MALDI-TOF Mass Spectrometry in Microbiology
Caister Academic Press
Markus Kostrzewa and Sören Schubert
Bruker Daltonik GmbH, Bremen, Germany and Max von Pettenkofer-Institut, Munich, Germany; respectively
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Recent years have seen a phenomenal increase in the use of MALDI-TOF mass spectrometry (MALDI-TOF MS) in microbiology laboratories. The introduction of this technology to microbiology has been a major success and MALDI-TOF MS is now used for routine diagnostic or diagnostic-like purposes in clinic, veterinary, pharma and food microbiology laboratories. It has also evolved into a powerful tool for the analysis of organisms in the environment and for research into microbial communities. The throughput capabilities, accuracy and low running costs of a MALDI-TOF MS system enable analyses at a scale which was not possible until recently.
In this timely and up-to-date book, experts in the field provide an overview of the application of MALDI-TOF MS in key areas of microbiology and discuss the impact this modern technology is having on laboratory practice and patient outcome. Several chapters cover applications in clinical and veterinary diagnostic laboratories, food microbiology, environmental microbiology and strain collections. Further chapters discuss the utilization of MALDI-TOF MS for the analysis of challenging microbial groups such as yeast and anaerobic bacteria. In addition, new applications such as microbial typing, DNA analysis and the detection of antibiotic resistance are also covered. The final chapter provides a valuable overview of potential future trends and developments in MALDI-TOF MS and assesses the impact of the technology in microbiology.
This authoritative volume will be indispensable for all microbiology laboratories.
Table of contents
Introduction. A Personal Vision of the MALDI-TOF-MS Journey from Obscurity to Frontline Diagnostics
Haroun N. Shah
1. Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for the Clinical Laboratory
Chris D. Doern, Robert C. Jerris and Mark D. Gonzalez
In the last decade, few technologies have had a greater impact on clinical microbiology than matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). To say it has revolutionized the discipline would be an understatement. The ability to cost-effectively and rapidly identify microorganisms from broth and plated media by MALDI-TOF MS is replacing the more arduous, and time consuming biochemical and antigen-based identification methods as well as some genetic-sequence-based methodologies. In this chapter we succinctly review the role of MALDI-TOF MS in the routine laboratory, focusing on the key functions in pre-analytical, analytical and post-analytical perspectives. We acknowledge the thousands of papers and investigators that have contributed to our knowledge on this subject.
2. Analysis of Anaerobes and Some Other Fastidious Bacteria
Anaerobes, similar to some other fastidious, slow-growing bacteria belonging to the HACEK group have special place among human pathogenic bacteria. They are important pathogens, however due to their special requirements to be isolated from clinical samples, such as strict anaerobic or CO2 -rich environment for their long incubation time, makes their identification time-consuming. In this chapter several applications of the mass spectrometry will be discussed beside the rapid identification of these bacteria. It has been shown since the first studies that the database developments are mandatory including not only reference strains of the different well known or newly accepted species, but including the mass spectra of well characterised clinical isolates may improve the performance of the MALDI-TOF MS identification of these bacteria. Mass spectrometry-based typing of Bacteroides fragilis strains may help to detect clinical isolates belonging to division II, which harbour resistance gene against carbapenems. Propionibacterium acnes phylotypes could also be distinguished by looking for peak variations of the mass spectra of the isolates belonging to this species. Direct identification of anaerobic bacteria from positive blood cultures should be evaluated further as well as the possibilities to use MALDI-TOF MS for antibiotic resistance determination of these bacteria.
3. Identification, Typing and Susceptibility Testing of Fungi (incl. Yeasts) by MALDI-TOF MS
Anna Kolecka, Maurizio Sanguinetti, Teun Boekhout and Brunella Posteraro
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has developed into a robust tool for the routine identification of many microbes, including bacteria, yeasts and filamentous fungi, which remain relevant for maintaining the quality of the human lifestyle, e.g. related to health, food, industry, ecology, water quality, waste management, and others. The number of correct and erroneous results depends mainly on the sample preparation method, the number of reference spectra present in the databases and the cut-off values used. These studies confirmed that MALDI-TOF MS platform outperformed traditional identification methods for fungi. Examples will be given on the use of MALDI-TOF MS for the identification of yeasts and filamentous fungi, including strain typing and susceptibility testing.
4. Molecular Typing of Bacteria/Fungi Using MALDI-TOF MS
Silpak Biswas, Frédérique Gouriet and Jean-Marc Rolain
Traditional microbial typing technologies for the characterization of pathogenic microorganisms and the monitoring of their global spread are often difficult to standardize and lack sufficient ease of use. Timely reporting of strain typing information is essential for the early initiation of infection control measures to prevent a further dissemination of the pathogen and for epidemiological purposes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become an important method in clinical microbiology laboratories for identifying and typing bacteria and fungi. With the exception of a few difficult strains, MALDI-TOF MS using standardized procedures allows the accurate identification of Gram-positive and Gram-negative bacterial strains at the species and sub-species levels. Recent studies have shown that MALDI-TOF MS also has the potential to accurately identify filamentous fungi. MALDI-TOF MS is a powerful tool for species and subspecies classification and rapid identification and typing of pathogenic microbes. Furthermore, the implementation of specific databases of well-known pathogens will likely increase the role of MALDI-TOF MS for typing in the future.
5. Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry for Determination of Resistance to Antibiotics
Jaroslav Hrabák, Monika Dolejská and Costas C. Papagiannitsis
Antibiotic resistance is a growing problem of current medicine worldwide, having significant impact on human health and economy. Rapid determination of susceptibility to antimicrobials in bacteria including detection of resistance mechanisms are crucial not only for successful treatment of infectious diseases in individual patients, but also for prevention of nosocomial acquisition and further transmission of multidrug resistant bacteria. For that reason, there is an urgent need to develop highly sensitive and automatized methods that can be implemented in microbiology diagnostic laboratories. The current research demonstates that MALDI-TOF MS is becoming a very promising tool for detection of antibiotic resistance. Several techniques have been developed up to know, some of them, such as MALDI-TOF detection of carbapenemases, have been already introduced into routine diagnostics. Pioneering works using MALDI-TOF for qualitative or quantitative evaluation of susceptibility or resistance as well as several techniques for detection clinically relevant pathogens (e.g., methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci) or specific antibacterial activity (e.g., beta-lactamase, ribosomal RNA methyltransferase) have been published and highlight many pitfalls during the early method development. Introduction of novel techniques using MALDI-TOF MS approaches as well as optimalization, validation and full automatization of these methods, increasing their reproducibility and decreasing the costs, are urgently needed. It is quite evident that MALDI-TOF MS technology will play an important role in routine diagnostics of antibacterial susceptibility as well as in epidemiological surveillance, overall, improving the quality of health care.
6. Application of MALDI-TOF MS in Veterinary and Food Microbiology
Claudia Hess, Merima Alispahic and Michael Hess
Considering the huge potential of the technology MALDI-TOF MS in veterinary and food microbiology is still in its infancy. Characterization of bacteria with traditional methods in comparison to genetic analysis is still the predominant task performed in routine diagnostics. In this context, the reliable identification of pathogenic bacteria is of high relevance and the application of MALDI-TOF MS is a valuable addition to existing technologies. Furthermore, proteomic data have gained access to resolve taxonomy/phylogeny. In food microbiology the detection of zoonotic bacteria has a high priority and MALDI-TOF MS can already be applied on major food-borne bacteria, e.g. Campylobacter spp., Salmonella spp., Listeria spp. and E. coli. The diverse functions of lactobacilli and acetic acid bacteria, being essential for the fermentation process towards the spoilage of food products, prompted studies on these microorganisms. This resulted in the transformation of knowledge obtained from single bacteria to a microbial community in a certain product or production process. Independent of the organism and the targeted subject the establishment of biomarkers has a high priority for easy discrimination and identification. The increasing application of MALDI-TOF MS in routine veterinary or food microbiology asks for further development and extension of the technique which will be addressed in a separate chapter.
7. MALDI-TOF MS for Environmental Analysis, Microbiome Research and as a Tool for Biological Resource Centres
Markus Kostrzewa, Chantal Bizet and Dominique Clermont
The simplicity of sample preparation, speed of analysis, high-throughput capabilities and low analysis costs per sample have made MALDI-TOF MS profiling not only a successful first-line diagnostic method but also a favoured tool for research and analysis of environmental strains and microbial communities. Its differentiation potential, which is frequently higher than that of 16S rDNA sequence analysis, enables detection of new species and subspecies and the de-replication of large numbers of isolated microbes. Further, tracking of environmental strains seems to be feasible at least for particular species. Thereby, MALDI-TOF MS besides DNA based and biochemical methods becomes a further basic tool for research in environmental microbiology and phylogeny. These characteristics also have made MALDI-TOF MS fingerprinting a first-line method for species identification and confirmation in strain collections. Together with other complementary methods it is already established for one factor in the multidimensional approach for description of new species.
8. The World of Nucleic Acid Based Mass Spectrometry for Microbial and Viral Detection
Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has been applied to nucleic acid based research and specifically sequencing for over 25 years with increasing clinical use today. In contrast to whole cell MS, where cultured material is the source for the identification of a microbial sample based on a proteomic fingerprint, nucleic acid MS based microbial detection, identification and characterization uses PCR amplifications of specific genomic target regions of interest followed by the detection of single nucleotide polymorphisms via primer extension reactions or followed by comparative sequence analysis of mass peak pattern generated via in vitro transcription and base-specific cleavage. Applications include the detection and identification of both microbial and viral pathogens, tracking of transmissions and the characterization of genetic heterogeneity by variant analysis. Multiplexing levels of up to 40- 60 targets per reaction surpass real-time PCR applications, but fall short when compared to the potential of next-generation sequencing. The unique feature of mass spectrometry (MS) though is the detection of various biomarker molecules including proteins, lipids, small molecules, carbohydrates and nucleic acids on one single platform.
9. Future Trends and Perspectives of MALDI-TOF Mass Spectrometry in the Microbiology Laboratory
Sören Schubert and Markus Kostrzewa
Within less than a decade Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a gold standard for microbial identification in clinical microbiology laboratories. Besides identification of microorganisms the typing of single strains as well as the antibiotic and antimycotic resistance testing has come into focus in order to speed up the microbiological diagnostic. However, the full potential of MALDI-TOF MS has not been tapped yet and future technological advancements will certainly expedite this method towards novel applications and enhancement of current practice. So, the following chapter shall be rather a brainstorming and forecast of how MALDI-TOF MS will develop to influence clinical diagnostics and microbial research in the future. It shall open up the stage for further discussions and does not claim for overall validity.
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(EAN: 9781910190418 9781910190425 Subjects: [bacteriology] [bacteriology] [bacteriology] [environmental microbiology] [medical microbiology] [microbiology] [molecular microbiology] )