Evaluation of MBT STAR-Cepha and MBT STAR-Carba kits for the detection of extended-spectrum β-lactamases and carbapenemase producing microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Rapid and simple detection of extended-spectrum β-lactamase (ESBL) and carbapenemase is essential for antimicrobial treatment and infection control. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based MBT STAR-Cepha and MBT STAR-Carba kits have been developed with simplified MBT STAR-BL operations. However, the utility of these kits has not been fully examined in clinical microbiology laboratories. In this study, we evaluated the utility of MALDI-TOF MS-based MBT STAR-Cepha and MBT STAR-Carba kits to detect ESBL and carbapenemase-producing bacteria, and compared it with the conventional broth microdilution test and PCR amplification assay. We found that the MBT STAR-Cepha kit efficiently distinguished resistant strains of third-generation cephalosporin susceptibility phenotypes and non-SHV-type ESBL producers. In the receiver operating characteristic analysis, the area under the receiver operating characteristic curve (AUC) for detecting third-generation cephalosporin resistance using the MBT STAR-Cepha kit was 0.97-1.00, but the AUC for detecting ESBL producers was 0.64. In addition, we showed that the MBT STAR-Carba kit enabled the accurate detection of antimicrobial resistance by IMP-type carbapenemase producers. The AUC for detecting carbapenemase producers was 1.00. The results suggested that the target bacterial strains, antimicrobial susceptibility phenotypes, and resistance genes were important for the utility of the MALDI-TOF MS-based MBT STAR-Cepha and MBT STAR-Carba kits in bacterial routine diagnostics.

Ota Y ，Furuhashi K ，Hirai N ，Ishikawa J ，Nagura O ，Yamanaka K ，Maekawa M ... -  《-》

Utility of the MBT STAR-Cepha kit in detecting extended-spectrum β-lactamase producers in clinical urine samples and positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Urinary tract infection (UTI) and bloodstream infection (BSI) caused by extended-spectrum β-lactamase (ESBL)-producing bacteria are important healthcare problems. Appropriate infection management requires the direct detection of organisms in clinical specimens. We assessed the capability of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based MBT STAR-Cepha kit to detect ESBL producers in clinical urine and blood samples. Over a 1-year period, 90 urine samples and 55 positive monomicrobial (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, or Proteus mirabilis) blood cultures were collected from patients with UTI or BSI at Hamamatsu University Hospital. β-lactamase activity in these samples was directly detected using the MBT STAR-Cepha kit, and the results were compared with those of antimicrobial susceptibility testing and polymerase chain reaction detection assay for the isolates. In receiver operating characteristic curve analysis, the kit assay showed low accuracy in detecting ESBL producers in urine samples (area under the curve [AUC], 0.69). Meanwhile, the AUC for detecting all ESBL-producing bacteria in positive blood cultures was 0.81. The kit assay detected cefotaxime (CTX) resistance (mainly in CTX-M-type ESBL producers) with high accuracy in positive blood cultures; however, it did not accurately detect ESBL producers in urine samples and CTX-susceptible isolates with other ESBL-associated genes (e.g., TEM and SHV types) in positive blood cultures. MBT STAR-Cepha testing can accurately discriminate CTX-resistant ESBL producers in BSI cases and thus can contribute to effective infection management. The results suggest that different sample types, antibiotic resistance profiles, and resistance genes can affect the kit performance.

Ota Y ，Furuhashi K ，Hirai N ，Nagao Y ，Ishikawa J ，Nagura O ，Yamashita K ，Maekawa M ... -  《-》

Detection of extended-spectrum β-lactamases producing Enterobacteriaceae using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based MBT STAR-BL software module with β-lactamase inhibition assay depends on the bacterial stra

Rapid and sensitive detection of extended-spectrum β-lactamases (ESBLs) is essential for infection control and antimicrobial treatment. Recently, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based MBT STAR-BL software module has been used for detecting β-lactamase activity; however, this system cannot differentiate ESBL producing bacteria from other third-generation cephalosporin-resistant strains. In this study, we utilized a MALDI-TOF MS-based MBT STAR-BL method to identify ESBL activity with β-lactamase inhibitors. A cefotaxime (CTX) hydrolysis assay, β-lactamase inhibition, clavulanic acid (CVA), and sulbactam (SBT) were used for detecting ESBL producers with the MBT STAR-BL software module. This software module automatically calculated the logRQ values in each assay. logRQ is the logarithm of the ratio of the summed hydrolyzed peak intensities to the summed non-hydrolyzed peak intensities and measured the efficiency of antibiotic hydrolysis. We divided the logRQ level of the β-lactamase inhibition assay by the logRQ value in the CTX hydrolysis assay, and we used this logRQ ratio as a measure of β-lactamase inhibition efficiency. We assessed the logRQ ratio calculated by this novel method for detecting ESBL producers in 132 Enterobacteriaceae. We performed the MALDI-TOF MS-based MBT STAR-BL approach with β-lactamase inhibitors for detecting ESBL producers and showed that the results of the inhibition assay with β-lactamase inhibitors depended on types of bacterial species. Furthermore, we improved elapsed times and accuracy in MBT STAR-BL methods by using proper β-lactamase inhibitors against specific bacterial strains to compare with the conventional standard lab method. The results suggest that the target bacterial species and β-lactamase inhibitors used were important for the utility of the MALDI-TOF MS-based MBT STAR-BL software module.

Ota Y ，Furuhashi K ，Nagao Y ，Nanba T ，Yamanaka K ，Ishikawa J ，Nagura O ，Iwaizumi M ，Hamada E ，Maekawa M ... -  《-》

Performance Evaluation of MALDI-TOF MS MBT STAR-BL Versus In-House Carba NP Testing for the Rapid Detection of Carbapenemase Activity in Escherichia coli and Klebsiella pneumoniae Strains.

Akyar I ，Kaya Ayas M ，Karatuna O 《-》

Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Cordovana M ，Kostrzewa M ，Sóki J ，Witt E ，Ambretti S ，Pranada AB ... -  《-》