The Serratia marcescens enzyme (SME) is a chromosomally encoded carbapenemase with no known optimal treatment. Various β-lactam/β-lactamase inhibitors and comparators were evaluated against 8 SME producers via broth microdilution. Four isolates were subsequently tested via time-kill analyses. All isolates were resistant to imipenem, imipenem-relebactam, and meropenem but susceptible to ceftazidime, ceftazidime-avibactam, and meropenem-vaborbactam.
KEYWORDS: Serratia marcescens, imipenem-relebactam, meropenem-vaborbactam, SME, carbapenem-resistant Enterobacteriaceae
ABSTRACT
The Serratia marcescens enzyme (SME) is a chromosomally encoded carbapenemase with no known optimal treatment. Various β-lactam/β-lactamase inhibitors and comparators were evaluated against 8 SME producers via broth microdilution. Four isolates were subsequently tested via time-kill analyses. All isolates were resistant to imipenem, imipenem-relebactam, and meropenem but susceptible to ceftazidime, ceftazidime-avibactam, and meropenem-vaborbactam. Ceftazidime, imipenem-relebactam, and meropenem-vaborbactam were bactericidal against 3, 0, and 4 isolates, respectively. Meropenem-vaborbactam may be a potential option for severe SME-producing infections.
INTRODUCTION
The Serratia marcescens enzyme (SME) is an Ambler class A carbapenemase that was first identified in 1982 from two clinical S. marcescens strains in England (1) and has since been reported elsewhere in Europe (2, 3), North America (4–6), and South America (7, 8). Although the prevalence of SME-producing S. marcescens currently appears low, it is likely underestimated because none of the commercially available carbapenemase detection methods include blaSME (9). In addition to being infrequently encountered, SME-harboring S. marcescens isolates generally display a unique phenotypic profile, demonstrating resistance to all β-lactams, including carbapenems, with the exception of extended-spectrum cephalosporins (e.g., ceftazidime and cefepime) (4, 9). Despite this baseline susceptibility, the development of resistance to these β-lactams during therapy and subsequent clinical failure have been documented in AmpC- and SME-producing S. marcescens (10). As a result, there is a need to identify alternative agents with adequate activity against these pathogens. The activities of relebactam and vaborbactam have been demonstrated against other Ambler class A carbapenemases but have not been well described against SME producers (11, 12). The objective of this work was to compare the in vitro activity of imipenem-relebactam, meropenem-vaborbactam, and comparator agents against clinical SME-producing S. marcescens strains.
(Results of this study were presented in part at ASM Microbe 2019, San Francisco, CA [13].)
Eight SME-producing S. marcescens isolates were obtained from the CDC and FDA Antibiotic Resistance Isolate Bank (14). Analytical grade ampicillin, avibactam, aztreonam, cefazolin, cefepime, ceftazidime, gentamicin, imipenem, levofloxacin, meropenem, sulfamethoxazole, trimethoprim (Sigma-Aldrich, St. Louis, MO), relebactam, and vaborbactam (MedChemExpress, Monmouth Junction, NJ) were obtained commercially. MICs were determined in triplicate via reference broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines (15). Avibactam (4 mg/liter), relebactam (4 mg/liter), and vaborbactam (8 mg/liter) were tested at fixed concentrations. Susceptibilities were determined based on approved CLSI breakpoints. Imipenem- or meropenem-based combinations were interpreted using the approved breakpoints for the carbapenem alone (16).
Complete genomes of the eight S. marcescens strains were downloaded from the NCBI nucleotide database, and β-lactam resistance genes were identified by BLAST searching against the ResFinder 3.1 (17) and CARD-RGI (18) databases. To assess genetic relatedness, a pangenome database was built via the Build_PGAdb module of the PGAdb-builder (19) and uploaded to the Build_wgMLSTtree module. The uploaded genomes were compared with the built allele database using BLASTN23 with default parameters and applied to create a whole-genome multilocus sequence type (wgMLST) tree (20).
