NDM-5-producing K. pneumoniae strains from Chinese hospitals (5,6). The bla NDM-5 -harboring plasmid (named pNDM5-NJ-IncX3) was 39,520 bp in length and closely related (100% nucleotide identity and 79% query coverage) to pNDM5-IncX3  (accession no. KU761328), previously described in the two aforementioned NDM-5-producing K. pneumoniae strains from China (6). No other resistance genes besides mcr-1 and bla NDM-5 were observed in plasmid pMCR1-NJ-IncX4 or pNDM5-NJ-IncX3, respectively. Strain MCR1_NJ was shown by MLST to be a single-locus variant of ST405, associated with E. coli phylogroup D (17). ST405 is classified as one of the main extraintestinal pathogenic E. coli (ExPEC) lineages (18), and is associated with the global spread of extended-spectrum ␤-lactamases, most notably CTX-M-15 (18)(19)(20). AmpC cephamycinases and NDM metallo-␤-lactamases have also been reported in ST405 (20), including NDM-1 (21) and NDM-4 (22). Consequently, ST405 may also be involved in the global dissemination of NDM-producing E. coli strains (22). Moreover, whereas strain MCR1_NJ was susceptible to various antibiotics, including gentamicin and trimethoprim-sulfamethoxazole, studies of ST405 strains from multiple countries suggest that they are typically resistant to these as well (20). Worrisomely, ST405 has been frequently associated with community onset urinary tract infections (23)(24)(25). Dissemination of mcr-1 within this global lineage may therefore contribute to further spread of polymyxin resistance within ESBL-and carbapenemaseproducing E. coli (and other Enterobacteriaceae) strains.
In summary, we report the isolation and identification of an E. coli strain harboring both mcr-1 and bla NDM-5 from urine of a patient without recent travel outside the United States. This strain was isolated in August 2014, highlighting an earlier presence of mcr-1 within the region than previously known and raising the likelihood of ongoing undetected transmission. Active surveillance efforts involving all polymyxin-and carbapenem-resistant organisms are imperative in order to determine mcr-1 prevalence and prevent further dissemination.
Accession number(s). The complete nucleotide sequences of plasmids pMCR1-NJ-IncX4 and pNDM5-NJ-IncX3 have been deposited as GenBank accession no. KX447768 and KX447767, respectively. The draft genome sequence of E. coli strain MCR1_NJ was deposited as GenBank accession no. MAJK00000000.

ACKNOWLEDGMENTS
This study was supported by grants from the National Institutes of Health (grants R01AI090155 to B.N.K. and R21AI117338 to L.C.). B.N.K. discloses that he holds two patents that focus on using DNA sequencing to identify bacterial pathogens.

FUNDING INFORMATION
This work, including the efforts of Barry N Kreiswirth, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (R01AI090155). This work, including the efforts of Liang Chen, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (R21AI117338).