SummaryBackground Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it diffi cult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the fi rst structured survey on the occurrence of carbapenemaseproducing Klebsiella pneumoniae and Escherichia coli in European hospitals.

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.

Carbapenemase-producing bacteria have now spread all over the world. Infections caused by those bacteria are difficult to treat. Therefore, there is an urgent need for accurate and fast detection of carbapenemases in diagnostic laboratories. In this review, we summarize screening methods for suspected isolates, direct assays for confirmation of carbapenemase activity (e.g. the Carba NP test and matrix-assisted laser desorption ionization time-of-flight mass spectrometry carbapenem hydrolysis assay), inhibitor-based methods for carbapenemase classification, and molecular-genetic techniques for precise identification of carbapenemase genes. We also propose a workflow for carbapenemase identification in diagnostic laboratories.

SUMMARYMatrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied as an identification procedure in clinical microbiology and has been widely used in routine laboratory practice because of its economical and diagnostic benefits. The range of applications of MALDI-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology. The purpose of this review is to summarize the contribution of the studies already performed with MALDI-TOF MS concerning antibiotic resistance and to analyze future perspectives in this field. We believe that current research should continue in four main directions, including the detection of antibiotic modifications by degrading enzymes, the detection of resistance mechanism determinants through proteomic studies of multiresistant bacteria, and the analysis of modifications of target sites, such as ribosomal methylation. The quantification of antibiotics is suggested as a new approach to study influx and efflux in bacterial cells. The results of the presented studies demonstrate that MALDI-TOF MS is a relevant tool for the detection of antibiotic resistance and opens new avenues for both clinical and experimental microbiology.

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

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 NH 4 HCO 3 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. Nosocomial infections caused by carbapenem-resistant Enterobacteriaceae and Pseudomonas spp. are now emerging worldwide and are difficult to treat (1). Previous data have shown that strict epidemiological intervention based on the rapid detection of carbapenemase production can prevent the spread of those bacteria (2). Therefore, the introduction of rapid and sensitive methodologies for the detection of carbapenemase-producing bacteria is of utmost importance. Recently, two new highly sensitive and rapid methods for the direct detection of carbapenemase activity were developed. In 2011, two research groups demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can detect carbapenemase activity, based on mass spectral profiles obtained from carbapenem molecules (3, 4). The second method, the Carba NP test, is a biochemical method used in the detection of carbapenemase activity in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter species (5-7). This test is based on a decrease in pH resulting from the hydrolysis of the ␤-lactam ring of carbapenem molecules, which is detected using phenol red as a pH indicator.The aim of this study was to validate the efficiency of a MALDI-TOF MS meropenem hydrolysis assay and the Carba NP test for the detection of carbapenemase producers. The performances of both methods were compared with that of a recently published modification of the MALDI-TOF MS assay (MALDI-TOF BIC) in an effort to increase the reliability of detecting OXA-48 producers (8). Furthermore, the automatic interpretation of all methods was evaluated.The methods were tested against a group of 124 Enterobacteriaceae and 37 Pseudomonas aeruginosa isolates from collections of the Faculty of Medicine and University Hospital in Plzen (Czech Republic), the National Medicines Institute in Warsaw (Poland), the Robert Koch Institute in Wernigerode (Germany), and the University Hospital in Larissa (Greece). The isolates were previously characterized, as described below. All isolates were tested for extended-spectrum ␤-lactamase (ESBL) and AmpC expression by the ESBL double-disk synergy test (DDST) with cefotaxime, ceftazidime, aztreonam, and amoxicillin-clavulanate disks in the absence and presence of cloxacillin (250 g/ml) (9). Susceptibility to carbapenems was determined by using imipenem, merope...

Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.

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.