Two-site study on performances of a commercially available MALDI-TOF MS-based assay for the detection of colistin resistance in Escherichia coli.

Colistin is a last resort drug for the treatment of multiple drug-resistant (MDR) Gram-negative bacterial infections. Rapid methods to detect resistance are highly desirable. Here, we evaluated the performance of a commercially available MALDI-TOF MS-based assay for colistin resistance testing in Escherichia coli at two different sites. Ninety clinical E. coli isolates were provided by France and tested in Germany and UK using a MALDI-TOF MS-based colistin resistance assay. Lipid A molecules of the bacterial cell membrane were extracted using the MBT Lipid Xtract Kit™ (RUO; Bruker Daltonics, Germany). Spectra acquisition and evaluation were performed by the MBT HT LipidART Module of MBT Compass HT (RUO; Bruker Daltonics) on a MALDI Biotyper® sirius system (Bruker Daltonics) in negative ion mode. Phenotypic colistin resistance was determined by broth microdilution (MICRONAUT MIC-Strip Colistin, Bruker Daltonics) and used as a reference. Comparing the results of the MALDI-TOF MS-based colistin resistance assay with the data of the phenotypic reference method for the UK, sensitivity and specificity for the detection of colistin resistance were 97.1% (33/34) and 96.4% (53/55), respectively. Germany showed 97.1% (33/34) sensitivity and 100% (55/55) specificity for the detection of colistin resistance by MALDI-TOF MS. Applying the MBT Lipid Xtract™ Kit in combination with MALDI-TOF MS and dedicated software showed excellent performances for E. coli. Analytical and clinical validation studies must be performed to demonstrate the performance of the method as a diagnostic tool.

Rapid Simultaneous Testing of Multiple Antibiotics by the MALDI-TOF MS Direct-on-Target Microdroplet Growth Assay.

Accelerating antimicrobial susceptibility testing (AST) is a priority in the development of novel microbiological methods. The MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA) has recently been described as a rapid phenotypic AST method. In this proof-of-principle study, we expanded this method to simultaneously test 24 antimicrobials. An Enterobacterales panel was designed and evaluated using 24 clinical isolates. Either one or two (only for antimicrobials with the EUCAST "I" category) breakpoint concentrations were tested. Microdroplets containing bacterial suspensions with antimicrobials and growth controls were incubated directly on the spots of a disposable MALDI target inside a humidity chamber for 6, 8 or 18 h. Broth microdilution was used as the standard method. After 6 and 8 h of incubation, the testing was valid (i.e., growth control was successfully detected) for all isolates and the overall categorical agreement was 92.0% and 92.7%, respectively. Although the overall assay performance applying short incubation times is promising, the lower performance with some antimicrobials and when using the standard incubation time of 18 h indicates the need for thorough standardization of assay conditions. While using "homebrew" utensils and provisional evaluation algorithms here, technical solutions such as dedicated incubation chambers, tools for broth removal and improved software analyses are needed.

MALDI-TOF Mass Spectrometry-Based Optochin Susceptibility Testing for Differentiation of Streptococcus pneumoniae from other Streptococcus mitis Group Streptococci.

Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL; correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study; however, confirmation and further improvements are warranted.

Study on carbapenemase-producing bacteria by matrix-assisted laser desorption/ionization approach.

The development of new techniques for the detection of carbapenemase activity is of great importance since the increased incident of resistance against carbapenems represents a serious threat to global public health. In this context, the matrix-assisted laser desorption/ionization approach already demonstrated to be a reliable tool for rapid carbapenemase detection. As a newly developed test, there is still a lack of in-depth analysis of its robustness and possible wider application. The main goal of this study was to evaluate the potential for using the design MBT STAR-Carba assay as the pre-characterization method for Enterobacterales and P. aeruginosa strains in terms of the produced classes of carbapenemases using modified procedure parameters-various suspension densities and incubation times. Moreover, its usefulness for the in-depth analysis and characterization of metallo-ß-lactamases (MBL) was tested by applying inhibition assays. In this study, the designed assay proved to be a sensitive tool for the detection of carbapenemase hydrolytic activity, which can be successfully used to partially classify the class of carbapenemase present. Additionally, the use of defined high concentration suspensions would allow to shorten the incubation time to 1 minute for certain strains. Considering that the assay was also suitable to investigate the effect of different inhibitors on the MBL activity, it demonstrates far higher discriminatory potential than only a rapid routine carbapenemase detection tool and could be used as a susceptibility assay.

Development of a MALDI-TOF MS-based screening panel for accelerated differential detection of carbapenemases in Enterobacterales using the direct-on-target microdroplet growth assay.

Carbapenemase-producing bacteria are a growing issue worldwide. Most phenotypic detection methods are culture-based, requiring long incubation times. We present a phenotypic screening panel for detection of carbapenem non-susceptibility and differentiation of carbapenemase classes and AmpC, the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). It was validated on 7 reference strains and 20 challenge Enterobacterales isolates. Broth microdilution (BMD) and combination disk test (CDT) were also performed, as well as PCR as reference method. The panel based on the synergy between meropenem and carbapenemase inhibitors, determined by incubating these substances with bacterial suspension on a MALDI-TOF MS target and subsequently assessing bacterial growth on the target's spots by MS. After 4 hours of incubation, DOT-MGA correctly identified KPC, MBL and OXA (100% agreement with PCR). Detection of AmpC coincided with BMD and CDT but agreement with PCR was low, not ruling out false negative PCR results. DOT-MGA delivered more accurate results than BMD and CDT in a significantly shorter time, allowing for detection of carbapenem non-susceptibility, MIC determination and carbapenemase differentiation in one step.

Detection of Methicillin Resistance in Staphylococcus aureus From Agar Cultures and Directly From Positive Blood Cultures Using MALDI-TOF Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay.

Matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) was recently described as a novel method of phenotypic antimicrobial susceptibility testing (AST). Here, we developed the application of MALDI-TOF MS-based DOT-MGA for Gram-positive bacteria including AST from agar cultures and directly from positive blood cultures (BCs) using the detection of methicillin resistance as example. Consecutively collected, a total of 14 methicillin-resistant Staphylococcus aureus (MRSA) and 14 methicillin-susceptible S. aureus (MSSA) clinical isolates were included. Furthermore, a collection of MRSA challenge strains comprising different SCCmec types, mec genes, and spa types was tested. Blood samples were spiked with MRSA and MSSA and positive BC broth processed by three different methods: serial dilution of BC broth, lysis/centrifugation, and differential centrifugation. Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 µl with and without cefoxitin at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard as a quality control (QC). Spectra were acquired and evaluated using MALDI Biotyper software. First, tests were considered as valid, if the growth control achieved an identification score of ≥1.7. For valid tests, same score criterion was used for resistant isolates when incubated with cefoxitin. An identification score <1.7 after incubation with cefoxitin defined susceptible isolates. On-target protein extraction using formic acid considerably improved detection of methicillin resistance in S. aureus and DOT-MGA showed feasible results for AST from agar cultures after 4 h incubation time. Comparing the different processing methods of positive BC broth, lysis/centrifugation method with a final dilution step 10-1 of the 0.5 McFarland suspension resulted in best test performance after 4 h incubation time. Overall, 96.4% test validity, 100% sensitivity, and 100% specificity were achieved for detection of methicillin resistance in clinical isolates. All strains of the MRSA challenge collection were successfully tested as methicillin-resistant. This first study on Gram-positive organisms showed feasibility and accuracy of MALDI-TOF MS-based DOT-MGA for rapid AST of S. aureus from agar cultures and directly from positive BCs.

Detection of Colistin Resistance in Escherichia coli by Use of the MALDI Biotyper Sirius Mass Spectrometry System.

