Third-generation cephalosporin resistance among Enterobacteriaceae, mediated by the spread of extended-spectrum β-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone β-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections.
KEYWORDS: AAI101, ESBL, Enterobacteriaceae, carbapenem, cefepime, enmetazobactam, extended-spectrum beta-lactamase, quality control
ABSTRACT
Third-generation cephalosporin resistance among Enterobacteriaceae, mediated by the spread of extended-spectrum β-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone β-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections. Using the Clinical and Laboratory Standards Institute (CLSI) M23 tier 2 study design, broth microdilution MIC and disk diffusion quality control (QC) ranges were determined for cefepime-enmetazobactam. Enmetazobactam was tested at a fixed concentration of 8 μg/ml in the MIC assay, and a cefepime-enmetazobactam disk mass of 30/20 μg was used in the disk diffusion assay. Escherichia coli ATCC 25922, E. coli ATCC 35218, E. coli NCTC 13353, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853 were chosen as reference strains. The CTX-M-15-producing E. coli NCTC 13353 isolate is recommended for routine testing to control for inhibition of ESBL activity by enmetazobactam. Broth microdilution MIC QC ranges spanned 3 to 4 doubling dilutions and contained 99.6% to 100.0% of obtained MIC values for the five reference strains. Disk diffusion yielded inhibition zone diameter QC ranges that spanned 7 mm and encompassed 97.1% to 100.0% of the obtained values. Quality control ranges were approved by the CLSI in 2017 (broth microdilution MIC) and 2019 (disk diffusion). The established QC ranges will ensure that appropriate assay performance criteria are attained using CLSI reference methodology when determining the susceptibility of clinical isolates to cefepime-enmetazobactam.
INTRODUCTION
Extended-spectrum β-lactamases (ESBLs) are a diversified group of enzymes that confer resistance to third- and fourth-generation cephalosporins (1). The prevalence of ESBL-producing Enterobacteriaceae has risen globally (2–5), prompting the World Health Organization to list these pathogens as a priority for development of new therapies (6). Using carbapenems, a “last resort” class of β-lactams, to treat serious infections caused by ESBL-producing Enterobacteriaceae (7) promotes the emergence and dissemination of carbapenem-resistant pathogens (2, 8, 9). Efforts to limit resistance development in Gram-negative pathogens recognize the importance of developing new “carbapenem-sparing” options as empirical therapy for ESBL-producing Enterobacteriaceae (10, 11).
Although piperacillin-tazobactam has been a β-lactam/β-lactamase inhibitor (BL/BLI) mainstay for treating serious infections caused by ESBL-producing Enterobacteriaceae, its continued appropriateness has been questioned due to microbiological concerns and inconsistent clinical efficacy (10, 12–14). Outcomes in a recent randomized clinical trial did not support using piperacillin-tazobactam rather than meropenem for treatment of bloodstream infections caused by ceftriaxone-resistant isolates of Escherichia coli and Klebsiella pneumoniae (15). These results, considered along with the precepts of antibiotic stewardship, underscore the need for carbapenem-sparing therapies for empirical treatment of infections caused by microorganisms expressing contemporary ESBLs (11).
Enmetazobactam (formerly AAI101) is an investigational penicillanic acid sulfone BLI active against a wide range of β-lactamases, particularly ESBLs (TEMs, SHVs, and CTX-Ms) (16). The combination of cefepime with enmetazobactam has in vitro activity comparable to that of meropenem and is more potent than piperacillin-tazobactam against clinical isolates of ESBL-producing Enterobacteriaceae collected in surveillance programs (17). Cefepime-enmetazobactam has entered phase 3 pivotal studies for complicated urinary tract infections, including acute pyelonephritis, attributed to Enterobacteriaceae (ClinicalTrials registration number NCT03687255). In the present study, quality control (QC) ranges for cefepime-enmetazobactam antimicrobial susceptibility testing by broth microdilution and disk diffusion (18–20) were determined.
(Data in this study were presented at the 28th European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], Madrid, Spain, 21 to 24 April 2018 [21], and the 29th ECCMID, Amsterdam, Netherlands, 13 to 16 April 2019 [22].)
MATERIALS AND METHODS
Bacterial isolates and culture media.
Bacterial strains were subcultured on Mueller-Hinton agar (MHA) overnight at 35°C. Isolates used to select the appropriate cefepime-enmetazobactam disk mass included a challenge panel of 58 recent geographically diverse clinical isolates consisting of 20 E. coli isolates, 36 K. pneumoniae isolates, and 2 Proteus mirabilis isolates expressing defined β-lactamases including ESBLs (CTX-M-15, SHV-11, SHV-12, SHV, and TEM-1), OXA-1/30, OXA-48, and KPC-2, of which 37.9% (22 of 56) were resistant to meropenem. An additional test panel of 518 clinical isolates (all obtained during 2016 from a worldwide surveillance program) was included consisting of 21 Citrobacter freundii isolates, 21 Citrobacter koseri isolates, 28 Klebsiella aerogenes isolates, 77 Enterobacter cloacae isolates, 103 E. coli isolates, 27 Klebsiella oxytoca isolates, 101 K. pneumoniae isolates, 27 Morganella morganii isolates, 25 P. mirabilis isolates, 21 Proteus vulgaris isolates, 21 Providencia rettgeri isolates, 21 Providencia stuartii isolates, and 25 Serratia marcescens isolates and expressing a diversity of ESBLs (CTX-M-14, CTX-M-15, CTX-M-27, CTX-M-55, CTX-M-91, SHV-2a, SHV-12, VEB-1), carbapenemases (KPC-2, KPC-3, OXA-48), metallo-β-lactamases (IMP-27, NDM-1, VIM-1), AmpC (ACT-16, ACT-17, ACT-18, ACT-47, CMY-2, CMY-86), and other β-lactamases, such as DHA-1, SHV-11, SHV-28, TEM-1, OXA1/30, OXA-9, and OXA-10.
For the M23 tier 2 QC studies, the QC reference strains used were E. coli ATCC 25922 (constitutive low-level EC-5 narrow-spectrum AmpC expression) (23, 24), E. coli ATCC 35218 (non-ESBL, TEM-1 β-lactamase-producing), E. coli NCTC 13353 (CTX-M-15, ESBL-producing), K. pneumoniae ATCC 700603 (SHV-18, OXA-2 genotype, ESBL-producing), and P. aeruginosa ATCC 27853 (inducible AmpC [PDC-5] β-lactamase-producing) (25). For broth microdilution MIC studies, the three lots of cation-adjusted Mueller-Hinton broth (CAMHB) used were from Difco (lot number 5181782; Becton, Dickinson, Franklin Lakes, NJ), BD/BBL (lot number 5257869; Becton, Dickinson), and Oxoid (lot number 1433705; Thermo Fisher Scientific, Waltham, MA). For disk diffusion studies, the three lots of MHA were from Remel (lot number 348358; Thermo Fisher Scientific), BD/BBL (lot number 8123531; Becton, Dickinson), and Hardy Diagnostics (lot number 417498; Hardy Diagnostics, Santa Maria, CA).
Broth microdilution susceptibility testing.
Bacterial inocula were quantified by serial dilution plating. Broth microdilution MIC testing was performed according to CLSI M07 (18) and M100 (19) guidelines in CAMHB for both cefepime and the combination of cefepime-enmetazobactam. Use of enmetazobactam at a fixed concentration of 8 μg/ml was derived from the exposure-response relationship described in an in vivo infection model (26). MIC panels were manufactured by Thermo Fisher Scientific (Oakwood Village, OH) and shipped frozen to participating laboratories.
