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

Abstract

bWith 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 ␤-lactam and two non-␤-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. The early initiation of adequate antibiotic therapy is known to be an important parameter for the outcome of septic patients (1-3). Therefore, empirical treatment must be administered as soon as sepsis is diagnosed; however, with the growing burden of antimicrobial resistance, affecting the sepsis-causing pathogen with an empirical therapy is becoming increasingly difficult. Prediction of antibiotic resistance at the earliest time point is necessary to ensure the best available treatment for the patient and to avoid the extensive use of broad-spectrum therapy. One approach to shorten the time to result for antibiotic susceptibility testing (AST) is the use of matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) for identification and resistance testing. The most common approach using mass spectrometry is the detection of ␤-lactamases with a functional assay that measures their hydrolysis products (4-9). This test provides results within 1 to 3 h, but, by its very nature, it is limited to the detection of ␤-lactamase-mediated resistance. Recently, Lange et al. (10) described a MALDI-TOF MS method that allows for resistance testing independent of the underlying mechanism. The MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) relies on a semiquantitative measurement of bacterial proteins. Bacterial cells are incubated in culture medium with or without antibiotics. Subsequently, the cells are lysed and spiked with an internal standard. Mass spectra from the two setups are acquired and normalized, and their peak intensit...

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for the Rapid Detection of Antimicrobial Resistance Mechanisms and Beyond

“…Jung et al also evaluated this method for detecting resistance against cefotaxime, piperacillin-tazobactam, gentamicin, and ciprofloxacin (115), by analyzing a series of 30 spiked blood cultures and 99 patient-derived blood cultures. Bacterial cells extracted from positive blood culture bottles were used to prepare an inoculum of 5 ϫ 10 6 CFU/ml.…”

Using the NCBI AMRFinder Tool to Determine Antimicrobial Resistance Genotype-Phenotype Correlations Within a Collection of NARMS Isolates

“…The gold standard for measuring antimicrobial susceptibility is based on standardized dilution- or diffusion-based in vitro antimicrobial susceptibility testing (AST) methods, where extensive research and testing have been performed to correlate AST measurements with clinical outcomes (1) Increasingly, molecular methods are being used in resistance surveillance and in some cases also to guide clinical therapy. These range from PCR detection of known resistance elements (2) to mass spectrometry-based methods (3-7). Whole genome shotgun sequencing (WGS) has been integrated into the clinical and public health settings, though the use of WGS has focused primarily on outbreak identification and tracking (8,9).…”

Determinación de fenotipos de resistencia a antimicrobianos en aislamientos clínicos bacterianos mediante espectrometría de masas MALDI-TOF cuantitativa

“…Ocurre algo similar en el caso de piperacilina-tazobactam, con la diferencia con otros estudios (Jung et al 2016) de que en nuestro caso, los resultados son favorables en el sentido de que no obtenemos falsos sensibles, todas las cepas resistentes fueron informadas como resistentes por nuestro sistema si mantenemos el valor en 0.33, y tan sólo encontraríamos 2 cepas mal clasificadas si aumentamos ese valor a 0.64. Debo destacar que ambos casos se dieron en hemocultivos simulados y no en cepas procedentes de pacientes, donde se observó una clara diferencia entre susceptibles y resistentes, ya que aun aumentando el valor a 0.64, todas las cepas resistentes tenían valores superiores a 0.9.…”

“…Para cefoxitina, 3 cepas fueron mal clasificadas como resistentes siendo sensibles, de manera que la sensibilidad se vio disminuida a un 89,26%. A pesar de ello, los resultados son muy prometedores.Otra alternativa propuesta recientemente es la espectrometría de masas MALDI-TOF semicuantitativa(Langue et al, 2014;Jung et al, 2016, Sparbier et al, 2016. Esta metodología también se basa en la incubación del microorganismo en presencia y ausencia de los antimicrobianos a analizar, y la cuantificación del crecimiento bacteriano en ambas circunstancias.…”

“…productora de carbapenemasas, y recientemente se ha probado con otros microorganismos contra varios antibióticos(Jung et al, 2016), e incluso para las micobacterias(Ceyssens et al, 2017), con buenos resultados. En el último estudio realizado con bacterias, se extrajeron células bacterianas de los frascos de cultivo de sangre, y se usaron para preparar una suspensión bacteriana equivalente a alrededor de 5x10 6 UFC / ml(Jung et al, 2016).Evitamos este paso en nuestro método porque previamente hemos observado que, en las enterobacterias, la concentración bacteriana en la positivización del hemocultivo es de alrededor de 10 6 UFC / ml.Al igual que el trabajo llevado a cabo por Jung et al, nuestro método se basa en las modificaciones de perfil de los picos condicionadas por el crecimiento del microorganismo en contacto con concentraciones específicas de antibióticos. La suma de los valores de los picos que usamos es conceptualmente similar al AUC utilizado por estos autores, y el crecimiento relativo (RG) definido por ellos también es conceptualmente similar a las tasas entre los picos de los valores de suma que usamos.…”