Mauro Tonolla

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 (Crohns 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.

Composition of bacterial and archaeal communities in freshwater sediments with different contamination levels (Lake Geneva, Switzerland)

The aim of this study was to compare the composition of bacterial and archaeal communities in contaminated sediments (Vidy Bay) with uncontaminated sediments (Ouchy area) of Lake Geneva using 16S rRNA clone libraries. Sediments of both sites were analysed for physicochemical characteristics including porewater composition, organic carbon, and heavy metals. Results show high concentrations of contaminants in sediments from Vidy. Particularly, high contents of fresh organic matter and nutrients led to intense mineralisation, which was dominated by sulphate-reduction and methanogenesis. The bacterial diversity in Vidy sediments was significantly different from the communities in the uncontaminated sediments. Phylogenetic analysis revealed a large proportion of Betaproteobacteria clones in Vidy sediments related to Dechloromonas sp., a group of dechlorinating and contaminant degrading bacteria. Deltaproteobacteria, including clones related to sulphate-reducing bacteria and Fe(III)-reducing bacteria (Geobacter sp.) were also more abundant in the contaminated sediments. The archaeal communities consisted essentially of methanogenic Euryarchaeota, mainly found in the contaminated sediments rich in organic matter. Multiple factor analysis revealed that the microbial community composition and the environmental variables were correlated at the two sites, which suggests that in addition to environmental parameters, pollution may be one of the factors affecting microbial community structure.

Evidence for anaerobic oxidation of methane in sediments of a freshwater system (Lago di Cadagno).

Anaerobic oxidation of methane (AOM) has been investigated in sediments of a high alpine sulfate-rich lake. Hot spots of AOM could be identified based on geochemical and isotopic evidence. Very high fractionation of methane (α=1.031) during oxidation was observed in the uppermost sediment layers, where methane is oxidized most likely with sulfate-containing bottom waters. However, we could not exclude that other electron acceptors such as iron, or manganese might also be involved. Light carbon isotope values (δ¹³C = -10‰ vs. Vienna Pee Dee Belemnite [VPDB]) of sedimentary carbonates at 16-20 cm sediment depth are indicative of a zone where methane was oxidized and the resulting bicarbonate ions were used for carbonate precipitation. 16S rRNA gene analysis revealed the presence of sequences belonging to the marine benthic groups B, C, and D and to the recently described clade of AOM-associated archaea (AAA). Catalyzed reporter deposition-FISH analysis revealed a high abundance of Deltaproteobacteria, especially of free-living sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus branch of Deltaproteobacteria in the AOM zone. Here, loose aggregations of AAA cells were found, suggesting that AAA might be responsible for oxidation of methane in Lake Cadagno sediments.

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry.

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.

European Surveillance for West Nile Virus in Mosquito Populations

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

Application of Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Identification and Clustering Analysis of Pantoea Species

ABSTRACT Pantoea agglomerans is an ecologically diverse taxon that includes commercially important plant-beneficial strains and opportunistic clinical isolates. Standard biochemical identification methods in diagnostic laboratories were repeatedly shown to run into false-positive identifications of P. agglomerans, a fact which is also reflected by the high number of 16S rRNA gene sequences in public databases that are incorrectly assigned to this species. More reliable methods for rapid identification are required to ascertain the prevalence of this species in clinical samples and to evaluate the biosafety of beneficial isolates. Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) methods and reference spectra (SuperSpectrum) were developed for accurate identification of P. agglomerans and related bacteria and used to detect differences in the protein profile within variants of the same strain, including a ribosomal point mutation conferring streptomycin resistance. MALDI-TOF MS-based clustering was shown to generally agree with classification based on gyrB sequencing, allowing rapid and reliable identification at the species level.

