https://orcid.org/0000-0003-0511-5053
Abstract
Antibiotic resistance is often spread through bacterial populations via conjugative plasmids. However, plasmid transfer is not well recognized in clinical settings because of technical limitations, and health care–associated infections are usually caused by clonal transmission of a single pathogen. In 2015, multiple species of carbapenem-resistant Enterobacteriaceae (CRE), all producing a rare carbapenemase, were identified among patients in an intensive care unit. This observation suggested a large, previously unrecognized plasmid transmission chain and prompted our investigation.
Electronic medical record reviews, infection control observations, and environmental sampling completed the epidemiologic outbreak investigation. A laboratory analysis, conducted on patient and environmental isolates, included long-read whole-genome sequencing to fully elucidate plasmid DNA structures. Bioinformatics analyses were applied to infer plasmid transmission chains and results were subsequently confirmed using plasmid conjugation experiments.
We identified 14 Verona integron-encoded metallo-ß-lactamase (VIM)-producing CRE in 12 patients, and 1 additional isolate was obtained from a patient room sink drain. Whole-genome sequencing identified the horizontal transfer of blaVIM-1, a rare carbapenem resistance mechanism in the United States, via a promiscuous incompatibility group A/C2 plasmid that spread among 5 bacterial species isolated from patients and the environment.
This investigation represents the largest known outbreak of VIM-producing CRE in the United States to date, which comprises numerous bacterial species and strains. We present evidence of in-hospital plasmid transmission, as well as environmental contamination. Our findings demonstrate the potential for 2 types of hospital-acquired infection outbreaks: those due to clonal expansion and those due to the spread of conjugative plasmids encoding antibiotic resistance across species.
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Each year, approximately 2.8 million illnesses and 35 000 deaths in the United States are attributable to antibiotic-resistant infections [1]. Due to decreasing susceptibility to other broad-spectrum antimicrobials, carbapenems have become the treatment of choice for infections caused by highly antibiotic-resistant bacteria [2]. The expanded use of carbapenem antimicrobials has coincided with a rise of carbapenem-resistant phenotypes, especially among the Enterobacteriaceae [3].
Enterobacteriaceae develop carbapenem resistance through several pathways, including the expression of a carbapenemase or the overproduction of certain beta-lactamases in combination with porin mutations and/or augmented efflux pump activity [4]. The rapid dissemination of carbapenem resistance is also driven by the horizontal spread of resistance genes on conjugative plasmids, including among carbapenem-resistant Enterobacteriaceae (CRE) strains that have become successful in clinical settings [5]. Therefore, outbreak investigations and surveillance studies are increasingly focusing on plasmids that facilitate inter- and intra-species transfers of antimicrobial resistance [6, 7].
While Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase in the United States [4], metallo-ß-lactamases (MBL) are increasing. These MBL enzymes include New Delhi metallo-β-lactamase (NDM) [8], recently associated with duodenoscope exposure in the United States [9], and Verona integron-encoded metallo-ß-lactamase (VIM) [10], among others. The VIM enzymes form 1 of the largest groups of MBLs, comprising 51 reported variants (https://www.ncbi.nlm.nih.gov/bioproject/305729). VIM and its associated Class 1 integrons are associated with numerous broad-range plasmids, including incompatibility group (Inc) N, IncHI2, and IncA/C2, further facilitating their spread [11–13]. The production of VIM in Enterobacteriaceae has been relatively rare in the United States, with only 57 reported instances as of December 2017. Like other CRE, VIM producers have been associated with more complicated patient courses, including higher relapse rates and a need for prolonged durations of antimicrobial therapy [14], and are an emerging threat to public health. The first VIM-producing CRE identified in the United States occurred in an adult patient in 2006 [15], and a second, travel-associated VIM isolate was reported in 2010 [16].
From August 2015 through May 2016, 12 patients colonized with 5 different species of VIM-producing CRE were identified in an intensive care unit (ICU) at a tertiary-care hospital in Kentucky [17]. Colonization with a VIM-producing CRE had been identified only once previously at this hospital, in an adult patient in 2013, as reported to the Kentucky Department for Public Health. The combination of a rare carbapenemase in CRE presenting in a single facility among a diverse set of pathogens prompted the use of next-generation sequencing to characterize these VIM-producing CRE to better understand the driving forces behind this cluster.