Time-kill experiments were performed as previously described against a subset of four isolates with various phenotypic profiles to ceftazidime and meropenem-vaborbactam (21). Ceftazidime, imipenem, imipenem-relebactam, meropenem, and meropenem-vaborbactam were tested alone at 0.25×, 1×, and 4× the MIC unless any of these concentrations exceeded the respective drug’s free maximum concentration (fCmax) value, in which case the fCmax was used. Ceftazidime-avibactam was not tested in time-kill experiments because all isolates were susceptible to ceftazidime alone at baseline. The fCmax values utilized were 25 mg/liter for imipenem (22) and 45 mg/liter for meropenem (23). For meropenem-vaborbactam, an MIC of 0.03 mg/liter was assumed for time-kill experiments for isolates with MICs of ≤0.03 mg/liter.
All eight strains carried the SME-4 resistance gene and one or more AmpC genes and were phenotypically resistant to both carbapenems but susceptible to ceftazidime (Tables 1 and 2). Only two pairs of isolates (SM-3/SM-4 and SM-5/SM-8) had <10-bp differences in the core genome. When combined with ceftazidime, imipenem, or meropenem, vaborbactam produced the largest fold reduction in MICs compared with avibactam and relebactam (Table 2). Neither carbapenem was bactericidal alone against any strain at the highest concentration tested (4× MIC or fCmax), whereas ceftazidime alone was bactericidal against three of four strains at 4× MIC (Fig. 1). The addition of relebactam to imipenem did not restore bactericidal activity or produce synergy against any strain, whereas meropenem-vaborbactam was bactericidal and synergistic against all strains tested (Fig. 1).
TABLE 1.
Genotypic and phenotypic susceptibility of tested agents against 8 clinical SME-producing S. marcescens isolates
| Isolate | β-Lactamasea | MIC (mg/liter) ofb: |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| AMP | ATM | CFZ | CAZ | CFP | GEN | IMI | LFX | MER | TMP-SMZc | ||
| SM-1 | SME-4, SRT-2 | ≥256 | 64 | ≥256 | 1 | 0.25 | 1 | ≥256 | 0.125 | 128 | 0.5 |
| SM-2 | SME-4, SRT-1, SRT-2 | ≥256 | 64 | ≥256 | 0.5 | 0.25 | 1 | ≥256 | 0.125 | 128 | 0.5 |
| SM-3 | SME-4, SRT-2 | ≥256 | 32 | ≥256 | 0.125 | 0.25 | 0.5 | ≥256 | 0.125 | 128 | 0.5 |
| SM-4 | SME-4, SRT-2 | ≥256 | 64 | ≥256 | 1 | 1 | 1 | ≥256 | 0.125 | 128 | 0.5 |
| SM-5 | SME-4, SRT-2 | ≥256 | 64 | ≥256 | 0.5 | 1 | 1 | ≥256 | 0.25 | 128 | 1 |
| SM-6 | SME-4, SRT-2 | ≥256 | 128 | ≥256 | 1 | 0.25 | 1 | ≥256 | 0.125 | 128 | 0.25 |
| SM-7 | SME-4, SST-1 | ≥256 | 64 | ≥256 | 0.5 | 0.125 | 1 | ≥256 | 0.125 | 128 | 0.5 |
| SM-8 | SME-4, SRT-2 | ≥256 | 64 | ≥256 | 0.25 | 0.125 | 0.5 | ≥256 | 0.125 | ≥256 | 0.5 |
SME, S. marcescens enzyme; SRT, class C cephalosporinase from S. marcescens hydrolyzing the 2-carboxypenam T-5575; SST, produced by β-lactam-susceptible strain of S. marcescens.
AMP, ampicillin; ATM, aztreonam; CFZ, cefazolin; CAZ, ceftazidime; CFP, cefepime; GEN, gentamicin; IMI, imipenem; LFX, levofloxacin; MER, meropenem; TMP-SMZ, trimethoprim-sulfamethoxazole.
MIC of trimethoprim component only.
TABLE 2.
