A MALDI-ToF mass spectrometry database for identification and classification of highly pathogenic bacteria

MALDI-TOF mass spectrometry: the quantum leap

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the identification of highly pathogenic bacteria

Recently, MALDI-TOF MS devices designed for use in clinical laboratories have been commercially introduced in various large centres worldwide. All published studies conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria and yeasts in a clinical microbiological laboratory. Although all data show that MALDI-TOF MS correctly identifies the great majority of isolates processed routinely, it cannot yet identify every such isolate. Until today, MALDI-TOF MS is inappropriate for the identification of Shigella species, pneumococci and viridans streptococci. Database upgrades and sample enrichment are essential elements to refine the MALDI-TOF MS technique, allowing the method to increase its power.For the identification of a significant proportion of yeasts, an extraction method prior to analysis in the mass spectrometer is mandatory to obtain appropriate spectra. Because of the low marginal costs, and the extreme speed of MALDI-TOF MS, the technique can improve laboratory efficiency when used early in identification protocols. Lengthier, more labour-intensive, and costlier techniques can be reserved for the minority of isolates not identified with high confidence by MALDI-TOF MS.MALDI-TOF MS also has the potential to directly identify pathogens in biological fluids, such as urine samples and blood cultures. For this application however, further well-designed prospective studies are warranted. The potential for identification at the serotype or strain level, and antibiotic resistance profiling within minutes make MALDI-TOF mass spectrometry an ongoing revolution in the clinical microbiology laboratory.

4 - Mass Spectrometry Applications in Infectious Disease and Pathogens Identification

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a reliable technique, is rapid and precise in the identification of pathogens, and is essential in clinical microbiology laboratories. The aim of this project is to evaluate the identification performance of the MALDI-TOF MS Smart MS 5020 system (Zhuhai DL Biotech) by comparing the identification results with those of the MALDI-TOF MS Biotyper Microflex LT system (Bruker Daltonics). A total of 612 clinical isolates were analyzed in parallel: 494 routine species and 118 species not commonly isolated in routine practice, previously isolated and frozen in our hospital's strain collection. This collection included gram-negative bacteria, gram-positive bacteria, anaerobes, and fungi. Concordance at genus and species level identification was evaluated, and 16S rRNA gene sequencing was performed for strains unidentified or with discordant results. Both systems, Smart MS 5020 and Biotyper Microflex LT, achieved high identification rates (100% and 98.9%, respectively). The concordance between the systems was 98.9% at the genus level and 97.2% at the species level. Correct species-level identification was 96.9% for the Smart MS 5020 and 96.6% for the Biotyper Microflex LT. The MALDI-TOF MS Smart MS 5020 and Biotyper Microflex LT systems offer high efficacy in identifying a wide variety of clinical isolates, both demonstrating excellent performance.IMPORTANCEAccurate identification of microorganisms in clinical microbiology laboratories is essential for the effective diagnosis and treatment of patients. This study evaluates the performance of the Smart MS 5020 matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) system in comparison to the widely used Biotyper Microflex LT system. As a new entrant to the market, independent validation of the Smart MS 5020 system is crucial for laboratories considering its implementation. By analyzing 612 clinical isolates, including both routine and rare species, this study provides valuable data on the concordance rates between the two systems. The results confirm the Smart MS 5020 high accuracy and reinforce the reliability of MALDI-TOF MS technology. The high concordance rate between the two systems establishes the Smart MS 5020 as a promising, cost-effective alternative for routine diagnostics. These results expand the evidence base on MALDI-TOF MS platforms and can inform procurement decisions in clinical laboratories worldwide.