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in less than 15 min but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF MS system (Bruker Daltonics). We optimized the sample preparation protocol by using a set of 6 mobile colistin resistance (MCR) protein-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR protein producers, 12 colistin-resistant isolates that tested negative for commonly encountered mcr genes (i.e., likely chromosomally resistant isolates), and 9 polymyxin-susceptible isolates. We calculated polymyxin resistance ratio (PRR) values from the acquired spectra; PRR values of 0, indicating polymyxin susceptibility, were obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains, independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test adapted for the routine MALDI Biotyper Sirius system provides an unbiased, fast, reliable, cost-effective, and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

Anidulafungin Susceptibility Testing of Candida glabrata Isolates from Blood Cultures by the MALDI Biotyper Antibiotic (Antifungal) Susceptibility Test Rapid Assay.

Echinocandins are the recommended first-line antifungals for treatment of invasive candidiasis. The increasing number of Candida glabrata strains resistant against echinocandins is an emerging health care concern. The rapid detection of resistant C. glabrata isolates is an urgent requirement for clinical laboratories. In this study, we developed the MALDI Biotyper antibiotic (antifungal) susceptibility test rapid assay (MBT ASTRA) for the rapid detection of anidulafungin-resistant C. glabrata isolates directly from positive blood cultures. Of 100 C. glabrata strains, MBT ASTRA classified 69 as susceptible and 29 as resistant. Microdilution assays performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, used as a standard reference, identified 65 susceptible, 9 intermediate, and 26 resistant isolates. Sequencing of hot spot 1 and hot spot 2 regions of the FKS1 and FKS2 genes classified 86 susceptible and 14 resistant isolates. The MBT ASTRA had sensitivity and specificity of 80% and 95%, respectively, compared to the microdilution method. Positive and negative agreement of MBT ASTRA was calculated at 100% and 80%, respectively, compared with the molecular sequencing approach. Together, these results revealed a high accuracy of MBT ASTRA compared to microdilution according to the CLSI and PCR analysis, resulting in a categorical agreement of 90% and 83%, respectively. The validity of MBT ASTRA was 98%. Importantly, MBT ASTRA provided antifungal susceptibility testing (AFST) within 6 h that was both accurate and reliable compared to the other two approaches, which require at least 24 h or are costly. Therefore, this method has the potential to facilitate clinical AFST rapidly at low sample costs for clinical labs already equipped with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Candida auris Identification and Rapid Antifungal Susceptibility Testing Against Echinocandins by MALDI-TOF MS.

Candida auris was first reported in an ear swab from Japan in 2009; it then promptly spread over five continents and turned into a global nosocomial problem. The main challenges faced by many researchers are the mis-identification by conventional methods in clinical laboratories and failure in treatment. About 90% of C. auris strains are intrinsically resistant to fluconazole (FLU), and it is developing resistance to multiple classes of available antifungals. Echinocandins are the most potent class of antifungals against C. auris; however, reduced susceptibility to one or many echinocandin drugs has been recently observed. Thus, the main issues addressed in this paper are the fast and accurate identification of C. auris derived from Sabouraud dextrose agar and blood culture bottles as well as the rapid antifungal susceptibility test by MALDI-TOF MS. This study successfully identified all isolates of C. auris (n = 50) by MALDI-TOF MS, with an average log score of ≥ 2. An accuracy of 100% was found on both agar plate and blood culture bottles. MALDI Biotyper antibiotic susceptibility test-rapid assay (MBT ASTRA) was used for rapid antifungal susceptibility testing (AFST). A comparison between MBT ASTRA and the Clinical and Laboratory Standards Institute guidelines (CLSI) detected a sensitivity and specificity of 100% and 98% for anidulafungin, and 100% and 95.5% for micafungin, respectively. A categorical agreement of 98% and 96% was calculated for the two methods. For caspofungin, sensitivity and specificity of 100 and 73% were found, respectively, with a categorical agreement of 82%. MBT ASTRA has the great potential to detect C. auris isolates non-susceptible against echinocandin antifungals within 6 h, which makes it a promising candidate for AFST in clinical laboratories in the future.