Disk diffusion testing and selection of cefepime-enmetazobactam disk mass.
Disk diffusion testing on MHA was performed according to CLSI M02 (20) and M100 (19) guidelines. Bacterial inocula were quantified by serial dilution plating. A CLSI M23 tier 1 study (27) was performed to identify the appropriate cefepime-enmetazobactam disk mass from disks impregnated with either 30/10 μg, 30/15 μg, 30/20 μg, or 30/30 μg of the combination. Disks of cefepime-enmetazobactam were prepared by spotting 20 μl of 50× drug stock solution (cefepime or enmetazobactam) onto sterile Taxo blank disks (lot number 231039; Becton, Dickinson), which were air-dried in a laminar flow hood in the dark before adding the second drug. Control disks containing solvent only were prepared and tested for inhibitory activity. Pending use, impregnated disks were stored at −20°C for up to 2 weeks. Reference broth microdilution MIC values and inhibition zone diameters (determined in duplicate) were obtained concurrently, using identical inocula, for the challenge panel of 58 Enterobacteriaceae isolates spanning a wide range of cefepime-enmetazobactam MIC values (enmetazobactam fixed at 8 μg/ml). Data were examined in scatterplot format, and the error rate-bounded method was used to identify the disk mass minimizing the number of very major (false susceptible), major (false resistant), and minor (misclassification in the range of 1 or 2 doubling dilutions above or below the intermediate MIC [i.e., I + 1, I + 2, I − 1, or I − 2]) discrepancy errors.
Further analysis of cefepime-enmetazobactam disk masses was performed using the test panel of 518 Enterobacteriaceae clinical isolates. For each isolate, broth microdilution MIC and inhibition zone diameters (tested in duplicate) were obtained concurrently, and the error rate-bounded method was used to select the final disk mass. QC strains E. coli ATCC 25922, E. coli ATCC 35218, E. coli NCTC 13353, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853 were used to ensure appropriate assay performance according to CLSI MIC and disk diffusion QC ranges for cefepime (lot number 6307919; Becton, Dickinson, Sparks, MD), meropenem (lot number 7019661; Becton, Dickinson), and piperacillin-tazobactam (lot number 7019661; Becton, Dickinson).
CLSI M23 tier 2 QC range study design.
The CLSI M23 tier 2 guidelines (27) were followed to establish QC ranges for broth microdilution MIC and disk diffusion assays. For broth microdilution MIC QC ranges, eight participating laboratories (exceeding the recommended testing at seven sites and allowing for exclusion of a data set from one laboratory if meeting statistical outlier criteria as described in reference 28) performed 10 MIC replicates in the three different media lots from three manufacturers for the five QC strains, totaling 240 MIC determinations per strain (a minimum of 210 MIC determinations are required). Each MIC replicate utilized an individually prepared inoculum suspension. Susceptibility testing was performed over a minimum of 3 days, with up to four replicates tested per day. Appropriate assay performance was verified by comparing cefepime MIC values for E. coli ATCC 25922, E. coli NCTC 13353, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853 to the established QC ranges reported in CLSI M100 (29). For disk diffusion QC ranges, 10 replicates of two lots of cefepime-enmetazobactam 30/20-μg disks (lot numbers 3044 and 3045; Oxoid Ltd, Basingstoke, UK) were tested on MHA from three different sources (2 × 3 × 10 = 60 inhibition zone diameters per laboratory) in each of the 8 participating laboratories, for a total of 480 inhibition zone diameters per QC strain (a minimum of 420 inhibition zone diameters are required). Two lots of cefepime 30-μg disks (lot number 8030809; Becton, Dickinson and lot number 2288845; Oxoid) were used. One lot of piperacillin-tazobactam 100/10-μg disks (lot number 8052746, Becton, Dickinson) also was tested (1 × 3 × 10 = 30 inhibition zone diameters per laboratory × 8 sites = 240 total inhibition zone diameter values per QC strain). Appropriate assay performance was assessed by comparing inhibition zone diameters obtained for cefepime and piperacillin-tazobactam disks to established QC ranges. Testing was performed over a minimum of 3 days, with no more than four replicates tested per day. Each replicate utilized an individually prepared inoculum suspension (minimum of five inoculum verifications per organism per participating laboratory). Three laboratories participated in both broth microdilution and disk diffusion M23 QC testing.
Cefepime-enmetazobactam 30/20-μg disks from a second supplier (Liofilchem S.r.l., Roseto degli Abruzzi, Italy) became available for testing after completing the M23 tier 2 studies. Performance of the Liofilchem disks was assessed for the five QC strains following CLSI tier 1 guidelines, in which 10 replicates of a single disk lot on three different sources of MHA (1 × 3 × 10 = 30 inhibition zone diameters per QC strain) were tested in a single laboratory over 3 days and with three inoculum preparations. Cefepime-enmetazobactam 30/20-μg disks from Oxoid and cefepime 30-μg disks (Becton, Dickinson; used for QC purposes) were tested concurrently to assess appropriate assay performance.
Analysis of QC reference ranges.
Inhibition zone diameter ranges for each QC reference strain were determined using the Gavan statistic (30) and RangeFinder (28) statistical program. RangeFinder also determines if the central tendencies (mean, median, and mode) of data sets obtained from individual laboratories are statistical outliers (28) and should be excluded from analysis. Only the CLSI-accepted QC ranges are presented in the text.
RESULTS
Determination of broth microdilution MIC QC ranges.
Cefepime-enmetazobactam (fixed enmetazobactam concentration of 8 μg/ml) broth microdilution MIC QC ranges for the five QC reference strains were established following a CLSI M23 tier 2 study design that included eight participating laboratories (Table 1). Overall, cefepime-enmetazobactam MIC determinations demonstrated acceptable intra- and interlaboratory reproducibility, as 99.2 to 100% of all reported values for the five QC strains were within a span of ≤3 doubling dilutions (Tables 2 to 6). The commercial source of CAMHB had no meaningful impact on MIC determinations for cefepime or cefepime-enmetazobactam, as the median and modal values for all strains varied by no more than a single doubling dilution. For E. coli ATCC 25922, cefepime-enmetazobactam MIC values were within a three log2 dilution range (0.03 to 0.12 μg/ml) (Table 2). All cefepime MIC values for this isolate were within the CLSI-approved four doubling dilution QC range of 0.016 to 0.12 μg/ml (data not shown), confirming appropriate assay performance. For TEM-1-producing strain E. coli ATCC 35218, which is highly susceptible to cefepime, MIC values obtained for cefepime with or without enmetazobactam were within the established cefepime CLSI four doubling dilution range of 0.008 to 0.06 μg/ml (Table 3). For CTX-M-15-producing strain E. coli NCTC 13353, which is highly resistant to cefepime, cefepime-enmetazobactam MIC values were within a three doubling dilution range (0.03 to 0.12 μg/ml) (Table 4). All cefepime MICs determined against E. coli NCTC 13353 were ≥64 μg/ml, affirming ESBL expression in this strain. The MIC data set collected in one participating laboratory for this isolate was determined to be a statistical outlier for the mean, median, and modal MIC values based on the RangeFinder program and was excluded from the analysis. For SHV-18-producing strain K. pneumoniae ATCC 700603, which encodes an ESBL that inefficiently hydrolyzes cefepime (31), cefepime-enmetazobactam MIC values were within a three doubling dilution range (0.12 to 0.5 μg/ml) (Table 5) that overlapped the four doubling dilution range of MICs obtained for cefepime (0.25 to 2 μg/ml; data not shown); all cefepime MICs were within the CLSI-approved range. For P. aeruginosa ATCC 27853, including enmetazobactam had no effect on the QC range relative to that of cefepime alone, as cefepime MICs with or without enmetazobactam were within the CLSI-approved cefepime QC range of 0.5 to 4 μg/ml (Table 6; data not shown).
