Direct β-Lactam Inactivation Method: a New Low-Cost Assay for Rapid Detection of Carbapenemase- or Extended-Spectrum-β-Lactamase-Producing Enterobacterales Directly from Positive Blood Culture Bottles

Abstract

We validate and evaluate a new phenotypic assay, named the direct β-lactam inactivation method (dBLIM), for the rapid and simultaneous detection of carbapenemase or extended-spectrum-cephalosporinase activity directly from Enterobacterales (EB)-positive blood cultures (BCs). It originates from the carbapenem inactivation method (CIM), an inexpensive and highly sensitive assay for carbapenemase activity detection. dBLIM cutoff values to detect extended-spectrum β-lactamase (ESBL) and carbapenemase activities resulted in diameters of ≤12 mm for a 5-μg-cefotaxime disk and for a 10-μg-meropenem disk. dBLIM assessment was determined with both aerobic and anaerobic BC bottles spiked with 422 characterized EB strains, classifiable into the following 4 phenotypic groups: (i) ESBL/AmpC-type β-lactamase (ACBL)/carbapenemase (CARB)-nonproducing (np-ESBL/ACBL/CARB) EB (n = 116), (ii) ESBL-producing EB (n = 111), (iii) AmpC-β-lactamase-producing EB (n = 33), and (iv) carbapenemase-producing EB (n = 162). No false-positive results were obtained in any of the np-ESBL/ACBL/CARB EB, ESBL, and AmpC groups, demonstrating an overall assay specificity of 100%. There were no significant discrepancies in dBLIM performance between aerobic and anaerobic BCs across all groups, except with VIM-type carbapenemase-expressing EB. Interestingly, among BCs spiked with blaVIM-harboring EB, the sensitivity rates of the assay in anaerobic and aerobic bottles were 53.6% and 100%, respectively. In contrast, dBLIM performance was deemed excellent for the KPC, OXA-48, and NDM carbapenemase producers regardless of the type of bottle being tested, with a sensitivity rate ranging between 99% and 100%. Concerning the detection of the extended-spectrum cephalosporinases of the ESBL-producing and AmpC types, dBLIM sensitivities was 100% and 84 to 87%, respectively. dBLIM may be a cost-effective and highly robust phenotypic screening method for the reliable detection of carbapenemases or extended-spectrum cephalosporinases directly from BCs on the same day of bottle positivity detection.

A Lateral Flow Immunoassay for the Rapid Identification of CTX-M-Producing Enterobacterales from Culture Plates and Positive Blood Cultures

“…Easy-to-carry-out tests, mostly based on inhibition of ESBLs by clavulanic acid or tazobactam, are recommended [ 30 ]. A new phenotypic assay that originates from the carbapenem inactivation method (CIM) [ 31 ], named the direct β-lactam inactivation method (dBLIM), that allows detection of extended-spectrum-cephalosporinase activity directly from Enterobacterales in culture or from positive blood cultures, using a 5-µg- cefotaxime disk [ 32 ]. These tests have excellent performances, but their main pitfalls are limited sensitivity, the requirement of an overnight incubation and doesn’t give information about the resistance mechanisms or genotype, and thus does not provide any information about the possible threats of spreading.…”

Modern Blood Culture

“…Overall, both colorimetric and mCIM-based methods demonstrate high specificities (≥90%) and sensitivities (80%–100%) for the detection of β-lactamase and/or carbapenemase-producing Enterobacterales extracted directly from aerobic blood culture bottles. 71 , 72 , 73 , 74 Resulting times between 20 minutes to 6.5 hours have been reported for colorimetric methods, and between 8 to 24 hours for mCIM-based methods. Although these early results are generally promising, more studies must be done to standardize methodology and result interpretation before clinical use.…”

“…Because these assays are relatively cheap and simple, many have explored their use directly on positive blood cultures to test for the presence of β-lactamase and/or carbapenemase-producing gram-negative bacteria, which are of great clinical concern internationally. 71 , 72 , 73 , 74 Colorimetric methods usually start with microbial extraction from positive blood cultures using lysis centrifugation or a short subculture on solid media. The ideal choice of hemolytic agent varies depending on the assay.…”

“…Variations of CLSI’s modified carbapenem inactivation method (mCIM) have also been used for bacteria with ESBL and carbapenemase activity directly from positive blood cultures. 71 , 73 , 74 Briefly, bacteria are extracted from blood cultures and incubated with either a meropenem or a cephalosporin disk at 35°C to 37°C for approximately 2 hours. Zinc supplementation can be used for metallo-β-lactamase activity detection.…”

Detection of Multidrug-Resistant Enterobacterales—From ESBLs to Carbapenemases

“…While the principle of this assay is applicable for the detection of ESBLs and carbapenemases in Gram-negative bacteria including nonfermenters, it is most commonly used for carbapenemase detection in Enterobacterales . Both cultured bacteria and blood-culture fluid can be used for the β-lactam inactivation assay [ 56 , 122 ].…”