Rapid and reproducible MALDI-TOF-based method for detection Vancomycin-resistant Enterococcus faecium using classifying algorithms

Vancomycin-resistant Enterococcus faecium has become a health threat over the last 20 years due to its ability to rapidly spread and cause outbreaks in hospital settings. Although MALDI-TOF MS has already demonstrated its usefulness for accurate identification of E. faecium, its implementation for antimicrobial resistance detection is still under evaluation. The reproducibility of MALDI-TOF MS for peak analysis and its performance for correct discrimination of vancomycin susceptible isolates (VSE) from those hosting the VanA and VanB resistance mechanisms was evaluated in this study. For the first goal, intra-spot, inter-spot -technical- and inter-day -biological- reproducibility was assayed. The capability of MALDI-TOF to discriminate VSE isolates from VanA VRE and VanB VRE strains was carried out on protein spectra from 178 E. faecium unique clinical isolates -92 VSE, 31 VanA VRE, 55 VanB VRE-, processed with Clover MS Data Analysis software. Unsupervised (Principal Component Analysis –PCA-) and supervised algorithms (Support Vector Machine -SVM-, Random Forest -RF- and Partial Least Squares-Discriminant Analysis -PLS-DA-) were applied. The reproducibility assay showed lower variability for normalized data (p<0.0001) and for the peaks within the 3000-9000 m/z range. Besides, 80.9%, 79.21% and 77.53% VSE vs VRE (VanA + VanB) discrimination was achieved by applying SVM, RF and PLS-DA, respectively. Correct differentiation of VanA from VanB VRE isolates was obtained by SVM in 86.65% cases. The implementation MALDI-TOF MS and peak analysis could represent a rapid and effective tool for VRE screening. However, further improvements are needed to increase the accuracy of this approach.

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From days to hours: Can MALDI-TOF MS system replace both conventional and molecular typing methods with new cut off level for Vancomycin Resistant Enterococcus faecium

Journal of Microbiological Methods ◽

10.1016/j.mimet.2018.12.015 ◽

2019 ◽

Vol 162 ◽

pp. 62-68 ◽

Cited By ~ 2

Author(s):

Sumeyra Savas ◽

Gulsen Hazirolan ◽

Alper Karagoz ◽

Mehmet Parlak

Keyword(s):

Enterococcus Faecium ◽

Molecular Typing ◽

Vancomycin Resistant Enterococcus ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Typing Methods ◽

Vancomycin Resistant ◽

Tof Ms

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Antimicrobial resistance classification using MALDI-TOF-MS is not that easy: lessons from vancomycin-resistant Enterococcus faecium

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2019.10.027 ◽

2020 ◽

Vol 26 (3) ◽

pp. 391-393

Author(s):

M. Brackmann ◽

S.L. Leib ◽

M. Tonolla ◽

N. Schürch ◽

M. Wittwer

Keyword(s):

Antimicrobial Resistance ◽

Enterococcus Faecium ◽

Vancomycin Resistant Enterococcus ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Vancomycin Resistant ◽

Tof Ms

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Performance of Five Commercial Identification Platforms for Identification of Staphylococcus delphini

Journal of Clinical Microbiology ◽

10.1128/jcm.00721-19 ◽

2019 ◽

Vol 57 (11) ◽

Cited By ~ 2

Author(s):

Matthew C. Canver ◽

Tsigereda Tekle ◽

Samantha T. Compton ◽

Katrina Callan ◽

Eileen M. Burd ◽

...

Keyword(s):

Distinct Species ◽

Human Infection ◽

Species Level ◽

Accurate Identification ◽

Content Type ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Staphylococcus Intermedius ◽

Animal Pathogens ◽

Tof Ms

ABSTRACT The Staphylococcus intermedius group (SIG) is a collection of coagulase-positive staphylococci consisting of four distinct species, namely, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus intermedius, and Staphylococcus pseudintermedius. SIG members are animal pathogens and rare causes of human infection. Accurate identification of S. pseudintermedius has important implications for interpretation of antimicrobial susceptibility testing data and may be important for other members of the group. Therefore, we sought to evaluate the performance of five commercially available identification platforms with 21 S. delphini isolates obtained from a variety of animal and geographic sources. Here, we show that automated biochemical platforms were unable to identify S. delphini to the species level, a function of its omission from their databases, but could identify isolates to the SIG level with various degrees of success. However, all automated systems misidentified at least one isolate as Staphylococcus aureus. One matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system was able to identify S. delphini to the species level, suggesting that MALDI-TOF MS is the best option for distinguishing members of the SIG. With the exception of S. pseudintermedius, it is unclear if other SIG members should be routinely identified to the species level; however, as our understanding of their role in animal and human diseases increases, it may be necessary and important to do so.

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Accurate identification of Culicidae at aquatic developmental stages by MALDI-TOF MS profiling

Parasites & Vectors ◽

10.1186/s13071-014-0544-0 ◽

2014 ◽

Vol 7 (1) ◽

Cited By ~ 47

Author(s):

Constentin Dieme ◽

Amina Yssouf ◽

Anubis Vega-Rúa ◽

Jean-Michel Berenger ◽

Anna-Bella Failloux ◽

...

Keyword(s):

Developmental Stages ◽

Accurate Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Discrimination between Asian populations of the parasitoid wasp Ganaspis cf. brasiliensis using a simple MALDI-TOF MS-based method for use with insects

Biology Methods and Protocols ◽

10.1093/biomethods/bpz002 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 3

Author(s):

Michael A Reeve ◽

M Lukas Seehausen

Keyword(s):

Reference Sample ◽

Principal Component ◽

Parasitoid Wasp ◽

Test Sample ◽

Invasive Pest ◽

Drosophila Suzukii ◽

Maldi Tof Ms ◽

Chinese Populations ◽

Maldi Tof ◽

Tof Ms

Abstract The fruit fly Drosophila suzukii has recently become an invasive pest insect of significant economic impact in Europe and the USA. In contrast to other Drosophila species, D. suzukii is able to infest intact fruit by means of a saw-like ovipositor, which allows females to deposit eggs beneath the skin of the fruit. Classical biological control using the parasitoid wasp Ganaspis cf. brasiliensis is currently being researched as an environmentally sustainable option for the control of D. suzukii. In particular, the host specificity of this parasitoid has been assessed for populations from different regions in China and Japan. In order to study the relationship between the differences in specificity and molecular variations, we have adapted a matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method, originally developed for use with plant material, to discriminate between example populations of G. cf. brasiliensis. We have employed a combination of principal component analysis and blind-tested comparison between reference sample MALDI-TOF MS spectra and test sample spectra to discriminate, on the basis of the acid-soluble insect protein spectra generated, between four populations of G. cf. brasiliensis (originally collected from Tokyo and Hasuike in Japan and Dali and Ximing in China). MALDI-TOF MS analysis is able to discriminate with 100% accuracy between populations G. cf. brasiliensis. The Chinese populations were observed to be similar, but the Tokyo population is slightly different and the Hasuike population is significantly different from the other populations. The Tokyo population appears more closely related to the Chinese populations than the Hasuike population, even though both originate from Japan.

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Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from “Pseudo-Cronobacter” to New Enterobacteriaceae Genera

Applied and Environmental Microbiology ◽

10.1128/aem.00234-17 ◽

2017 ◽

Vol 83 (13) ◽

Cited By ~ 6

Author(s):

Barbora Svobodová ◽

Jiří Vlach ◽

Petra Junková ◽

Ludmila Karamonová ◽

Martina Blažková ◽

...

