The essence on mass spectrometry based microbial diagnostics

Kliem, Magdalena; Sauer, Sascha

doi:10.1016/j.mib.2012.02.006

Cited by 65 publications

(55 citation statements)

References 48 publications

(43 reference statements)

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“…atrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid, robust, and cost-effective technology, which is emerging as a routine bacterial identification method in clinical diagnostic laboratories (1)(2)(3). Despite its utility, there are concerns with the safety of sample processing and the quality of spectral databases for the identification of security-sensitive biological agents (SSBAs).…”

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Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

2016

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bWe examined the utility of a single matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry method for the identification of security-sensitive biological agents (risk group 3 bacterial pathogens). The goal was 2-fold: to verify a method for inclusion into our scope of accreditation, and to assess the biological safety of extractions. We developed our sample flow to include a tube-based chemical extraction, followed by filtration, before processing on MALDI-TOF MS instruments in a containment level 2 laboratory. Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid, robust, and cost-effective technology, which is emerging as a routine bacterial identification method in clinical diagnostic laboratories (1-3). Despite its utility, there are concerns with the safety of sample processing and the quality of spectral databases for the identification of security-sensitive biological agents (SSBAs). As our laboratory predominately works with SSBA bacterial pathogens (the risk group 3 [RG-3] pathogens: Francisella tularensis, Yersinia pestis, Bacillus anthracis, Brucella species, and Burkholderia pseudomallei), our goal was to verify the usefulness and safety of a MALDI-TOF MS method for inclusion into our scope of accreditation (ISO 17025). We chose to pursue a full-tube-based protein extraction method involving cellular disruption and filtration, rather than the direct-colony plate (smear) method (4, 5), in an effort to mitigate the risk of viable organisms.Over a period of 1 year, 146 bacterial samples (including 57 SSBA samples; Table 1) were processed as previously described (6). The bacteria were previously identified by standard genotypic and phenotypic methods. In brief, all cultures were chemically extracted in a microcentrifuge tube using 70% ethanol, 70% formic acid, and acetonitrile, and all SSBA bacteria were extracted in a biosafety level 3 (BSL-3) laboratory. SSBA extracts from sporeforming bacteria were filtered through a 0.1 M microcentrifuge filtration unit (EMD Millipore, Etobicoke, Ontario, Canada), as recommended by Dauphin and Bowen (7). The viability of the extract was performed by inoculating 10% of the extract onto both sheep's blood agar (SBA) (or cysteine heart agar [CHA] for F. tularensis) and into 2 ml of tryptic soy broth (TSB), and incubating at 35°C and 5% CO 2 for 48 h. After 48 h, the TSB was further subcultured to SBA or CHA and monitored for an additional 72 h. Any bacterial growth was tested with agent-specific assays to rule out the SSBA, indicating a contaminant. After a lack of growth was confirmed, extracts were removed from the BSL-3 laboratory, and 1.5 l of the extract was applied (at a minimum of four replicate spots) to an MSP-96 MALDI target plate (Bruker Daltonics, East Milton, Ontario, Canada), with an overlay of 1.5 l of ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (50% acetonitrile and 2.5% trifluoroacetic acid). Mass spectra were acquired with the FlexControl software (version 3.0...

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Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

2016

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“…Several groups in the mid-1990s: (including Edward-Jones et al, 2000;Holland et al, 1996Holland et al, , 1999Krishnamurphy et al, 1996Krishnamurphy et al, , 1999Wall et al, 1999) Recent studies have shown that whole cell (WC) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reduce the average identification time after growth to 10 min or less for identifying clinical microbiological isolates (Croxatto et al, 2011;Dubois et al, 2012;Giebel et al, 2010;Kliem and Sauer, 2012;Martiney et al, 2012;Risch et al, 2010;Safferet et al, 2012;Seibold et al, 2012;Welkner and Moore, 2011). However it must be emphasized that prior growth is still required which takes, from 5-48 hours for rapidly growing organisms and or in extreme cases up to 12 weeks (e.g.…”

Section: Introductionmentioning

confidence: 99%

“…Simply put, bacterial colonies are directly streaked onto a MALDI target plate, coated with a matrix and put into a mass spectrometer where mass spectra of the samples are generated and then analyzed by compatible software (Kliem, 2012). The mass spectrum of that sample is then compared to other spectra stored in a database.…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometry and tandem mass spectrometry characterization of protein patterns, protein markers and whole proteomes for pathogenic bacteria

Intelicato-Young

Fox

2013

Journal of Microbiological Methods

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“…This rapid and accurate method can be easily applied to identify bacteria at the genus, species and, in some cases, the subspecies levels [5]. Therefore, MALDI-TOF MS represents a promising alternative to the standard phenotypic and molecular techniques carried out in diagnostic laboratories.…”

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confidence: 99%

“…Furthermore, a slight variation among replicates for each isolate was observed, which corroborates the importance of using more than one spot per sample and even more than one spot measure to ensure bacterial identification and its reproducibility. Although MALDI-TOF MS has been validated for the identification of several microorganisms [5,10], the technique still presents some limitations with regard to spectral variation and reproducibility. The main causes for this are bacterial growth conditions and protein extraction methods [10].…”

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Application of protein profiling of virulent Haemophilus parasuis by MALDI-TOF mass spectrometry

Moreno

Silva

Gomes

et al. 2016

J Infect Dev Ctries

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The essence on mass spectrometry based microbial diagnostics

Cited by 65 publications

References 48 publications

Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

Mass spectrometry and tandem mass spectrometry characterization of protein patterns, protein markers and whole proteomes for pathogenic bacteria

Application of protein profiling of virulent Haemophilus parasuis by MALDI-TOF mass spectrometry

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