Diagnóstico rápido proteómico (MALDI-TOF MS) para la determinación de bacterias resistentes a los antibióticos

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007P01[Resumen] La espectrometría de masas es una técnica de análisis que permite caracterizar analitos midiendo las relaciones de masas de las moléculas analizadas. La evolución de la tecnología ha permitido llegar al MALDI-TOF MS, que es una variante que permite la detección de mezclas proteicas complejas con mínimas fragmentaciones, lo que permite su aplicación en microbiología en la identificación de microorganismos. Siguiendo con el desarrollo de nuevas aplicaciones de esta tecnología, la detección de moléculas como los antibióticos para la detección de mecanismos de resistencia a los antimicrobianos es la base de esta tesis. Nos hemos basado en la aplicación de un ensayo de resistencia funcional, basado en la medida de la acción enzimática de la bacteria con mecanismos de resistencia específicos (fundamentalmente β-lactamasas) sobre el antibiótico y el seguimiento de este en el espectro de masas. Los antibióticos estudiados son β-lactámicos, incluyendo carbapenémicos y quinolonas. Hemos conseguido resultados satisfactorios en la aplicación de esta técnica tanto en bacterias aisladas en cultivo como en el hemocultivo. La principal ventaja de esta metodología es la obtención de resultados 24 h antes que los métodos fenotípicos, con bajo coste y con valores de sensibilidad y especificidad similares a los métodos moleculares.[Resumo] A espectrometría de masas é unha técnica de análise que permite caracterizar analitos medindo as relacións de masas das moléculas analizadas. A evolución da tecnoloxía permitiu chegar ao MALDI-TOF MS, que é unha variante que permite a detección de mesturas proteicas complexas con mínimas fragmentacións, o que permite a súa aplicación en microbiología na identificación de microorganismos. Seguindo co desenvolvemento de novas aplicacións desta tecnoloxía, a detección de moléculas como os antibióticos para a detección de mecanismos de resistencia aos antimicrobianos é a base desta tese. Baseámonos na aplicación dun ensaio de resistencia funcional, baseado na medida da acción enzimática da bacteria sobre o antibiótico e o seguimento deste no espectro de masas. Os antibióticos estudados son os β-lactámicos, carbapenémicos e quinolonas. Conseguimos resultados satisfactorios na aplicación desta técnica tanto en bacterias en cultivo como no hemocultivo. A principal vantaxe desta metodoloxía é a obtención de resultados 24 h antes que os métodos fenotípicos, con baixo custo e con valores de sensibilidade e especificidade similares aos métodos moleculares.[Abstract] Mass spectrometry is an analytical technique that allows analytes to be characterized by measuring the mass ratios of the molecules. Evolution of the technology has enabled getting at MALDI-TOF MS, that is variant that allows detection of complex protein mixtures with minimum fragmentations. This leads into its application in microbiology in the identification of microorganisms. Following with the development of new applications of this technology, the basis of this thesis is the detection of molecules as antibiotics for antimicrobial resistance detection. We have based our study on the application of a functional assay that measures the enzymatic activity in the antibiotic and further tracing of the mass spectrum generated. The studied antibiotics were β-lactams, carbapenems and quinolones. We have reached satisfactory results in the application of this technique either in bacterial cultures as in blood cultures. The main advantage of the methodology is the possibility of obtaining results 24 h before than phenotypic methods, with low cost and with sensitivity and specificity values in the range of molecular methods

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Lowenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score >= 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification

Rapid Detection of KPC-Producing Enterobacterales Susceptible to Imipenem/Relebactam by Using the MALDI-TOF MS MBT STAR-Carba IVD Assay

KPC-producing Enterobacterales represent a serious public health concern. Limited therapeutic options are available for treatment, however, the novel combination of imipenem/relebactam represents a promising alternative. To preserve the activity of this new antibiotic combination, only targeted treatments will be recommended, and rapid tests to detect susceptible bacteria are therefore urgently needed. Here, we propose a MALDI-TOF-based method using the MBT STAR-Carba IVD assay, Bruker Daltonik, to detect KPC-producing Enterobacterales susceptible to imipenem/relebactam in a random selection of 143 clinical isolates previous molecular characterized, carrying 97 bla KPC, 1 bla GES, 12bla VIM, 4bla IMP, 3bla NDM, and 26bla OXA- 48 -like. Species identification was confirmed by MALDI-TOF MS. The molecular characterization of the isolates was performed by the Xpert Carba-R Assay and the results were used as gold standard. Besides, all isolates were submitted to imipenem and imipenem/relebactam microdilution susceptibility testing. The assay showed an overall sensitivity and specificity to detect class A-producing Enterobacterales susceptible to imipenem/relebactam of 98% (96/98) and 93% (42/45), respectively. This MALDI-TOF-based methodology, with a turnaround time of less than 1 h, is a reliable test for detecting imipenem/relebactam activity and its inclusion in routine laboratory screening would facilitate the correct use of this new combination of antimicrobials as a targeted treatment

