Epidemiology, clinical characteristics, outcome and biofilm forming properties in candidaemia: A single-centre retrospective 4-year analysis from Hungary.

BACKGROUND: Candidaemia is a life-threatening disease that is associated with high mortality, especially in intensive care units (ICUs). The number of comprehensive studies dealing with the epidemiologic characteristics of biofilm-related properties is limited. OBJECTIVE: This study evaluated the clinical characteristics of candidaemia, to assess the biofilm-forming properties of isolates, and to identify the risk factors of mortality. PATIENTS AND METHODS: A total of 149 candidaemia episodes from the University of Debrecen, Clinical Centre, between January 2020 and December 2023 were investigated retrospectively. The susceptibility of Candida isolates to fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin was evaluated and compared to the susceptibility of 1-day-old biofilms. Multivariate logistic regression analysis was applied to identify the independent predictors of 30-day mortality rate. RESULTS: The most common Candida species was Candida albicans (41%), followed by C. parapsilosis (20%), C. glabrata (14%), C. tropicalis (13%), rare Candida species (7%), and C. krusei (5%). Sixty-six percent of Candida isolates were biofilm formers and 44% had high metabolic activity. The 30-day mortality rate was 52%, which was higher in ICUs (65%). The logistic regression analysis revealed several factors significantly influencing mortality including ICU admission (odds ratio [OR] 2.99, 95% confidence interval [CI] 1.17-8.04, p = 0.025), fluconazole treatment (OR 4.12, 95% CI 1.62-11.42, p = .004), and pneumonia (OR 0.261, 95% CI 0.1-0.67, p = .006). CONCLUSIONS: This comprehensive analysis supports the better characterisation of candidaemia in healthcare settings, which ultimately may reduce mortality among patients.

First Synthesis of DBU-Conjugated Cationic Carbohydrate Derivatives and Investigation of Their Antibacterial and Antifungal Activity.

The emergence of drug-resistant bacteria and fungi represents a serious health problem worldwide. It has long been known that cationic compounds can inhibit the growth of bacteria and fungi by disrupting the cell membrane. The advantage of using such cationic compounds is that the microorganisms would not become resistant to cationic agents, since this type of adaptation would mean significantly altering the structure of their cell walls. We designed novel, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)-derived amidinium salts of carbohydrates, which may be suitable for disturbing the cell walls of bacteria and fungi due to their quaternary ammonium moiety. A series of saccharide-DBU conjugates were prepared from 6-iodo derivatives of d-glucose, d-mannose, d-altrose and d-allose by nucleophilic substitution reactions. We optimized the synthesis of a d-glucose derivative, and studied the protecting group free synthesis of the glucose-DBU conjugates. The effect of the obtained quaternary amidinium salts against Escherichia coli and Staphylococcus aureus bacterial strains and Candida albicans yeast was investigated, and the impact of the used protecting groups and the sugar configuration on the antimicrobial activity was analyzed. Some of the novel sugar quaternary ammonium compounds with lipophilic aromatic groups (benzyl and 2-napthylmethyl) showed particularly good antifungal and antibacterial activity.

Transcriptional control of transglutaminase 2 expression in mouse apoptotic thymocytes.

Transglutaminase 2 (TGM2) is a ubiquitously expressed multifunctional protein, which participates in various biological processes including thymocyte apoptosis. As a result, the transcriptional regulation of the gene is complex and must depend on the cell type. Previous studies from our laboratory have shown that in dying thymocytes the expression of Tgm2 is induced by external signals derived from engulfing macrophages, such as retinoids, transforming growth factor (TGF)-ß and adenosine, the latter triggering the adenylate cyclase signaling pathway. The existence of TGF-ß and retinoid responsive elements in the promoter region of Tgm2 has already been reported, but the intergenic regulatory elements participating in the regulation of Tgm2 have not yet been identified. Here we used publicly available results from DNase I hypersensitivity analysis followed by deep sequencing and chromatin immunoprecipitation followed by deep sequencing against CCCTC-binding factor (CTCF), H3K4me3, H3K4me1 and H3K27ac to map a putative regulatory element set for Tgm2 in thymocytes. By measuring eRNA expressions of these putative enhancers in retinoid, rTGF-ß or dibutiryl cAMP-exposed thymocytes we determined which of them are functional. By applying ChIP-qPCR against SMAD4, retinoic acid receptor, retinoid X receptor, cAMP response element binding protein, P300 and H3K27ac under the same conditions, we identified two enhancers of Tgm2, which seem to act as integrators of the TGF-ß, retinoid and adenylate cyclase signaling pathways in dying thymocytes. Our study describes a novel strategy to identify and characterize the signal-specific functional enhancer set of a gene by integrating genome-wide datasets and measuring the production of enhancer specific RNA molecules.