METHODS: CASE DEFINITION, FINDING, AND SERIES
A case was defined as a polymerase chain reaction (PCR)-confirmed blaVIM-containing CRE isolate if it was isolated from a clinical or surveillance culture from a patient who had a negative admission screening culture and was admitted to the affected hospital on or after 1 August 2015.
Suspected cases were initially identified through perirectal screening that included admission and weekly multidrug-resistant organism surveillance cultures. CRE were tested for blaVIM using the Verigene nucleic acid test (Nanosphere, Chicago, IL) and VIM-positive isolates were submitted to the Centers for Disease Control and Prevention (CDC) for PCR confirmation and whole-genome sequence analysis, as described below. A review of infection control records over the prior year at the hospital was completed to identify historical cases.
Common upstream and downstream referral facilities from Hospital A were identified using Centers for Medicare and Medicaid Services claims data from fee-for-service beneficiaries. Point prevalence perirectal surveillance of high-risk patients (extended length of stay, devices in place, or on a ventilator) was conducted at the 4 facilities (including tertiary-care hospitals and long-term, acute-care hospitals) identified as most connected in the referral network of the hospital, including both upstream and downstream patient transfers. The activities involved in this investigation underwent human subjects review at the CDC and were determined to constitute an urgent public health response; therefore, the investigation was exempt from institutional review board approval.
Environmental Assessment and Antimicrobial Resistance Testing
Environmental samples were collected from sinks, drains, environmental service carts, and surfaces in rooms that housed affected patients and in common areas of affected units. Samples were collected using Sponge-Sticks (3M, St. Paul, MN) and EnviroMax Plus foam paddle swabs (Puritan Medical, Guilford, ME), then processed as described previously [18]. Dilutions were plated onto CHROMagar KPC (DRG International, Springfield, NJ), Trypticase Soy Agar with 5% sheep’s blood (TSA II, BD, Sparks, MD), and MacConkey II (BD, Sparks, MD), and were cultured overnight at 35˚C.
Environmental isolates were screened for antimicrobial resistance using 10 µg meropenem disks placed between the first and second quadrant of growth. Suspect colonies were selected within a 21 mm zone, then screened for MBL activity by comparing imipenem broth microdilution minimum inhibitory concentrations in the presence (32 to 0.25 mg/ml) and absence (64 to 0.5 mg/ml) of metal chelators (0.2 mM ethylene diamine tetra-acetic acid and 0.02 mM phenanthroline). Resistance determinants were identified from cultured isolates by multiplex PCR for the detection of blaOXA-48, blaVIM, blaNDM, and blaKPC [19].
Isolate Preparation and Genomic Analysis
A detailed description of molecular methods is provided in the Supplemental Appendix. In brief, 19 isolates, including 14 from 12 case patients, 2 environmental isolates, and 3 historical specimens, underwent short-read sequencing using a MiSeq (Illumina, San Diego, CA). A subset of 6 isolates was selected for long-read sequencing on a RSII instrument (Pacific Biosciences, Menlo Park, CA). Clean short-reads were assembled de novo into contigs using SPAdes 3.9.0 [20]. Acquired antimicrobial resistance genes were detected using SSTAR [21] and the ResFinder database [22]. PacBio reads were assembled using HGAP and were subsequently polished with Quiver [23]. Known resistance mechanisms were determined as described for Illumina contigs. Clean Illumina reads from all IncA/C2 plasmid replicon-containing isolates were mapped to the core plasmid genome using lyve-SET1.1.4f [24]. Raw sequencing reads, PacBio assemblies, and drug susceptibility data were placed under National Center for Biotechnology Information BioProject PRJNA302185.