TABLE 1.
CLSI-approved broth microdilution MIC QC ranges determined for cefepime and cefepime-enmetazobactam (fixed enmetazobactam concentration of 8 μg/ml) against selected reference strains
| Reference strain | Cefepime-enmetazobactam |
Cefepime |
||||
|---|---|---|---|---|---|---|
| MIC QC rangea | No. of doubling dilutions in range | % Of values in rangeb | MIC QC range | No. of doubling dilutions in range | % Of values in range | |
| E. coli ATCC 25922 | 0.03/8–0.12/8 | 3 | 100.0 | 0.016–0.12c | 4 | 100.0 |
| E. coli ATCC 35218 | 0.008/8–0.06/8 | 4 | 100.0 | 0.008–0.06 | 4 | 100.0 |
| E. coli NCTC 13353 | 0.03/8–0.12/8d | 3 | 100.0 | ≥64c | 100.0 | |
| K. pneumoniae ATCC 700603 | 0.12/8–0.5/8 | 3 | 100.0 | 0.25–2c | 4 | 100.0 |
| P. aeruginosa ATCC 27853 | 0.5/8–2/8 | 3 | 99.6 | 0.5–4c | 4 | 100.0 |
QC ranges were approved at the June 2017 meeting of the CLSI Subcommittee on Antimicrobial Susceptibility Testing.
Percentage of values in range determined from 240 replicates performed in 8 laboratories.
Current CLSI QC range (35).
Excluding data from one laboratory (statistical outlier).
TABLE 2.
Inter- and intralaboratory comparisons of cefepime-enmetazobactam MICs (fixed enmetazobactam concentration of 8 μg/ml) and inhibition zone diameters with 30/20-μg disks for E. coli ATCC 25922 obtained in the CLSI M23 tier 2 study to establish QC ranges
| Assay Measurea | No. of occurrences by medium lotb
|
No. of occurrences by disk lot |
No. of occurrences by laboratoryc
|
Total no. of occurrences | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | A | B | A | B | C | D | E | F | G | H | ||
| MIC (μg/ml) | ||||||||||||||
| 0.03 | 41 | 23 | 3 | 19 | 9 | 3 | 7 | 3 | 10 | 3 | 13 | 67 | ||
| 0.06 | 39 | 57 | 75 | 11 | 21 | 27 | 22 | 26 | 20 | 27 | 17 | 171 | ||
| 0.12 | 2 | 1 | 1 | 2 | ||||||||||
| IZD (mm) | ||||||||||||||
| 31 | 1 | 1 | 1 | 1 | ||||||||||
| 32 | ||||||||||||||
| 33 | 16 | 8 | 4 | 14 | 14 | 4 | 1 | 4 | 10 | 9 | 28 | |||
| 34 | 27 | 28 | 29 | 39 | 45 | 8 | 10 | 15 | 10 | 2 | 17 | 22 | 84 | |
| 35 | 50 | 69 | 48 | 86 | 81 | 23 | 19 | 19 | 28 | 32 | 17 | 3 | 26 | 167 |
| 36 | 49 | 36 | 48 | 72 | 61 | 20 | 31 | 14 | 14 | 25 | 10 | 16 | 3 | 133 |
| 37 | 10 | 13 | 20 | 17 | 26 | 5 | 11 | 3 | 1 | 37 | 17 | 43 | ||
| 38 | 6 | 2 | 3 | 4 | 7 | 11 | 11 | |||||||
| 39 | 2 | 4 | 5 | 6 | 5 | 11 | 2 | |||||||
| 40 | 2 | 1 | 1 | 2 | ||||||||||
| Total | 80/160 | 80/160 | 80/160 | 240 | 240 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 240/480 |
| Mean | 0.04/35.2 | 0.05/35.3 | 0.06/35.5 | 35.3 | 35.4 | 0.04/35.2 | 0.05/35.4 | 0.06/35.3 | 0.05/35.0 | 0.06/35.4 | 0.05/34.8 | 0.06/37.3 | 0.05/34.4 | 0.05/35.3 |
| Median | 0.03/35 | 0.06/35 | 0.06/35 | 35 | 35 | 0.03/35 | 0.06/36 | 0.06/35 | 0.06/35 | 0.06/35 | 0.06/35 | 0.06/37 | 0.06/34 | 0.06/35 |
| Mode | 0.03/35 | 0.06/35 | 0.06/35,36 | 35 | 35 | 0.03/35 | 0.06/36 | 0.06/35 | 0.06/35 | 0.06/35 | 0.06/34,35 | 0.06/37 | 0.06/35 | 0.06/35 |
| Geometric mean | 0.04/35.2 | 0.05/35.2 | 0.06/35.5 | 35.3 | 35.3 | 0.04/35.2 | 0.05/35.3 | 0.06/35.3 | 0.05/35.0 | 0.06/35.4 | 0.05/34.7 | 0.06/37.3 | 0.04/34.4 | 0.05/35.3 |
| Range | 2/7 | 2/7 | 3/10 | 10 | 8 | 2/5 | 2/3 | 2/5 | 3/7 | 3/4 | 2/5 | 2/6 | 2/4 | 3/10 |
Values in bold are results from the broth microdilution MIC assay. Enmetazobactam was included in the combination at a fixed concentration of 8 μg/ml. IZD, inhibition zone diameter.
The medium used for broth microdilution MIC determinations was cation-adjusted Mueller-Hinton broth from (A) Difco (lot number 5181782), (B) BD (lot number 5257869), and (C) Oxoid (lot number 1433705). The medium used in the disk diffusion assay was Mueller-Hinton agar from (A) Remel (lot number 348358), (B) BBL (lot number 8123531), and (C) Hardy Diagnostics (lot number 417498).
The eight qualified participating laboratories are coded A through H. Only three of the laboratories participated in both the broth microdilution MIC and disk diffusion M23 QC studies.
TABLE 3.