Comparative transcriptional analysis of Candida auris biofilms following farnesol and tyrosol treatment.

Candida auris is frequently associated with biofilm-related invasive infections. The resistant profile of these biofilms necessitates innovative therapeutic options, where quorum sensing may be a potential target. Farnesol and tyrosol are two fungal quorum-sensing molecules with antifungal effects at supraphysiological concentrations. Here, we performed genome-wide transcript profiling with C. auris biofilms following farnesol or tyrosol exposure using transcriptome sequencing (RNA-Seq). Since transition metals play a central role in fungal virulence and biofilm formation, levels of intracellular calcium, magnesium, and iron were determined following farnesol or tyrosol treatment using inductively coupled plasma optical emission spectrometry. Farnesol caused an 89.9% and 73.8% significant reduction in the calcium and magnesium content, respectively, whereas tyrosol resulted in 82.6%, 76.6%, and 81.2% decrease in the calcium, magnesium, and iron content, respectively, compared to the control. Genes involved in biofilm events, glycolysis, ergosterol biosynthesis, fatty acid oxidation, iron metabolism, and autophagy were primarily affected in treated cells. To prove ergosterol quorum-sensing molecule interactions, microdilution-based susceptibility testing was performed, where the complexation of farnesol, but not tyrosol, with ergosterol was impeded in the presence of exogenous ergosterol, resulting in a minimum inhibitory concentration increase in the quorum-sensing molecules. This study revealed several farnesol- and tyrosol-specific responses, which will contribute to the development of alternative therapies against C. auris biofilms. IMPORTANCE: Candida auris is a multidrug-resistant fungal pathogen, which is frequently associated with biofilm-related infections. Candida-derived quorum-sensing molecules (farnesol and tyrosol) play a pivotal role in the regulation of fungal morphogenesis and biofilm development. Furthermore, they may have remarkable anti-biofilm effects, especially at supraphysiological concentrations. Innovative therapeutic approaches interfering with quorum sensing may be a promising future strategy against C. auris biofilms; however, limited data are currently available concerning farnesol-induced and tyrosol-related molecular effects in C. auris. Here, we detected several genes involved in biofilm events, glycolysis, ergosterol biosynthesis, fatty acid oxidation, iron metabolism, and autophagy, which were primarily influenced following farnesol or tyrosol exposure. Moreover, calcium, magnesium, and iron homeostasis were also significantly affected. These results reveal those molecular and physiological events, which may support the development of novel therapeutic approaches against C. auris biofilms.

Total transcriptome analysis of Candida auris planktonic cells exposed to tyrosol.

Tyrosol, a secondary metabolite of Candida species, regulates fungal morphogenesis, and its application may represent a novel innovative therapy against emerging multi-resistant fungal superbug such as Candida auris. In the current study, the effects of tyrosol on growth, redox homeostasis, intracellular microelement contents and activities of virulence-related enzymes released by C. auris were examined. To gain further information about the effect of tyrosol exposure, we revealed gene transcriptional changes using total transcriptome sequencing (RNA-Seq). At a concentration of 15 mM, tyrosol significantly decrease the growth of fungal cells within 2 h of its addition (5.6 × 107±1.2 × 107 and 2.5 × 107±0.6 × 107 colony forming unit/mL for control and tyrosol-treated cells, respectively). Furthermore, it enhanced the release of reactive oxygen species as confirmed by a dichlorofluorescein (DCF) assay (7.3 ± 1.8 [nmol DCF (OD640)-1] versus 16.8 ± 3.9 [nmol DCF (OD640)-1]), which was coincided with elevated superoxide dismutase, catalase and glutathione peroxidase activities. Tyrosol exerted in a 37%, 25%, 34% and 55% decrease in intracellular manganese, iron, zinc and copper contents, respectively, compared to control cells. The tyrosol treatment led to a 142 and 108 differentially transcripted genes with at least a 1.5-fold increase or decrease in transcription, respectively. Genes related to iron and fatty acid metabolism as well as nucleic acid synthesis were down-regulated, whereas those related to the antioxidative defence, adhesion and oxoacid metabolic processes were up-regulated. This study shows that tyrosol significantly influences growth, intracellular physiological processes and gene transcription in C. auris, which could highly support the development of novel treatment approaches against this important pathogen.