RESULTS
Case Finding and Case Series
From August 2015 through May 2016, 14 CRE carrying blaVIM-1 were identified in 12 patients from perirectal swabs. No patients had a symptomatic infection, suggesting that all positive cultures represented colonization. The epidemic curve by hospital location is shown in Figure 1A. In the first 5 weeks of the outbreak, VIM-producing isolates represented 4 CRE species, including 2 different strains of Klebsiella pneumoniae, but subsequent isolates were exclusively Enterobacter hormaechei (Figure 1B). In total, the most prevalent species was E. hormaechei (n = 10; 71%) followed by K. pneumoniae (n = 2; 12%), Escherichia coli (n = 1; 7%), and Raoultella ornithinolytica (n = 1; 7%). There were 2 patients who were colonized with 2 different bacterial species from the same culture site. Demographics of the 12 case-patients are shown in Table 1. No patients reported health-care exposure outside the United States, and a chart review did not reveal any known risk factors for acquisition, such as prior, major health-care exposure or long-term care. Among colonized infants, all 5 spent time on the neonatal ICU/intermediate care unit (neonatal ward) during their hospitalizations. The neonatal ward is arranged into pods of between 2 and 6 neonate beds; all 5 case-infants spent time in pods that other case-infants once occupied, and 2 case-infants overlapped temporally in the same pod before positive culture.
Demographic and Clinical Characteristics of Affected Patients
| Demographic and Clinical Characteristics | Case Patients, n = 12 |
|---|---|
| Male, n (%) | 7 (58%) |
| Infants less than 1 year, n (%) | 5 (42%) |
| Adults over 18 years, n (%) | 7 (58%) |
| Age at first positive culture | |
| Mean age of infants, days (range) | 29 (9–54) |
| Mean age of adults, years (range) | 52 (41–68) |
| Length of stay | |
| Median length of stay to first positive culture, days (range) | 16.5 (8–54) |
| Hospital location | |
| Neonatal ICU or intermediate care ward, n (%) | 5 (42%) |
| Adult trauma/surgical ICU or intermediate care ward, n (%) | 4 (33%) |
| Other, n (%) | 3 (25%) |
| Demographic and Clinical Characteristics | Case Patients, n = 12 |
|---|---|
| Male, n (%) | 7 (58%) |
| Infants less than 1 year, n (%) | 5 (42%) |
| Adults over 18 years, n (%) | 7 (58%) |
| Age at first positive culture | |
| Mean age of infants, days (range) | 29 (9–54) |
| Mean age of adults, years (range) | 52 (41–68) |
| Length of stay | |
| Median length of stay to first positive culture, days (range) | 16.5 (8–54) |
| Hospital location | |
| Neonatal ICU or intermediate care ward, n (%) | 5 (42%) |
| Adult trauma/surgical ICU or intermediate care ward, n (%) | 4 (33%) |
| Other, n (%) | 3 (25%) |
Abbreviation: ICU, intensive care unit.
Demographic and Clinical Characteristics of Affected Patients
| Demographic and Clinical Characteristics | Case Patients, n = 12 |
|---|---|
| Male, n (%) | 7 (58%) |
| Infants less than 1 year, n (%) | 5 (42%) |
| Adults over 18 years, n (%) | 7 (58%) |
| Age at first positive culture | |
| Mean age of infants, days (range) | 29 (9–54) |
| Mean age of adults, years (range) | 52 (41–68) |
| Length of stay | |
| Median length of stay to first positive culture, days (range) | 16.5 (8–54) |
| Hospital location | |
| Neonatal ICU or intermediate care ward, n (%) | 5 (42%) |
| Adult trauma/surgical ICU or intermediate care ward, n (%) | 4 (33%) |
| Other, n (%) | 3 (25%) |
| Demographic and Clinical Characteristics | Case Patients, n = 12 |
|---|---|
| Male, n (%) | 7 (58%) |
| Infants less than 1 year, n (%) | 5 (42%) |
| Adults over 18 years, n (%) | 7 (58%) |
| Age at first positive culture | |
| Mean age of infants, days (range) | 29 (9–54) |
| Mean age of adults, years (range) | 52 (41–68) |
| Length of stay | |
| Median length of stay to first positive culture, days (range) | 16.5 (8–54) |
| Hospital location | |
| Neonatal ICU or intermediate care ward, n (%) | 5 (42%) |
| Adult trauma/surgical ICU or intermediate care ward, n (%) | 4 (33%) |
| Other, n (%) | 3 (25%) |
Abbreviation: ICU, intensive care unit.
A–C, Epidemic curve of Verona integron-encoded metallo-ß-lactamase-producing carbapenem-resistant Enterobacteriaceae by hospital location, organism species, and plasmid variant, respectively. Abbreviation: Epi week, epidemiological week; Inc, incompatibility group.