Rapid Detection of Extended-Spectrum ß-Lactamases (ESBL) and AmpC ß-Lactamases in Enterobacterales: Development of a Screening Panel Using the MALDI-TOF MS-Based Direct-on-Target Microdroplet Growth Assay.

Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum ß-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against ß-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of ß-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC ß-lactamase inhibitors and cephalosporins, which indicates ß-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without ß-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed. Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT. Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC ß-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.

Rapid detection of tetracycline resistance in bovine Pasteurella multocida isolates by MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA).

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8-101.5) and 100% (95% CI = 100-100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 93.5% (95% CI = 86.3-100.6) and 96.3% (95% CI = 91.3-101.3) respectively, classifying 95% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

Rapid Direct Susceptibility Testing from Positive Blood Cultures by the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay.

The recently developed direct-on-target microdroplet growth assay (DOT-MGA) allows rapid universal antimicrobial susceptibility testing (AST) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Here, we investigated a direct application of this method on positive blood cultures (BCs) for the acceleration of sepsis diagnostics. Blood samples spiked with meropenem-nonsusceptible and meropenem-susceptible Enterobacterales isolates were inoculated into Bactec Plus Aerobic/F bottles and incubated in the Bactec automated system. Positive-BC broth was processed using four different methods, filtration/dilution, dilution, lysis/centrifugation, and differential centrifugation. For both dilution-based methods, AST was performed from 1:100, 1:1,000, and 1:10,000 dilutions of positive-BC broth in cation-adjusted Mueller-Hinton broth (CA-MHB). For both centrifugation-based methods, a 0.5 McFarland standard turbidity suspension was prepared from a bacterial pellet and adjusted to a final inoculum of 5 × 105 CFU/ml in CA-MHB. Six-microliter microdroplets with or without meropenem at the breakpoint concentration were spotted in triplicate onto a MALDI-TOF MS target, followed by incubation in a humidity chamber for 3 or 4 h and subsequent broth removal. Spectra were evaluated by MALDI Biotyper software. The test was considered valid if the growth control without antibiotic achieved an identification score of ≥1.7. For samples with meropenem, successful identification (score, ≥1.7) was interpreted as a nonsusceptible result, whereas failed identification (score, <1.7) defined susceptibility. The best test performance was achieved with the lysis/centrifugation method after a 4-h incubation. At this time point, 96.3% validity, 91.7% sensitivity, and 100% specificity were reached. This study demonstrated the feasibility and accuracy of a rapid DOT-MGA from positive BCs. Parallel to susceptibility determination, this method provides simultaneous species identification.

Proof of Concept for MBT ASTRA, a Rapid Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)-Based Method To Detect Caspofungin Resistance in Candida albicans and Candida glabrata.

The incidence of candidemia caused by Candida albicans and Candida glabrata is constantly increasing and is accompanied by the rising use of the few available antifungals. The widespread use of echinocandins and azoles for the treatment of invasive candidemia has enhanced the development of antifungal resistance, resulting in an increasing health care problem. Hence, the rapid detection of resistant strains is required. This study aimed to evaluate the detection of C. albicans and C. glabrata strains resistant to caspofungin by the matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA). This novel semiquantitative technique facilitates the detection of caspofungin-resistant strains within 6 h. MBT ASTRA results were compared to the data obtained by the use of Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical isolates of C. albicans (n = 58) and C. glabrata (n = 57) were analyzed by MBT ASTRA and the CLSI microdilution method. Antifungal susceptibility testing against caspofungin by the CLSI microdilution method classified the C. albicans isolates into 36 susceptible and 22 resistant strains and the C. glabrata isolates into 5 susceptible, 33 resistant, and 19 intermediate strains. For C. albicans, the comparison of MBT ASTRA and the CLSI method revealed an excellent categorical agreement of 100%. A sensitivity and a specificity between MBT ASTRA and the CLSI microdilution method of 94% and 80%, respectively, were detected for C. glabrata strains, based on categorical agreement. In conclusion, the results obtained by MBT ASTRA indicate that this is a very promising approach for the rapid detection of Candida isolates resistant to caspofungin.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria.

