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.

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.

Genomic epidemiology of antifungal resistance in human and avian isolates of Candida albicans: a pilot study from the One Health perspective.

Stress-induced genomic changes in Candida albicans contribute to the adaptation of this species to various environmental conditions. Variations of the genome composition of animal-origin C. albicans strains are largely unexplored and drug resistance or other selective pressures driving the evolution of these yeasts remained an intriguing question. Comparative genome analysis was carried out to uncover chromosomal aneuploidies and regions with loss of heterozygosity (LOH), two mechanisms that manage genome plasticity. We detected aneuploidy only in human isolates. Bird-derived isolates showed LOH in genes commonly associated with antifungal drug resistance similar to human isolates. Our study suggests that environmental fungicide usage might exert selective pressure on C. albicans infecting animals, thus contributing to the spread of potentially resistant strains between different hosts.

Unveiling the Hungarian landscape of laboratory and clinical management capacities for invasive fungal infections: navigating the frontlines against fungal menaces.

Background: Antifungal diagnostic capacity has been documented in various countries, there is a lack of comprehensive research on clinical mycology diagnostics and treatment in Hungary. Methods: We conducted an online survey encompassing questions that explored various aspects of the mycology diagnostic and antifungal therapy-related information. The survey aimed to gather details about institutional profiles, perceptions of invasive fungal infections (IFIs), and access to microscopy, culture, serology, antigen detection, molecular testing, and therapeutic drug monitoring. Results: As of May 2023, a total of 17 institutions responded to the questionnaire. Seven participants categorized the institutional incidence of IFI as 'very low', four as 'low', and six as 'mild'. The majority of centers identified Candida spp. (94%) and Aspergillus spp. (82%) as the most prevalent fungal pathogens. Nearly half of the laboratories (47%) reported using matrix-assisted laser desorption/ionization-time of flight mass spectrometry for identification. All institutions had access to microscopy and culture-based diagnostic approaches. A significant number of centers had access to antigen detection (71%) and various molecular assays (59%). Regarding antifungal agents, all reporting sites used at least one triazole, with voriconazole (77%) being the most common mold-active azole. Furthermore, 71% of the centers applied at least one formulation of amphotericin B, and 65% to one echinocandin. However, only 18% of the centers used 5-flucytosine. Conclusion: Resource availability for diagnosing and treating IFI in Hungary varies across hospitals based on location. Surveys help identify gaps and limitations in this area. To address these challenges, interregional cooperation within Hungary could be a facilitating strategy.

METHODS: We did an online survey with questions about how hospitals in Hungary handle fungal infections. We wanted to know about the hospitals' characteristics, how they see these infections, and what tools they use for diagnosis and treatment. RESULTS: As of May 2023, we got responses from 17 hospitals. Some said they hardly ever see these infections, while others said they see them a bit more. Most hospitals found Candida and Aspergillus as the most common fungal culprits. Many used a tool called MALDI-TOF MS for identification. All of them had ways to look at samples under a microscope and grow them in a dish. Many hospitals had tests to look for certain things in the blood (71%), and they also used different genetic tests (59%). When it came to medicines, they all had at least one kind of medicine called a triazole, with voriconazole being the most common one. They also had amphotericin B and echinocandins. But only a few had a medicine called 5-flucytosine. CONCLUSION: Hospitals in Hungary differ in how they handle fungal infections. Doing surveys like this can help find problems and limits. To fix these issues, hospitals in different parts of Hungary can work together.

Exploring how Hungary deals with serious fungal infections: facing fungal threats head-on Background: While various countries have looked into their ability to diagnose fungal infections, there hasn't been a comprehensive study on how Hungary deals with diagnosing and treating these infections.

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.

In Vitro Killing Activities of Anidulafungin and Micafungin with and without Nikkomycin Z against Four Candida auris Clades.

Candida auris is a multidrug-resistant pathogen against which echinocandins are the drug of choice. However, information on how the chitin synthase inhibitor nikkomycin Z influences the killing activities of echinocandins against C. auris is currently lacking. We determined the killing activities of anidulafungin and micafungin (0.25, 1, 8, 16 and 32 mg/L each) with and without nikkomycin Z (8 mg/L) against 15 isolates representing four C. auris clades (South Asian n = 5; East Asian n = 3; South African n = 3; South American n = 4, two of which were of environmental origin). Two and one isolates from the South Asian clade harbored mutations in the hot-spot 1 (S639Y and S639P) and 2 (R1354H) regions of the FKS1 gene, respectively. The anidulafungin, micafungin and nikkomycin Z MIC ranges were 0.015-4, 0.03-4 and 2->16 mg/L, respectively. Anidulafungin and micafungin alone exerted weak fungistatic activity against wild-type isolates and the isolate with a mutation in the hot-spot 2 region of FKS1 but was ineffective against the isolates with a mutation in the hot-spot 1 region. The nikkomycin Z killing curves were always similar to their respective controls. Twenty-two of sixty (36.7%) anidulafungin plus nikkomycin Z and twenty-four of sixty (40%) micafungin plus nikkomycin Z combinations produced at least 100-fold decreases in the CFUs (synergy), with a 41.7% and 20% fungicidal effect, respectively, against wild-type isolates. Antagonism was never observed. Similar results were found with the isolate with a mutation in hot-spot 2 of FKS1, but the combinations were ineffective against the two isolates with prominent mutations in hot-spot 1 of FKS1. The simultaneous inhibition of ß-1,3 glucan and chitin synthases in wild-type C. auris isolates produced significantly greater killing rates than either drug alone. Further studies are warranted to verify the clinical efficacy of echinocandin plus nikkomycin Z combinations against echinocandin susceptible C. auris isolates.

