The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

• Klíčová slova
• Raman spectroscopy, biofilm, cryo-SEM, sample preparation, scanning electron microscopy,
• MeSH
• bakteriální infekce diagnóza   mikrobiologie   MeSH
• biofilmy růst a vývoj   MeSH
• Candida parapsilosis izolace a purifikace   patogenita   ultrastruktura   MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• Ramanova spektroskopie MeSH
• Staphylococcus epidermidis izolace a purifikace   patogenita   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Candida spp. is able to form a biofilm, which is considered resistant to the majority of antifungals used in medicine. The aim of this study was to evaluate the in vitro activity of micafungin against Candida spp. biofilms at different stages of their maturation (2, 6, and 24 h). We assessed the inhibitory effect of micafungin against 78 clinical isolates of Candida spp., growing as planktonic or sessile cells, by widely recommended broth microdilution method. The in vitro effect on sessile cells viability was evaluated by colorimetric reduction assay. All examined strains were susceptible or intermediate to micafungin when growing as planktonic cells. At the early stages of biofilm maturation, from 11 (39.3%) to 20 (100%), tested strains, depending on the species, exhibited sessile minimal inhibitory concentrations (SMICs) of micafungin at ≤ 2 mg/L. For 24-h-old Candida spp. biofilms, from 3 (10.7%) to 20 (100%) of the tested strains displayed SMICs of micafungin at ≤ 2 mg/L. Our findings confirm that micafungin exhibits high potential anti-Candida-biofilm activity. However, this effect does not comprise all Candida species and strains. All strains were susceptible or intermediate to micafungin when growing as planktonic cells, but for biofilms, micafungin displays species- and strain-specific activity. Paradoxical growth of C. albicans and C. parapsilosis was observed. Antifungal susceptibility testing of Candida spp. biofilms would be the best solution, but to date, no reference method is available. The strongest antibiofilm activity of micafungin is observed at early stages of biofilm formation. Possibly, micafungin could be considered as an effective agent for prevention of biofilm-associated candidiasis, especially catheter-related candidaemia.

• Klíčová slova
• Antifungal drugs, Candidiasis, Echinocandin, Paradoxical growth, Sessile cells,
• MeSH
• antifungální látky farmakologie   MeSH
• biofilmy účinky léků   MeSH
• Candida albicans účinky léků   růst a vývoj   fyziologie   MeSH
• Candida glabrata účinky léků   růst a vývoj   fyziologie   MeSH
• Candida parapsilosis účinky léků   růst a vývoj   fyziologie   MeSH
• echinokandiny farmakologie   MeSH
• kandidóza mikrobiologie   MeSH
• lidé MeSH
• lipopeptidy farmakologie   MeSH
• micafungin MeSH
• mikrobiální testy citlivosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antifungální látky MeSH
• echinokandiny MeSH
• lipopeptidy MeSH
• micafungin MeSH

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is, currently, used as a rapid and reliable tool in microbial diagnostics. The discriminatory power of the method extends its applicability also beyond species level. This study examined the possibility to use MALDI-TOF MS to differentiate between Candida parapsilosis sensu stricto biofilm-positive (n = 12) and biofilm-negative (n = 9) strains. The results indicated a grouping trend within MALDI-TOF mass spectra belonging to each of the tested groups. However, these trends were eclipsed by mass spectral variations resulting from limited repeatability of the method, making its application for the selected purpose impossible. Improvement in the discriminatory power of the method was not obtained neither by using different matrices (α-cyano-4-hydroxycinnamic acid, ferulic acid, 5-chloro-2-mercaptobenzothionazole) for MALDI-TOF MS analysis nor by testing different culture conditions (cultivation length, culture media).

• MeSH
• biofilmy růst a vývoj   MeSH
• Candida chemie   fyziologie   MeSH
• lidé MeSH
• mikrobiologické techniky metody   MeSH
• reprodukovatelnost výsledků MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made.

• MeSH
• biofilmy * MeSH
• Candida klasifikace   cytologie   ultrastruktura   MeSH
• Ramanova spektroskopie * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A case report of ventriculoperitoneal shunt infection caused by Candida lusitaniae in a 6-year-old patient with cerebral astrocytoma and obstructive hydrocephalus is presented briefly with emphasis on the course of antifungal treatment. Seven isolates recovered subsequently from the cerebrospinal fluid were studied retrospectively. To confirm identity, isolates were typed using pulsed-field gel electrophoresis and melting curve of random amplified polymorphic DNA (McRAPD). Further, the ability to form biofilm and its susceptibility to systemic antifungals were evaluated. Using McRAPD, identity of C. lusitaniae isolates showing slight microevolutionary changes in karyotypes was undoubtedly confirmed; successful application of numerical interpretation of McRAPD for typing is demonstrated here for the first time. The strain was also recognized as a strong biofilm producer. Moreover, minimum biofilm inhibitory concentrations were very high, in contrast to low antifungal minimum inhibitory concentrations of isolates. It can be concluded that McRAPD seems to be a simple and reliable method not only for identification but also for typing of yeasts. A ventriculoperitoneal shunt colonized by C. lusitaniae was revealed as the source of this nosocomial infection, and the ability of the strain to form biofilm on its surface likely caused treatment failure.

• MeSH
• antifungální látky farmakologie   MeSH
• astrocytom mozkomíšní mok   komplikace   farmakoterapie   mikrobiologie   patologie   chirurgie   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• Candida účinky léků   genetika   izolace a purifikace   MeSH
• denaturace nukleových kyselin MeSH
• dítě MeSH
• hydrocefalus mozkomíšní mok   komplikace   farmakoterapie   mikrobiologie   patologie   chirurgie   MeSH
• infekce spojené se zdravotní péčí mozkomíšní mok   komplikace   farmakoterapie   mikrobiologie   patologie   chirurgie   MeSH
• kandidóza mozkomíšní mok   komplikace   farmakoterapie   mikrobiologie   patologie   chirurgie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mykologické určovací techniky MeSH
• nádory mozku mozkomíšní mok   komplikace   farmakoterapie   mikrobiologie   patologie   chirurgie   MeSH
• neúspěšná terapie MeSH
• pulzní gelová elektroforéza MeSH
• technika náhodné amplifikace polymorfní DNA * MeSH
• ventrikuloperitoneální zkrat škodlivé účinky   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antifungální látky MeSH

The possible presence of biofilms was examined in mucosal specimens of 15 patients, undergoing functional endoscopic sinus surgery or a modified Caldwell-Luc approach for chronic rhinosinusitis (CRS). Biofilms were found in 7 of the 15 patients, positive cultures being obtained in most samples, which supports the role of biofilms as an important factor in the pathogenesis of CRS.

• MeSH
• biofilmy růst a vývoj   MeSH
• chronická nemoc MeSH
• dospělí MeSH
• endoskopie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nosní sliznice mikrobiologie   ultrastruktura   MeSH
• rýma mikrobiologie   chirurgie   MeSH
• sinusitida mikrobiologie   chirurgie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH