Evaluation of Metallo-β-Lactamase Susceptibility Testing in a Physiologic Medium

Asempa, Tomefa E; Bajor, Hannah; Mullins, John R.; Hartnett, Janice; Nicolau, David P.

doi:10.1128/spectrum.01670-21

Cited by 3 publications

(2 citation statements)

References 24 publications

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“…This raises the possibility that other metal ions might allow for relevant hydrolytic activity of MBLs in human infections in the absence of zinc. Although a recent study reported lower meropenem MICs in AST with urine as test medium compared to AST with CAMHB ( Asempa et al, 2021b ), another investigation showed that meropenem AST with urine resulted in resistant MICs in vitro , suggesting that MBL-mediated, clinical resistance in urinary tract infections is possible ( Hobson et al, 2020 ). Finally, additional host and pathogen factors that regulate zinc bioavailability and have not yet been explored are conceivable and could play a significant role for treatment success ( Asempa et al, 2021a ).…”

Section: Discussionmentioning

confidence: 99%

Increased zinc levels facilitate phenotypic detection of ceftazidime-avibactam resistance in metallo-β-lactamase-producing Gram-negative bacteria

et al. 2022

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Ceftazidime-avibactam is one of the last resort antimicrobial agents for the treatment of carbapenem-resistant, Gram-negative bacteria. Metallo-β-lactamase-producing bacteria are considered to be ceftazidime-avibactam resistant. Here, we evaluated a semi-automated antimicrobial susceptibility testing system regarding its capability to detect phenotypic ceftazidime-avibactam resistance in 176 carbapenem-resistant, metallo-β-lactamase-producing Enterobacterales and Pseudomonas aeruginosa isolates. Nine clinical isolates displayed ceftazidime-avibactam susceptibility in the semi-automated system and six of these isolates were susceptible by broth microdilution, too. In all nine isolates, metallo-β-lactamase-mediated hydrolytic activity was demonstrated with the EDTA-modified carbapenemase inactivation method. As zinc is known to be an important co-factor for metallo-β-lactamase activity, test media of the semi-automated antimicrobial susceptibility testing system and broth microdilution were supplemented with zinc. Thereby, the detection of phenotypic resistance was improved in the semi-automated system and in broth microdilution. Currently, ceftazidime-avibactam is not approved as treatment option for infections by metallo-β-lactamase-producing, Gram-negative bacteria. In infections caused by carbapenem-resistant Gram-negatives, we therefore recommend to rule out the presence of metallo-β-lactamases with additional methods before initiating ceftazidime-avibactam treatment.

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Section: Discussionmentioning

confidence: 99%

Increased zinc levels facilitate phenotypic detection of ceftazidime-avibactam resistance in metallo-β-lactamase-producing Gram-negative bacteria

et al. 2022

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“…Unlike the previous study ( Oho et al, 2021 ), we did not find supplementation of culture media with zinc sulfate to improve the detection of metallo-β-lactamases (MBLs) of NDM- and VIM-type (data not show). This may be explained by the use in our study of ertapenem instead of imipenem disks or by the cultivation of isolates on Mueller-Hinton agar which contains zinc ions in concentrations sufficient for MBLs to exert their hydrolytic activity ( Asempa et al, 2021 ). Our study, however, did not include IMP-type MBLs that were lacking in our collection, which may be considered as a limitation of the study.…”

