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Whole-cell fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in combination with a dedicated bioinformatic software tool (MALDI Biotyper 2.0) was used to identify 152 staphylococcal strains corresponding to 22 staphylococcal species. Spectra of the 152 isolates, previously identified at the species level using a sodA gene-based oligonucleotide array, were analyzed against the main spectra of 3,030 microorganisms. A total of 151 strains out of 152 (99.3%) were correctly identified at the species level; only one strain was identified at the genus level. The MALDI-TOF MS method revealed different clonal lineages of Staphylococcus epidermidis that were of either human or environmental origin, which suggests that the MALDI-TOF MS method could be useful in the profiling of staphylococcal strains. The topology of the dendrogram generated by the MALDI Biotyper 2.0 software from the spectra of 120 Staphylococcus reference strains (representing 36 species) was in general agreement with that inferred from the 16S rRNA gene-based analysis.

Our findings indicate that the MALDI-TOF MS technology, associated with a broad-spectrum reference database, is an effective tool for the swift and reliable identification of Staphylococci. Most staphylococci are harmless and reside normally on the skin and mucous membranes of humans and other organisms (,, ). Staphylococcal strains are isolated from various food products in which they are involved in fermentation (, ). Staphylococcus species can cause a wide variety of diseases in humans and other animals (,, -, ). Aureus is a major pathogen in human infections (). Several other Staphylococcus species have also been implicated in human infections, notably S.

Saprophyticus, S. Epidermidis, S. Lugdunensis, and S. Schleiferi (,,, ).

Coagulase-negative staphylococci (CoNS) have emerged as predominant pathogens in hospital-acquired infections (,,, ). One of the major challenges of daily diagnostic work is therefore to identify Staphylococcus species. Several manual and automated methods based on phenotypic characteristics have been developed for the identification of Staphylococci (, ). Unfortunately, these systems have their limitations, mostly due to phenotypic differences between strains from the same species (,,, ). Over the last 10 years, many genotypic methods based on the analysis of selected DNA targets have been designed for species-level identification of most common isolated CoNS (,, ). The sequence polymorphism of the sodA gene has significant discriminatory power () and allows the development of assays based on DNA chip technologies (“Staph array”) (). Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using protein “fingerprints” was used for the identification of microorganisms (,,,,,,, ).

In the present study, we assessed the ability of the MALDI Biotyper system (Bruker Daltonique, Wissembourg, France) to identify Staphylococcus species of clinical and environmental origins previously identified by s odA gene-based oligonucleotide array (). Sample preparation. Sample preparation for MALDI-TOF MS was performed as previously described (). Briefly, a few colonies of a fresh overnight culture grown on Columbia blood agar at 37°C under aerobic conditions were suspended in 300 μl distilled water to which 900 μl absolute ethanol was added.

The mixture was centrifuged at 12,000 × g for 2 min, and the supernatant was discarded. One Last Tour A Live Soundtrack Скачать. Ten microliters of formic acid (70%) was added to the pellet and mixed thoroughly by pipetting before the addition of 10 μl of acetonitrile to the mixture. The mixture was centrifuged at 12,000 × g for 2 min. One microliter of the supernatant was placed onto a steel target plate and air dried at room temperature. Each sample was overlaid with 2 μl saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% trifluoroacetic acid and air dried at room temperature. MALDI-TOF MS measurements were performed with a Bruker Ultraflex II MALDI-TOF/TOF (tandem TOF) instrument equipped with 200-Hz Smartbeam laser technology. Spectra were recorded in the positive linear mode within a mass range of 2,000 to 20,000 Da.

Five hundred laser shots were recorded for each spectrum. Data analysis. Raw spectra of the Staphylococcus strains were analyzed by MALDI Biotyper 2.0 software (Bruker Daltonique) with default settings. The whole process from MALDI-TOF MS measurement to identification was performed automatically without any user intervention. The peak lists generated were used for matches against the reference library directly using the integrated pattern-matching algorithm of the software. The software provides a log score, and the cutoff log score of 2 was used to validate identification at the species level, as recommended by the manufacturer. For strain classification, the creation of the dendrogram is based on cross-wise minimum spanning tree (MSP) matching.

