Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory

Rodríguez‐Sánchez, Belén; Marı́n, Mercedes; Sánchez‐Carrillo, Carlos; Cercenado, Emilia; Ruíz, Adrián; Rodríguez‐Créixems, Marta; Bouza, Emilio

doi:10.1016/j.diagmicrobio.2014.01.021

Cited by 37 publications

(25 citation statements)

References 21 publications

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“…While acetonitrile enhances protein solubility, 98% formic acid hastens extraction (3-5 min) and improves log-scores as were seen in our study. Similar efficacy of this method has been noted in bacterial studies [18,19]. We found Sabouraud's dextrose agar as an M A N U S C R I P T…”

Section: All Three Extraction Methods Correctly Identified the Candidsupporting

confidence: 58%

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections

Ghosh

Paul

Sood

et al. 2015

Clinical Microbiology and Infection

110

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Few studies have systematically standardised and evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of yeasts from bloodstream infections. This is rapidly becoming pertinent for early identification of yeasts and appropriate antifungal therapy. We used 354 yeast strains identified by polymerase chain reaction (PCR) sequencing for standardisation and 367 blind clinical strains for validation of our MALDI-TOF MS protocols. We also evaluated different sample preparation methods and found the on-plate formic acid extraction method as most cost- and time-efficient. The MALDI-TOF assay correctly identified 98.9% of PCR-sequenced yeasts. Novel main spectrum projections (MSP) were developed for Candida auris, C. viswanathii and Kodamaea ohmeri, which were missing from the Bruker MALDI-TOF MS database. Spectral cut-offs computed by receiver operating characteristics (ROC) analysis showed 99.4% to 100% accuracy at a log score of ≥ 1.70 for C. tropicalis, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. albicans, C. rugosa, C. guilliermondii, C. lipolytica, C. metapsilosis, C. nivariensis. The differences in the species-specific scores of our standardisation and blind validation strains were not statistically significant, implying the optimal performance of our test protocol. The MSPs of the three new species also were validated. We conclude that MALDI-TOF MS is a rapid, accurate and reliable tool for identification of bloodstream yeasts. With proper standardisation, validation and regular database expansion, its efficiency can be further enhanced.

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Section: All Three Extraction Methods Correctly Identified the Candidsupporting

confidence: 58%

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections

Ghosh

Paul

Sood

et al. 2015

Clinical Microbiology and Infection

110

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“…Two gene regions were targeted for NTM identification: the 5= end of the 16S rRNA gene with the universal primers E8F (5=-AGAGTTTGATCCTGGCTCAG-3=) and E533R (5=-TTACCGCGGCTGCTGGCA-3=) (6) and a 439-bp fragment within the hsp65 gene using the primers TB11 (5=-ACCAACGATGGTGTGTCCAT) and TB12 (5=-CTTGTCGA ACCGCATACCCT), as described by Telenti et al (7). The amplification, purification, and sequencing process was carried out as previously described by Rodríguez-Sánchez et al (8). Interpretation of sequencing results was performed considering CLSI recommendations for the genus Mycobacterium (9).…”

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confidence: 99%

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Nontuberculous Mycobacteria from Clinical Isolates

et al. 2015

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dMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of nontuberculous mycobacterial (NTM) isolates was evaluated in this study. Overall, 125 NTM isolates were analyzed by MALDI-TOF and GenoType CM/AS. Identification by 16S rRNA/hsp65 sequencing was considered the gold standard. Agreements between MALDI-TOF and GenoType CM/AS with the reference method were, respectively, 94.4% and 84.0%. In 17 cases (13.6%), results provided by GenoType and MALDI-TOF were discordant; however, the reference method agreed with MALDI-TOF in 16/17 cases (94.1%; P ‫؍‬ 0.002). Identification of nontuberculous mycobacteria (NTM) by conventional phenotypic tests requires long incubation periods that may take up to 12 weeks. The GenoType Mycobacterium CM/AS (Hain Lifescience GmbH, Nehren, Germany) is a test based on the amplification of a 23S rRNA gene region followed by reverse hybridization with specific probes that allows the identification of 40 of the most common NTM. However, the hybridization step requires high sequence homology, and even point mutations in the target regions may hamper the hybridization step, leading to unreliable results (1). Matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) has been recently applied to the identification of a wide range of microorganisms (2), mainly clinically significant bacteria and fungi. Experience with MALDI-TOF for the rapid identification of NTM is very limited, mainly because of the number of identified species and because database information is scarce (3-5). In this study, a large collection of NTM clinical isolates and reference strains were analyzed using MALDI-TOF technology in comparison with the GenoType Mycobacterium CM/AS procedure and the reference procedure, 16S rRNA/hsp65 gene sequencing.From January 2010 to September 2014, all the NTM with purportedly clinical significance were grown on Lowenstein-Jensen agar and simultaneously identified by GenoType CM/AS assay (Hain Lifescience, GmbH, Nehren, Germany) and with MALDI-TOF MS technology (Bruker Daltonics, Bremen, Germany). Ten reference isolates were also included in this study as positive controls (Table 1). All the strains were also analyzed by 16S rRNA/ hsp65 sequencing as the reference method in order to resolve possible discrepancies.Genotypic identification. Two gene regions were targeted for NTM identification: the 5= end of the 16S rRNA gene with the universal primers E8F (5=-AGAGTTTGATCCTGGCTCAG-3=) and E533R (5=-TTACCGCGGCTGCTGGCA-3=) (6) and a 439-bp fragment within the hsp65 gene using the primers TB11 (5=-ACCAACGATGGTGTGTCCAT) and TB12 (5=-CTTGTCGA ACCGCATACCCT), as described by Telenti et al. (7). The amplification, purification, and sequencing process was carried out as previously described by . Interpretation of sequencing results was performed considering CLSI recommendations for the genus Mycobacterium (9). Results obtained by 16S rRNA/hsp65 sequencing were considered the final microorganism identification.GenoType CM/AS iden...

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“…Mass spectrometry is a fast and less labor-intensive technique for bacterial identification. The approximate cost was reported to be 0.5 to 1.00 USD per sample [50]. However, pretreatment of chemicals, temperature, and media composition may affect the quality of spectra.…”

Section: Discussionmentioning

confidence: 99%

Tracking of Intentionally Inoculated Lactic Acid Bacteria Strains in Yogurt and Probiotic Powder

2019

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The present work aimed at tracking intentionally inoculated lactic acid bacteria (LAB) strains in yogurt and probiotic powder. Leuconostoc (Leu.) mesenteroides (11251), Lactobacillus (L.) brevis (B151), and Lactobacillus plantarum (LB41K) strains were tracked in yogurt, and L. plantarum (LB41P) was tracked in a commercial probiotic powder. The yogurt was intentionally inoculated with the selected bacterial strains. Two types of yogurt with known and unknown bacterial pools were utilized. The standard 16S rRNA gene sequencing was used to evaluate the initial screening. The molecular typing tools, random amplified polymorphic DNA (RAPD), repetitive element palindromic PCR (rep-PCR), and comparative gene sequence analysis of selected housekeeping loci were used to track the inoculated dubious strains. Out of 30 random selections for each inoculation, the developed method identified seven (11251), nine (B151), and five (LB41K) colonies in the yogurt. The validation was performed by identifying 7 colonies (LB41P) out of 30 in the probiotic powder. The DNA banding profiles and the gene sequence alignments led to the identification of the correct inoculated strains. Overall, the study summarizes the use of molecular tools to identify the deliberately inoculated LAB strains. In conclusion, the proposed polyphasic approach effectively tracked the intentionally inoculated strains: Leu. mesenteroides, L. brevis, and L. plantarum (LB41K) in yogurt and L. plantarum (LB41P) in probiotic powder. The study demonstrates how to track industrially relevant misused LAB strains in marketable food products.

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Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory

Cited by 37 publications

References 21 publications

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Nontuberculous Mycobacteria from Clinical Isolates

Tracking of Intentionally Inoculated Lactic Acid Bacteria Strains in Yogurt and Probiotic Powder

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