Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically relevant bacteria

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens.

Methodology/Principal Findings

We compared the identification efficiency of MALDI-TOF MS with that of Phoenix®, API® and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7% of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63% of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn''s kappa values: 0.9 to 0.93).

Conclusions/Significance

MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria.     

Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry- and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.     

Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era

The isolation of anaerobes from patients has declined in recent years, whereas their detection by molecular techniques has increased. In the present work, we analyzed the application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing to routine identification of anaerobes in clinical microbiology laboratory. We identified 544 isolates of 79 species by routine culture from deep samples in our hospital. MALDI-TOF MS allowed identification of 332 isolates (61%). The remaining 212 (39%) were identified by 16S rRNA gene sequencing, allowing identification of 202 at the species level. The most common anaerobes were Propionibacterium spp. (12%), Finegoldia magna (4%), Fusobacterium spp. (6%) and Bacteroides spp. (6%). However, among the 79 identified species, seven were new species or genera, including two Prevotella conceptionensis, a species previously detected by our team by amplification and sequencing, five Anaerococcus sp. and one Prevotella sp. Beyond the identification of these new species, we also identified several uncommon or previously not described associations between species and specific pathologic conditions. MALDI-TOF MS-based identification, which will become more effective with future spectra database improvement, will be likely responsible of a burden of emerging anaerobes in clinical microbiology.     

Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods

In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥1,5 and ≥1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥1,5 for genus level and ≥1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories.     

Identification and Cluster Analysis of Streptococcus pyogenes by MALDI-TOF Mass Spectrometry

Background

Whole-cell matrix–assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been successfully applied for bacterial identification and typing of many pathogens. The fast and reliable qualities of MALDI-TOF MS make it suitable for clinical diagnostics. MALDI-TOF MS for the identification and cluster analysis of Streptococcus pyogenes, however, has not been reported. The goal of our study was to evaluate this approach for the rapid identification and typing of S. pyogenes.

Methods

65 S. pyogenes isolates were obtained from the hospital. The samples were prepared and MALDI-TOF MS measurements were conducted as previously reported. Identification of unknown spectra was performed via a pattern recognition algorithm with a reference spectra and a dendrogram was constructed using the statistical toolbox in Matlab 7.1 integrated in the MALDI Biotyper 2.0 software.

Results

For identification, 61 of 65 S. pyogenes isolates could be identified correctly by MALDI-TOF MS with BioType 2.0 when compared to biochemical identification (API Strep), with an accuracy of 93.85%. In clustering analysis, 44 of 65 isolates were in accordance with those established by M typing, with a matching rate of 67.69%. When only the M type prevalence in China was considered, 41 of 45 isolates were in agreement with M typing, with a matching rate of 91.1%.

Conclusions

It was here shown that MALDI-TOF MS with Soft Biotype 2.0 and its database could facilitate rapid identification of S. pyogenes. It may present an attractive alternative to traditional biochemical methods of identification. However, for classification, more isolates and advances in the MALDI-TOF MS technology are needed to improve accuracy.     

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.     

Automated protein identification by the combination of MALDI MS and MS/MS spectra from different instruments

The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.     

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at H?pital Principal de Dakar,Senegal (West Africa)

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, “Necropsobacter massiliensis.” The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.     

Optimization of MALDI-TOF MS for strain level differentiation of Arthrobacter isolates

Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be a rapid and sensitive method for characterization of bacteria, but it has not yet become a routine microbiological procedure. Currently there are no standardized protocols that would allow development of large libraries of reproducible protein profiles from a broad range of microorganisms to use for identification purposes. Important variables that may affect spectrum quality are MALDI matrices, solvents, cell growth condition, and culture age. In the present study our aim was to: (1) to determine optimal sample preparation and MALDI conditions for discrimination at the strain level; (2) to determine if changes in growth cycle correlated with MALDI spectrum changes; and (3) to compare level of isolate discrimination based on their MALDI spectra versus their 16S rRNA gene sequence. Using 16 strains of the Gram positive bacterium Arthrobacter, optimal spectra were obtained using two-layer sample application of intact cells grown on solid surface overlaid with a matrix consisting of sinapinic acid (SA) or alpha-cyano-hydroxy-cinnaminic acid (CHCA) in 50:50 acetonitrile:water solvent with 2% trifluoroacetic acid. Spectrum changes paralleled the coccus-rod-coccus growth cycle indicative of Arthrobacter. Strain differences based on their MALDI profiles (using Pearson coefficient and UPGMA) corresponded with their 16S rRNA gene phylogeny but it had greater discrimination.     