MALDI-TOF MS of Trichoderma: a model system for the identification of microfungi

This investigation aimed to assess whether MALDI-TOF MS analysis of the proteome could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of peptide mass fingerprints would reveal apomorphies that could be useful in diagnosing species in this genus. One hundred and twenty nine morphologically and genetically well-characterized strains of Hypocrea and Trichoderma, belonging to 25 species in 8 phylogenetic clades, were analyzed by MALDI-TOF MS mass spectrometry. The resulting peak lists of individual samples were submitted to single-linkage cluster analysis to produce a taxonomic tree and were compared to ITS and tef1 sequences from GenBank. SuperSpectra™ for the 13 most relevant species of Trichoderma were computed. The results confirmed roughly previously defined clades and sections. With the exceptions of T. saturnisporum (Longibrachiatum Clade) and T. harzianum (Harzianum Clade), strains of individual species clustered very closely. T. polysporum clustered distantly from all other groups. The MALDI-TOF MS analysis accurately reflected the phylogenetic classification reported in recent publications, and, in most cases, strains identified by DNA sequence analysis clustered together by MALDI-TOF MS. The resolution of MALDI-TOF MS, as performed here, was roughly equivalent to ITS rDNA. The MALDI-TOF MS technique analyzes peptides and represents a rough equivalent to sequencing, making this method a useful adjunct for determination of species limits. It also allows simple, reliable, and quick species identification, thus representing a valid alternative to gene sequencing for species diagnosis of Trichoderma and other fungal taxa.

Spatio‐temporal distribution of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland)

Abstract In situ hybridization was used to study the spatio-temporal distribution of phototrophic sulfur bacteria in the permanent chemocline of meromictic Lake Cadagno, Switzerland. At all four sampling times during the year the numerically most important phototrophic sulfur bacteria in the chemocline were small-celled purple sulfur bacteria of two yet uncultured populations designated D and F. Other small-celled purple sulfur bacteria (Amoebobacter purpureus and Lamprocystis roseopersicina) were found in numbers about one order of magnitude lower. These numbers were similar to those of large-celled purple sulfur bacteria (Chromatium okenii) and green sulfur bacteria that almost entirely consisted of Chlorobium phaeobacteroides. In March and June when low light intensities reached the chemocline, cell densities of all populations, with the exception of L. roseopersicina, were about one order of magnitude lower than in August and October when light intensities were much higher. Most populations were evenly distributed throughout the whole chemocline during March and June, while in August and October a microstratification of populations was detected suggesting specific eco-physiological adaptations of different populations of phototrophic sulfur bacteria to the steep physico-chemical gradients in the chemocline of Lake Cadagno.

Identification of dermatophytes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this study we evaluated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of dermatophytes in diagnostic laboratories. First, a spectral database was built with 108 reference strains belonging to 18 species of the anamorphic genera Epidermophyton, Microsporum and Trichophyton. All strains were well characterized by morphological criteria and ITS sequencing (gold standard). The dendrogram resulting from MALDI-TOF mass spectra was almost identical with the phylogenetic tree based on ITS sequencing. Subsequently, MALDI-TOF MS SuperSpectra were created for the identification of Epidermophyton floccosum, Microsporium audouinii, M. canis, M. gypseum (teleomorph: Arthroderma gypseum), M. gypseum (teleomorph: A. incurvatum), M. persicolor, A. benhamiae (Tax. Entity 3 and Am-Eur. race), T. erinacei, T. interdigitale (anthropophilic and zoophilic populations), T. rubrum/T. violaceum, T. tonsurans and T. terrestre. Because T. rubrum and T. violaceum did not present enough mismatches, a SuperSpectrum covering both species was created, and differentiation between them was done by comparison of eight specific peptide masses. In the second part of this study, MALDI-TOF MS with the newly created SuperSpectra was tested using 141 clinical isolates representing nine species. Analyses were done with 3-day-old cultures. Results were compared to morphological identification and ITS sequencing; 135/141 (95.8%) strains were correctly identified by MALDI-TOF MS compared to 128/141 (90.8%) by morphology. Therefore, MALDI-TOF MS has proven to be a useful and rapid identification method for dermatophytes.

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 Cohens 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.