Inter- and intralaboratory comparisons of cefepime-enmetazobactam MICs (enmetazobactam concentrations fixed at 8 μg/ml) and inhibition zone diameters with 30/20-μg disks for E. coli ATCC 35218 obtained in the CLSI M23 tier 2 study to establish QC ranges
| Assay measurea | No. of occurrences by medium lotb
|
No. of occurrences by disk lot |
No. of occurrences by laboratoryc
|
Total no. of occurrences | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | A | B | A | B | C | D | E | F | G | H | ||
| MIC (μg/ml) | ||||||||||||||
| 0.008 | 49 | 9 | 5 | 8 | 4 | 6 | 6 | 4 | 7 | 49 | ||||
| 0.016 | 59 | 5 | 41 | 20 | 11 | 9 | 12 | 9 | 12 | 9 | 23 | 105 | ||
| 0.03 | 21 | 26 | 37 | 1 | 12 | 13 | 14 | 15 | 12 | 17 | 84 | |||
| 0.06 | 2 | 2 | 2 | |||||||||||
| IZD (mm) | ||||||||||||||
| 32 | 3 | 3 | 2 | 3 | 5 | 2 | 1 | 5 | 8 | |||||
| 33 | 11 | 12 | 4 | 12 | 15 | 7 | 5 | 3 | 2 | 10 | 27 | |||
| 34 | 31 | 40 | 29 | 50 | 50 | 27 | 8 | 9 | 12 | 2 | 19 | 23 | 100 | |
| 35 | 38 | 51 | 44 | 67 | 66 | 22 | 23 | 13 | 14 | 23 | 16 | 6 | 16 | 133 |
| 36 | 61 | 39 | 60 | 79 | 81 | 2 | 25 | 28 | 25 | 35 | 14 | 25 | 6 | 160 |
| 37 | 15 | 15 | 17 | 26 | 21 | 4 | 3 | 6 | 9 | 25 | 47 | |||
| 38 | 1 | 4 | 3 | 2 | 1 | 4 | 5 | |||||||
| Total | 80/160 | 80/160 | 80/160 | 240 | 240 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 240/480 |
| Mean | 0.02/35.2 | 0.016/35.0 | 0.024/35.4 | 35.2 | 35.1 | 0.013/34.3 | 0.020/35.4 | 0.018/35.3 | 0.020/35.6 | 0.019/35.6 | 0.018/35.2 | 0.021/36.5 | 0.013/34.1 | 0.017/35.2 |
| Median | 0.016/35 | 0.008/35 | 0.016/36 | 35 | 35 | 0.016/34 | 0.016/35 | 0.016/36 | 0.016/36 | 0.02/36 | 0.016/35 | 0.03/36 | 0.016/34 | 0.016/35 |
| Mode | 0.016/36 | 0.008/35 | 0.016/36 | 36 | 36 | 0.016/34 | 0.03/36 | 0.03/36 | 0.03/36 | 0.03/36 | 0.016, 0.03/34 | 0.03/36, 37 | 0.016/34 | 0.016/36 |
| Geometric mean | 0.019/35.2 | 0.012/35.0 | 0.022/35.4 | 35.2 | 35.1 | 0.013/34.2 | 0.020/35.4 | 0.017/35.3 | 0.020/35.3 | 0.019/35.5 | 0.018/35.1 | 0.021/36.4 | 0.014/34.1 | 0.017/35.2 |
| Range | 2/7 | 3/6 | 3/7 | 7 | 7 | 3/5 | 4/4 | 3/7 | 3/5 | 3/3 | 3/5 | 3/4 | 2/5 | 4/7 |
Values in bold are results from the broth microdilution MIC assay. Enmetazobactam was included in the combination at a fixed concentration of 8 μg/ml.
The medium used for broth microdilution MIC determinations was cation-adjusted Mueller-Hinton broth from (A) Difco (lot number 5181782), (B) BD (lot number 5257869), and (C) Oxoid (lot number 1433705). The medium used in the disk diffusion assay was Mueller-Hinton agar from (A) Remel (lot number 348358), (B) BBL (lot number 8123531), and (C) Hardy Diagnostics (lot number 417498).
The eight qualified participating laboratories are coded A through H. Only three laboratories participated in both broth microdilution MIC and disk diffusion M23 QC studies.
TABLE 4.
Inter- and intralaboratory comparisons of cefepime-enmetazobactam MICs (fixed enmetazobactam concentration of 8 μg/ml) and inhibition zone diameters with 30/20-μg disks for E. coli ATCC 13353 obtained in the CLSI M23 tier 2 study to establish QC ranges
| Assay measurea | No. of occurrences by medium lotb
|
No. of occurrences by disk lot |
No. of occurrences by laboratoryc
|
Total no. of occurrences | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | A | B | A | B | C | D | E | F | G | H | ||
| MIC (μg/ml) | ||||||||||||||
| 0.03 | 2 | 1 | 1 | 2 | ||||||||||
| 0.06 | 67 | 66 | 64 | 29 | 24 | 29 | 29 | 30 | 1 | 28 | 28 | 197 | ||
| 0.12 | 1 | 4 | 6 | 6 | 1 | 2 | 2 | 11 | ||||||
| 0.25 | 18 | |||||||||||||
| 0.5 | 11 | |||||||||||||
| IZD (mm) | ||||||||||||||
| 27 | 4 | 6 | 2 | 8 | 10 | |||||||||
| 28 | 14 | 11 | 9 | 2 | 14 | 25 | ||||||||
| 29 | 47 | 46 | 30 | 1 | 11 | 1 | 14 | 11 | 25 | 93 | ||||
| 30 | 70 | 73 | 10 | 17 | 23 | 21 | 27 | 20 | 16 | 9 | 143 | |||
| 31 | 67 | 77 | 9 | 32 | 22 | 21 | 17 | 21 | 18 | 4 | 144 | |||
| 32 | 31 | 23 | 10 | 4 | 13 | 8 | 19 | 54 | ||||||
| 33 | 7 | 4 | 4 | 7 | 11 | |||||||||
| Total | 80/160 | 80/160 | 80/160 | 240 | 240 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 240/480 |
| Mean | 0.06/30.4 | 0.06/29.8 | 0.07/30.4 | 30.3 | 30.2 | 0.06/29.3 | 0.07/30.9 | 0.06/30.3 | 0.06/31.0 | 0.06/30.0 | 0.34/30.4 | 0.07/31.3 | 0.07/28.8 | 0.06/30.2 |
| Median | 0.06/31 | 0.06/30 | 0.06/30.5 | 30 | 30 | 0.06/29 | 0.06/31 | 0.06/30 | 0.06/31 | 0.06/30 | 0.25/30 | 0.06/31 | 0.06/29 | 0.06/30 |
| Mode | 0.06/31 | 0.06/29 | 0.06/31 | 30 | 31 | 0.06/29 | 0.06/31 | 0.06/30 | 0.06/30, 31 | 0.06/30 | 0.25/31 | 0.06/32 | 0.06/29 | 0.06/31 |
| Geometric mean | 0.06/30.4 | 0.06/29.8 | 0.06/30.4 | 30.2 | 30.2 | 0.06/29.2 | 0.07/30.8 | 0.06/30.3 | 0.06/31.0 | 0.06/30.0 | 0.31/30.4 | 0.06/31.3 | 0.06/28.8 | 0.06/30.2 |
| Range | 3/7 | 2/6 | 2/7 | 7 | 7 | 2/5 | 2/4 | 2/4 | 2/5 | 1/4 | 4/4 | 2/4 | 2/5 | 3/7 |
Values in bold are results determined from the broth microdilution MIC assay. Enmetazobactam was included in the combination at a fixed concentration of 8 μg/ml. Values in bold italics are MIC results that have been excluded from the analysis as the data set determined in the participating laboratory (F) was deemed to be a statistical outlier.
The medium used for broth microdilution MIC determinations was cation-adjusted Mueller-Hinton broth from (A) Difco (lot number 5181782), (B) BD (lot number 5257869), and (C) Oxoid (lot number 1433705). The medium used in the disk diffusion assay was Mueller-Hinton agar from (A) Remel (lot number 348358), (B) BBL (lot number 8123531), and (C) Hardy Diagnostics (lot number 417498).
The eight qualified participating laboratories are coded A through H. Only three laboratories participated in both broth microdilution MIC and disk diffusion M23 QC studies.
TABLE 5.