Concentration-Dependent Antibacterial Activity of Chitosan on Lactobacillus plantarum.

The antimicrobial effect of chitosan and synthetic chitosan derivatives has been confirmed on many Gram-positive and Gram-negative bacteria and fungi. The tests were carried out on pathogenic microorganisms, so the mechanism and concentration dependence of the inhibitory effect of chitosan were revealed. We conducted our tests on a probiotic strain, Lactobacillus plantarum. Commercially available chitosan derivatives of different molecular weights were added to L. plantarum suspension in increasing concentrations. The minimum inhibitory concentration (MIC) value of chitosan was determined and confirmed the viability decreasing effect at concentrations above the MIC with a time-kill assay. The release of bacterium cell content was measured at 260 nm after treatment with 0.001-0.1% concentration chitosan solution. An increase in the permeability of the cell membrane was observed only with the 0.1% treatment. The interaction was also investigated by zeta potential measurement, and the irreversible interaction and concentration dependence were established in all concentrations. The interaction of fluorescein isothiocyanate (FITC) labeled low molecular weight chitosan and bacterial cells labeled with membrane dye (FM® 4-64) was confirmed by confocal microscopy. In conclusion, the inhibitory effect of chitosan was verified on a probiotic strain, which is an undesirable effect in probiotic preparations containing chitosan additives, while the inhibitory effect experienced with pathogenic strains is beneficial.

Synergistic Interaction of Caspofungin Combined with Posaconazole against FKS Wild-Type and Mutant Candida auris Planktonic Cells and Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to cause biofilm-associated outbreaks, where frequently indwelling devices are the source of infections. The number of effective therapies is limited; thus, new, even-combination-based strategies are needed. Therefore, the in vitro efficacy of caspofungin with posaconazole against FKS wild-type and mutant Candida auris isolates was determined. The interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss model, and a LIVE/DEAD assay. Planktonic minimum inhibitory concentrations (pMICs) for the caspofungin-posaconazole combination showed a 4- to 256-fold and a 2- to 512-fold decrease compared to caspofungin and posaconazole alone, respectively. Sessile minimum inhibitory concentrations (sMICs) for caspofungin and posaconazole in combination showed an 8- to 128-fold and a 4- to 512-fold decrease, respectively. The combination showed synergy, especially against biofilms (FICIs were 0.033-0.375 and 0.091-0.5, and Bliss cumulative synergy volumes were 6.96 and 32.39 for echinocandin-susceptible and -resistant isolates, respectively). The caspofungin-exposed (4 mg/L) C. auris biofilms exhibited increased cell death in the presence of posaconazole (0.03 mg/L) compared to untreated, caspofungin-exposed and posaconazole-treated biofilms. Despite the favorable effect of caspofungin with posaconazole, in vivo studies are needed to confirm the therapeutic potential of this combination in C. auris-associated infections.

Physiological and transcriptional profiling of surfactin exerted antifungal effect against Candida albicans.

