Instant screening and verification of carbapenemase activity in Bacteroides fragilis in positive blood culture, using matrix-assisted laser desorption ionization–time of flight mass spectrometry

Abstract

Rapid identification of isolates in positive blood cultures are of great importance to secure correct treatment of septicaemic patients. As antimicrobial resistance is increasing, rapid detection of resistance is crucial. Carbapenem resistance in Bacteroides fragilis associated with cfiA-encoded class B metallo-beta-lactamase is emerging. In our study we spiked blood culture bottles with 26 B. fragilis strains with various cfiA-status and ertapenem MICs. By using main spectra specific for cfiA-positive and cfiA-negative B. fragilis strains, isolates could be screened for resistance. To verify strains that were positive in the screening, a carbapenemase assay was performed where the specific peaks of intact and hydrolysed ertapenem were analysed with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). We show here that it is possible to correctly identify B. fragilis and to screen for enzymic carbapenem resistance directly from the pellet of positive blood cultures. The carbapenemase assay to verify the presence of the enzyme was successfully performed on the pellet from the direct identification despite the presence of blood components. The result of the procedure was achieved in 3 h. Also the Bruker mass spectrometric b-lactamase assay (MSBL assay) prototype software was proven not only to be based on an algorithm that correlated with the manual inspection of the spectra, but also to improve the interpretation by showing the variation in the dataset.

Rapid detection ofcfiAmetallo-β-lactamase-producingBacteroides fragilisby the combination of MALDI-TOF MS and CarbaNP

“…The mechanism of cfiA activation in these strains is unknown. A small number of isolates with similar characteristics has been previously described 14 33. Three strains with multiple cfiA gene copies have been previously reported, but correlation between copy number and the degree of resistance is unclear 7…”

“…Nonetheless, the approach has only been evaluated with bacterial collections containing a relatively small number of cfiA -positive isolates 10–12. Recent investigations have further shown that MALDI-TOF MS could be used to detect MBL activity mediated by cfiA in 3 hours 13 14. In addition, the performance of two commercial tests—double-ended Etest±EDTA (bioMerieux) and the KPC/MBL Confirm Kit—in detecting cfiA MBL in B. fragilis has also been evaluated 15.…”

Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae

“…In 2011, two different groups demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was able to detect by-products resulting from the hydrolysis of a carbapenem in the presence of a bacterial extract of CPE (13,14). Since then, several investigators have improved the sensitivity of the method as well as the interpretation of the results (15)(16)(17)(18)(19).…”

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

“…For the latter case, it is important to note that some variants show low enzymatic activity and hence require an extended incubation time (4 hr) to avoid false-negative results [ 49 50 ]. Further, the assay has been applied to samples obtained from positive blood culture, potentially avoiding a cultivation step using solid growth media [ 51 52 ]. Multiplexing, i.e., an assay with multiple antibiotics, may be feasible when lysates of bacteria are used [ 45 ].…”