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Clin Microbiol Infect 2012; 18: 475-484 Abstract Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory. [ABSTRACT FROM AUTHOR]

As a result of variable expression of biochemical characters, misidentification by conventional phenotypic means often occurs with clinical isolates belonging to Staphylococcus species. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of 450 blood isolates of the most relevant staphylococcal species, using sequence analysis of the rpoB gene as the reference method. A correct species identification by MALDI-TOF was obtained in 99.3% (447/450), with only three isolates being misidentified. In addition, MALDI-TOF correctly identified all the staphylococcal subspecies studied, including Staphylococcus capitis subsp. capitis and subsp. urealyticus, Staphylococcus cohnii subsp. urealyticus, Staphylococcus hominis subsp. novobiosepticus and subsp. hominis, Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus schleiferi subsp. schleiferi and Staphylococcus sciuri subsp. sciuri. Thus, MALDI-TOF MS-based species identification of staphylococci can be routinely achieved without any substantial costs for consumables or the time needed for labour-intensive DNA sequence analysis. [ABSTRACT FROM AUTHOR]

Summary: In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems. [Copyright &y& Elsevier]

Objectives Biofilm formation (BF) by fungal isolates may dramatically complicate infection. We determined the ability of Candida parapsilosis isolates from single fungaemia episodes to form biofilms and we analysed biofilm subgroups for antifungal susceptibility and pathogenic potential. We then correlated BF with clinical characteristics and outcomes of the episodes. Methods BF was measured using the crystal violet biomass assay. Antifungal susceptibility of preformed biofilms was assessed, and virulence was studied using the Galleria mellonella model. A retrospective analysis of patients' clinical records was performed. Results Of 190 patient-unique isolates, 84, 38 and 68 were identified as having high BF (HBF), moderate BF (MBF) or low BF (LBF), respectively. Among 30 randomly selected isolates, nine (eight HBF and one MBF), six (all HBF) and one (HBF) isolates had elevated sessile minimum inhibitory concentrations to fluconazole, anidulafungin or amphotericin B; all HBF and MBF isolates had elevated voriconazole sessile minimum inhibitory concentrations. G. mellonella killing rates of HBF isolates were significantly greater than MBF (or LBF) isolates (50% vs. 20%, 2 days from infection). By comparing HBF/MBF (106 patients) and LBF (84 patients) groups, we found that HBF/MBF patients had more central venous catheter-related fungaemias (62/106 (58.5%) vs. 29/84 (34.5%), p 0.001) and were more likely to die at 30 days from fungaemia onset (61/106 (57.5%) vs. 28/84 (33.3%), p 0.01). In the HBF/MBF group, azole antifungal therapy and central venous catheter removal were significantly associated with a higher and lower 30-day mortality rate, respectively. Conclusions C. parapsilosis BF influences the clinical outcome in patients with fungaemia. [ABSTRACT FROM AUTHOR]