Inter- and intralaboratory comparisons of cefepime-enmetazobactam MICs (enmetazobactam concentration fixed at 8 μg/ml) and inhibition zone diameters with 30/20-μg disks for K. pneumoniae ATCC 700603 obtained in the CLSI M23 tier 2 study to establish QC ranges
| Assay measurea | No. of occurrences by medium lotb
|
No. of occurrences by disk lot |
No. of occurrences by laboratoryc
|
Total no. of occurrences | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | A | B | A | B | C | D | E | F | G | H | ||
| MIC (μg/ml) | ||||||||||||||
| 0.12 | 10 | 3 | 4 | 12 | 2 | 1 | 2 | 17 | ||||||
| 0.25 | 62 | 68 | 69 | 17 | 23 | 27 | 26 | 27 | 24 | 27 | 28 | 199 | ||
| 0.5 | 8 | 9 | 7 | 1 | 7 | 1 | 4 | 3 | 5 | 3 | 24 | |||
| IZD (mm) | ||||||||||||||
| 23 | 1 | 1 | 1 | 1 | ||||||||||
| 24 | ||||||||||||||
| 25 | ||||||||||||||
| 26 | 1 | 4 | 1 | 5 | 1 | 6 | 6 | |||||||
| 27 | 8 | 19 | 4 | 18 | 13 | 14 | 7 | 6 | 4 | 31 | ||||
| 28 | 36 | 53 | 31 | 57 | 63 | 26 | 35 | 12 | 21 | 8 | 1 | 17 | 120 | |
| 29 | 46 | 39 | 46 | 66 | 65 | 12 | 18 | 20 | 10 | 22 | 23 | 9 | 17 | 131 |
| 30 | 37 | 32 | 41 | 51 | 59 | 2 | 22 | 25 | 10 | 15 | 16 | 20 | 110 | |
| 31 | 17 | 9 | 26 | 28 | 24 | 6 | 16 | 1 | 14 | 15 | 52 | |||
| 32 | 12 | 4 | 6 | 11 | 11 | 9 | 12 | 1 | 22 | |||||
| 33 | 2 | 5 | 4 | 3 | 7 | 7 | ||||||||
| Total | 80/160 | 80/160 | 80/160 | 240 | 240 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 240/480 |
| Mean | 0.26/30.4 | 0.27/29.8 | 0.27/30.4 | 30.3 | 30.2 | 0.19/27.8 | 0.29/28.2 | 0.24/29.4 | 0.27/30.4 | 0.27/28.7 | 0.27/29.6 | 0.27/30.8 | 0.24/28.9 | 0.26/29.2 |
| Median | 0.25/31 | 0.25/30 | 0.25/30.5 | 30 | 30 | 0.25/28 | 0.25/28 | 0.25/29 | 0.25/30 | 0.25/29 | 0.25/29 | 0.25/31 | 0.25/29 | 0.25/29 |
| Mode | 0.25/31 | 0.25/29 | 0.25/31 | 30 | 31 | 0.25/28 | 0.25/28 | 0.25/30 | 0.25/30 | 0.25/29 | 0.25/29 | 0.25/30 | 0.25/30 | 0.25/29 |
| Geometric mean | 0.24/30.4 | 0.25/29.8 | 0.26/30.4 | 30.2 | 30.2 | 0.19/27.8 | 0.29/28.2 | 0.24/29.4 | 0.27/30.4 | 0.27/28.6 | 0.27/29.6 | 0.27/30.8 | 0.24/28.8 | 0.25/29.2 |
| Range | 3/7 | 3/6 | 3/7 | 7 | 7 | 3/5 | 2/3 | 3/4 | 2/4 | 2/5 | 3/4 | 2/6 | 2/10 | 3/11 |
Values in bold are results from the broth microdilution MIC assay. Enmetazobactam was included in the combination at a fixed concentration of 8 μg/ml.
The medium used for broth microdilution MIC determinations was cation-adjusted Mueller-Hinton broth from (A) Difco (lot number 5181782), (B) BD (lot number 5257869), and (C) Oxoid (lot number 1433705). The medium used in the disk diffusion assay was Mueller-Hinton agar from (A) Remel (lot number 348358), (B) BBL (lot number 8123531), and (C) Hardy Diagnostics (lot number 417498).
The eight qualified participating laboratories are coded A through H. Only three laboratories participated in both broth microdilution MIC and disk diffusion M23 QC studies.
TABLE 6.
Inter- and intralaboratory comparisons of cefepime-enmetazobactam MICs (fixed enmetazobactam concentration of 8 μg/ml) and inhibition zone diameters with 30/20-μg disks for P. aeruginosa ATCC 27853 obtained in the CLSI M23 tier 2 study to establish QC ranges
| Assay measurea | No. of occurrences by medium lotb
|
No. of occurrences by disk lot |
No. of occurrences by laboratoryc
|
Total no. of occurrences | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | A | B | A | B | C | D | E | F | G | H | ||
| MIC (μg/ml) | ||||||||||||||
| 0.5 | 3 | 1 | 1 | 2 | 3 | 5 | ||||||||
| 1 | 63 | 68 | 64 | 22 | 25 | 29 | 26 | 20 | 30 | 23 | 20 | 195 | ||
| 2 | 14 | 11 | 14 | 8 | 5 | 1 | 2 | 9 | 7 | 7 | 39 | |||
| 4 | 1 | 1 | 1 | |||||||||||
| IZD (mm) | ||||||||||||||
| 26 | 1 | 2 | 1 | 2 | 3 | |||||||||
| 27 | 8 | 12 | 4 | 13 | 11 | 19 | 2 | 2 | 1 | |||||
| 28 | 32 | 40 | 38 | 55 | 55 | 30 | 29 | 1 | 5 | 20 | 25 | |||
| 29 | 40 | 46 | 44 | 59 | 71 | 8 | 27 | 16 | 7 | 31 | 4 | 9 | 28 | |
| 30 | 52 | 37 | 46 | 68 | 67 | 2 | 39 | 24 | 21 | 20 | 23 | 6 | ||
| 31 | 20 | 21 | 22 | 35 | 28 | 4 | 24 | 1 | 13 | 21 | ||||
| 32 | 5 | 1 | 6 | 7 | 5 | 3 | 3 | 6 | ||||||
| 33 | 2 | 1 | 2 | 1 | 2 | 1 | ||||||||
| Total | 80/160 | 80/160 | 80/160 | 240 | 240 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 30/60 | 240/480 |
| Mean | 1.2/30.4 | 1.1/29.8 | 1.2/30.4 | 29.3 | 29.2 | 1.1/27.8 | 1.1/28.2 | 1/29.4 | 1/30.4 | 1.3/28.7 | 1/29.6 | 1.2/30.8 | 1.1/28.9 | 1.1/29.2 |
| Median | 1/31 | 1/30 | 1/30.5 | 29 | 29 | 1/28 | 1/28 | 1/29 | 1/30 | 1/29 | 1/29 | 1/31 | 1/29 | 1/29 |
| Mode | 1/31 | 1/29 | 1/31 | 30 | 29 | 1/28 | 1/28 | 1/30 | 1/30 | 1/29 | 1/29 | 1/30 | 1/30 | 1/29 |
| Geometric mean | 1.1/30.4 | 1.1/29.8 | 1.1/30.4 | 29.3 | 29.2 | 1.2/27.8 | 1.1/28.2 | 1/29.4 | 1/30.4 | 1.3/28.6 | 1/29.6 | 1.2/30.8 | 1.1/28.8 | 1.1/29.2 |
| Range | 3/8 | 3/8 | 4/6 | 8 | 8 | 2/5 | 2/3 | 2/4 | 3/4 | 3/5 | 1/4 | 2/6 | 3/10 | 4/11 |
Values in bold are results from the broth microdilution MIC assay. Enmetazobactam was included in the combination at a fixed concentration of 8 μg/ml.
The medium used for broth microdilution MIC determinations was cation-adjusted Mueller-Hinton broth from (A) Difco (lot number 5181782), (B) BD (lot number 5257869), and (C) Oxoid (lot number 1433705). The medium used in the disk diffusion assay was Mueller-Hinton agar from (A) Remel (lot number 348358), (B) BBL (lot number 8123531), and (C) Hardy Diagnostics (lot number 417498).