Section: Resultsmentioning

confidence: 99%

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate

et al. 2022

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MALDI-TOF mass spectrometry has become widely used in clinical microbiology and has proved highly accurate for detection of carbapenemases in Gram-negative bacteria. However, the use of carbapenem-hydrolysis assays in routine diagnostics is hampered by the need for antibiotic substances and for making their fresh solutions each time an assay is conducted. Here, we evaluated the use of commercial antibiotic susceptibility-testing disks as source of ertapenem substrate in MALDI-TOF MS-based assay for detection of carbapenemase-producing Enterobacterales (CPE). The assay was validated on 48 CPE isolates of 8 different species expressing NDM-, VIM-, KPC- and OXA-48-type carbapenemases and exhibiting various levels of resistance to carbapenems (MIC range: 0.25– > 32 mg/l), as well as on 48 carbapenemase-non-producing isolates. The assay conditions were optimized as follows: 10-μl loopful of bacterial colonies was suspended in 150 μl 0.01 M Na-PBS buffer, pH 7.4, a 10 μg ertapenem susceptibility-testing disk was immersed in the suspension and incubated 3 h at 35°C, after which supernatant was obtained by centrifugation and applied on a target plate with alpha-cyano-4-hydroxycinnamic acid matrix. Mass spectra were analyzed between 440 and 560 m/z. Carbapenemase activity was detected in all tested CPE isolates by the appearance of m/z peaks corresponding to ertapenem hydrolysis products: [Mh + H]+:494.2, [Mh + Na]+:516.2, [Mh + 2Na]+:538.2, [Mh/d + H]+:450.2, [Mh/d + Na]+:472.2, and simultaneous decrease or loss of peaks of intact antibiotic: [M + H]+:476.2, [M + Na]+:498.1, [M + 2Na]+:520.1. No hydrolysis peaks or loss of intact ertapenem peaks were observed for carbapenemase-negative strains. We therefore report the development of a sensitive, specific and cost-effective MALDI-TOF MS-based assay for detection of CPE, which makes use of antibiotic disks readily available in most laboratories.

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Cefepime in vivo activity against carbapenem-resistant Enterobacterales that test as cefepime susceptible or susceptible-dose dependent in vitro: implications for clinical microbiology laboratory and clinicians

Fouad

Gill

Simner

et al. 2023

Journal of Antimicrobial Chemotherapy

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Background Carbapenem-resistant Enterobacterales (CRE) are a public health concern. Among these isolates, there are reports of isolates that test as cefepime susceptible or susceptible-dose dependent (SDD) in vitro despite presence of a carbapenemase. This study aimed to evaluate the pharmacokinetic/pharmacodynamic profile of cefepime against carbapenemase-producing (CP-CRE) and non-producing (non-CP-CRE) isolates with a range of cefepime MICs. Methods Reference broth microdilution and modified carbapenem inactivation method (mCIM) were performed on genotypically characterized clinical CRE isolates. Ultimately, CP-CRE (n = 21; blaKPC) and non-CP-CRE (n = 19) isolates with a distribution of cefepime MICs (≤0.5 to >256 mg/L) were utilized in the murine thigh infection model. Mice were treated with cefepime human-simulated regimens (HSRs) representative of a standard dose (1 g q12h 0.5 h infusion) or the SDD dose (2 g q8h 0.5 h infusion). Efficacy was assessed as the change in bacterial growth at 24 h compared with 0 h control, where ≥1 log bacterial reduction is considered translational value for clinical efficacy. Results Among both cohorts of CRE isolates, i.e. CP-CRE and non-CP-CRE, that tested as SDD to cefepime in vitro, 1 log bacterial reduction was not attainable with cefepime. Further blunting of cefepime efficacy was observed among CP-CRE isolates compared with non-CP-CRE across both susceptible and SDD categories. Conclusions Data indicate to avoid cefepime for the treatment of serious infections caused by CRE isolates that test as cefepime susceptible or SDD. Data also provide evidence that isolates with the same antibiotic MIC may have different pharmacokinetic/pharmacodynamic profiles due to their antimicrobial resistance mechanism.

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Evaluation of Metallo-β-Lactamase Susceptibility Testing in a Physiologic Medium

Cited by 3 publications

References 24 publications

Increased zinc levels facilitate phenotypic detection of ceftazidime-avibactam resistance in metallo-β-lactamase-producing Gram-negative bacteria

Increased zinc levels facilitate phenotypic detection of ceftazidime-avibactam resistance in metallo-β-lactamase-producing Gram-negative bacteria

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate

Cefepime in vivo activity against carbapenem-resistant Enterobacterales that test as cefepime susceptible or susceptible-dose dependent in vitro: implications for clinical microbiology laboratory and clinicians

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