Similar MSPs result in a high matching score value. Each MSP is matched against all MSPs of the analyzed set. The list of score values is used to calculate normalized distance values between the analyzed species, resulting in a matrix of matching scores. The visualization of the respective relationship between the MSPs is displayed in a dendrogram using the standard settings of the MALDI Biotyper 2.0 software. Species with distance levels under 500 have been described as reliably classified ().

Classification of staphylococcal reference strains. A score-oriented dendrogram was generated on the basis of the 120 reference strains in the Bruker MALDI Biotyper 2.0 database (Fig.

This dendrogram revealed, with the default critical distance level of 500, 4 of the 11 cluster groups defined by Poyart et al. From the phylogenetic analysis based on the sodA gene (): S. Lugdunensis, and S. Auricularis (Fig. These cluster groups were also in accordance with those established by the phylogenetic analysis of the 16S rRNA gene (). With a critical distance level of 750, the dendrogram revealed overall the cluster groups of 16S rRNA genes, with some variations (Fig. The 16S rRNA gene-based cluster group S.

Haemolyticus including S. Haemolyticus and S. Hominis was divided into branches which linked at a distance level of 850. Unlike in other phylogenetic analyses, in which S. Kloosii belonged to S.

Saprophyticus cluster group, this species was clustered within the S. Simulans group in the MALDI-TOF MS-based dendrogram (,, ). Inside the S. Saprophyticus cluster group, the two strains S.

Toad For Sql Server License Serial Keygen. Equorum subsp. Equorum DSM 20675 and DSM 20674 belonged to two distinct clusters. It is noteworthy that the S.

Lutrae and S. Schleiferi species each constituted a well-defined clade inside the S. Intermedius cluster group, like S. Cohnii, and S. Arlettae in the S.

Saprophyticus cluster group and S. Capitis in the S. Epidermidis cluster group.

Classification of staphyloccal reference strains. Shown is a score-oriented dendrogram of staphylococcal reference strains included in the database. The terms “500-level cg” and “750-level cg” define cluster groups (cg).

Although some differences were observed, the topology of the MALDI-TOF MS-based phylogenetic tree was in more general agreement with that inferred from the analysis of the 16S rRNA gene sequences than that based on the sodA gene (, ). The proteins analyzed by MALDI Biotyper correspond to predominant proteins, such as ribosomal proteins. The probable coevolution of the ribosomal proteins and rRNAs may explain the similarities between 16S rRNA gene and MALDI-TOF-based dendrograms (). MALDI-TOF MS could thus be a valuable tool in phyloproteomics.

It might serve as a technique for protein profiling, which is recommended as an additional test for the description of new staphylococcal species (). Biodiversity of S.

Epidermidis species. The dendrogram of Fig. Shows protein “fingerprint” heterogeneity of clinical and environmental (salt meat originated) S. Epidermidis strains. Clinical isolates are gathered within two cluster groups and two isolated strains, whereas meat origin isolates displayed one distinct cluster group and one isolated strain.

Diversity within the S. Epidermidis species has been observed for human origin S. Epidermidis and animal origin S. Epidermidis (). The MALDI-TOF MS method might have enough discriminatory power to group isolates below the species level. MALDI-TOF MS associated with MALDI Biotyper software appears a reliable and accurate tool for the identification of Staphylococcus species.

However, further studies are required to test this technology with a large collection of staphylococci of diverse origins. The speed and the simplicity of sample preparation and result acquisition associated with minimal consumable costs make this method well suited for routine and high-throughput use. Hence, it is an excellent alternative to traditional methods in food processing and medical care laboratories. It may also be used for the analysis of clonal and/or taxonomic relationships.