Escherichia coli Harboring mcr-1 in a Cluster of Liver Transplant Recipients: Detection through Active Surveillance and Whole-Genome Sequencing

mcr-1, a plasmid-associated gene for colistin resistance, was first described in China in 2015, but its spread in the United States is unknown. We report detection of mcr-1-carrying Escherichia coli ST117 in a cluster of three liver transplant recipients.

ABSTRACT

mcr-1, a plasmid-associated gene for colistin resistance, was first described in China in 2015, but its spread in the United States is unknown. We report detection of mcr-1-carrying Escherichia coli ST117 in a cluster of three liver transplant recipients.

INTRODUCTION

Polymyxins are treatments of “last resort” for multidrug-resistant Gram-negative bacterial (MDR-GNB) infections. mcr-1, a plasmid-associated gene for polymyxin resistance, was first described in China in 2015, with widespread dissemination noted (1, 2), but only 53 U.S. cases have been reported to date (https://www.cdc.gov/drugresistance/biggest-threats/tracking/mcr.html). We report a cluster of Escherichia coli sequence type (ST) 117 harboring mcr-1 in three liver transplant recipients after an initial case of infection in 2015 (3).

The index case (patient 1) had bloodstream and biliary fluid infection with mcr-1-carrying E. coli (3). The remaining three patients carried mcr-1-positive E. coli in stool samples collected during the prospective liver transplant (PLT) study but did not experience infections (4). We conducted an epidemiological analysis in order to discern possible transmission events (Fig. 1).

FIG 1.

Epidemiological analysis of cluster of patients colonized and infected with health care-associated mcr-1-carrying E. coli. Patient 1 was the index case and had infection with mcr-1-carrying E. coli in biliary fluid and blood isolates following elective biliary surgery. Subsequently, closely related mcr-1-carrying E. coli was detected in fecal samples of patients 2 to 4, who had all undergone liver transplantation. Endoscopy performed on the same day appeared to be a common epidemiological link for patients 1, 2, and 4 (indicated with dotted lines). No such link was found for patient 3. LTx, liver transplant.

Patient 1 underwent elective biliary tree excision and Roux-en-Y hepaticojejunostomy in May 2015 for recurrent episodes of cholangitis on a background of sickle-cell anemia and cholecystectomy. Intraoperative biliary cultures grew mcr-1-positive E. coli. The patient became febrile the day after surgery, and blood cultures also grew mcr-1-positive E. coli. The patient was successfully treated with meropenem. Patients 2 to 4 had undergone liver transplant: two patients in November 2014 and one in January 2015. Patients 2 to 4 regularly provided fecal samples as part of the PLT study (Fig. 1) and were noted to have new onset MDR E. coli colonization at days 241, 275, and 322 posttransplant, respectively, prompting WGS and retrospective detection of mcr-1.

Patient 2 underwent same-day endoscopic retrograde cholangiopancreatography (ERCP), as did patient 1, in March 2015, and the first available subsequent stool sample was positive for mcr-1 6 months later (Fig. 1). Patient 3 underwent gastroduodenoscopy the same day patient 2 underwent ERCP in September 2015. Of note, patient 3 had a stool sample negative for mcr-1 6 days prior to the endoscopy, followed by detection of mcr-1 7 days following the endoscopy, suggesting acquisition around the time of endoscopy. Patient 4 had no obvious epidemiologic links to explain mcr-1 acquisition. No overlap was noted between this patient and other cluster patients with respect to endoscopy procedures, outpatient visits, or radiology investigations. The patients all lived in different zip codes. During 2.5 years of follow-up, patients 2 to 4 did not develop any mcr-1-associated infections.

Multilocus sequence typing indicated that all isolates were ST117. Patients 1 and 4 had two isolates each available for analysis, while patients 2 and 3 had single isolates. Cultured isolates underwent identification and susceptibility testing with Vitek-2 (bioMérieux). We performed MIC determination for colistin and polymyxin B with broth microdilution according to established guidelines (5). Tigecycline susceptibility testing was performed with Etest (bioMérieux). Details of whole-genome sequencing (WGS) are provided in the supplemental material.

The following MIC ranges were noted on a Vitek-2 instrument (bioMérieux, Durham, NC): ceftriaxone, 2 to 8 μg/ml; cefepime, <1 μg/ml; aztreonam, >64 μg/ml; ertapenem, <0.5 μg/ml; meropenem, <0.25 μg/ml; amikacin, <2 μg/ml; tobramycin, <1 μg/ml; gentamicin, <1 μg/ml; levofloxacin, >8 μg/ml; and tigecycline, 0.38 to 0.75 μg/ml. In addition to mcr-1, isolates harbored resistance genes to beta-lactams (bla_SHV-12 and bla_TEM-1D), aminoglycosides [aph(3′)-IIa, aph(6)-Ic, aadA2, aph(3′)-Ia, strA, and strB], sulfonamides (sul1 and sul2), trimethoprim (dfrA12), chloramphenicol (catA1), macrolides (mphA), and tetracycline [tet(A)]. Of note, bla_SHV-12 codes for an extended-spectrum beta-lactamase (6). Full isolate antimicrobial susceptibilities, resistance genes, and plasmid replicons are shown in Tables S2 to S4 in the supplemental material. Within-patient pairwise single nucleotide variant (SNV) distances of patient 1 and patient 4 isolates were 15 and 3 SNV (Table S1). Pairwise SNV distances of isolates from different patients ranged from 7 to 28 SNVs. Phylogenetic relationships are shown in Fig. 2. The mcr-1 gene was carried on an IncX4 plasmid in all isolates. The plasmid replicon content was similar for the isolates.

FIG 2.

Phylogenetic tree of mcr-1-carrying E. coli isolates. The genome of isolate NR2148 (GenBank accession no. CP019903.1) was the reference. Different patients are represented by colored nodes (red, patient 1; green, patient 2; orange, patient 3; blue, patient 4). The epidemiological associations for the shared endoscopy location are indicated by the colored fields.

We then conducted a retrospective molecular survey using mcr-1 PCR to evaluate for possible spread of mcr-1 (1). First, we tested all 197 E. coli isolates resistant to third-generation cephalosporins cultured from PLT study patients from 2015 to 2017. We then tested 187 discarded surveillance rectal swabs of consecutive patients admitted to medical and surgical intensive care units (ICUs) in 2017. Finally, we tested 123 stored clinical E. coli isolates resistant to third-generation cephalosporins collected from 2011 to 2016. No further cases of mcr-1-carrying isolates were detected (n = 507).

The detection of this cluster demonstrates the potential for silent dissemination of mcr-1 in a U.S. hospital setting through asymptomatic colonization and suggests a possible role for same-day endoscopy-related transmission, independent of using the same endoscope. This represents the earliest documented health care-associated cluster of mcr-1 in the United States and predates a recent report from 2017 (7). Both reports implicate endoscopy as a potential route for transmission. The current report also highlights the difficulty of instituting surveillance measures for mcr-1-harboring isolates when endemicity is low. While infective episodes have the greatest impact on patient outcomes, detection of silent colonization may play an important role in stemming the spread of mcr-1.

Our epidemiological analysis showed the possible role of endoscopy, with three patients having same-day endoscopies. However, the same endoscopes were not used during same-day procedures. Other mechanisms, such as a common environmental source, may have contributed. A previous outbreak of KPC-2 Klebsiella pneumoniae implicated a positioning pillow (8). Although endoscopy location was the clearest association, there were multiple other possible contributors since these patients had frequent radiological investigations, were cared for by the same caregivers, and had admissions to the same hospital wards. This may explain the acquisition in patient 4. Unfortunately, mcr-1 was only detected several years after sample collection, limiting our ability to conduct a real-time investigation. Our isolate collections also focused only on E. coli isolates resistant to third-generation cephalosporins. While mcr-1 colonization was found in these patients due to participation in a prospective study, colonization in other patients may have gone undetected. Nevertheless, our molecular survey of over 500 samples did not detect additional cases, either before or since (through 2017), demonstrating that mcr-1 has not yet become widespread. Furthermore, colonization in all three liver transplant recipients was not associated with clinical infection.

Genomic analysis confirmed that isolates belonged to ST117, were related, and carried mcr-1 on near-identical IncX4 plasmids. The distance of 15 SNVs between isolates from the index patient was high and suggests possible within-host evolution through long-term colonization. This relatively high SNV distance has direct implications for defining related clusters and spread. Conversely, the reference isolate was less distant from isolate 35479 from patient 2 (7 SNVs), suggesting transmission.

Our findings highlight the need for ongoing surveillance of mcr-1 and other forms of transferrable resistance to polymyxins in the United States. Our analysis suggested endoscopy location as an epidemiologic link but did not implicate endoscopes directly. Although we documented acquisition of mcr-1 colonization in several liver transplant recipients through active surveillance of fecal MDR-GNB carriage and WGS, this is a highly resource-intensive approach and is not applicable on a larger scale. A more viable alternative may be to implement PCR screening of discarded specimens (e.g., stool samples or rectal swabs) (9). In order to stop further spread of emerging forms of polymyxin resistance, future surveillance approaches need to recognize that clinical isolates may only represent the “tip of the iceberg” formed by the burden of asymptomatic colonization.