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.     

Accurate Identification of Common Pathogenic Nocardia Species: Evaluation of a Multilocus Sequence Analysis Platform and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Species identification of Nocardia is not straightforward due to rapidly evolving taxonomy, insufficient discriminatory power of conventional phenotypic methods and also of single gene locus analysis including 16S rRNA gene sequencing. Here we evaluated the ability of a 5-locus (16S rRNA, gyrB, secA1, hsp65 and rpoB) multilocus sequence analysis (MLSA) approach as well as that of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in comparison with sequencing of the 5’-end 606 bp partial 16S rRNA gene to provide identification of 25 clinical isolates of Nocardia. The 5’-end 606 bp 16S rRNA gene sequencing successfully assigned 24 of 25 (96%) clinical isolates to species level, namely Nocardia cyriacigeorgica (n = 12, 48%), N. farcinica (n = 9, 36%), N. abscessus (n = 2, 8%) and N. otitidiscaviarum (n = 1, 4%). MLSA showed concordance with 16S rRNA gene sequencing results for the same 24 isolates. However, MLSA was able to identify the remaining isolate as N. wallacei, and clustered N. cyriacigeorgica into three subgroups. None of the clinical isolates were correctly identified to the species level by MALDI-TOF MS analysis using the manufacturer-provided database. A small “in-house” spectral database was established incorporating spectra of five clinical isolates representing the five species identified in this study. After complementation with the “in-house” database, of the remaining 20 isolates, 19 (95%) were correctly identified to species level (score ≥ 2.00) and one (an N. abscessus strain) to genus level (score ≥ 1.70 and < 2.00). In summary, MLSA showed superior discriminatory power compared with the 5’-end 606 bp partial 16S rRNA gene sequencing for species identification of Nocardia. MALDI-TOF MS can provide rapid and accurate identification but is reliant on a robust mass spectra database.     

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species.     

Rapid identification of clinical isolates of Bacteroides species by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry

The objective of this study was to compare MALDI-TOF MS and Rapid ID 32A with 16S rRNA gene sequencing, the reference method for identification of Bacteroides species. Results show that MALDI-TOF MS can be a good option for identification of Bacteroides species, especially if the database is expanded.     

Identification of Staphylococcus intermedius Group by MALDI-TOF MS

The Staphylococcus intermedius Group includes S. intermedius, S. pseudintermedius and S. delphini, coagulase-positive bacteria commonly isolated from animals. The identification of organisms belonging to this group is presently carried out using molecular methods. This study assessed the suitability of MALDI-TOF MS for their identification. 69 strains of different biological and geographic origins, identified by partial hsp60 gene sequencing as S. intermedius (n = 15), S. pseudintermedius (n = 32) and S. delphini (n = 22), were analyzed by MALDI-TOF MS. The estimated sensitivity, specificity and efficiency were calculated. In addition we computed the agreement between the outcome of MALDI-TOF MS identification and partial hsp60 gene sequencing. The sensitivity of MALDI-TOF MS was higher for S. intermedius [0.95 (95% CI: 0.68-0.99)], than for S. pseudintermedius [0.78 (95% CI: 0.60-0.90)] and S. delphini [0.64 (95% CI: 0.41-0.83)], whereas the specificity was 1 for S. intermedius and S. delphini and 0.97 (95% CI: 0.86-0.99) for S. pseudintermedius. The Cohen's kappa coefficient indicated almost perfect agreement between MALDI-TOF MS and hsp60 gene sequencing for the identification of S. intermedius [0.96 (95% CI: 0.87-1.04)], and substantial agreement for S. delphini and S. pseudintermedius [0.70 (95% CI: 0.52-0.89) and 0.76 (95% CI: 0.616-0.92), respectively]. The overall efficiency of the proteomic identification ranged between 0.88 (95% CI: 0.78-0.95) for S. pseudintermedius and S. delphini and 0.99 (95% CI: 0.92-0.99) for S. intermedius. MALDI-TOF MS is thus a valuable and reliable tool for the rapid and accurate identification of bacteria belonging to the S. intermedius Group.     

