Progressive Development of Cefiderocol Resistance in Escherichia coli During Therapy is Associated With an Increase in bla_NDM-5 Copy Number and Gene Expression

Abstract

Background

As cefiderocol is increasingly being prescribed in clinical practice, it is critical that we understand key mechanisms contributing to acquired resistance to this agent.

Methods

We describe a patient with acute lymphoblastic leukemia and a New Delhi metallo-ß-lactamase (NDM)–5–producing Escherichia coli intra-abdominal infection in whom resistance to cefiderocol evolved approximately 2 weeks after the start of treatment. Through whole-genome sequencing (WGS), messenger RNA expression studies, and ethylenediaminetetraacetic acid inhibition analysis, we investigated the role of increased NDM-5 production and genetic mutations contributing to the development of cefiderocol resistance, using 5 sequential clinical E. coli isolates obtained from the patient.

Results

In all 5 isolates, bla_NDM-5 genes were identified. The minimum inhibitory concentrations for cefiderocol were 2, 4, and >32 μg/mL for isolates 1–2, 3, and 4–5, respectively. WGS showed that isolates 1–3 contained a single copy of the bla_NDM-5 gene, whereas isolates 4 and 5 had 5 and 10 copies of the bla_NDM-5 gene, respectively, on an IncFIA/FIB/IncFII plasmid. These findings were correlated with those of bla_NDM-5 messenger RNA expression analysis, in which isolates 4 and 5 expressed bla_NDM-5 1.7- and 2.8-fold, respectively, compared to, isolate 1. Synergy testing with the combination of ceftazidime-avibactam and aztreonam demonstrated expansion of the zone of inhibition between the disks for all isolates. The patient was successfully treated with this combination and remained infection free 1 year later.

Conclusions

The findings in our patient suggest that increased copy numbers of bla_NDM genes through translocation events are used by Enterobacterales to evade cefiderocol-mediated cell death. The frequency of increased bla_NDM-5 expression in contributing to cefiderocol resistance needs investigation.

In a patient infected with a New Delhi metallo-ß-lactamase–producing Escherichia coliisolate, whole-genome sequencing, messenger RNA expression studies, and ethylenediaminetetraacetic acid inhibition work uncovered the mechanisms that led to relatively rapid development of cefiderocol resistance.

Carbapenem-resistant Enterobacterales (CRE) infections are associated with significant morbidity and mortality rates [1]. Perhaps the CRE infections that pose the greatest clinical management dilemmas are those caused by metallo-β-lactamase (MBL)–producing Enterobacterales, most commonly New Delhi–MBL (NDM)–producing Enterobacterales. Delays in identifying NDM production translate to delays in effective antibiotic initiation, ultimately resulting in poor patient outcomes. Cefiderocol (a novel siderophore-conjugated cephalosporin), in contrast with other US Food and Drug Administration–approved novel β-lactam agents, has in vitro activity against NDM-producing organisms [2–4].

Our understanding of the mechanisms of resistance of the Enterobacterales to cefiderocol are not well defined and include reports of mutations in 2-component regulatory systems (eg, TonB, BaeSR, and OmpR/EnvZ); iron transport-related proteins (eg, Fiu, Cir) [5, 6]; as well as deletions, insertions, and amino acid substitutions in or proximal to the omega loop of AmpC enzymes [7, 8]. There have been observations that NDM-producing Enterobacterales isolates have higher cefiderocol MIC₉₀s than isolates that produce serine β-lactamases, although this is not always associated with frank cefiderocol resistance [9, 10]. In one surveillance study including 151 international CRE isolates, cefiderocol was active against 98% of all isolates [9]. On closer inspection, cefiderocol was active against 100% of the 75 Klebsiella pneumoniae carbapenemase (KPC)–producing Enterobacterales isolates and the 32 OXA-48–like-producing isolates but only 58% of the 12 NDM-producing Enterobacterales isolates, using cefiderocol MICs ≤4 μg/mL as indicative of susceptibility [9, 11].

