Subject(s)
Acetyltransferases/analysis , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Enterobacteriaceae/drug effects , Enterobacteriaceae/enzymology , Quinolones/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Acetylation , Acetyltransferases/genetics , Ciprofloxacin/pharmacology , Drug Resistance, Bacterial/genetics , Enterobacteriaceae/isolation & purification , Norfloxacin/pharmacology , Plasmids/genetics , Time FactorsABSTRACT
Objectives: Development of a novel MALDI-TOF MS-based method for the rapid detection of ESBL-producing Enterobacteriaceae. Methods: The method was evaluated in terms of sensitivity, specificity and turnaround time regarding the antibiotic used (cefotaxime, ceftazidime, ceftriaxone, cefpodoxime or cefepime) and the performance of the automated MBT STAR-BL software (Bruker Daltonik GmbH, Germany) relative to qualitative interpretation of spectra for detecting ß-lactamase resistance by MALDI-TOF MS (Bruker Daltonik) in a collection of 11 isogenic Escherichia coli control strains expressing different ß-lactamases. Finally, for clinical validation, ß-lactamase activity was determined under previously evaluated conditions in 100 clinical isolates previously characterized by PCR and sequencing. Results: Clinical validation of the assay showed 100% sensitivity and specificity for detecting ß-lactam resistance in 30 min by measuring hydrolysis of ceftriaxone (0.50 mg/mL) with the automated MBT STAR-BL software. Regarding the antibiotics evaluated, ceftriaxone yielded 70% more positive results than cefotaxime, 80% more than ceftazidime and 20% more than cefpodoxime, with 100% specificity. Cefepime revealed 100% sensitivity, but only 27% specificity. For the same incubation time, the automated software yielded on average 41% more positive results in relation to detecting resistance than qualitative interpretation of spectra. Conclusions: Our clinical validation of the method proved it to be highly reliable, simple to perform and time saving, transforming ß-lactam resistance detection by MALDI-TOF MS into a ready-to-use technique in clinical laboratories.
Subject(s)
Bacteriological Techniques/methods , Enterobacteriaceae Infections/diagnosis , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/enzymology , Enterobacteriaceae/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , beta-Lactamases/analysis , Anti-Bacterial Agents/pharmacology , Automation, Laboratory/methods , Carbapenems/pharmacology , Early Diagnosis , Enterobacteriaceae/drug effects , Humans , Sensitivity and Specificity , beta-Lactam ResistanceABSTRACT
Detection of carbapenemase-producing bacteria directly from blood cultures is a major challenge, as patients with bacteraemia are critically ill. Early detection can be helpful for selection of the most appropriate antibiotic therapy as well as adequate control of outbreaks. In the current study, a novel matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF)-based method was developed for the rapid, automated detection of carbapenemase-producing Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii directly from blood cultures. Carbapenemase activity was determined in 30 min by measuring hydrolysis of imipenem (0.31 mg/mL) in blood cultures spiked with a series of 119 previously characterised isolates, 81 of which carried a carbapenemase enzyme (10 blaKPC, 10 blaVIM, 10 blaNDM, 10 blaIMP, 26 blaOXA-48-type, 9 blaOXA-23, 1 blaOXA-237, 3 blaOXA-24 and 2 blaOXA-58). Twenty blood cultures obtained from bacteraemic patients carrying blaOXA-48-producing isolates were also analysed using the same protocol. Analysis was performed using MALDI-TOF Biotyper® Compass software, which automatically provides a result of sensitivity or resistance, calculated as the logRQ or ratio of hydrolysis of the antibiotic. This assay is simple to perform, inexpensive, time saving, universal for Gram-negative bacilli, and highly reliable (overall sensitivity and specificity of 98% and 100%, respectively). Moreover, the protocol could be established as a standardised method in clinical laboratories as it does not require specialised training in mass spectrometry.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacteremia/microbiology , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/microbiology , Imipenem/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , beta-Lactam Resistance , Automation, Laboratory/methods , Blood Culture , Humans , Hydrolysis , Microbial Sensitivity Tests/methods , Time FactorsABSTRACT
A rapid and sensitive (100%) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect OXA-48-type producers, using 161 previously characterized clinical isolates. Ertapenem was monitored to detect carbapenem resistance, and temocillin was included in the assay as a marker for OXA-48-producers. Structural analysis of temocillin is described. Data are obtained within 60 min.
Subject(s)
Bacterial Typing Techniques , Enterobacteriaceae/classification , Enterobacteriaceae/enzymology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , beta-Lactamases/biosynthesis , Anti-Bacterial Agents/pharmacology , Enterobacteriaceae/drug effects , Ertapenem , Humans , Sensitivity and Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , beta-Lactams/pharmacologyABSTRACT
Carbapenem-resistant Acinetobacter baumannii isolates were obtained from 50 patients between July 2011 and July 2012 at the University Hospital A Coruña (NW Spain). These multidrug-resistant isolates, which belonged to a single clone, remained only susceptible to tigecycline, minocycline, and colistin and produced the carbapenem-hydrolyzing oxacillinase, OXA-23. This is the first reported outbreak of OXA-23-producing A. baumannii isolates in Spain.