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

Universal protocol for the rapid automated detection of carbapenem-resistant Gram-negative bacilli directly from blood cultures by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS).

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.

Evaluation of a Semiquantitative Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method for Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures.

With the increasing prevalence of multidrug-resistant Gram-negative bacteria, rapid identification of the pathogen and its individual antibiotic resistance is crucial to ensure adequate antiinfective treatment at the earliest time point. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for the identification of bacteria directly from the blood culture bottle has been widely established; however, there is still an urgent need for new methods that permit rapid resistance testing. Recently, a semiquantitative MALDI-TOF mass spectrometry-based method for the prediction of antibiotic resistance was described. We evaluated this method for detecting nonsusceptibility against two ß-lactam and two non-ß-lactam antibiotics. A collection of 30 spiked blood cultures was tested for nonsusceptibility against gentamicin and ciprofloxacin. Furthermore, 99 patient-derived blood cultures were tested for nonsusceptibility against cefotaxime, piperacillin-tazobactam, and ciprofloxacin in parallel with MALDI-TOF mass spectrometry identification from the blood culture fluid. The assay correctly classified all isolates tested for nonsusceptibility against gentamicin and cefotaxime. One misclassification for ciprofloxacin nonsusceptibility and five misclassifications for piperacillin-tazobactam nonsusceptibility occurred. Identification of the bacterium and prediction of nonsusceptibility was possible within approximately 4 h.

Evaluation of different pretreatment protocols to detect accurately clinical carbapenemase-producing Enterobacteriaceae by MALDI-TOF.

OBJECTIVES: Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications. METHODS: The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the ß-lactam common structural motif, which can be detected using MALDI-TOF MS. RESULTS: A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed. CONCLUSIONS: Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.

Vimentin fragments are potential markers of rheumatoid synovial fibroblasts.

OBJECTIVES: To study the protein expression differences between primary fibroblasts explanted from synovial membranes of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Fibroblast cultures were obtained from 10 patients with RA and 5 patients with OA. After two-dimensional gel electrophoresis, proteins were excised and identified using peptide mass fingerprint. Expression of selected proteins was subsequently examined by immunoblot. Furthermore, we examined the cellular lysates for the presence of citrullinated proteins. RESULTS: The study was designed to compare expression changes of the common proteins detected in all studied fibroblast cultures (i.e. detected in all patients samples). We totally identified 191 shared proteins between RA and OA fibroblasts. A significant difference was defined as at least 2-fold upregulation or 0.6-fold downregulation of protein expression. The most obvious alteration observed in RA was the appearance of several vimentin fragments not present in OA. We did not detect citrullinated proteins in lysates from RA fibroblasts. This corroborates the current assumption that fibroblasts are not able to citrullinate proteins by themselves and that invading macrophages play a central role in this process. CONCLUSIONS: We demonstrated that fibroblasts from patients with RA, despite being grown under identical conditions, preserve a particular feature and generate vimentin fragments not present in fibroblasts from OA. Elevated levels of different vimentin fragments have been recently reported in several rheumatic conditions. Further studies are needed to elucidate the pathogenic mechanisms induced by vimentin fragments in RA.

MBT-ASTRA: A suitable tool for fast antibiotic susceptibility testing?

The increasing resistance to antibiotics is an urgent health care problem. Detection of resistant microorganisms is the pre-requisite for initiating an adequate therapy and implementing respective hygiene measures. Depending on the species and the method employed for analysis, the time to result of antibiotic resistance testing ranges between five and 24h. As MALDI-TOF MS has become an established tool for the fast species identification in microbiological laboratories a time gap between the results of species identification and the information about antibiotic susceptibility arises. Here, we present a semi-quantitative MALDI-TOF MS-based approach for the detection of resistance in different species against different antibiotics.