In a follow-up study investigating resistance mechanisms in 9 Enterobacterales clinical isolates with cefiderocol MICs >4 μg/mL from this international collection, 5 of the 9 isolates contained bla_NDM genes [12]. In another international cohort, cefiderocol was active against 86% of 79 NDM-producing isolates [10]. Both of these studies included isolates not previously exposed to cefiderocol therapy; the incidence of NDM-producing Enterobacterales not susceptible to cefiderocol after cefiderocol clinical use is unknown. We report findings in a patient with an NDM-producing Enterobacterales infection in whom resistance to cefiderocol evolved approximately 2 weeks after the start of cefiderocol therapy. Using whole-genome sequencing (WGS), messenger RNA (mRNA) expression studies, and ethylenediaminetetraacetic acid (EDTA) inhibition analysis, we investigated the role of increased NDM production and genetic mutations contributing to the development of cefiderocol resistance.

PATIENT AND METHODS

Clinical Case Presentation

A 35-year-old woman living in the United Arab Emirates with T-cell acute lymphoblastic leukemia after hematopoietic stem cell transplantation was on a flight to the United States to seek medical care for a relapse of her leukemia. During the flight, she became febrile and tachycardic and experienced lower abdominal pain. In the emergency department, after her arrival to the Johns Hopkins Hospital, a blood culture was obtained, and cefepime treatment was promptly initiated (Figure 1). Escherichia coli, susceptible to all antibiotics tested, was identified in blood culture bottles. A urine culture obtained 3 days later revealed a carbapenem-resistant E. coli with a cefiderocol MIC of 2 μg/mL (herein referred to as day 1 and isolate 1). Several intra-abdominal abscesses, the largest approximately 6cm in diameter, were observed on imaging. Drainage of her intra-abdominal collections was performed on day 3, and a carbapenem-resistant E. coli with a cefiderocol MIC of 2 μg/mL was again isolated (isolate 2; Table 1).

Figure 1.

Timeline of the clinical course, culture data, and antibiotic treatment data for a patient infected with a New Delhi metallo-ß-lactamase (NDM)–producing Escherichia coli; bla_NDM copy numbers harbored on the IncFIA/IncFIB/IncFII plasmid are included in parentheses after the genes. Abbreviation: MIC, minimum inhibitory concentration; ST, sequence type.

Table 1.

Antimicrobial Susceptibility Testing Results for 5 Escherichia coli Isolates Producing New Delhi Metallo-ß-Lactamase, Recovered From a Patient Before and During Treatment With Cefiderocol^a

Isolate	Day of Culture	Length of FDC Exposure, d	Source	ATM	CAZ	CAZ-AVI	CRO	CST	FDC	FEP	IMI	IMI-REL	MER	MER-VAB	MIN	PLZ	TGC
1	1	0	Urine	>16	>16	>16/4	>32	≤0.25	2	>16	8	>16/4	8	>16/8	4	0.5	≤1
2	3	0	Pelvic abscess	>16	>16	>16/4	>32	≤0.25	2	>16	>8	>16/4	>8	>16/8	4	0.5	≤1
3	22	19	Urine	>16	>16	>16/4	>32	≤0.25	4	>16	8	>16/4	>8	>16/8	8	0.5	2
4	22	19	Urine	>16	>16	>16/4	>32	≤0.25	>32	>16	>8	>16/4	>8	>16/8	>8	0.5	8
5	27	24	Blood	>16	>16	>16/4	>32	≤0.25	>32	>16	>8	>16/4	>8	>16/8	>8	1	8

Abbreviations: ATM, aztreonam; AVI, avibactam; CAZ, ceftazidime; CRO, ceftriaxone; CST, colistin; FDC, cefiderocol; FEP, cefepime; IMI, imipenem; MER, meropenem; MIN, minocycline; PLZ, plazomicin; REL, relebactam; TGC, tigecycline; TZP, piperacillin-tazobactam; VAB, vaborbactam.

Results shown for antibiotics represent minimum inhibitory concentrations (MICs), given in micrograms per milliliter as determined by the GN7F and MDRGN2F panels. For all isolates, the MIC for amikacin was ≤8 μg/mL; for ciprofloxacin, ≥2 μg/mL; for gentamicin, ≤2 μg/mL; and for trimethoprim-sulfamethoxazole, >2/38 μg/mL.

After identification of the NDM enzyme by the Carba 5 lateral flow assay (NG Biotech) from isolate 1, the patient was transitioned to cefiderocol and gentamicin on day 3, and cefiderocol monotherapy, administered intravenously using the standard dosing of 2g every 8 hours, given over 3 hours, was started on day 7. Chemotherapy was initiated the same day. On subsequent imaging, the patient’s intra-abdominal collections gradually resolved without additional surgical intervention. Tigecycline was added to cefiderocol beginning on day 17, owing to new fevers.

On day 22, an NDM-producing E. coli isolate was recovered from a urine culture and the cefiderocol MIC increased to >32 μg/mL (isolate 4) after 19 days of cefiderocol exposure. The patient’s antibiotic regimen was adjusted to include both cefiderocol and tigecycline. An NDM-producing E. coli isolate (cefiderocol MIC >32 μg/mL) was recovered from her bloodstream on day 27 (isolate 5), while she was still receiving cefiderocol and tigecycline. She was transitioned to a combination of ceftazidime-avibactam (CAZ-AVI) and aztreonam on day 36, and antibiotic therapy was discontinued on day 65. One year after discontinuation of antibiotic therapy, the patient remains infection free and has since successfully undergone a second hematopoietic stem cell transplantation.

Antimicrobial Susceptibility Testing

Five clinical E. coli isolates were obtained from the patient over a 27-day period and were identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (Bruker Daltonics). Initial antimicrobial susceptibility testing (AST) results were determined using the BD Phoenix Automated System (BD Diagnostics). Additional antimicrobial agents that were tested include cefiderocol, tested with the disk diffusion method (Hardy Diagnostics), tigecycline, tested with Etest (bioMérieux), and colistin, tested with the disk broth elution method [13]. As per routine Johns Hopkins Hospital diagnostic practices, the modified carbapenem inactivation method was performed to identify carbapenemase production, and the Carba 5 lateral flow assay identified specific carbapenemase families from nonblood sources. The GenMark Dx ePlex Blood Culture Identification Gram-Negative Panel identified carbapenemase genes associated with gram-negative organisms recovered from the bloodstream. Isolates were stored at −80°C in glycerol until further testing was performed.

Frozen isolates were subcultured twice using tryptic soy agar with 5% sheep blood. AST was determined using lyophilized Sensititer broth microdilution GN7F and MDRGN2F panels (Thermo Fisher Scientific). Clinical and Laboratory Standards Institute (CLSI) interpretive criteria were applied to determine antimicrobial susceptibility criteria for most agents [11]. For agents for which CLSI criteria were not available for the Enterobacterales, (ie, tigecycline and eravacycline), Food and Drug Administration susceptibility test interpretive criteria were applied [14]. For all AST studies, quality control organisms were evaluated each day of testing.

To determine whether in vitro synergy was present between CAZ-AVI and aztreonam (ATM), as this combination can be effective at inhibition of NDM-induced β-lactam hydrolysis [15], a 0.5 McFarland standard was prepared from fresh cultured growth of the recovered E. coli, and inoculated to form a lawn on cation-adjusted Mueller-Hinton agar plates. Disks containing CAZ-AVI and ATM (both Becton Dickinson) were placed 15mm (edge-to-edge distance) and incubated overnight at 37°C. The agents were considered synergistic if the zones of inhibition around the disks were enhanced in the presence of each other (ie, between the 2 disks). Of note, there is no CLSI-standardized approach to identifying in vitro synergy between CAZ-AVI and ATM.

NDM Inhibition

In addition to using WGS to identify mutations in antimicrobial resistance target genes and the acquisition of antimicrobial resistance genes during cefiderocol exposure, described below, we explored whether inhibition of NDM production could restore cefiderocol activity. EDTA is a chelator known to bind to zinc, which is required for MBLs, including NDM-5, to be active [16]. To observe the impact of NDM inhibition on cefiderocol results, we performed cefiderocol disk diffusion testing with and without EDTA. We placed two 30-μg cefiderocol disks (Hardy Diagnostics), one supplemented with 10 μL of 0.5 mmol/L EDTA (Sigma-Aldrich), on the surface of a Mueller-Hinton agar plate inoculated with a lawn of recovered E. coli and incubated for 18–24 hours at 35°C ± 2°C [11]. A 0.5-mmol/L concentration of EDTA was added; prior work has not demonstrated inhibition of gram-negative organism growth with this concentration of EDTA [11, 17]. The presence of an expanded zone of inhibition with the addition of EDTA (>3mm) was interpreted as circumstantial evidence that NDM production was contributing to cefiderocol resistance.