Matrix-assisted laser desorption ionization time of flight mass spectrometry and diagnostic testing for prosthetic joint infection in the clinical microbiology laboratory

First introduced into clinical microbiology laboratories in Europe, MALDI-TOF MS is being rapidly embraced by laboratories around the globe. Although it has multiple applications, its widespread adoption in clinical microbiology relates to its use as an inexpensive, easy, fast, and accurate method for identification of grown bacteria and fungi based on automated analysis of the mass distribution of bacterial proteins. This review provides a historical perspective on this new technology. Modern applications in the clinical microbiology laboratory are reviewed with a focus on the most recent publications in the field. Identification of aerobic and anaerobic bacteria, mycobacteria, and fungi are discussed, as are applications for testing urine and positive blood culture bottles. The strengths and limitations of MALDI-TOF MS applications in clinical microbiology are also addressed. MALDI-TOF MS is a tool for rapid, accurate, and cost-effective identification of cultured bacteria and fungi in clinical microbiology. The technology is automated, high throughput, and applicable to a broad range of common as well as esoteric bacteria and fungi. MALDI-TOF MS is an incontrovertibly beneficial technology for the clinical microbiology laboratory.

ABSTRACTKlebsiella variicola is a pathogen that is increasingly recognized as being associated with human infections, but the methods available to clinical microbiology laboratories for accurate identification are limited. In this study, we assessed the accuracy of identification of K. variicola by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry using genetic identification with multiplex PCR as the reference method. Antimicrobial susceptibilities and virulence of K. variicola strains were also investigated. Fifty-five Klebsiella pneumoniae, 26 K. variicola, and 2 Klebsiella quasipneumoniae clinical strains were used for evaluation. Both MALDI Biotyper with library version 9 and Klebsiella MALDI TypeR, a web-based species identification tool using MALDI-TOF data, accurately identified all K. variicola strains. In addition, two strains of K. quasipneumoniae were accurately identified with Klebsiella MALDI TypeR. Whole-genome sequencing confirmed the accurate identification to the subspecies level by Klebsiella MALDI TypeR for four strains (two strains each of K. variicola subsp. variicola and K. quasipneumoniae subsp. similipneumoniae). While 13 strains, 3 strains, and 1 strain of K. pneumoniae showed nonsusceptibility to ampicillin-sulbactam, ceftriaxone, and meropenem, respectively, all strains of K. variicola were susceptible to all tested antimicrobial agents. Although two K. variicola strains were positive for the string test, no K. variicola strains harbored any of the genes associated with hypervirulence of K. pneumoniae. Accurate identification of the K. pneumoniae complex, including K. variicola, by MALDI-TOF in clinical microbiology laboratories is expected to clarify the clinical characteristics of each species in the future.IMPORTANCE Recent widespread use of bacterial whole-genome sequencing analysis has resulted in the proposal of novel bacterial species and reclassification of taxonomy. Accurate methods for identification of bacterial species in clinical microbiology laboratories are essential to accumulate information on the clinical characteristics of each bacterial species. Klebsiella variicola is a member of the Klebsiella pneumoniae complex, and its association with human infections has been increasingly recognized, but accurate identification methods approved for use in clinical microbiology laboratories have been limited thus far. The findings of the present study suggest that K. variicola can be accurately identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry using updated library or web-based identification tools. Accurate identification will promote exploration of clinical characteristics of K. variicola.

Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in the last decade has revealed itself as a valid support in the workflow in the clinical microbiology laboratory for the identification of bacteria and fungi, demonstrating high reliability and effectiveness in this application. Its use has reduced, by 24 h, the time to obtain a microbiological diagnosis compared to conventional biochemical automatic systems. MALDI-TOF MS application to the detection of pathogens directly in clinical samples was proposed but requires a deeper investigation, whereas its application to positive blood cultures for the identification of microorganisms and the detection of antimicrobial resistance are now the most useful applications. Thanks to its rapidity, accuracy, and low price in reagents and consumables, MALDI-TOF MS has also been applied to different fields of clinical microbiology, such as the detection of antibiotic susceptibility/resistance biomarkers, the identification of aminoacidic sequences and the chemical structure of protein terminal groups, and as an emerging method in microbial typing. Some of these applications are waiting for an extensive evaluation before confirming a transfer to the routine. MALDI-TOF MS has not yet been used for the routine identification of parasites; nevertheless, studies have been reported in the last few years on its use in the identification of intestinal protozoa, Plasmodium falciparum, or ectoparasites. Innovative applications of MALDI-TOF MS to viruses’ identification were also reported, seeking further studies before adapting this tool to the virus’s diagnostic. This mini-review is focused on the MALDI-TOF MS application in the real life of the diagnostic microbiology laboratory.