Given the risk of Candida albicans overgrowth in the gut, novel complementary therapies should be developed to reduce fungal dominancy. This study highlights the antifungal characteristics of a Bacillus subtilis-derived secondary metabolite, surfactin with high potential against C. albicans. Surfactin inhibited the growth of C. albicans following a 1-hour exposure, in addition to reduced adhesion and morphogenesis. Specifically, surfactin did not affect the level of reactive oxygen species but increased the level of reduced glutathione. Surprisingly, ethanol production was increased following 2 h of surfactin exposure. Surfactin treatment caused a significant reduction in intracellular iron, manganese and zinc content compared to control cells, whereas the level of copper was not affected. Alongside these physiological properties, surfactin also enhanced fluconazole efficacy. To gain detailed insights into the surfactin-related effects on C. albicans, genome-wide gene transcription analysis was performed. Surfactin treatment resulted in 1390 differentially expressed genes according to total transcriptome sequencing (RNA-Seq). Of these, 773 and 617 genes with at least a 1.5-fold increase or decrease in transcription, respectively, were selected for detailed investigation. Several genes involved in morphogenesis or related to metabolism (e.g., glycolysis, ethanol and fatty acid biosynthesis) were down-regulated. Moreover, surfactin decreased the expression of ERG1, ERG3, ERG9, ERG10 and ERG11 involved in ergosterol synthesis, whereas genes associated with ribosome biogenesis and iron metabolism and drug transport-related genes were up-regulated. Our data demonstrate that surfactin significantly influences the physiology and gene transcription of C. albicans, and could contribute to the development of a novel innovative complementary therapy.

Transcriptional Profiling of the Candida auris Response to Exogenous Farnesol Exposure.

The antifungal resistance threat posed by Candida auris necessitates bold and innovative therapeutic options. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment regimens. To gain further insights into the farnesol-related effect on C. auris, genome-wide gene transcription analysis was performed using transcriptome sequencing (RNA-Seq). Farnesol exposure resulted in 1,766 differentially expressed genes. Of these genes, 447 and 304 genes with at least 1.5-fold increase or decrease in transcription, respectively, were selected for further investigation. Genes involved in morphogenesis, biofilm events (maturation and dispersion), gluconeogenesis, iron metabolism, and regulation of RNA biosynthesis showed downregulation, whereas those related to antioxidative defense, transmembrane transport, glyoxylate cycle, fatty acid ß-oxidation, and peroxisome processes were upregulated. In addition, farnesol treatment increased the transcription of certain efflux pump genes, including MDR1, CDR1, and CDR2. Growth, measured by the change in the number of CFU, was significantly inhibited within 2 h of the addition of farnesol (5.8 × 107 ± 1.1 × 107 and 1.1 × 107 ± 0.3 × 107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (P < 0.001). In addition, farnesol treatment caused a significant reduction in intracellular iron (152.2 ± 21.1 versus 116.0 ± 10.0 mg/kg), manganese (67.9 ± 5.1 versus 18.6 ± 1.8 mg/kg), and zinc (787.8 ± 22.2 versus 245.8 ± 34.4 mg/kg) (P < 0.05 to 0.001) compared to untreated control cells, whereas the level of cooper was significantly increased (274.6 ± 15.7 versus 828.8 ± 106.4 mg/kg) (P < 0.001). Our data demonstrate that farnesol significantly influences the growth, intracellular metal ion contents, and gene transcription related to fatty acid metabolism, which could open new directions in developing alternative therapies against C. auris. IMPORTANCE Candida auris is a dangerous fungal pathogen that causes outbreaks in health care facilities, with infections associated with a high mortality rate. As conventional antifungal drugs have limited effects against the majority of clinical isolates, new and innovative therapies are urgently needed. Farnesol is a key regulator molecule of fungal morphogenesis, inducing phenotypic adaptations and influencing biofilm formation as well as virulence. Alongside these physiological modulations, it has a potent antifungal effect alone or in combination with traditional antifungals, especially at supraphysiological concentrations. However, our knowledge about the mechanisms underlying this antifungal effect against C. auris is limited. This study has demonstrated that farnesol enhances the oxidative stress and reduces the fungal survival strategies. Furthermore, it inhibits manganese, zinc transport, and iron metabolism as well as increases fungal intracellular copper content. In addition, metabolism was modulated toward ß-oxidation. These results provide definitive explanations for the observed antifungal effects.

