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.     

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.     

Diversity of Acetobacter pasteurianus Strains Isolated From Solid-State Fermentation of Cereal Vinegars

Vinegar production is based on the acetification process by indigenous acetic acid bacteria (AAB). Among vinegar technologies, solid-state fermentation (SSF) processes are widespread in Asian countries to produce vinegar at small-scale. In this study, 21 AAB strains isolated from Chinese cereal vinegars produced by SSF collected in different regions of China were characterized by enterobacterial repetitive intergenic consensus (ERIC)–PCR fingerprinting. Isolates exhibited high degree of phenotypic variability as well as suitable traits for their uses as selected strains in SSF vinegar production (growth modality by superficial biofilm, no production of cellulose, ability to growth on ethanol media). 16S rRNA gene sequencing analysis of representative strains showed that strains of Acetobacter pasteurianus have a close association to cereal vinegars, whereas Gluconacetobacter europaeus population is not favoured. Selection of single or multiple strains culture within A. pasteurianus species was predicted in view of their application in SSF technology. This seems to be the first report showing phenotypic and genetic variability of AAB strains involved in SSF processes. Results can be exploited for the implementation of large-scale SSF processes by selected strains for vinegar production and other innovative biotechnological applications.     

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.     

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.     

Genetic variability of plant-associated proteobacteria isolated from Slovak (Central European) territory

Genetic variability of 17 wild strains of γ-proteobacteria isolated from different host plants, locations and seasons, identified via routine diagnostics as Erwinia amylovora, was analysed. The ERIC-PCR confirmed the genetic homogeneity among 15 virulent strains while 2 avirulent strains were genetically distinct. These two avirulent strains differed in their antibiotic susceptibility from all virulent strains. The only avirulent Ra1051/98 strain showed significant ampicillin resistance. All 15 virulent strains were confirmed via the ERIC-PCR and MALDI-TOF MS, as closely related to the reference strain NCPPB 683 of E. amylovora. Avirulent strains were identified as Rahnella aquatilis and L. quercina subsp. britannica (formerly Brenneria quercina), by MALDI-TOF MS and the identification was confirmed by 16S rRNA gene sequence analysis. For the first time, L. quercina subsp britannica was identified in the fire blight-symptomatic samples in Central Europe, and for the first time this bacterium was isolated from the host different than oak.     

Polyphasic characterization of a novel species in the Lactobacillus casei group from cow manure of Taiwan: Description of L. chiayiensis sp. nov.

Two Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strains, NCYUAS^T and BCRC 18859 (=NRIC 1947), were isolated from cow manure of Taiwan and coconut juice of Philippines, respectively. Comparative sequence analysis of 16S rRNA gene revealed that the novel strains were members of the genus Lactobacillus. These two strains had 100% of 16S rRNA gene sequence similarity and 98.6% of average nucleotide identity (ANI) value based on whole genome sequences. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (99.6% similarity), Lactobacillus paracasei subsp. paracasei (99.1%), L. paracasei subsp. tolerans (99.1%), Lactobacillus rhmnosus (99.0%) and ‘Lactobacillus zeae’ (99.7%) were the closest neighbors to these novel strains. The results of phenotypic and chemotaxonomic characterization, multilocus sequence analysis (MLSA) based on the sequences of three housekeeping genes (dnaK, pheS and yycH), whole-genome sequence (WGS)-based comparison by ANI and in silico DNA–DNA hybridization (isDDH), species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses demonstrated that the novel two strains represented a single, novel species within the L. casei group, for which the name Lactobacillus chiayiensis sp. nov., is proposed. The type strain is NCYUAS^T (=BCRC 81062^T = NBRC 112906^T).     

Genotypic and Phenotypic Applications for the Differentiation and Species-Level Identification of Achromobacter for Clinical Diagnoses

The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected ‘house-keeping’ genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data.     

Typing of nitrogen-fixing Frankia strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was evaluated as a technique to characterize strains of the nitrogen-fixing actinomycete Frankia. MALDI-TOF MS reliably distinguished 37 isolates within the genus Frankia and assigned them to their respective host infection groups, i.e., the Alnus/Casuarina and the Elaeagnus host infection groups. The assignment of individual strains to sub-groups within the respective host infection groups was consistent with classification based on comparative sequence analysis of nifH gene fragments, confirming the usefulness of MALDI-TOF MS as a rapid and reliable tool for the characterization of Frankia strains.     

