Caroline Massonet

Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region

BackgroundThe number of patients with yeast infection has increased during the last years. Also the variety of species of clinical importance has increased. Correct species identification is often important for efficient therapy, but is currently mostly based on phenotypic features and is sometimes time-consuming and depends largely on the expertise of technicians. Therefore, we evaluated the feasibility of PCR-based amplification of the internally transcribed spacer region 2 (ITS2), followed by fragment size analysis on the ABI Prism 310 for the identification of clinically important yeasts.ResultsA rapid DNA-extraction method, based on simple boiling-freezing was introduced. Of the 26 species tested, 22 could be identified unambiguously by scoring the length of the ITS2-region. No distinction could be made between the species Trichosporon asteroides and T. inkin or between T. mucoides and T. ovoides. The two varieties of Cryptococcus neoformans (var. neoformans and var. gattii) could be differentiated from each other due to a one bp length difference of the ITS2 fragment. The three Cryptococcus laurentii isolates were split into two groups according to their ITS2-fragment lengths, in correspondence with the phylogenetic groups described previously. Since the obtained fragment lengths compare well to those described previously and could be exchanged between two laboratories, an internationally usable library of ITS2 fragment lengths can be constructed.ConclusionsThe existing ITS2 size based library enables identification of most of the clinically important yeast species within 6 hours starting from a single colony and can be easily updated when new species are described. Data can be exchanged between laboratories.

Comparison of VITEK 2 with ITS2-Fragment Length Polymorphism Analysis for Identification of Yeast Species

ABSTRACT A total of 61 clinical yeast isolates of Candida, Cryptococcus, Blastoschizomyces, and Saccharomyces spp. were used to compare two identification techniques, VITEK 2 and ITS2-fragment length polymorphism analysis (ITS2-FLP), with ID32C as the reference method. ID32C identified 58 isolates correctly. ITS2-FLP with Instagene DNA extraction identified 59 isolates. ITS2-FLP combined with boiling-freezing DNA extraction identified 55 isolates. VITEK 2 identified 41 isolates correctly.

The role of σB in persistence of Staphylococcus epidermidis foreign body infection

Staphylococcal biofilm formation depends on the transcription factor sigma(B). We further investigated the role of sigma(B) in biofilm formation and persistence in vitro and in vivo in a subcutaneous rat model. As expected, expression of all sigma(B) operon genes was transiently higher in the first 6 h of biofilm formation compared to planktonic bacteria, concurrent with a temporary upregulation of icaA and aap expression. However, we also observed a second upregulation of sigB expression in biofilm more than 2 days old without upregulation of icaA or aap. Biofilm formation by Staphylococcus epidermidis strains 8400 and 1457 was compared to that of isogenic mutants with inactivation of rsbU, of rsbUVW and of the entire sigma(B) operon. Both wild-type strains and the constitutively sigB-expressing rsbUVW mutant showed a strong biofilm-positive phenotype. The rsbUVW mutant biofilm was, however, thinner and more evenly spread than the wild-type biofilm. Inactivation of SigB in the rsbUVWsigB mutant or mutation of the positive regulator RsbU reduced both the number of sessile bacteria and polysaccharide intercellular adhesin (PIA) synthesis. These differences between the wild-types and their respective mutants appeared after 6 h in in vitro biofilms but only after 4 days in in vivo biofilms. Our results provide additional evidence for a role for sigma(B) in biofilm formation. They also suggest a role for sigma(B) in biofilm maturation and stability that is independent of PIA or accumulation-associated protein (Aap) and point to significant differences in the temporal development between in vitro and in vivo biofilms.

Effect of iron on the expression of sirR and sitABC in biofilm-associated Staphylococcus epidermidis

BackgroundDifferent gene expression patterns correlate with the altered phenotype in biofilm-associated bacteria. Iron and iron-linked genes are thought to play a key-role in biofilm formation. The expression of Fe-linked genes (sirR, sitABC operon) in Staphylococcus epidermidis, was compared in planktonic versus sessile bacteria in vitro and in vivo in a subcutaneous foreign body rat model.ResultsIn vitro in a Fe-limited environment, the planktonic form of S. epidermidis produces siderophores and grows slower than in Fe-rich environment. The expression of sirR in planktonic bacteria, in vitro, was not different in medium without Fe or with 1 μM FeCl3. High Fe concentrations (25 μM FeCl3) increased expression of sirR transiently during the early phase of incubation. Expression of sitC in vitro, in planktonic bacteria, was inversely correlated with sirR expression in medium with 25 μM FeCl3: sitC expression decreased for the first 3 hours followed by an up regulation.In sessile bacteria in vitro, sirR expression was high and independent of the Fe concentration. The expression of sitC was not inversely correlated to sirR expression.In vivo, expression levels of sirR and of sitABC were high during the initial phase after implantation and, after a transient decrease, remained stable over a period of two weeks.ConclusionOur data suggest that the expression of sirR and the regulatory effect of sirR on the sitABC operon are different in planktonic and sessile bacteria.