Clinical and epidemiological characteristics of pediatric COVID-19 patients in a community-based study / Az alapellátásban kezelt COVID-19-fertozött gyermekek tünettani és epidemiológiai jellemzoi.

Összefoglaló. Bevezetés: A COVID-19-pandémia kapcsán számos tanulmány vizsgálta a tünetek gyakoriságát és a járványterjedés jellemzoit gyermekkorban, kevés azonban az alapellátás adatait összefoglaló publikáció. Közleményünkben 12 házi gyermekorvosi praxis 545 SARS-CoV-2-fertozött betegének adatait elemeztük a 2. (n = 293) és a 3. (n = 252) járványhullámban. Célkituzés: A gyermekkori fertozések tünettanának és epidemiológiai jellemzoinek összehasonlítása korcsoportok és járványhullámok között. Módszer: Valamennyi alapellátó praxis egységes retrospektív adatgyujtést végzett ugyanazon paraméterek regisztrálásával. Eredmények: A 10 év alatti betegekben a láz, a nátha és a köhögés dominált (30-50%), míg a 10 év felettiekben magas arányban regisztráltunk általános tüneteket is (30-40% fejfájás, gyengeség, szaglászavar). A 2. hullámban a 11-18 évesek (68%), a 3. hullámban a 0-10 évesek (53%) voltak többségben. A 3. hullámban szignifikánsan emelkedett a légúti tünetek elofordulása, az általános tünetek gyakorisága jelentosen csökkent, és szignifikánsan nott a családon belüli expozíció aránya (36% vs. 58%) a 2. hullámmal összehasonlítva. A gyermekrol családtagra történo továbbterjedés 24% és 16% volt a két járványhullámban, és mértékét az életkor befolyásolta. Megbeszélés: A klinikai kép az életkorral és a feltételezett vírusvariánssal mutatott összefüggést: 10 év alatt a légúti tünetek domináltak, 10 év felett szignifikánsan több általános tünetet regisztráltunk a 0-10 évesekhez képest. A 3. járványhullámban az alfa-variáns terjedésével gyakoribbá váltak a légúti tünetek, az iskolabezárások következtében megváltozott az életkori megoszlás, és megemelkedett a családi expozíció okozta fertozések aránya. A fertozés továbbadása háztartáson belül mindkét hullámban alacsony maradt. Következtetés: A COVID-19 klinikai megjelenését és terjedési jellemzoit jelentosen befolyásolta az érintett gyermekpopuláció életkori összetétele, a cirkuláló vírusvariáns és az aktuális korlátozó intézkedések. Orv Hetil. 2021; 162(44): 1751-1760. INTRODUCTION: During the COVID-19 pandemic, a large number of publications examined the frequency of symptoms and the mode of transmission in childhood but only a few community-based studies have been published. In our paper, 545 pediatric COVID-19 patients' data were collected by 12 primary care pediatricians in the second (n = 293) and third (n = 252) waves of the pandemic. OBJECTIVE: To compare the frequency of symptoms and household transmission in different age groups and between the two waves. METHOD: Patients' data and disease characteristics were recorded retrospectively in the same manner by all participating pediatricians. RESULTS: In patients of <10 years of age, fever, rhinorrhea and cough were registered the most frequently (30-50%), in contrast to patients of >10 years, where high frequency of general symptoms was found (30-40% headache, weakness, anosmia). In the third wave, the ratio of the age group 11-18 years declined from 68% to 47%, the frequency of respiratory symptoms increased significantly, while the ratio of general symptoms decreased. Household exposition was more frequent in the third wave (36% vs. 58%), while the transmission rate from children to family members was 24% and 16%, respectively, and it varied with age. DISCUSSION: Clinical manifestation showed relation to age and virus variant: the older age associated with higher frequency of general symptoms and the spread of the alpha variant led to the predominance of respiratory symptoms over general complaints. Prolonged school closures affected the age distribution and increased the frequency of household exposition. Secondary household transmission remained low. CONCLUSION: Clinical and epidemiological characteristics of pediatric COVID-19 disease were highly influenced by age, dominant virus variant and mitigation measures. Orv Hetil. 2021; 162(44): 1751-1760.