Keyword(s):

Intact Cell ◽

Foodborne Pathogen ◽

Principal Component ◽

Powdered Infant Formula ◽

Content Type ◽

Reliable Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Maldi Biotyper ◽

Tof Ms

ABSTRACT In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter. Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method. IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter. While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

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Implementation of MALDI-TOF Mass Spectrometry and Peak Analysis: Application to the Discrimination of Cryptococcus neoformans Species Complex and Their Interspecies Hybrids

Journal of Fungi ◽

10.3390/jof6040330 ◽

2020 ◽

Vol 6 (4) ◽

pp. 330

Author(s):

Margarita E. Zvezdanova ◽

Manuel J. Arroyo ◽

Gema Méndez ◽

Jesús Guinea ◽

Luis Mancera ◽

...

Keyword(s):

Mass Spectrometry ◽

Support Vector Machine ◽

Cryptococcus Neoformans ◽

Support Vector ◽

Its2 Region ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Interspecies Hybrids ◽

Correct Discrimination ◽

Tof Ms

Matrix-assisted laser desorption–ionization/time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for the rapid identification of microorganisms. Although most bacteria, yeasts and filamentous fungi can be accurately identified with this method, some closely related species still represent a challenge for MALDI-TOF MS. In this study, two MALDI-TOF-based approaches were applied for discrimination at the species-level of isolates belonging to the Cryptococcus neoformans complex, previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region: (i) an expanded database was built with 26 isolates from the main Cryptococcus species found in our setting (C. neoformans, C. deneoformans and AFLP3 interspecies hybrids) and (ii) peak analysis and data modeling were applied to the protein spectra of the analyzed Cryptococcus isolates. The implementation of the in-house database did not allow for the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of supervised classifiers (partial least squares-discriminant analysis and support vector machine) in a two-step analysis allowed for the 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. In addition, PCA analysis prior to support vector machine (SVM) provided 98.45% correct discrimination of the three analyzed species in a one-step analysis. This novel method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.

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Reagent-Free Identification of Clinical Yeasts by Use of Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Journal of Clinical Microbiology ◽

10.1128/jcm.01739-18 ◽

2019 ◽

Vol 57 (5) ◽

Author(s):

Lisa M. T. Lam ◽

Philippe J. Dufresne ◽

Jean Longtin ◽

Jacqueline Sedman ◽

Ashraf A. Ismail

Keyword(s):

Fourier Transform ◽

Ftir Spectroscopy ◽

Fourier Transform Infrared ◽

Attenuated Total Reflectance ◽

Reference Database ◽

Accurate Identification ◽

Total Reflectance ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

ABSTRACT Invasive fungal infections by opportunistic yeasts have increased concomitantly with the growth of an immunocompromised patient population. Misidentification of yeasts can lead to inappropriate antifungal treatment and complications. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is a promising method for rapid and accurate identification of microorganisms. ATR-FTIR spectroscopy is a standalone, inexpensive, reagent-free technique that provides results within minutes after initial culture. In this study, a comprehensive spectral reference database of 65 clinically relevant yeast species was constructed and tested prospectively on spectra recorded (from colonies taken from culture plates) for 318 routine yeasts isolated from various body fluids and specimens received from 38 microbiology laboratories over a 4-month period in our clinical laboratory. ATR-FTIR spectroscopy attained comparable identification performance with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). In a preliminary validation of the ATR-FTIR method, correct identification rates of 100% and 95.6% at the genus and species levels, respectively, were achieved, with 3.5% unidentified and 0.9% misidentified. By expanding the number of spectra in the spectral reference database for species for which isolates could not be identified or had been misidentified, we were able to improve identification at the species level to 99.7%. Thus, ATR-FTIR spectroscopy provides a new standalone method that can rival MALDI-TOF MS for the accurate identification of a broad range of medically important yeasts. The simplicity of the ATR-FTIR spectroscopy workflow favors its use in clinical laboratories for timely and low-cost identification of life-threatening yeast strains for appropriate treatment.

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