Of 7 adult patients, 4 (57%) spent time on the adult trauma/surgical ICU/intermediate care unit (adult trauma/surgical ward) during their hospitalizations; 2 spent time in the same adult trauma/surgical ward room, separated by 4 months. The remaining 3 adult patients (43%) were never admitted to the adult trauma/surgical ward.
We identified 4 referral network facilities in the region as highly connected to the affected hospital, and these facilities agreed to colonization screening. In each facility, 10 high-risk patients underwent perirectal colonization testing, for a total of 40 patients tested; none were positive for VIM-producing CRE.
Infection Control Observations and Environmental Assessment
Multiple lapses in infection control were observed, including adherence to hand hygiene and the use of personal protective equipment. Lapses were corrected when observed, and systematic changes were recommended based on these observations. Recommendations implemented by the facility included health-care personnel education, hand hygiene monitoring, increased surveillance, cohorting of affected patients, and increased attention to environmental services practices.
Initial environmental sampling yielded 2 VIM-producing carbapenem-resistant isolates in the adult trauma/surgical ward. A VIM-1–producing Citrobacter amalonaticus was isolated from a case-patient room sink drain and a likely unrelated VIM-2 (10% amino acid dissimilarity with VIM-1 [25])-producing Pseudomonas putida was isolated from the surface of an environmental service cart on the same ward. No additional VIM-2–producing bacteria were identified during the study.
Characterization of VIM CRE
The presence of blaVIM-1 was confirmed in 18 isolates, including 14 outbreak samples from 12 patients, 3 historical patient isolates from 2013, and 1 environmental specimen. Several early findings in the investigation prompted a further analysis with long-read sequencing to retrieve fully closed, high-quality plasmid sequences and evaluate alternative mechanisms for the spread of blaVIM-1. These findings included the presence of blaVIM-1 across multiple species and strains, 100% concordance between the presence and absence of blaVIM-1 and the IncA/C2 plasmid replicon marker gene, and a lack of concordance between the presence of blaVIM-1 and all other replicons (Supplementary Table S1; Supplementary Data S1). We observed extensive genetic diversity among all VIM-producing species, including 8 and 3 different strain types of E. hormaechei and K. pneumoniae, respectively (Supplementary Table S1). Among the 18 VIM-producing isolates, numerous other resistance mechanisms were detected, including genes conferring resistance to aminoglycosides, sulfonamides, fluoroquinolones, and trimethoprim (Supplementary Data S1). A fraction of VIM producers harbored a complete Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) defense mechanism (n = 7; 39%), which could provide immunity against plasmid introduction (Supplementary Data S1).
For 6 VIM-positive isolates, we assembled full-length, circular plasmid structures using PacBio single molecule real-time long-read sequencing technology. Computational screening of these plasmid sequences confirmed that blaVIM-1 was located on a plasmid of IncA/C2. In addition, each IncA/C2 plasmid carried blaVIM-1 on a previously described and widely distributed 4482 base pair (bp) Class 1 In1209 integron that shared 100% sequence similarity with all other In1209 integrons in this data set [26]. The In1209 integron also carried additional resistance genes aacA7, aadA1, and dfrA1, conferring resistance to aminoglycosides and trimethoprim (Figure 2). Additionally, all 6 isolates that underwent PacBio sequencing contained numerous plasmids of various incompatibility groups harboring a vast array of antimicrobial resistance genes (Supplementary Data S2). All further discussion of plasmid characteristics will be limited to these 6 IncA/C2 plasmids harboring blaVIM-1, unless otherwise stated.
Molecular structure of the In1209 integron identified among all incompatibility group A/C2 outbreak plasmids. Resistance genes, except blaVIM, are in orange.
Incompatibility Group A/C2 Plasmid Classification
An assessment of the rhs gene and other features classified all 6 IncA/C2 plasmids as Type 1 [27]; Type 1 is the most common IncA/C2 group, which often harbors other beta-lactamase genes, including blaCMY and blaNDM. Moreover, these plasmids were part of plasmid Multilocus Sequence Typing group ST1.1, the largest sub-group worldwide, and were not previously described to harbor blaVIM (Supplementary Results; Supplementary Table S2). All 6 IncA/C2 plasmids from this outbreak were most closely related to historical IncA/C2 plasmid pRMH760 from Australia isolated in 1997 (KF976462.2), and plasmid pKPC_CAV1344 isolated in the US in 2010 (CP011622), which contained a blaKPC (Supplementary Results; Supplementary Figure S1).
