Percorrer por autor "Bujdáková, Helena"
A mostrar 1 - 8 de 8
Resultados por página
Opções de ordenação
- Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosisPublication . Štefánek, Matúš; Wenner, Sigurd; Borges, Vítor; Pinto, Miguel; Gomes, João Paulo; Rodrigues, João; Faria, Isabel; Pessanha, Maria Ana; Martins, Filomena; Sabino, Raquel; Veríssimo, Cristina; Nogueira, Isabel D.; Carvalho, Patrícia Almeida; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism’s distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.
- Catheter related bloodstream infection caused by E. cloacae and Candida parapsilosis: Are biofilms guilty?Publication . Štefánek, Matúš; Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel D.; Pinto, Miguel; Faria, Isabel; Pessanha, Maria Ana; Veríssimo, Cristina; Sabino, Raquel; Rodrigues, João; Matias, Rui; Carvalho, Patrícia Almeida; Gomes, João Paulo; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections is a public health concern in the context of healthcare associated infections (HAI) such as catheter related bloodstream infections (CRBSI). Here the dynamics of two top ten etiological agents of CRBSI, Enterobacter cloacae and Candida parapsilosis isolated from a CRBSI’s patient, were studied to get insights on the role played by biofilms on this HAI. Antimicrobial susceptibility of CVC and HC’s isolates was evaluated according to EUCAST guidelines. Single and/or mixed biofilms assembled on different materials in Mueller-Hinton broth with 2% glucose were assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification purposes and to assess microorganisms distribution within the biofilm (3D reconstruction) complemented with Focus Ion Beam (FIB)-SEM to assess biofilms assembled on the inner/outer CVC’s surfaces (tomograms). Whole-genome sequencing (WGS) was performed for all isolates. All isolates were antimicrobial resistant. Of note E.cloacae resistance to collistin and an additional resistance of the CVC compared to HC-isolate (ceftolozame-tazobactam) probably linked to a mutation in rpoB gene. Candida resistance to fluconazol might be explained by ERG11 gene mutation. Enterobacter and Candida assembled biofilms on glass, polystyrene and polyurethane being mixed biofilms denser when both microorganism were present from the beginning. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Using environmental isolates of the same species we showed that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), denser mixed biofilms richer in EPS were observed. A distinct phenotype was present on the patient’s CVC by SEM and FIB/SEM. WGS confirmed the genetic identity of the pair CVC/HC isolates, while corroborating the virulence potential and observed antimicrobial resistant character of the studied CRBSI-driving pathogens. The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 GenePublication . Štefánek, Matúš; Garaiová, Martina; Valček, Adam; Jordao, Luisa; Bujdáková, HelenaThis work presents a comparative analysis of two clinical isolates of C. parapsilosis, isolated from haemoculture (HC) and central venous catheter (CVC). Both strains harboured Y132F and R398I mutations in the gene ERG11 associated with resistance to fluconazole (FLC). Differences between the HC and CVC isolates were addressed in terms of virulence, resistance to FLC, and lipid distribution. Expression of the ERG6 and ERG9 genes, lipid analysis, fatty acid composition, and lipase activity were assessed via qPCR, thin-layer chromatography/high-performance liquid chromatography, gas chromatography, and spectrophotometry, respectively. Regulation of the ERG6 and ERG9 genes did not prove any impact on FLC resistance. Analysis of lipid metabolism showed a higher accumulation of lanosterol in both the isolates regardless of FLC presence. Additionally, a decreased level of triacylglycerols (TAG) with an impact on the composition of total fatty acids (FA) was observed for both isolates. The direct impact of the ERG11 mutations on lipid/FA analysis has not been confirmed. The higher lipase activity observed for C. parapsilosis HC isolate could be correlated with the significantly decreased level of TAG. The very close relatedness between both the isolates suggests that one isolate was derived from another after the initial infection of the host.
- Effect of Quorum Sensing Molecule Farnesol on Mixed Biofilms of Candida albicans and Staphylococcus aureusPublication . Gaálová-Radochová, Barbora; Kendra, Samuel; Jordao, Luisa; Kursawe, Laura; Kikhney, Judith; Moter, Annette; Bujdáková, HelenaThe natural bioactive molecule farnesol (FAR) is widely studied mainly for its antibiofilm and antimicrobial properties. In addition, it increases the effectiveness of some antimicrobial substances, which makes it interesting for the development of combined therapy. In the present work, the effect of FAR either alone or in combination with oxacillin (OXA) on mixed biofilms formed by clinically relevant pathogens, Candida albicans and Staphylococcus aureus, was studied. S. aureus isolates used for biofilm formation originated from blood cultures and central venous catheters (CVC) were characterized in terms of antimicrobial resistance. The minimal biofilm inhibitory concentration (MBIC50) for FAR of 48 h mixed biofilms formed by the C. albicans and methicillin-sensitive S. aureus (MSSA) was determined to be 125 M, and for the mixed biofilms with methicillin-resistant S. aureus (MRSA) was determined to be 250 M. Treatment of mixed biofilms with OXA (2 mg/mL) showed 4% inhibition; however, the combination of OXA (2 mg/mL) and FAR (300 M) resulted in 80% inhibition of biofilms. In addition, planktonic cells of S. aureus exhibited an increased susceptibility to OXA, cefoxitin and kanamycin in the presence of FAR (150 and 300 M). Scanning electron microscopy (SEM) micrographs confirmed patchy biofilm and lack of candidal hyphae in the samples treated with FAR and FAR/OXA in comparison to control and mixed biofilms treated only with OXA. Intriguingly, in a pilot experiment using fluorescence in situ hybridization (FISH), considerable differences in activity (as indicated by ribosome content) of staphylococcal cells were detected. While the activity rate of the staphylococci in mixed biofilms treated with FAR was high, no FISH-positive signal for staphylococcal cells was found in the biofilm treated with FAR/OXA.
- Effect of quorum sensing molecule farnesol on mixed biofilms of Candida anbicans and Staphylococcus aureusPublication . Gaálová, Barbora; Kapustová, Magdaléna; Jordão, Luísa; Dizová, Stanislava; Bujdáková, HelenaSignalling molecules are extensively studied in recent years because of their potential to control pathogens. Microorganisms naturally produce quorum sensing molecules (QS) as a tool for intra- and inter-species communication. Farnesol is synthesized as a by product of the ergosterol pathway of the yeast Candida albicans and in a concentration dependent maner blocks the morphological shift from the yeast to the hyphal form. The aim of this work was to study the effect of farnesol (30-300 microM) and the combination of farnesol with antibiotics (oxacillin, ciprofloxacin) on mixed biofilms formed by C. albicans/ S. aureus. For this purpose, clinical strains of S. aureus from blood cultures and central venous cathethers were collected. Overall, 8 MRSA and 3 MSSA were identified by PCR by detection of FemA, MecA genes. Strains were characterized according to selected virulence factors associated with resistance to antibiotics and disinfectants or biofilm formation (Nor, Qac genes coding for efflux pumps, Ica operon). The efficacy of farnesol to single and mixed biofilms was determined using different approaches, the total biomass stainning (crystal violet), metabolic activity meassuring (MTT test), colony forming units counting (CFU/ml) and microscopy (SEM). The concentration of 300 microMwas determined as the MIC50 of farnesol inhibiting mixed biofilm formation. This concentration did not show mutagenic potential according to the Ames test and was not cytotoxic for HeLa cell line. SEM definitely confirmed that farnesol inhibited hyphae of C. albicans that are a suitable substrate for adherence of staphilococci. Testing a combination of farnesol with oxacillin by E-test revealed an enhancing susceptibility of MRSA and MSSA to oxacillin. This effect was already observed and it can be associated with the inhibition of cell wall biosynthesis through the reduction of free C55 lipid carrier with subsequent retardation of murein monomer precursor transport across the cell membrane. We can conclude, that farnesol has multiple effects on mixed biofilms.