Culturomics has permitted discovery of hundreds of new bacterial species isolated from the human microbiome. Profiles generated by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry have been added to the mass spectrometer database used in clinical microbiology laboratories. We retrospectively collected raw data from MALDI-TOF mass spectrometry used routinely in our laboratory in Marseille, France, during January 2012–March 2018 and analyzed 16S rDNA sequencing results from misidentified strains. During the study period, 744 species were identified from clinical specimens, of which 21 were species first isolated from culturomics. This collection involved 105 clinical specimens, accounting for 98 patients. In 64 cases, isolation of the bacteria was considered clinically relevant. MALDI-TOF mass spectrometry was able to identify the species in 95.2% of the 105 specimens. While contributing to the extension of the bacterial repertoire associated with humans, culturomics studies also enlarge the spectrum of prokaryotes involved in infectious diseases.

Urinary tract infections are one of the most common bacterial infections and rapid diagnosis of the infection is essential for appropriate antibiotic therapy. The goal of our study was to identify urinary pathogens directly by MALDI-TOF MS and to perform antibiotic susceptibility tests in order to shorten the period spent for culturing.Urine samples submitted for culture to the Clinical Microbiology Laboratory were enrolled in this study. Urine samples were screened for leukocyte and bacteria amount by flow cytometry. Samples with bacterial load of 106-107/mL were tested directly by MALDI-TOF MS and antibiotic susceptibility tests (AST) were performed.In total, 538 positive urine samples were evaluated in our study. MALDI-TOF MS identified the microorganism directly from the urine sample in 91.8% of these samples and the concordance rate of conventional identification and direct detection was 95.8% for Gram-negatives at the genus and species level. Escherichia coli (n:401) was the most frequently isolated microorganism, followed by Klebsiella pneumoniae (n:57). AST results were generated for 111 of these urine samples and the concordance was 90% and 87% for E. coli and K. pneumoniae, respectively.Our results showed that screening of urine samples with flow cytometry to detect positive samples and identification of uropathogens directly by MALDI-TOF MS with an accuracy of over 90% can be a suitable method particularly for Gram-negative bacteria in clinical microbiology laboratories.

BackgroundNon-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed.MethodologyWe set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 105 colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours.ConclusionsOur data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.

MALDI-TOF MS has revolutionized the identification of microorganisms and has become an indispensable part of routine diagnostics in the clinical microbiological laboratory. However, application of this technique in microbial surveillance outside of clinical settings is limited. In this study, we have evaluated the performance of a Bruker MALDI Biotyper System for the identification of bacteria isolated from the hand palms of fresh produce handlers and their surrounding environments in a wholesale fresh produce market in Doha, Qatar. The accuracy was verified against the results obtained by bacterial 16S rRNA gene sequencing. A total of 105 isolates were tested, of which 67 (64%) isolates were identified by MALDI-TOF MS and 101 isolates (96%) were identified by 16S rRNA gene sequencing, either at the genus level or species level. However, MALDI-TOF MS identified more isolates (41%) at the species level than 16S rRNA gene sequencing (28%). MALDI-TOF MS was particularly useful in the species level identification of Enterobacteriaceae. MALDI-TOF MS successfully identified most known human pathogens in a rapid and cost-effective manner but failed to identify a significant number of isolates that were of environmental origin, suggesting room for further expansion of the reference database.

The present study compared two chemical-based methods, namely, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, to understand the misidentification of Exophiala dermatitidis and Exophiala phaeomuriformis. The study utilized 44 E. dermatitidis and 26 E. phaeomuriformis strains, which were partially treated with strong acids and bases for further evaluation. MALDI-TOF MS and ATR-FTIR spectroscopy data of the two Exophiala species were compared. Data groupings were observed for the chromic acid- and nitric acid-treated species when the black yeast sources were categorized as creosoted-oak sleepers, concrete sleepers, or dishwasher isolates. The MALDI-TOF MS data for the metalloenzyme-containing regions were consistent with the ATR-FTIR spectroscopy data. These results indicated that environmental isolates might contain metals not found in human isolates and might interfere with chemical-based identification methods. Therefore, MALDI-TOF MS reference libraries should be created for clinical strains and should exclude petroleum-associated environmental isolates.