During the outbreak, 2 highly related IncA/C2 sub-groups were identified (Figure 1C) through granular plasmid genome characterization (Figure 3). Of the sequenced plasmids, 3 were approximately ~160 Kb and contained a 3366 bp Class 1 In27 integron that carried aadA2 (aminoglycoside resistance) and dfrA12 (dihydrofolate reductase, trimethoprim resistance). The remaining 3 plasmids were slightly larger (~164 Kb) and lacked the 3366 bp In27 integron, but instead harbored a 2197 bp Class 1 In7 integron structure carrying aadB (aminoglycoside resistance) that was absent in the first group of plasmids. Both integrons were conserved and shared 100% sequence similarity within their respective plasmid sub-groups. The 164 Kb plasmids also carried a blaTEM-1B beta-lactamase transported by a Tn3 element (5216 bp) that were absent from the 160 Kb plasmids. After aligning all 6 IncA/C2 plasmid sequences with each other, we were able to fully differentiate the 2 plasmid sub-groups by the presence and absence of transposable elements and their associated antimicrobial resistance genes, mentioned above (Figure 4).
A representative for each subgroup of the IncA/C2 plasmid. Genes are denoted as arrows. Conjugal transfer genes are depicted in yellow, the blaVIM gene is in dark blue, the associated In1209 integron is in light blue, the blaTEM-1B gene and associated mobile elements are in purple, the In7 integron is in red, and the In27 integron is in green. Abbreviation: Inc, incompatibility group.
Circular BLAST alignment of all 6 outbreak IncA/C2 plasmids from PacBio long-read sequencing. The white areas indicate the absence of a certain genomic region. Sample identifiers starting with ‘N’ are from neonatal case-patients, starting with ‘A’ are from adult case-patients, and ‘2013’ denotes a historical adult patient isolate from 2013. Lower case letters at the end of an isolate identifier indicates multiple isolates from the same patient. Abbreviations: BLAST, Basic Local Alignment Search Tool; Inc, incompatibility group.
Phylogenetic Analysis and Incompatibility Group A/C2 Conjugation
An estimated 157 Kb genomic region was shared among all 6 IncA/C2 plasmids (core plasmid genome). After mapping Illumina short reads from all 18 VIM- and IncA/C2-positive isolates against this core, the genetic diversity of all plasmids ranged between 0 and 16 single-nucleotide polymorphisms (SNPs) (data not shown). These SNPs grouped all 18 IncA/C2 plasmids in 2 distinct clades, representing the 160Kb- and 164Kb-sized plasmids. No further transmission events were inferred from the short-read data. However, we tested the conjugation capacities of both IncA/C2 plasmid variants for all 5 CRE species involved in this outbreak and observed conjugation efficiencies ranging from 1.3 × 10−2 to 2.1 × 10−5 conjugants/recipient colony forming units (Supplementary Results; Supplementary Table S3).
Discussion
This investigation represents the largest known outbreak of VIM-producing CRE in the United States to date, comprising a diverse set of bacterial species, and was driven by horizontal plasmid transmission. In accordance with recent trends, we leveraged genomics data for the investigation of horizontal plasmid transmission between bacterial species. During the course of the investigation, we demonstrated that blaVIM-1, a rare carbapenemase gene among CRE in the United States, inserted itself into a broad host range IncA/C2 plasmid and spread within a tertiary-care hospital. Plasmids of IncA/C2 are strongly associated with resistance to clinically relevant, third-generation cephalosporins and carbapenems, and are therefore of great concern to public health [28, 29].
Reported health care–associated outbreaks are generally caused by a single, clonal strain. However, in this case the range of genera and strains of the same species (e.g., 8 strains among 10 E. hormaechei isolates) carrying blaVIM-1 pointed to an alternate path of transmission: namely, a promiscuous plasmid. In recent years there has been growing interest in conjugative plasmids, due to their importance in the dissemination of antibiotic resistance in pathogenic bacteria [7, 30, 31].
