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.

IntroductionMeropenem bactericidal activity depends on the time when the free drug concentrations remain above the minimum inhibitory concentration of pathogens. The goal of this study was to compare clinical and bacteriological efficacy of continuous meropenem infusion versus bolus administration in critically ill patients with severe infection, and to evaluate the safety of both dosing regimens.MethodsPatients admitted to the interdisciplinary Intensive Care Unit (ICU) who suffered from severe infections and received meropenem were randomized either in the Infusion group (n = 120) or in the Bolus group (n = 120). Patients in the Infusion group received a loading dose of 2 g of meropenem followed by a continuous infusion of 4 g of meropenem over 24 hours. Patients in the Bolus group were given 2 g of meropenem over 30 minutes every 8 hours. Clinical and microbiological outcome, safety, meropenem-related length of ICU and hospital stay, meropenem-related length of mechanical ventilation, duration of meropenem treatment, total dose of meropenem, and ICU and in-hospital mortality were assessed.ResultsClinical cure at the end of meropenem therapy was comparable between both groups (83.0% patients in the Infusion vs. 75.0% patients in the Bolus group; P = 0.180). Microbiological success rate was higher in the Infusion group as opposed to the Bolus group (90.6% vs. 78.4%; P = 0.020). Multivariate logistic regression identified continuous administration of meropenem as an independent predictor of microbiological success (OR = 2.977; 95% CI = 1.050 to 8.443; P = 0.040). Meropenem-related ICU stay was shorter in the Infusion group compared to the Bolus group (10 (7 to 14) days vs. 12 (7 to 19) days; P = 0.044) as well as shorter duration of meropenem therapy (7 (6 to 8) days vs. 8 (7 to 10) days; P = 0.035) and lower total dose of meropenem (24 (21 to 32) grams vs. 48 (42 to 60) grams; P < 0.0001). No severe adverse events related to meropenem administration in either group were observed.ConclusionsContinuous infusion of meropenem is safe and, in comparison with higher intermittent dosage, provides equal clinical outcome, generates superior bacteriological efficacy and offers encouraging alternative of antimicrobial therapy in critically ill patients.

e 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 ␤-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. Detection of carbapenemases has been one of the challenges in clinical microbiology diagnostics (9). Recently, new matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assays for ␤-lactamase activity have been developed independently by at least three groups (2, 7, 12). These techniques are based on the detection of ␤-lactams and their degradation products. A similar assay was validated to detect carbapenemases in Acinetobacter baumannii (8).However, for some ␤-lactams, like meropenem, visualization of degradation products by MALDI-TOF MS seemed to be problematic (7,12). This might be due to binding of the molecules to cell lysate components. Here we describe a modification of one of these protocols (7) that allows the detection of degradation products and shortening of the turnaround time to ca. 2.5 h. The modified assay was validated with NDM-1-, VIM-1-, KPC-2-, KPC-3-, and OXA-48/-162-producing members of the Enterobacteriaceae and NDM-1-producing A. baumannii isolates.(The data included in this article were presented in part as a poster at the 22nd European Congress of Clinical Microbiology

The aim of this study was to characterize the first cases and outbreaks of OXA-48-like-producing Enterobacteriaceae recovered from hospital settings in the Czech Republic. From 2013 to 2015, 22 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, and 1 Enterobacter cloacae isolate producing OXA-48-like carbapenemases were isolated from 20 patients. Four of the patients were colonized or infected by two or three different OXA-48-like producers. The K. pneumoniae isolates were classified into nine sequence types (STs), with ST101 being predominant (n ϭ 8). The E. coli isolates were of different STs, while the E. cloacae isolate belonged to ST109. Twenty-four isolates carried bla OXA-48 , while two isolates carried bla OXA-181 or bla . Almost all isolates (n ϭ 22) carried bla OXA-48 -positive plasmids of a similar size (ϳ60 kb), except the two isolates producing OXA-181 or OXA-232. In an ST45 K. pneumoniae isolate and an ST38 E. coli isolate, S1 nuclease profiling plus hybridization indicated a chromosomal location of bla . Sequencing showed that the majority of bla OXA-48 -carrying plasmids exhibited high degrees of identity with the pOXA-48-like plasmid pE71T. Additionally, two novel pE71T derivatives, pOXA-48_30715 and pOXA-48_30891, were observed. The bla OXA-181 -carrying plasmid was identical to the IncX3 plasmid pOXA181_EC14828, while the bla OXA-232 -carrying plasmid was a ColE2-type plasmid, being a novel derivative of pOXA-232. Finally, sequencing data showed that the ST45 K. pneumoniae and ST38 E. coli isolates harbored the IS1R-based composite transposon Tn6237 containing bla OXA-48 integrated into their chromosomes. These findings underlined that the horizontal transfer of pOXA-48-like plasmids has played a major role in the dissemination of bla in the Czech Republic. In combination with the difficulties with their detection, OXA-48 producers constitute an important public threat.