Analysis of the Degradation of Broad-Spectrum Cephalosporins by OXA-48-Producing Enterobacteriaceae Using MALDI-TOF MS

The objective of the study was to evaluate the activity of OXA-48 against different broad-spectrum cephalosporins and to identify the reaction products by MALDI-TOF MS. The action of OXA-48 on cefotaxime, ceftazidime, and ceftriaxone was assessed by this method, using an Escherichia coli J53 transconjugant carrying only the ~62 Kb IncL plasmid containing the blaOXA-48 gene, and the same strain without any plasmid was included as a negative control. In addition, a collection of 17 clinical OXA-48-producing Enterobacteriaceae, which were susceptible to broad-spectrum cephalosporins, was evaluated. MALDI-TOF MS-based analysis of the E. coli transconjugant carrying the blaOXA-48-harboring plasmid, and also the clinical isolates, showed degradation of cefotaxime into two inactive compounds—decarboxylated and deacetylated cefotaxime (~370 Da) and deacetyl cefotaxime (~414 Da), both with the hydrolyzed beta-lactam ring. Reaction products were not obtained when the experiment was performed with ceftriaxone or ceftazidime. From a clinical point of view, our study supports the idea that the efficacy of cefotaxime against OXA-48-producing Enterobacteriaceae is doubtful, in contrast to ceftazidime and ceftriaxone which could be valid choices for treating infections caused by these bacteria. However, further clinical studies confirming this hypothesis are required

Multicenter evaluation of rapid BACpro® II for the accurate identification of microorganisms directly from blood cultures using MALDI-TOF MS

The identification of microorganisms directly from blood cultures using MALDI-TOF MS has been shown to be the most impacting application of this methodology. In this study, a novel commercial method was evaluated in four clinical microbiology laboratories. Positive blood culture samples (n = 801) were processed using a rapid BACpro® II kit and then compared with the routine gold standard. A subset of monomicrobial BCs (n = 560) were analyzed in parallel with a Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) and compared with the rapid BACpro® II kit. In addition, this kit was also compared with two different in-house methods. Overall, 80.0% of the monomicrobial isolates (609/761; 95% CI 71.5–88.5) were correctly identified by the rapid BACpro® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper® Kit showed that the rapid BACpro® II kit generated higher rates of correct species-level identification for all categories (p > 0.0001), except for yeasts identified with score values > 1.7. It also proved superior to the ammonium chloride method (p > 0.0001), but the differential centrifugation method allowed for higher rates of correct identification for Gram negative bacteria (p > 0.1). The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods

Interplay between OXA-10 β-Lactamase Production and Low Outer-Membrane Permeability in Carbapenem Resistance in Enterobacterales

The OXA-10 class D β-lactamase has been reported to contribute to carbapenem resistance in non-fermenting Gram-negative bacilli; however, its contribution to carbapenem resistance in Enterobacterales is unknown. In this work, minimum inhibitory concentrations (MICs), whole genome sequencing (WGS), cloning experiments, kinetic assays, molecular modelling studies, and biochemical assays for carbapenemase detection were performed to determine the impact of OXA-10 production on carbapenem resistance in two XDR clinical isolates of Escherichia coli with the carbapenem resistance phenotype (ertapenem resistance). WGS identified the two clinical isolates as belonging to ST57 in close genomic proximity to each other. Additionally, the presence of the blaOXA-10 gene was identified in both isolates, as well as relevant mutations in the genes coding for the OmpC and OmpF porins. Cloning of blaOXA-10 in an E. coli HB4 (OmpC and OmpF-deficient) demonstrated the important contribution of OXA-10 to increased carbapenem MICs when associated with porin deficiency. Kinetic analysis showed that OXA-10 has low carbapenem-hydrolysing activity, but molecular models revealed interactions of this β-lactamase with the carbapenems. OXA-10 was not detected with biochemical tests used in clinical laboratories. In conclusion, the β-lactamase OXA-10 limits the activity of carbapenems in Enterobacterales when combined with low permeability and should be monitored in the future

Multicenter Performance Evaluation of MALDI-TOF MS for Rapid Detection of Carbapenemase Activity in Enterobacterales: The Future of Networking Data Analysis With Online Software.

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: "software design," "intercenter evaluation," and "clinical validation." First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy