Martin Welker

Proteomics for routine identification of microorganisms

The invention of MALDI‐TOF‐MS enormously contributed to the understanding of protein chemistry and cell biology. Without this technique proteomics would most likely not be the important discipline it is today. Besides ‘true’ proteomics, MALDI‐TOF‐MS was applied for the analysis of microorganisms for their taxonomic characterization from its beginning. This approach has since been developed as a diagnostic tool readily available for routine, high‐throughput analysis of microbial isolates from clinical specimens by intact‐cell mass spectrometry (ICMS), the direct analysis of whole bacterial cell without a preceding fractionation or separation by chromatography or electrophoresis. ICMS exploits the reproducibility of mass fingerprints for individual bacterial and fungal strains as well as the high similarity of mass fingerprints within a species. Comparison of mass spectral data to genomic sequences emphasized the validity of peak patterns as taxonomic markers. Supported by comprehensive databases, MALDI‐TOF‐MS‐based identification has been widely accepted in clinical laboratories within only a few years.

Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01–10.24; p = 0.023; CI 95%: 1.20–12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS.

Streptococcus equi includes very important animal and human pathogens. S. equi subsp. equi (SEE) is a highly pathogenic equine specific subspecies, while S. equi subsp. zooepidemicus (SEZ) and S. equi subsp. ruminatorum are opportunistic pathogens of various animal species and humans. Due to great phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies in horses. Superspectra created in this study enabled correct identification of 86 strains belonging to different subspecies of S. equi, isolated from various hosts, infection sites and years. In general, higher average identification accuracy was achieved for SEE (99.0±3.0%) than for SEZ (93.3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method with spectra analyses using the SARAMIS database. Additionally, first results on subtyping of SEZ indicated that a more refined discrimination, for example for epidemiological surveys, may be possible.

Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

ABSTRACT We describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization–time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers.

The Role of New Technologies in Medical Microbiological Research and Diagnosis

Microbial culture is exemplified by the Petri dish, a tool that (one century after its invention) still remains one of the “gold standards” for microbiological analysis. However, current trends towards automation, massively paralleled assays, and miniaturization (as well as the observation that we still cannot culture most microorganisms), suggest that new ideas in microbial culture are required. In the Petri dish, nutrient containing agar is typically used as the matrix on which microorganisms are cultured. However, new materials such as nanofibres and nanoporous materials may be better choices as supporting matrixes. Further, emerging techniques in microengineering and the fabrication of low cost materials are helping to create new porous disposables that are of sufficiently low cost that they may be used in the routine microbiology laboratory. These disposables are in turn allowing the development of novel miniature culture methods to take place, methods such as microchemostats, cages for growing microorganisms, and “habitats on a chip”. One particularly useful porous ceramic is Porous Aluminium Oxide (PAO), which can be utilized to generate highly subdivided culture chips that possess up to one million separate, miniaturized, growth areas. Indeed, this material has applications in microbiological diagnostics, microbiological research and industrial microbiology. In this chapter, the applications, advantages, and limitations of porous matrixes and accompanying culture chips will be examined. It is expected that these advances will yield significant improvements in microbial culture when compared to the classical Petri dish.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement.

Fusobacterium nucleatum Subspecies Identification by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT We explored the use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for identification of Fusobacterium nucleatum subspecies. MALDI-TOF MS spectra of five F. nucleatum subspecies (animalis, fusiforme, nucleatum, polymorphum, and vincentii) were analyzed and divided into four distinct clusters, including subsp. animalis, nucleatum, polymorphum, and fusiforme/vincentii. MALDI-TOF MS with the modified SARAMIS database further correctly identified 28 of 34 F. nucleatum clinical isolates to the subspecies level.

Molecular characterization of microcystin-producing cyanobacteria from Romanian fresh waters

In this study the molecular analysis of 24 potentially toxic cyanobacterial strains of genus Microcystis isolated from Romania is presented. The toxic potential was assessed by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), which indicated that five out of the 24 strains are able to produce microcystins. Beside this, the strains were shown to produce various peptides, including aeruginosins, microginins or cyanopeptolins. Identification of potentially toxic Microcystis strains by PCR was performed using primers described in known literature. These primers occasionally generated amplification products in non-microcystin-producing strains. New primers were designed, and the amplification of a mcyD gene fragment exclusively in microcystin-producing strains certified the high specificity of these new primers for microcystin-producing cells. All of the five microcystin-producing strains were shown to possess four characteristic parts of the gene cluster responsible for microcystin synthesis (mcyA, mcyB, mcyD and mcyE). The 16S–23S rDNA ITS sequence was used to confirm the taxonomic identification of the strains and their relationship to strains from outside Romania.