WGS and Analysis

Genomic DNA was extracted from the 5 E. coli isolates using the PowerBiofilm Kit (Qiagen). WGS was conducted using Illumina MiSeq short-read sequencing (Illumina) and long-read nanopore sequencing, as described elsewhere [18]. WGS sequencing analysis focused on comparing isolates 2–5 with isolate 1 (ie, index NDM-producing isolate) and the E. coli American Type Culture Collection 25922 reference genome, using multiple sequence alignment to identify missense mutations resulting in changes to amino acid composition that may have contributed to changes in cefiderocol MICs. Efforts focused on examining E. coli resistance targets described for earlier siderophore-antibiotic conjugates and/or cefiderocol [3]. These include insertions, deletions, and frameshift mutations in penicillin-binding protein 3 (PBP3), 2-component regulatory systems or iron transport–related proteins (eg, TonB, CirA, Fiu, BaeSR, ExbD, EnvZ, OmpR, and YicM), [5, 6] as well as deletions, insertions, and amino acid substitutions in or proximal to the omega loop of AmpC [7, 8]. Bioinformatics analyses were performed by Ares Genetics and are summarized in the Supplementary Materials. Sequencing reads and de novo whole-genome assemblies were deposited to the National Center for Biotechnology Information, under BioProject PRJNA762553.

Expression Studies

We investigated whether changes in bla_NDM-5 gene expression were associated with increased cefiderocol MICs for isolates 2–5 compared with the index isolate (isolate 1), as described in the Supplementary Materials. Fold changes in bla_NDM-5 gene expression between isolates 2–5 and isolate 1 were calculated from normalized ratios. Each investigation occurred in triplicate, and the average value was used for the analysis. The Centers for Disease Control and Prevention Antibiotic Resistance Bank isolates 0150 and 0151 NDM-5–producing E. coli were included as positive controls [19].

RESULTS

Antimicrobial Susceptibility Testing

The 5 isolates were resistant to all traditional β-lactams (eg, meropenem) as well as novel β-lactam-β-lactam inhibitor combinations (eg, meropenem-vaborbactam; Table 1). Cefiderocol MICs gradually increased from 2 μg/mL in isolates 1–2 to 4 μg/mL in isolate 3 and ≥32 μg/mL in isolates 4–5. The shift from susceptibility to resistance to cefiderocol occurred after 19 days of cefiderocol therapy. Synergy testing with the combination of CAZ-AVI and ATM was conducted for all isolates, and the combination of these agents demonstrated expansion of the zone of inhibition between the disks for all isolates. A similar trend in increasing MICs to tigecycline and minocycline were observed in isolates collected 5 days after the start of tigecycline therapy (Table 1).

WGS Analysis

The sequence types (STs), plasmids and antimicrobial resistance genes associated with the isolates are summarized Supplementary Table 2. All 5 isolates belonged to ST167 and contained a bla_NDM-5 gene identified on a multireplicon IncFIA/IncFIB/IncFII plasmid. The bla_NDM-5 region was highly conserved across all isolates, with an average nucleotide identity of 99.7%, and was located in a structure that comprised IS6-IS30-bla_NDM-5-ble_MBL-trpF-dsbD-IS91 (Figure 2). Isolates 1–3 contained 1 copy of the bla_NDM-5 gene on the IncFIA/FIB/IncFII plasmid, whereas isolates 4 and 5 had increased quantities of the bla_NDM-5 gene at 5 and 10 copies, respectively. Isolates 1–5 harbored bla_CTX-M-15 and bla_OXA-1, and isolate 5 further harbored bla_TEM-1B.

Figure 2.

IncFIA/IncFIB/IncFII consensus plasmid and characteristics of the translocatable unit containing bla_NDM-5 identified in isolates 1–5.

Core genome analysis revealed that isolates 1–5 differ on average by 137 single-nucleotide polymorphisms (SNPs; range 8–307) and 510 indels (insertion or deletion mutations; range, 171–1077). Notably, the number of SNPs and indels is not distributed uniformly across isolates 1–5. There was no correlation between the number of variations and the MIC of cefiderocol (Supplementary Table 3). Variant mapping of isolates 2–5 against isolate 1 revealed additional findings. Isolate 5 contained 19 SNPs in envZ (a sensor kinase gene involved in iron homeostasis [20]); 12 SNPs in marR (a component of the multiple antibiotic resistance regulator [MarR], a transcriptional regulator that oversees an operon that encodes a drug efflux pump [21]); and 31 SNPs in aroP (which encodes a permease necessary for cell transportation and metabolism [22]). SNPs were not identified in these genes in isolates 1–2, and 0–1 SNPs were identified in these genes in isolates 3–5 (Supplementary Table 2). No mutations in outer membrane proteins or PBP3 were identified.