MICs of various β-lactam/β-lactamase inhibitor combinations against 8 clinical SME-producing S. marcescens isolates
| Isolate | MIC (mg/liter) ofa: |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CAZ | CAZ-AVI | CAZ-REL | CAZ-VAB | IMI | IMI-AVI | IMI-REL | IMI-VAB | MER | MER-AVI | MER-REL | MER-VAB | |
| SM-1 | 1 | 0.25 | 0.25 | 0.25 | ≥256 | 8 | 128 | 2 | 128 | 2 | 8 | 0.125 |
| SM-2 | 0.5 | 0.125 | 0.25 | 0.25 | ≥256 | 8 | ≥256 | 4 | 128 | 1 | 64 | ≤0.03 |
| SM-3 | 0.125 | 0.125 | 0.25 | 0.125 | ≥256 | 8 | 64 | 2 | 128 | 2 | 64 | ≤0.03 |
| SM-4 | 1 | 0.25 | 0.25 | 0.125 | ≥256 | 8 | 128 | 2 | 128 | 2 | 16 | 0.125 |
| SM-5 | 0.5 | 0.125 | 0.5 | 0.5 | ≥256 | 8 | ≥256 | 2 | 128 | 1 | 16 | ≤0.03 |
| SM-6 | 1 | 0.125 | 0.25 | 0.25 | ≥256 | 8 | ≥256 | 2 | 128 | 16 | 32 | ≤0.03 |
| SM-7 | 0.5 | 0.125 | 0.25 | 0.25 | ≥256 | 16 | 64 | 2 | 128 | 0.25 | 1 | ≤0.03 |
| SM-8 | 0.25 | 0.06 | 0.25 | 0.25 | ≥256 | 8 | ≥256 | 2 | ≥256 | 16 | 32 | ≤0.03 |
Avibactam and relebactam were tested at a fixed concentration of 4 mg/liter; vaborbactam was tested at a fixed concentration of 8 mg/liter. CAZ, ceftazidime; AVI, avibactam; REL, relebactam; VAB, vaborbactam; IMI, imipenem; MER, meropenem.
FIG 1.
Mean log10 CFU/ml versus time profile for each drug at the highest concentration tested against four SME-producing S. marcescens strains. Ceftazidime and meropenem-vaborbactam are shown at 4× MIC, and imipenem, imipenem-relebactam, and meropenem are shown at fCmax. Curves represent average concentrations for triplicate experiments.
To our knowledge, the current study is the largest to date to report the susceptibility of SME-producing S. marcescens isolates to imipenem-relebactam and meropenem-vaborbactam; the first to directly compare the inhibitory activities of avibactam, relebactam, and vaborbactam by using the same β-lactam backbone; and the first to report on the time-killing profile of any antimicrobial agent against SME-producing S. marcescens isolates. Results of the current study suggest that imipenem is more efficiently hydrolyzed than meropenem by SME-4, which is in agreement with previous findings for SME-1 and SME-2 (4). Importantly, these data also suggest that vaborbactam has greater inhibitory activity than relebactam against SME. This may be related to the higher binding affinity and longer residence time of vaborbactam than relebactam against Ambler class A carbapenemases, such as the Klebsiella pneumoniae carbapenemase-1 (24, 25), which has the highest similarity to SME-1 (∼45% identical) (26). Further enzymatic characterization studies are warranted to confirm these findings. Limitations of our study include the 24-h static nature of time-kill experiments, the number of strains tested, and inclusion of only SME-4-producing strains. Additionally, the bactericidal activity of ceftazidime and meropenem-vaborbactam may have been underestimated in time-kill experiments by using multiplicative MICs in place of serum achievable concentrations, but this strategy allowed us to compare differences between S. marcescens strains that were not exclusively due to differences in the concentration-MIC ratio (27).
In the absence of clinical data, our results support meropenem-vaborbactam as a treatment option for severe or life-threatening SME-producing S. marcescens infections and demonstrate that relebactam does not reliably restore the activity of carbapenems against these pathogens. Further clinical investigation of extended-spectrum cephalosporins and meropenem-vaborbactam for SME-producing S. marcescens infections is warranted.
ACKNOWLEDGMENT
E.W. serves on the speaker’s bureau for Melinta Therapeutics and Astellas Pharma and on the advisory board for GenMark Diagnostics and Shionogi. All other authors have no conflicts of interest to declare.
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