A comparison of a matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH4HCO3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers.

• MeSH
• Electronic Data Processing MeSH
• Bacterial Proteins analysis   MeSH
• beta-Lactamases analysis   MeSH
• Bicarbonates * MeSH
• Hydrolysis MeSH
• Automation, Laboratory MeSH
• Humans MeSH
• Meropenem MeSH
• Buffers MeSH
• Sensitivity and Specificity MeSH
• Software MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Thienamycins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ammonium bicarbonate MeSH Browser
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• Bicarbonates * MeSH
• Meropenem MeSH
• Buffers MeSH
• Thienamycins MeSH

Carbapenemase-mediated resistance to carbapenems in Enterobacteriaceae has become the main challenge in the treatment and prevention of infections recently. The partially unnoticed spread of OXA-48-type carbapenemase producers is usually assigned to low minimum inhibitory concentrations (MICs) of carbapenems that OXA-48-producing isolates often display. Therefore, there is an urgent need of specific and sensitive methods for isolation and detection of OXA-48 producers in clinical microbiology diagnostics. The influence of bicarbonates on carbapenem MICs against carbapenemase-producing Enterobacteriaceae was tested. We also checked whether the addition of bicarbonates to liquid media supplemented with meropenem may facilitate the selective enrichment of various carbapenemase producers in cultures. Furthermore, the sensitivity of carbapenemase confirmation by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) and spectrophotometric hydrolysis assays upon the addition of NH4HCO3 was examined. The addition of NaHCO3 significantly increased MICs of ertapenem and meropenem for OXA-48 producers. Furthermore, liquid media supplemented with NaHCO3 and meropenem were reliable for the selective enrichment of carbapenemase producers. The presence of NH4HCO3 in buffers used in the spectrophotometric and MALDI-TOF MS carbapenemase detection increased the sensitivity of that assay. Our results demonstrate that bicarbonates in media or reaction buffers can enhance the sensitivity of screening methods and diagnostic tests for carbapenemase producers.

• MeSH
• Bacterial Proteins analysis   metabolism   MeSH
• beta-Lactamases analysis   metabolism   MeSH
• Enterobacteriaceae enzymology   isolation & purification   MeSH
• Enterobacteriaceae Infections diagnosis   microbiology   MeSH
• Bicarbonates * MeSH
• Culture Media chemistry   MeSH
• Microbial Sensitivity Tests methods   MeSH
• Buffers MeSH
• Sensitivity and Specificity MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• Bicarbonates * MeSH
• Culture Media MeSH
• Buffers MeSH

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a potentially useful tool for the detection of antimicrobial resistance, especially that conferred by β-lactamases. Here we describe a modification of a previously reported MALDI-TOF MS meropenem hydrolysis assay. The modified method was validated on 108 carbapenemase-producing members of the Enterobacteriaceae, two NDM-1-producing Acinetobacter baumannii isolates, and 35 carbapenem-resistant enterobacteria producing no carbapenemase. The detection of carbapenemases by MALDI-TOF MS seems to be a powerful, quick, and cost-effective method for microbiological laboratories.

• MeSH
• Acinetobacter baumannii enzymology   MeSH
• Bacterial Proteins metabolism   MeSH
• beta-Lactamases metabolism   MeSH
• Enterobacteriaceae enzymology   MeSH
• Enterobacteriaceae Infections microbiology   MeSH
• Hydrolysis MeSH
• Humans MeSH
• Meropenem MeSH
• Microbial Sensitivity Tests methods   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Thienamycins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH
• Names of Substances
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• Meropenem MeSH
• Thienamycins MeSH

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.

• MeSH
• Anti-Bacterial Agents analysis   MeSH
• Bacterial Proteins metabolism   MeSH
• beta-Lactamases metabolism   MeSH
• Enterobacteriaceae enzymology   MeSH
• Humans MeSH
• Meropenem MeSH
• Pseudomonas aeruginosa enzymology   MeSH
• Sensitivity and Specificity MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Thienamycins analysis   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• Meropenem MeSH
• Thienamycins MeSH

Carbapenemase-producing Gram-negative bacteria peak clinical interest due to their ability to hydrolyze most β-lactams, including carbapenems; moreover, their genes spread through bacterial populations by horizontal transfer. Bacteria with acquired carbapenemase have sporadically been reported in the Czech Republic, so far only in Enterobacteriaceae and Pseudomonas aeruginosa. In this study, we described the first finding of a KPC-2-producing strain of Klebsiella pneumoniae, which was isolated from a surgical wound swab, decubitus ulcer, and urine of a patient previously hospitalized in Greece. The patient underwent various antibiotic therapies including a colistin treatment. However, after approximately 20 days of the colistin therapy, the strain developed a high-level resistance to this drug. All the isolates were indistinguishable by pulsed field gel electrophoretic analysis and belonged to the international clone ST258, which is typical of KPC-producing K. pneumoniae isolates. The bla (KPC-2) gene was located on a Tn4401a transposon variant. The OmpK35 and OmpK36 genes analysis performed due to the high resistance level of the strains to β-lactams exhibited no changes in their sequence or in their expression when compared with carbapenem-susceptible isolates.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial drug effects   genetics   MeSH
• beta-Lactamases biosynthesis   genetics   MeSH
• Hospitalization MeSH
• Klebsiella pneumoniae drug effects   enzymology   genetics   isolation & purification   MeSH
• Colistin pharmacology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Porins genetics   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Greece MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• beta-lactamase KPC-2, Klebsiella pneumoniae MeSH Browser
• beta-Lactamases MeSH
• Colistin MeSH
• Porins MeSH