NDM Inhibition and Expression Studies

Figure 3 displays the zone diameters of cefiderocol disk testing alone compared with cefiderocol disks with the addition of EDTA. NDM-5 production was greatest for isolates 4 and 5 based on expression studies. Although cefiderocol susceptibility was not restored in either isolate, the zone diameter increased by 5–6mm with the addition of EDTA (Figure 3). These findings correlate with bla_NDM-5 mRNA expression analysis in which isolates 4 and 5, both with a cefiderocol MIC >32 μg/mL, expressed bla_NDM-5 1.7- and 2.8-fold, respectively, compared to isolate 1 (Figure 4).

Figure 3.

Evaluation of the zone diameters for 5 Escherichia coli isolates, using a 30-μg cefiderocol disk alone or with 10 μL of 0.5 mmol/L of ethylenediaminetetraacetic acid (EDTA) added.

Figure 4.

Expression of bla_NDM-5 in 4 Escherichia coli isolates, comparing isolates 2–5 with isolate 1, using bla_NDM-5 messenger RNA expression analysis and performed in triplicate. Abbreviation: MIC, minimum inhibitory concentration.

DISCUSSION

We report the case of a transplant recipient infected with an E. coli isolate harboring a bla_NDM-5 gene. The NDM-producing strain became progressively less susceptible to cefiderocol after approximately 2 weeks of cefiderocol therapy. Although initial E. coli isolates were susceptible to several β-lactam agents, treatment with cefepime and meropenem likely reduced the abundance of relatively susceptible E. coli isolates colonizing the patient’s intestinal tract and rapidly selected for overgrowth of an NDM-producing isolate (isolate 1–5). WGS analysis indicated an increase in the number of bla_NDM-5 copies that contributed to increased NDM production and reduced cefiderocol susceptibility. This is further supported by the successful expansion of cefiderocol zone diameters in the presence of EDTA and the association between increased bla_NDM-5 expression and increased cefiderocol MICs in mRNA expression analysis.

The concept of increased copies of β-lactamase genes rendering novel β-lactams ineffective when lower copy numbers of the same genes do not yield the same results is not unique to cefiderocol and NDM-carbapenemases. Resistance to CAZ-AVI and meropenem-vaborbactam in the presence of increased copy numbers of the bla_KPC gene has been reported [23–26]. As an example, in one study investigating the mechanisms contributing to meropenem-vaborbactam MIC increases in the Enterobacterales for isolates without mutations in ompK35 or ompK36 genes, 22 of 25 isolates demonstrated a 2–10-fold increase in the bla_KPC gene copy number [26]. Three potential explanations for the increased bla_KPC gene copy number were identified: (1) intracellular transposition of the bla_KPC-carrying TN4401 from a low-copy-number plasmid to a high-copy-number plasmid; (2) rearrangements of a bla_KPC-harboring plasmid, increasing either the copy number of bla_KPC per plasmid or the number of KPC-carrying plasmids; and (3) insertional activation of the repA2 gene, which oversees plasmid replication [26].

A similar phenomenon was described among porcine E. coli ST218 isolates, in which several copies of the bla_NDM-5 gene were identified among conjugative plasmids of multiple replicon types, including IncF type plasmids [27]. Interestingly, bla_NDM-5 was located in a similar translocatable unit IS300-IS5-bla_NDM-5-ble_MBL-trpF-dsbC-IS26-IS300 to the structure observed in our clinical isolates (IS6-IS30-bla_NDM-5-ble_MBL-trpF-dsbD-IS91). The investigators hypothesize that the translocatable unit may act as a reservoir for further dissemination to other plasmids or chromosomal sites. In the present case, it appears the selective pressures of therapy resulted in multiple translocation events of the unit within the same plasmid, resulting in increased copy numbers and expression of bla_NDM-5 contributing to cefiderocol resistance. Differences in bla_NDM-5 copy number and corresponding cefiderocol MICs for isolates 3 and 4 recovered on day 22 from urine likely reflect the evolution of different subpopulations of the ST167 E. coli. NDM-5 has enhanced carbapenemase activity compared with NDM-1, which may further contribute to the reduced activity of cefiderocol observed against our patient’s isolate [28].

