Improved Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)-Based Identification of Mycobacterium spp. by Use of a Novel Two-Step Cell Disruption Preparatory Technique

Abstract

Ireland bT he use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for microbial identification in clinical microbiology in recent years. This development can be attributed to its low operating cost and to its speed and reliability of identification for many bacteria and fungi. However, the use of MALDI-TOF MS for mycobacteriology has been less satisfactory, mainly owing to nonstandardized preparatory methods, poor reproducibility, and comparatively inferior results (1). The accurate and rapid identification of mycobacteria to the species level has implications for effective patient treatment and implementation of timely infection control measures.Our laboratory set about validating the Bruker Biotyper for the identification of mycobacteria using wild-type Mycobacterium spp. previously identified using genotype CM/AS and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing by the Irish Mycobacterial Reference Laboratory, St. James's Hospital, Dublin, Ireland, and Bruker's Mycobacteria Extract Method (MycoEX) version 3 (Fig. 1). Heat inactivation coupled with ethanol treatment (as detailed in Fig. 1) proved efficacious in the inactivation of mycobacteria; following extended incubation, there was no growth. Preliminary procedures using Bruker's recommended protocol yielded poor results. Therefore, we set about improving the results attained.Cell disruption has previously been shown to be a key step in liberating mycobacterial protein for analysis (2). We designed a two-step cell disruption protocol (Fig. 1) and found that the combined use of 0.5-mm-diameter silica/zirconia beads and sonication for 15 min produced greatly improved results (Table 1). A direct comparison between our method and MycoEX showed a marked improvement for Mycobacterium tuberculosis complex isolates using our method; 100% achieved successful identification to the genus level, with 85% achieving a reliable identification to the species level (log score of greater than 2.0). MycoEX resulted in successful identification to the genus level of 79% of isolates, with 62% achieving reliable identification to the species level. The number of results showing no identifications was reduced from 7 (22%) to 0 using the two-step cell disruption protocol. Similar improvements were shown for mycobacteria other than tuberculosis isolates, using the two-step cell disruption protocol; 87.5% achieved a log score of 1.7 or greater and 62.5% achieved a log score of greater than 2 compared to MycoEX, and 67% of isolates achieved a log score of 1.7 or greater, with 29% achieving a log score of greater than 2.0.In conclusion, using the described two-step cell disruption protocol, there was a 9-fold decrease in failures to achieve correct

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

“…As it allows pathogen-specific culture and protein extraction protocols (34,35), we first optimized our in-house mycobacterial cell disruption and protein extraction protocol for MALDI-TOF MS analysis. Upon comparison of various described protocols using M. tuberculosis H37Rv cells (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), we found that in our hands, bead beating in the presence of 1.2 ml of 70% ethanol gave the best output in terms of peak intensity and identification scores (Table S1).…”

“…Unlike most bacteria, mycobacteria require prior inactivation and disruption of the mycolic acid-rich cell wall, both for biosafety reasons and to liberate the cellular protein content. Numerous suitable procedures have been proposed, combining heat-killing, ethanol, sonication, and/or silica-based bead beating preceding the regular formic acid/acetonitrile extraction (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The reported success rates vary between 55% and 98% correct species-level identifications, but the large variety in strain collections, culture conditions, extraction protocols, hardware, and databases hinder data comparison between publications.…”

“…The reported success rates vary between 55% and 98% correct species-level identifications, but the large variety in strain collections, culture conditions, extraction protocols, hardware, and databases hinder data comparison between publications. However, most studies agree that a very reliable distinction between NTM and M. tuberculosis complex can be made using MALDI-TOF MS (23)(24)(25)(26)(27)(28), which is arguably the most important task of a clinical mycobacterial lab (29).…”

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

“…A similar prospective study on Vitek MS would be helpful to define real performances of IVD v3.0 from primary cultures of respiratory samples. Several authors recently described new extraction protocols (12,18,40,41), which could increase the ratio of mycobacterial proteins to patients' proteins and probably improve the identification rates at the time of detection.…”

“…This technique is quite simple and cost-effective (8) and allows rapid identification of organisms on the basis of spectral fingerprints produced by extracted proteins. It was initially evaluated with mycobacterial intact cells (9)(10)(11)(12), but several investigators worked on improving inactivation and protein extraction (13)(14)(15)(16)(17)(18)(19)(20). Most published studies assessed the performance of MALDI-TOF BioTyper (Bruker Daltonics, Bremen, Germany) (15,17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31).…”