This outbreak was characterized by 2 highly related plasmids that drove the spread of antibiotic resistance within a single facility. The IncA/C2 plasmids in this study contained numerous genes conferring antimicrobial resistance. Furthermore, the observed plasmid conjugation efficiencies demonstrated the mobile nature of these IncA/C2 plasmids, even when some recipient cells within this outbreak might contain active CRISPR systems known to ward off the transfer of such mobile elements into bacterial cells [32, 33]. This combination of multiple resistance mechanisms and the demonstrated mobile nature of this particular IncA/C2 plasmid within and across species barriers likely resulted in its occurrence among multiple host species.
Our findings in this outbreak investigation provide evidence of in-hospital transmission. It is also possible that an additional silent spread occurred but was not captured in our screening approach. Interventions included instituting enhanced infection control and prevention measures, as detailed in the 2015 CDC CRE Toolkit Update [34]. Given the promiscuity of this plasmid and the emerging nature of this resistance mechanism, the emphasis on early aggressive containment was imperative to preventing further spread.
These interventions appear to have halted transmission at the facility and, along with communication of the case-patients’ colonization status at transfer, appear to have limited further spread.
The results of this investigation also highlight the potential role of persistent environmental sources in the spread of CRE, since a VIM-producing Citrobacter carrying an outbreak-associated plasmid was identified from the sinks of affected units. Persistent bacteria in biofilms in the premise plumbing could have served as a reservoir for plasmid transmission. Although no point source was identified, a possible introduction was through a patient treated at the facility in 2013. Carbapenem-resistant organisms in sink drain biofilms have been noted during outbreaks in the past [35, 36], and biofilms provide a permissive environment for plasmid transfer between organisms [37]. Although the best methods for the eradication of sink drain biofilms are not known, practices like daily cleaning of sinks and surrounding surfaces, as well as eliminating the storage of equipment and medication preparation near sinks, can likely decrease the transmission risk. There are several limitations of our investigation. This was not a planned study, but was conducted as part of a public health investigation. Due to this, multiple interventions and evaluations were implemented over an extended time frame until the cessation of transmission was documented. In addition, there was no control group. Therefore, it is not possible to definitively determine the effect of our interventions. Point prevalence surveys done outside of the affected facility were limited in scope and the power to detect other VIM-producing CRE colonized or infected patients was small, especially considering that Centers for Medicare and Medicaid Services data may not be well suited to describe the movement of neonates across health-care facilities. Only 6 of 19 isolates underwent long-read sequencing; based on those results, it is possible that we missed additional diversity among the IncA/C2 plasmids, introducing gaps in our inferred transmission network. For instance, the investigation suggests that adults and neonatal patients all shared the same plasmid, but it seems implausible that much cross exposure occurred between these 2 groups. Additional sampling and long-read sequencing might have elucidated additional routes of transmission that were not identified using our approaches. We mapped short reads onto a long-read plasmid assembly; however, this approach may yield misleading results when plasmid plasticity is high [38]. Several integron mobilization events among A/C2 plasmids were identified in a relatively short period of time. Therefore, we cannot assume that our long-read plasmid assemblies represent a stable structure; this further limits our transmission inference investigation. Early recognition of this plasmid outbreak by long-read whole-genome sequencing represented an opportunity to implement control measures to prevent VIM-producing CRE from further regional spread. Our investigation highlights the horizontal spread of antibiotic resistance through mobile genetic elements and identifies a potential threat to efforts to contain antibiotic resistance. Improving our understanding of this mechanism of transmission will strengthen, accelerate, and likely alter public health responses and infection control practices.
Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgements. The authors thank the Kentucky Department for Public Health’s Katie Myatt, Kimberly Daniels, Lynn Roser, Kimberly Porter, Robert Brawley, Douglas Thoroughman, Kraig Humbaugh, and Rachel Zinner; and the Centers for Disease Control and Prevention staff, including Terri Forster, Hollis Houston, Kristen Allison Perry, Bette Jensen, James Matheson, Kaitlin Tagg, and Alicia Shams.
Disclaimer. The findings and conclusions are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
Financial support. This work was supported by the Centers for Disease Control and Prevention’s investments to combat antibiotic resistance and the Advanced Molecular Detection program at the Centers for Disease Control and Prevention. S. H. is supported by the Australian Postgraduate Award.
Potential conflicts of interest. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
References
Author notes
T. J. B. dM. and A. Q. Y. contributed equally to this work.
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