The eight qualified participating laboratories are coded A through H. Only three of the laboratories participated in both broth microdilution MIC and disk diffusion M23 QC studies.
Selection of cefepime-enmetazobactam disk mass.
A two-step approach was taken to select a suitable cefepime-enmetazobactam disk mass. Initially, four different cefepime-enmetazobactam disk masses were evaluated in a pilot study against a challenge panel of 58 Enterobacteriaceae isolates that express a range of β-lactamases including ESBLs (CTX-M, SHV, and TEM), AmpC, OXA, and KPC and with MIC values bracketing the projected susceptibility breakpoint for cefepime-enmetazobactam (susceptible-dose-dependent MIC of ≤8 μg/ml). When this evaluation was completed, two of the four cefepime-enmetazobactam disk masses from the pilot study were selected to assess their performance against a test panel of 518 contemporary Enterobacteriaceae clinical isolates. All cefepime-enmetazobactam disks contained a cefepime mass of 30 μg to match the cefepime mass used in commercially available disks approved by the CLSI and EUCAST. Inhibition zone diameters and MICs were correlated in scatterplots and error rates determined as described in CLSI document M23-04 (27).
Cefepime-enmetazobactam (fixed enmetazobactam concentration of 8 μg/ml) broth microdilution MIC values covered a range from 0.03 μg/ml to 32 μg/ml for the challenge panel of 58 isolates. Disk diffusion was performed concurrently, and results were presented in scatterplots for cefepime-enmetazobactam disk masses of 30/10 μg (see Fig. S1 in the supplemental material), 30/15 μg (see Fig. S2 in the supplemental material), 30/20 μg (see Fig. S3 in the supplemental material), and 30/30 μg (see Fig. S4 in the supplemental material).
When the CLSI cefepime breakpoint interpretive criteria (susceptible, ≤2 μg/ml; susceptible dose dependent, 4 to 8 μg/ml; resistant, ≥16 μg/ml) for Enterobacteriaceae were applied to the scatterplot of cefepime-enmetazobactam 30/20-μg disks, the proposed inhibition zone diameter breakpoints of ≥22 mm for susceptible and ≤18 mm for resistant resulted in no very major errors or major errors (Fig. S3). Adjusting the breakpoints for 30/10-μg disks to ≥20 mm for susceptible and ≤15 mm for resistant (Fig. S1), for 30/15-μg disks to ≥21 mm for susceptible and ≤16 mm for resistant (Fig. S2), and for 30/30-μg disks to ≥22 mm for susceptible and ≤17 mm for resistant (Fig. S4) yielded the lowest very major error rates attainable for each disk mass (1.7%, 1.7%, and 2.6%, respectively) yet still exceeded the limit of 1.5%. A single major error occurred with both the 30/10-μg and 30/15-μg disks. Results for all four cefepime-enmetazobactam disk masses exceeded the minor error rates for ≥I + 2 (50.0 to 100.0%) and I + 1 to I − 1 (43.3 to 59.4%), an observation likely due to the small sample size and clinically nonrepresentative composition of the challenge panel. Based on these results, 30/10-μg and 30/20-μg disk masses were selected for further analysis using a much larger and more clinically representative strain panel.
The cefepime-enmetazobactam (fixed enmetazobactam concentration of 8 μg/ml) MIC values determined for the clinically representative test set of 518 contemporary Enterobacteriaceae isolates ranged from 0.008 μg/ml to 128 μg/ml. From the scatterplot with 30/10-μg disks (see Fig. S5 in the supplemental material), a proposed breakpoint of ≥23 mm for susceptible and ≤18 mm for resistant resulted in no very major errors or major errors, whereas a high minor error rate of 68.8% (I + 1 to I − 1) exceeded the 40% limit. From the scatterplot with 30/20-μg disks (see Fig. S6 in the supplemental material), breakpoints of ≥25 mm for susceptible and ≤19 mm for resistant resulted in acceptable error rates, with no very major errors, no major errors, a minor error rate within the I + 1 to I − 1 range of 25.0% (<40% required), and a total minor error rate of 0.9%. Based on these results, the cefepime-enmetazobactam 30/20-μg disk mass was selected to establish QC ranges.
Determination of disk diffusion QC ranges.
A CLSI M23 tier 2 study with eight participating laboratories was used to establish cefepime-enmetazobactam 30/20-μg disk QC ranges for the five QC strains (Table 7). In each laboratory, 60 replicate inhibition zone diameters were determined for the QC strains and analyzed using RangeFinder and the Gavan statistics methods to determine appropriate QC ranges.
TABLE 7.
CLSI disk diffusion QC ranges determined for cefepime (30 μg) and cefepime-enmetazobactam (30/20 μg) against relevant reference strains
| Reference strain | Cefepime-enmetazobactam |
Cefepime |
||||
|---|---|---|---|---|---|---|
| Inhibition zone diameter QC range (mm)a | No. of mm | % Of values in rangeb | Inhibition zone diameter QC range (mm) | No. of mm | % Of values in range | |
| E. coli ATCC 25922 | 32–38 | 7 | 97.1 | 31–37c | 7 | 97.1 |
| E. coli ATCC 35218 | 32–38 | 7 | 100.0 | 31–37 | 7 | 100.0 |
| E. coli NCTC 13353 | 27–33 | 7 | 100.0 | 6–15c ,d | 10 | 99.5 |
| K. pneumoniae ATCC 700603 | 26–32 | 7 | 98.3 | 23–29c | 7 | 99.8 |
| P. aeruginosa ATCC 27853 | 26–32 | 7 | 99.4 | 25–31c | 7 | 99.2 |
QC ranges were approved at the January 2019 meeting of the CLSI Subcommittee on Antimicrobial Susceptibility Testing.
Percentage of values in range determined from 480 replicates performed in eight laboratories.
Current CLSI QC range (35).
Excluding data from one laboratory (statistical outlier).
For E. coli ATCC 25922, the geometric means of cefepime-enmetazobactam 30/20-μg disk inhibition zone diameters ranged from 34.4 mm to 37.3 mm among the participating laboratories (Table 2). A QC range of 32 to 38 mm is proposed, with 97.1% of obtained inhibition zone diameters occurring within these limits (Table 7). For E. coli ATCC 35218, the geometric means ranged from 34.1 mm to 36.4 mm (Table 3). All inhibition zone diameters for this isolate were within the proposed QC range of 32 to 38 mm (Table 7). The geometric means for E. coli NCTC 13353 ranged from 28.8 mm to 31.1 mm (Table 4); the proposed QC range for this isolate is 27 to 33 mm, with 100% of reported values occurring within this boundary (Table 7). For K. pneumoniae ATCC 700603, geometric means varied from 27.8 mm to 29.6 mm (Table 5). A QC range of 26 to 32 mm is proposed, with 98.3% of reported inhibition zone diameters occurring within these limits (Table 7). The geometric means for P. aeruginosa ATCC 27853 ranged from 27.7 mm to 30.5 mm (Table 6), and a QC range of 26 to 32 mm was calculated, encompassing 99.4% of replicate values obtained in the participating laboratories (Table 7). Across all participating laboratories, the source of commercially prepared MHA plates (from three different manufacturers) had minimal impact on inhibition zone diameters, with differences in geometric means of <1 mm for each of the five QC strains (Tables 2 to 6). Moreover, minimal variability was observed between the two disk lots from a single manufacturer (Oxoid, Thermo Fisher Scientific), as the geometric means for both lots differed by ≤0.1 mm for each QC strain (Tables 2 to 6).