Along with increased copy number of bla_NDM-5 genes, we identified mutations in other genes in our patient’s E. coli isolates. Isolate 5 contained a number of SNPs in envZ [20], marR [21], and aroP [22]. We were unable to find any reports in the literature of mutations in marR or aroP contributing to increasing cefiderocol MICs. However, mutations of envZ have been associated with a 4-fold increase in cefiderocol MICs [6]. EnvZ/OmpR is a 2-component signaling system in which EnvZ responds to cytoplasmic signals that arise from changes in the extracellular milieu and OmpR activates a stress response [29]. One of OmpR’s many functions is to oversee 2 genes that negatively regulate the iron transport genes cirA, fecA, and fepA [30]. Despite the logical association with envZ and cefiderocol activity, the role of envZ mutants causing resistance to cefiderocol in our patient’s isolates is unclear because only a single SNP was identified in isolate 4, which had a cefiderocol MIC of >32 μg/mL. Isolate 5 also had a cefiderocol MIC of >32 μg/mL and had 19 SNPs identified in envZ, leading us to postulate that this mutant gene at most contributed a minor role in the cefiderocol resistance exhibited by isolates 4 and 5.

Reports of mechanisms contributing to cefiderocol nonsusceptibility to the Enterobacterales are scarce and heterogenous. Deletions of both cirA and fiu, components of the bacterial iron transport system, have been associated with significant increases in cefiderocol MICs [31]. Mutations in the baeS gene, responsible for encoding a sensor kinase protein of the 2-component BaeSR signal transduction system, contribute to marked increases in cefiderocol MICs [6, 32]. Similarly, mutations in exbD, a component of TonB dependent energy transduction which affects the function of iron transporters has been associated with dramatic increases in cefiderocol MICs [6]. Finally, deletions, insertions, and amino acid substitutions in or proximal to the omega loop of chromosomal AmpC enzymes in the Enterobacter cloacae complex have been linked to increased cefiderocol MICs [7, 8]. We were unable to identify any of these resistance mutations in our patient’s isolates.

Interestingly, within 5 days after the addition of tigecycline to the patient’s treatment regimen, the tigecycline MICs increased from ≤1 to 8 μg/mL. The explanation for this increase is unclear, but at least one of two explanations is possible. First, variants of tetA have been shown to contribute to tigecycline resistance [33, 34]. We did not observe any tetA variants, but we did identify increased copy numbers of tetA in isolates 4 and 5 (2 copies each) which were the 2 isolates with tigecycline MICs of 8 μg/mL. It is unclear whether this copy number would be sufficient to lead to an 8-fold increase in tigecycline MICs. Alternatively, there is at least one published experience of the emergence of tigecycline resistance in an NDM-5–producing E. coli isolate that was presumed to have occurred in part due to dysregulation of the transcriptional activator MarA, which regulates expression of acrAB efflux pump genes [35]. MarA-induced overexpression of the AcrAB efflux pump has been previously linked to the emergence of tigecycline resistance [36]. We identified marR mutations (repressor of the marRAB operon) in isolate 5 (12 SNPs), but this does not explain resistance observed in isolate 4. Further investigation into whether there is any potential cross-resistance between cefiderocol and tigecycline is needed, although in our patient’s case we suspect that these were independent events.

This is a single patient case report, and our findings need to be replicated by others to determine the frequency of increased bla_NDM-5 expression in contributing to cefiderocol resistance. In addition, it is unknown how generalizable our findings are to other MBLs, such as VIM or IMP carbapenemases. Nonetheless, our patient’s case suggests that increased copy numbers of bla_NDM genes may be one of the several approaches used by the Enterobacterales to evade cefiderocol-mediated bacterial cell death.

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

Financial support. This work was supported by the National Institutes of Health (NIH; grant R21-AI153580 to P. J. S. and P. D. T.).

Potential conflicts of interest. P. J. S. reports grants and personal fees from Accelerate Diagnostics, OpGen, Roche Diagnostics, and BD Diagnostics; grants from bioMerieux, Affinity Biosensors, and Hardy Diagnostics; and personal fees from Roche Diagnostics, Shionogi, and GeneCapture, outside the submitted work. H. H. M. reports receipt of the following, all outside the submitted work: research contract, reagents, and equipment from DiaSorin Molecular; research contract, reagents, and equipment from BioRad; consulting fees from Hologic, AlphaSight, and Guidepoint; honoraria/payments for speaking, manuscript writing, or educational events from Qiagen and GenMark; and equipment, materials, drugs, medical writing, gifts, or other services from BioRad, Hologic, and DiaSorin for research. M. A. and S. B. are employees of Ares Genetics. 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.