Rapid Identification of Clinical Yeast Isolates by Mass Spectrometry

Historically, yeasts and molds have been identified by their microscopy and macroscopy appearance, detection of specific antigens and metabolites, the results of biochemical testing, and the presence of species-specific gene sequences. These methods are either nonspecific or, in the case of gene sequencing, technically demanding, time-consuming, and expensive. Recent studies have demonstrated that matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is a rapid, simple, inexpensive, and accurate tool for the identification of a wide spectrum of bacteria and fungi. There are also promising reports that this technology can be used for subtyping organisms and for performing antifungal susceptibility tests. This article reviews the applications of MALDI-TOF MS for routine clinical microbiology testing.     

Speeding up the screening of steroids in urine: Development of a user-friendly library

This work presents a novel database search engine – MLibrary – designed to assist the user in the detection and identification of androgenic anabolic steroids (AAS) and its metabolites by matrix assisted laser desorption/ionization (MALDI) and mass spectrometry-based strategies. The detection of the AAS in the samples was accomplished by searching (i) the mass spectrometric (MS) spectra against the library developed to identify possible positives and (ii) by comparison of the tandem mass spectrometric (MS/MS) spectra produced after fragmentation of the possible positives with a complete set of spectra that have previously been assigned to the software. The urinary screening for anabolic agents plays a major role in anti-doping laboratories as they represent the most abused drug class in sports. With the help of the MLibrary software application, the use of MALDI techniques for doping control is simplified and the time for evaluation and interpretation of the results is reduced. To do so, the search engine takes as input several MALDI-TOF–MS and MALDI-TOF–MS/MS spectra. It aids the researcher in an automatic mode by identifying possible positives in a single MS analysis and then confirming their presence in tandem MS analysis by comparing the experimental tandem mass spectrometric data with the database. Furthermore, the search engine can, potentially, be further expanded to other compounds in addition to AASs. The applicability of the MLibrary tool is shown through the analysis of spiked urine samples.     

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

ABSTRACT: BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.     

MALDI-TOF MS of phosphatidylethanolamines: Different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described.In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Identification of fungal microorganisms by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for fast identification and classification of microorganisms. In this regard, it represents a strong challenge to microscopic and molecular biology methods. Nowadays, commercial MALDI systems are accessible for biological research work as well as for diagnostic applications in clinical medicine, biotechnology and industry. They are employed namely in bacterial biotyping but numerous experimental strategies have also been developed for the analysis of fungi, which is the topic of the present review. Members of many fungal genera such as Aspergillus, Fusarium, Penicillium or Trichoderma and also various yeasts from clinical samples (e.g. Candida albicans) have been successfully identified by MALDI-TOF MS. However, there is no versatile method for fungi currently available even though the use of only a limited number of matrix compounds has been reported. Either intact cell/spore MALDI-TOF MS is chosen or an extraction of surface proteins is performed and then the resulting extract is measured. Biotrophic fungal phytopathogens can be identified via a direct acquisition of MALDI-TOF mass spectra e.g. from infected plant organs contaminated by fungal spores. Mass spectrometric peptide/protein profiles of fungi display peaks in the m/z region of 1000–20 000, where a unique set of biomarker ions may appear facilitating a differentiation of samples at the level of genus, species or strain. This is done with the help of a processing software and spectral database of reference strains, which should preferably be constructed under the same standardized experimental conditions.     

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.