- Experiences with evaluation of results from antimicrobial testing on microbial biofilms formed in vitroPublication . Barbora, Gaálová; Černáková, Lucia; Jordão, Luisa; Dohál, Matúš; Bujdáková, HelenaThis presentation summarizes some experiences and troubleshooting leading to the data validation extracted from experiments focused on antimicrobial testing using various antimicrobial agents, surfaces and microorganisms (the yeast Candida albicans, bacteria Streptococcus mutans, Staphylococcus aureus, and Escherichia coli). In screening of anti-biofilm activity when many samples are tested, 3 approaches are fundamental: determination of number of survived cells (CFU), measurement of metabolic activity, and microscopy. These techniques can be combined by different ways, using various substrates or dyes. An evaluation through metabolic activity, the most easy approach, uses spectral measurements at A560 (MTT assay) or A490 (XTT assay). Substrates used in both techniques are associated with activity of dehydrogenases and are reduced in final detected product. Methods are suitable for testing biofilms formed on stable materials (for example, single or dual biofilms formed by C. albicans and S. mutans on hydroxyapatite blocks). However, compounds participating in redox reactions can significantly diminish activity of both substrates. The example of such compound is the photoactive dye methylene blue that is promising compound used in nanotechnology. Similarly, colloidal dispersions containing clay minerals interact with substrates in respect to the charge. In testing of such materials, determination of CFU is the most common choice. Taking into account those limitations, challenging approach is the molecular quantification of live/dead cells in microbial biofilms by real-time PCR. The intercalating dyes ethidium monoazide and propidium monoazide can be selected in respect to microorganisms (for example, S. mutans vs. E. coli). Mentioned methods do not allow describe many details in biofilms. Microscopic techniques in combination with appropriate dyes (CLSM) or nucleotide probes (FISH), but also SEM can help to prove a real fitness and architecture of microbial consortia.
- Impact of farnesol and Corsodyl® on Candida albicans forming dual biofilm with Streptococcus mutansPublication . Černáková, Lucia; Jordao, Luisa; Bujdáková, HelenaObjective: This work studied the biofilm formed by Candida albicans and Streptococcus mutans on a hydroxyapatite surface after exposure to the quorum-sensing molecule farnesol (200 µM) in comparison with the diluted mouthwash Corsodyl® (0.0001% chlorhexidine digluconate). Materials and Methods: The cytotoxicity of farnesol was evaluated by Galleria melonella surviving assay. The viability of biofilm cells after exposure to farnesol and Corsodyl® was determined by colony-forming units. The morphology and structure of a dual-species biofilm was evaluated by scanning electron microscopy. Results: Farnesol did not exhibit a toxic effect on larval survival. While 200 µM farnesol effectively reduced the yeast-to-hyphae transition in the dual biofilm, it did not affect the growth of S. mutans. Additionally, despite the presence of farnesol, many blastospores were observed. Corsodyl® reduced S. mutans in the dual biofilm, but did not influence C. albicans. Conclusion: This study showed that 200 µM farnesol modulated C. albicans in a dual-species biofilm with S. mutans, but did not exhibit antimicrobial activity against S. mutans. Moreover, it seems that S. mutans provides conditions that support the growth of the yeast form of C. albicans. The mouthwash Corsodyl® reduces S. mutans, but was not effective against C. albicans.
- Opportunist Coinfections by Nontuberculous Mycobacteria and Fungi in Immunocompromised PatientsPublication . Joao, Ines; Bujdáková, Helena; Jordao, LuisaNontuberculous mycobacteria (NTM) and many fungal species (spp.) are commonly associated with opportunistic infections (OPIs) in immunocompromised individuals. Moreover, occurrence of concomitant infection by NTM (mainly spp. of Mycobacterium avium complex and Mycobacterium abscessus complex) and fungal spp. (mainly, Aspergillus fumigatus, Histoplasma capsulatum and Cryptococcus neoformans) is very challenging and is associated with poor patient prognosis. The most frequent clinical symptoms for coinfection and infection by single agents (fungi or NTM) are similar. For this reason, the accurate identification of the aetiological agent(s) is crucial to select the best treatment approach. Despite the significance of this topic it has not been sufficiently addressed in the literature. This review aims at summarizing case reports and studies on NTM and fungi coinfection during the last 20 years. In addition, it briefly characterizes OPIs and coinfection, describes key features of opportunistic pathogens (e.g., NTM and fungi) and human host predisposing conditions to OPIs onset and outcome. The review could interest a wide spectrum of audiences, including medical doctors and scientists, to improve awareness of these infections, leading to early identification in clinical settings and increasing research in the field. Improved diagnosis and availability of therapeutic options might contribute to improve the prognosis of patients’ survival.