Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as an Alternative to 16S rRNA Gene Sequencing for Identification of Difficult-To-Identify Bacterial Strains

Abstract

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.The technique matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories and has been shown to be a promising method to replace conventional phenotypic identification for the majority of bacterial pathogens routinely found in clinical samples (1,3,8,15,17).For a subset of bacterial isolates, conventional techniques which mainly rely on commercial biochemical kit tests fail to provide a definitive identification of these infrequent isolates. In those cases, alternative techniques are required. That could be an expanded battery of biochemical tests, high-performance liquid chromatography (HPLC), or sequencing of housekeeping bacterial genes such as 16S rRNA genes and comparing the obtained sequences to those in databases (4). Since 16S rRNA gene sequencing is both time-consuming and costly, we wanted to assess the potential of MALDI-TOF MS, which is based on the study of proteic profiles, to replace it.We investigated a collection of 1,405 isolates on which 16S rRNA gene sequencing was performed in the last 8 years in our laboratory due to a lack of satisfactory identification by conventional methods such as Vitek2 or the API system (bioMérieux, Marcy l'Etoile, France). We selected for each species a maximum of 5 isolates. This yielded a total of 433 bacterial isolates representing 207 different species from 84 different genera. For 23 isolates, no growth could be obtained from frozen stock. A total of 410 isolates were consequently further studied. Acquisition of protein mass spectra of strains by MALDI-TOF MS was done on a Microflex LT instrument (Bruker Daltonics, Leipzig, Germany) using the FlexControl 3.0 software (Bruker Daltonics) after a formic acid-acetonitrile extraction step was performed as previously described (1). Automated data analysis of raw spectra was performed by the BioTyper automation 2.0 software (Bruker Daltonics), using a library of 3,290 spectra (database update of 2 September 2008) with default settings. According to the criteria proposed by the manufacturer, a result was considered valid at the species level whenever the score value attributed by BioTyper was x Ն 2.0, valid at the genus level when the score was 1.7 Յ x Ͻ 2, and no reliable identification when the score was x Ͻ 1.7. 16S rRNA gene sequencing and identification were performed by standard methods (5).For routine isolates, MALDI-TOF MS has shown overall correct identification levels often greater than 85% (1,3,8,15,17). In the present study, MALDI-TOF MS yielded a score of...

Sepsityper® for rapid identification of microorganisms from positive blood cultures

“…No obstante hay que remarcar las limitaciones de esta tecnología en dos situaciones clínicas: la primera, de gran trascendencia, la bacteriemia por Streptococcus pneumoniae y la posibilidad de confusión por parte del sistema MALDITOF MS® con Streptococcus del grupo viridans (18,24). Este fallo, bien conocido del sistema MALDITOF-MS®, se debe según algunos autores, a la gran similitud a nivel proteómico de múltiples especies de streptococcus, hasta el punto de ser necesario hacer genotipado molecular de las mismas para su correcta diferenciación (25); la segunda, de mucha menor trascendencia clínica, es el caso de la asignación de especie para los estafilococos coagulasa negativos, en los que el porcentaje de concordancia en especie desciende al 86.5%. Hay que destacar, que en ningún caso hubo discordancias con la identificación de Staphylococcus aureus, y en todos los casos de discrepancia a nivel de especie no supuso ningún cambio en el manejo clíni-co del paciente.…”

“…Queda aún por esclarecer si la identificación correcta en estos casos es atribuible a errores en la tecnología MALDITOF-MS® o por el contrario se deben a errores en la identificación convencional. La secuenciación del ARN16S y el posterior análisis filogenético de las secuencias habría permitido establecer cuál de los dos sistemas estaba emitiendo el resultado correcto (25).…”

An Evaluation Of Direct Identification Of Pathogens From Blood Cultures By Maldi-Tof Mass Spectrometry

“…With a special set of MALDI Sepsityper is possible to identify bacteria directly from the positive blood culture (17)(18)(19)(20)(21)(22)(23)(24)(25). Most reports describe the blood culture must be grown in automated BACTEC system vials (26,27).…”

MALDI-TOF MS: Its Application in the Clinical Laboratory and a Paradigm Shift in Clinical Microbiology

“…However, as analysis of microorganisms by MALDI-TOF MS became more commonplace, it became apparent that the IC method was not always appropriate for all specimen types, in spite of its relative simplicity; problems with spectral generation from some microbes [12,13] and biosafety issues [14,15] and interpretation forms are shown in Fig. 1.…”