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
• automatizované zpracování dat MeSH
• bakteriální proteiny analýza   MeSH
• beta-laktamasy analýza   MeSH
• hydrogenuhličitany * MeSH
• hydrolýza MeSH
• laboratorní automatizace MeSH
• lidé MeSH
• meropenem MeSH
• pufry MeSH
• senzitivita a specificita MeSH
• software MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ammonium bicarbonate MeSH Prohlížeč
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• hydrogenuhličitany * MeSH
• meropenem MeSH
• pufry MeSH
• thienamyciny 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
• bakteriální proteiny analýza   metabolismus   MeSH
• beta-laktamasy analýza   metabolismus   MeSH
• Enterobacteriaceae enzymologie   izolace a purifikace   MeSH
• enterobakteriální infekce diagnóza   mikrobiologie   MeSH
• hydrogenuhličitany * MeSH
• kultivační média chemie   MeSH
• mikrobiální testy citlivosti metody   MeSH
• pufry MeSH
• senzitivita a specificita MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• hydrogenuhličitany * MeSH
• kultivační média MeSH
• pufry 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 enzymologie   MeSH
• bakteriální proteiny metabolismus   MeSH
• beta-laktamasy metabolismus   MeSH
• Enterobacteriaceae enzymologie   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• hydrolýza MeSH
• lidé MeSH
• meropenem MeSH
• mikrobiální testy citlivosti metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• meropenem MeSH
• thienamyciny 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
• antibakteriální látky analýza   MeSH
• bakteriální proteiny metabolismus   MeSH
• beta-laktamasy metabolismus   MeSH
• Enterobacteriaceae enzymologie   MeSH
• lidé MeSH
• meropenem MeSH
• Pseudomonas aeruginosa enzymologie   MeSH
• senzitivita a specificita MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• meropenem MeSH
• thienamyciny 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
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence účinky léků   genetika   MeSH
• beta-laktamasy biosyntéza   genetika   MeSH
• hospitalizace MeSH
• Klebsiella pneumoniae účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• kolistin farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• poriny genetika   MeSH
• regulace genové exprese u bakterií MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Řecko MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-lactamase KPC-2, Klebsiella pneumoniae MeSH Prohlížeč
• beta-laktamasy MeSH
• kolistin MeSH
• poriny MeSH