All of the inhibition zone diameters determined with cefepime-enmetazobactam 30/20-μg disks from a second manufacturer (Liofilchem S.r.l.) in the tier 1 study were within the approved CLSI ranges for the five QC reference strains (see Table S1 in the supplemental material). Geometric means for these disks, though smaller by 1.1 mm to 2.6 mm for the QC strains, were still within acceptable limits.
DISCUSSION
As increased usage of carbapenems to treat ESBL-producing pathogens has helped drive carbapenem resistance, “carbapenem-sparing” therapeutic options are required to stem resistance development and dissemination among Gram-negative pathogens (2, 32, 33). Development of the combination of cefepime with the novel BLI enmetazobactam aims to provide empiric and definitive therapy for infections caused by Enterobacteriaceae, particularly those expressing ESBLs. Whereas cefepime exhibits intrinsic activity against isolates expressing β-lactamases, AmpCs, and many OXAs, including OXA-48 (34), this fourth-generation cephalosporin remains susceptible to most ESBLs. Enmetazobactam inhibits a broad array of class A β-lactamases, including SHV, TEM, and CTX-M ESBLs (16), and cefepime-enmetazobactam exhibited similar potencies against both a collection of 1,696 recent clinical isolates of Enterobacteriaceae and a subset of 211 ESBL-producing Enterobacteriaceae, with an MIC90 of 0.25 μg/ml for both groups (17). That MIC QC ranges of cefepime-enmetazobactam (fixed enmetazobactam concentration of 8 μg/ml) for CTX-M-15-producing E. coli NCTC 13353 and E. coli ATCC 25922 are identical indicates that ESBL activity was completely suppressed by enmetazobactam. Differences in the diffusion properties of cefepime and/or enmetazobactam through agar, or reduced enzyme-inhibitor interactions in semisolid medium, or differences in physiological status or amplitude of expression of blaCTX-M-15 under liquid versus semisolid conditions may account for the smaller inhibition zone diameter range obtained for E. coli NCTC 13353 relative to that of E. coli ATCC 25922. An MIC QC range of ≥64 μg/ml was previously established for cefepime against E. coli NCTC 13353, and all cefepime MIC replicates in this study were determined to be >128 μg/ml. This strain has been recommended previously as a routine QC strain for testing cefepime-tazobactam (35, 36) and likewise serves as an appropriate control to assess the functionality of enmetazobactam in combination with cefepime. It is, therefore, recommended as a routine QC strain to assess cefepime-enmetazobactam performance in broth microdilution and disk diffusion assays.
The MIC QC ranges of cefepime-enmetazobactam (enmetazobactam concentration fixed at 8 μg/ml) for E. coli ATCC 25922, E. coli ATCC 35218, K. pneumoniae ATCC 700603, and P. aeruginosa ATCC 27853 were nearly identical to those for cefepime alone (Table 1); similarly, cefepime-enmetazobactam QC ranges were marginally smaller but overlapped those for cefepime alone (Tables 2). Enmetazobactam provided little additional benefit to the antimicrobial activity of cefepime against these four strains, and they are not advised for monitoring BLI activity of enmetazobactam despite a recommendation that E. coli ATCC 35218 and K. pneumoniae ATCC 700603 are suitable for routine testing of most BL/BLI combinations (19).
In conclusion, the proposed broth microdilution and disk diffusion QC ranges for cefepime-enmetazobactam were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at the June 2017 and January 2019 meetings, respectively. The approved QC ranges ensure that clinical laboratories can reliably and reproducibly assess appropriate assay performance of cefepime-enmetazobactam against several reference strains, most notably E. coli NCTC 13353. These results will help establish the susceptibility breakpoints for cefepime-enmetazobactam required to inform patient care.
Supplementary Material
ACKNOWLEDGMENTS
This study was funded by Allecra Therapeutics SAS (Saint-Louis, France).
A.B. and P.K. are employees of Allecra Therapeutics SAS. S.S. is a consultant for Allecra Therapeutics SAS and a shareholder in Allecra Therapeutics GmbH. M.D.H., K.A.F., A.A.W., and R.K.F. are employees of JMI Laboratories, Inc. (North Liberty, IA).
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/JCM.00607-19.
REFERENCES
- 1.Bradford PA. 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 14:933–951. doi: 10.1128/CMR.14.4.933-951.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Doi Y, Iovleva A, Bonomo RA. 2017. The ecology of extended-spectrum β-lactamases (ESBLs) in the developed world. J Travel Med 24:S44–S51. doi: 10.1093/jtm/taw102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Lob SH, Nicolle LE, Hoban DJ, Kazmierczak KM, Badal RE, Sahm DF. 2016. Susceptibility patterns and ESBL rates of Escherichia coli from urinary tract infections in Canada and the United States, SMART 2010–2014. Diagn Microbiol Infect Dis 85:459–465. doi: 10.1016/j.diagmicrobio.2016.04.022. [DOI] [PubMed] [Google Scholar]
- 4.Cassini A, Högberg LD, Plachouras D, Quattrocchi A, Hoxha A, Simonsen GS, Colomb-Cotinat M, Kretzschmar ME, Devleesschauwer B, Cecchini M, Ouakrim DA, Oliveira TC, Struelens MJ, Suetens C, Monnet DL, Strauss R, Mertens K, Struyf T, Catry B, Latour K, Ivanov IN, Dobreva EG, Tambic Andraševic A, Soprek S, Budimir A, Paphitou N, Žemlicková H, Schytte Olsen S, Wolff Sönksen U, Märtin P, Ivanova M, Lyytikäinen O, Jalava J, Coignard B, Eckmanns T, Abu Sin M, Haller S, Daikos GL, Gikas A, Tsiodras S, Kontopidou F, Tóth Á, Hajdu Á, Guólaugsson Ó, Kristinsson KG, Murchan S, Burns K, Pezzotti P, et al. . 2019. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European economic area in 2015: a population-level modelling analysis. Lancet Infect Dis 19:56–66. doi: 10.1016/S1473-3099(18)30605-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Temkin E, Fallach N, Almagor J, Gladstone BP, Tacconelli E, Carmeli Y. 2018. Estimating the number of infections caused by antibiotic-resistant Escherichia coli and Klebsiella pneumoniae in 2014: a modelling study. Lancet Glob Heal 6:e969–e979. doi: 10.1016/S2214-109X(18)30278-X. [DOI] [PubMed] [Google Scholar]
- 6.World Health Organization. 2017. Global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. World Health Organization, Geneva, Switzerland. [Google Scholar]
- 7.Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, Levin SA, Goossens H, Laxminarayan R. 2018. Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc Natl Acad Sci U S A 115:E3463–E3470. doi: 10.1073/pnas.1717295115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.World Health Organization. 2014. Antimicrobial resistance: global report on surveillance (2014). World Health Organization, Geneva, Switzerland. [Google Scholar]
- 9.Bush K. 2018. Past and present perspectives on β-lactamases. Antimicrob Agents Chemother 62:e01076-18. doi: 10.1128/aac.01076-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Tamma PD, Rodriguez BJ. 2017. The use of noncarbapenem β-lactams for the treatment of extended-spectrum β-lactamase infections. Clin Infect Dis 64:972–980. doi: 10.1093/cid/cix034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Hayden MK, Won SY. 2018. Carbapenem-sparing therapy for extended-spectrum β-lactamase-producing E. coli and Klebsiella pneumoniae bloodstream infection: the search continues. JAMA 320:979–981. doi: 10.1001/jama.2018.12565. [DOI] [PubMed] [Google Scholar]