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.     

Identification of Lactobacillus from the Saliva of Adult Patients with Caries Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. Bacteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification.     

Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to sodA sequence analyses

The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult.In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method.By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.     

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.     

Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene

Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S–23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNA^Ile and tRNA^Ala, the sequences of 16S–23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. aceti-specific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.     

Comparison of genotypic and biochemical characteristics of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> strains isolated from sour milk products

Overall, 72 strains of lactic acid thermophilic streptococci isolated from sour milk products manufactured in various regions of Russia and European countries were analyzed using classical microbiological and molecular biological methods. Physiological and biochemical properties and genetic diversity of these Streptococcus thermophilus strains were studied, and a comparative analysis of the nucleotide sequences of the 16S rRNA gene was conducted. It has been demonstrated that the homology of proximal parts of the 16S rRNA gene of all the strains studied towards one another and towards the reference strain ATCC19258 amounts to 100%. As for the sugar fermentation, some strains display the characteristics untypical of the S. thermophilus members. The data obtained suggest that it is preferable to use gene 16S rRNA sequencing data for identification of natural isolates of closely related lactic acid bacterial species; moreover, this method is recommended for a precise species identification of industrial bacterial strains used in the food industry.     

Rapid identification of Streptomyces isolates by MALDI-TOF MS

The recent emergence of multidrug-resistant bacteria over the last decade has led to a renewal in the discovery of new antimicrobial drugs. Streptomyces members are practically unlimited sources of new antibiotics. However, the identification of Streptomyces species is difficult and time-consuming. Therefore, there is a need for alternative methods for their rapid identification. In this study, an efficient protocol of identification using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was developed and applied for the rapid identification of Streptomyces isolates from the El Kala lakes in northeastern Algeria. A collection of 48 Streptomyces isolates were used for this study. The optimized procedure allowed us to obtain specific and reproducible protein spectra for each Streptomyces isolate tested. The spectra generated were used to build a preliminary local database based on their initial 16S rRNA identification. The blind test used for the identification of 20 Streptomyces strains already available in our created database and 20 unknown Streptomyces isolates showed that all (100%) of the Streptomyces strains listed in the database were rapidly (<30 min) identified with high scores of up to 2.8. Here, for the first time we showed that MALDI-TOF MS could be used as a cost-effective tool for the rapid identification of Streptomyces isolates.     

Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage

Background

Two important plant pathogenic bacteria Acidovorax oryzae and Acidovorax citrulli are closely related and often not easy to be differentiated from each other, which often resulted in a false identification between them based on traditional methods such as carbon source utilization profile, fatty acid methyl esters, and ELISA detection tests. MALDI-TOF MS and Fourier transform infrared (FTIR) spectra have recently been successfully applied in bacterial identification and classification, which provide an alternate method for differentiating the two species.

Results

Characterization and comparison of the 10 A. oryzae strains and 10 A. citrulli strains were performed based on traditional bacteriological methods, MALDI-TOF MS, and FTIR spectroscopy. Our results showed that the identity of the two closely related plant pathogenic bacteria A. oryzae and A. citrulli was able to be confirmed by both pathogenicity tests and species-specific PCR, but the two species were difficult to be differentiated based on Biolog and FAME profile as well as 16?S rRNA sequence analysis. However, there were significant differences in MALDI-TOF MS and FTIR spectra between the two species of Acidovorax. MALDI-TOF MS revealed that 22 and 18 peaks were specific to A. oryzae and A. citrulli, respectively, while FTIR spectra of the two species of Acidovorax have the specific peaks at 1738, 1311, 1128, 1078, 989?cm^-1 and at 1337, 968, 933, 916, 786?cm^-1, respectively.

Conclusions

This study indicated that MALDI-TOF MS and FTIR spectra may give a new strategy for rapid bacterial identification and differentiation of the two closely related species of Acidovorax.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.