- 12.Pilmis B, Jullien V, Tabah A, Zahar JR, Brun-Buisson C. 2017. Piperacillin–tazobactam as alternative to carbapenems for ICU patients. Ann Intensive Care 7:113. doi: 10.1186/s13613-017-0334-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Nguyen HM, Shier KL, Graber CJ. 2014. Determining a clinical framework for use of cefepime and β-lactam/β-lactamase inhibitors in the treatment of infections caused by extended-spectrum-β-lactamase-producing Enterobacteriaceae. J Antimicrob Chemother 69:871–880. doi: 10.1093/jac/dkt450. [DOI] [PubMed] [Google Scholar]
- 14.Ng TM, Khong WX, Harris PNA, De PP, Chow A, Tambyah PA, Lye DC. 2016. Empiric piperacillin-tazobactam versus carbapenems in the treatment of bacteraemia due to extended-spectrum beta-lactamase-producing Enterobacteriaceae. PLoS One 11:e0153696. doi: 10.1371/journal.pone.0153696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Harris P, Tambyah PA, Lye DC, Mo Y, Lee TH, Yilmaz M, Alenazi TH, Arabi Y, Falcone M, Bassetti M, Righi E, Rogers BA, Kanj S, Bhally H, Iredell J, Mendelson M, Boyles TH, Looke D, Miyakis S, Walls G, Al Khamis M, Zikri A, Crowe A, Ingram P, Daneman N, Griffin P, Athan E, Lorenc P, Baker P, Roberts L, Beatson SA, Peleg AY, Harris-Brown T, Paterson DL, MERINO Trial Investigators and the Australasian Society for Infectious Disease Clinical Research Network (ASID-CRN). 2018. Effect of piperacillin-tazobactam vs meropenem on 30-day mortality for patients with E. coli or Klebsiella pneumoniae bloodstream infection and ceftriaxone resistance. JAMA 320:984–994. doi: 10.1001/jama.2018.12163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Papp-Wallace KM, Bethel CR, Caillon J, Barnes MD, Potel G, Bajaksouzian S, Rutter JD, Reghal A, Shapiro S, Taracila MA, Jacobs MR, Bonomo RA, Jacqueline C. 2019. Beyond piperacillin-tazobactam: cefepime and AAI101 as a potent β-lactam-β-lactamase inhibitor combination. Antimicrob Agents Chemother 63:e00105-19. doi: 10.1128/AAC.00105-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Morrissey I, Magnet S, Hawser S, Shapiro S, Knechtle P. 15 April 2019. In vitro activity of cefepime-enmetazobactam against Gram-negative isolates collected from United States and European hospitals during 2014-2015. Antimicrob Agents Chemother doi: 10.1128/AAC.00514-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Clinical and Laboratory Standards Institute. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—10th ed CLSI document M07-A10 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 19.Clinical and Laboratory Standards Institute. 2018. Performance standards for antimicrobial susceptibility testing; 28th informational supplement. CLSI M100-S28 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 20.Clinical and Laboratory Standards Institute. 2018. Performance standards for antimicrobial disk susceptibility tests; approved standard—13th ed CLSI document M02-A10 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 21.Huband M, Rhomberg PR, Fedler KA, Flamm RK, Knechtle P, Shapiro S. 2018. Disk content assessment and proposed breakpoint interpretive criteria for cefepime in combination with AAI101, abstr P0157. Abstr 28th European Congress of Clinical Microbiology and Infectious Diseases, Madrid, Spain, 21 to 24 April 2018. [Google Scholar]
- 22.Huband MD, Fedler KA, Watters AA, Belley A, Knechtle P, Flamm RK. 2019. Cefepime-enmetazobactam (formerly AAI101; 30/20 μg) and cefepime (30 μg) disk diffusion quality control ranges using a CLSI M23 (2018) multi-laboratory study design, abstr P2778. Abstr 29th European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, Netherlands, 13 to 16 April 2019. [Google Scholar]
- 23.Tracz DM, Boyd DA, Bryden L, Hizon R, Giercke S, Van Caeseele P, Mulvey MR. 2005. Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT–PCR. J Antimicrob Chemother 55:768–772. doi: 10.1093/jac/dki074. [DOI] [PubMed] [Google Scholar]
- 24.Black JA, Moland ES, Thomson KS. 2005. AmpC disk test for detection of plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking chromosomal AmpC β-lactamases. J Clin Microbiol 43:3110–3113. doi: 10.1128/JCM.43.7.3110-3113.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Lahiri SD, Walkup GK, Whiteaker JD, Palmer T, McCormack K, Tanudra MA, Nash TJ, Thresher J, Johnstone MR, Hajec L, Livchak S, McLaughlin RE, Alm RA. 2015. Selection and molecular characterization of ceftazidime/avibactam-resistant mutants in Pseudomonas aeruginosa strains containing derepressed AmpC. J Antimicrob Chemother 70:1650–1658. doi: 10.1093/jac/dkv004. [DOI] [PubMed] [Google Scholar]
- 26.Crandon JL, Nicolau DP. 2015. In vivo activities of simulated human doses of cefepime and cefepime-AAI101 against multidrug-resistant gram-negative Enterobacteriaceae. Antimicrob Agents Chemother 59:2688–2694. doi: 10.1128/AAC.00033-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Clinical and Laboratory Standards Institute. 2018. Development of in vitro susceptibility testing criteria and quality control parameters; approved standard—4th ed CLSI document M23-04 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 28.Turnidge J, Bordash G. 2007. Statistical methods for establishing quality control ranges for antibacterial agents in clinical and laboratory standards institute susceptibility testing. Antimicrob Agents Chemother 51:2483–2488. doi: 10.1128/AAC.01457-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Clinical and Laboratory Standards Institute. 2017. Performance standards for antimicrobial susceptibility testing; 27th informational supplement. CLSI M100-S27 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 30.Gavan TL, Jones RN, Barry AL, Fuchs PC, Gerlach EH, Matsen JM, Reller LB, Thornsberry C, Thrupp LD. 1981. Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: a collaborative study. J Clin Microbiol 14:67–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Rasheed JK, Anderson GJ, Yigit H, Queenan AM, Doménech-Sánchez A, Swenson JM, Biddle JW, Ferraro MJ, Jacoby GA, Tenover FC. 2000. Characterization of the extended-spectrum β-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18. Antimicrob Agents Chemother 44:2382–2388. doi: 10.1128/aac.44.9.2382-2388.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Wilson A. 2017. Sparing carbapenem usage. J Antimicrob Chemother 72:2410–2417. doi: 10.1093/jac/dkx181. [DOI] [PubMed] [Google Scholar]
- 33.Joseph NM, Bhanupriya B, Shewade DG, Harish BN. 2015. Relationship between antimicrobial consumption and the incidence of antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae isolates. J Clin Diagn Res 9:DC08–DC12. doi: 10.7860/JCDR/2015/11029.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Endimiani A, Perez F, Bonomo RA. 2008. Cefepime: a reappraisal in an era of increasing antimicrobial resistance. Expert Rev Anti Infect Ther 6:805–824. doi: 10.1586/14787210.6.6.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Clinical and Laboratory Standards Institute. 2019. Performance standards for antimicrobial susceptibility testing. 29th informational supplement. CLSI M100-S29 Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 36.Riedel S, Huband MD, Sader HS, Flamm RK, Jones RN. 2017. Determination of disk diffusion and MIC quality control guidelines for high-dose cefepime-tazobactam (WCK 4282), a novel antibacterial combination consisting of a β-lactamase inhibitor and a fourth-generation cephalosporin. J Clin Microbiol 55:3130–3134. doi: 10.1128/JCM.00788-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.