The In Vitro Activity of Fluconazole, Amphotericin B and Echinocandins Against Cyberlindnera fabianii Planktonic Cells and Biofilms.

Until recently, little was known about the susceptibility pattern of Cyberlindnera fabianii (Cy. fabianii) planktonic cells and biofilms regarding the most frequently administered systemic antifungals, despite the high mortality rate and its potential role in catheter-related infections. In the current study, the activity of fluconazole, amphotericin B and echinocandins (anidulafungin, caspofungin and micafungin) was determined against planktonic and sessile cells of Cy. fabianii clinical isolates (n = 8). Planktonic minimum inhibitory concentrations (MICs) ranged from 1 to 2, from 0.25 to 1, from 0.015 to 0.06, from 0.03 to 0.12 and from 0.25 to 0.5 mg/l for fluconazole, amphotericin B, anidulafungin, caspofungin and micafungin, respectively. One-day-old biofilms were highly resistant to fluconazole (MIC ranged from 512 to > 512) compared to planktonic counterparts, but not to amphotericin B (MIC ranged from 0.25 to 2 mg/l) and echinocandins (MIC ranged from 0.06 to 2 mg/l). Based on the calculated planktonic killing rates, the highest activity was observed in the case of anidulafungin (k values ranged from 0.37 to 2.09), while micafungin, caspofungin, amphotericin B and fluconazole exerted 0.46-1.47, 0.14-0.86, -0.03 to 2.08 and -0.15 to 0.09 killing rate value ranges, respectively. The obtained in vitro planktonic and sessile susceptibility patterns suggest that echinocandins and amphotericin B may be the most reliable treatment option for the treatment of Cy. fabianii infections.

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.

Faecal Carriage of Carbapenem-Resistant Acinetobacter baumannii: Comparison to Clinical Isolates from the Same Period (2017-2019).

Increasing prevalence of A. baumannii was found in the faecal samples of inpatients without infection caused by A. baumannii (0.15%; 55/7806). The aim of the study was to determine whether there is a relationship between the clinical strains and the increased faecal occurrence. Characteristics of faecal and clinical isolates were compared between 2017 and 2019, and the direction of causality was assessed by Granger causality tests. In the case of the antibiotic resistance, faecal carriage of carbapenem-resistant Acinetobacter baumannii (CRAb) was Granger-caused by prevalence of CRAb in inpatients (F = 15.84, p < 0.001), but inpatient prevalence was not Granger-caused by CRAb faecal carriage (F = 0.03, p = 0.855). Whole genomes of 16 faecal isolates were sequenced by Illumina MiSeq; cgMLST types were determined. In faecal isolates, the occurrence of carbapenem resistance was lower than among the clinical isolates from the same period; only blaOXA-72 harbouring ST636 and ST492 were detected, and the blaOXA-23 harbouring ST2 and ST49 strains previously dominant in clinical isolates were absent. Carriage of blaOXA-72 was linked to pMAL-1-like and pA105-2-like plasmids in ST636 and ST492 isolates, respectively, both in clinical and faecal isolates. The new ST636 and ST492 strains may colonise the gut microbiota of the patients, which thus may play a role as a reservoir.

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.

In Vivo Efficacy of Amphotericin B against Four Candida auris Clades.

Candida auris is a multidrug-resistant fungus against which in some clinical situations amphotericin B (AMB) remains the alternative or first line drug. We compared daily 1 mg/kg of AMB efficacy in a neutropenic murine bloodstream infection model against 10 isolates representing four C. auris clades (South Asian n = 2; East Asian n = 2; South African n = 2; South American n = 4; two of which were of environmental origin). Five days of AMB treatment significantly increased the survival rates in mice infected with isolates of the East Asian clade, and 1 isolate each from the South African and South American clades (originated from bloodstream), but not in mice infected with the South Asian and 2 environmental isolates from the South American clades. AMB treatment decreased the fungal burden in mice infected with the 2 isolates each from East Asian and South African, and 1 out of 2 bloodstream isolates from South American clades in the hearts (p < 0.01), kidneys (p < 0.01) and brain (p < 0.05). AMB treatment, regardless of clades, significantly decreased colony forming units in the urine at day 3. However, histopathological examination in AMB-treated mice revealed large aggregates of yeast cells in the kidneys and hearts, and focal lesions in the cerebra and cerebelli, regardless of precise C. auris clade. Our clade-specific data confirm that the efficacy of AMB against C. auris is weak, explaining the therapeutic failures in clinical situations. Our results draw attention to the necessity to maximize the killing at the start of treatment to avoid later complications in the heart and central nervous system.

Heme Oxygenase-1 (HMX1) Loss of Function Increases the In-Host Fitness of the Saccharomyces 'boulardii' Probiotic Yeast in a Mouse Fungemia Model.

The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of S. 'boulardii'. Here in this study, the potential role of the gene heme oxygenase-1 (HMX1) in probiotic yeast bloodstream-derived infections was studied by generating marker-free HMX1 deletion mutants with CRISPR/Cas9 technology from both commercial and clinical S. 'boulardii' isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of HMX1 in S. 'boulardii' significantly (p < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of S. 'boulardii' strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination.

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.

Virulence Factors and in-Host Selection on Phenotypes in Infectious Probiotic Yeast Isolates (Saccharomyces 'boulardii').

Saccharomyces yeast probiotics (S. 'boulardii') have long been applied in the treatment of several gastrointestinal conditions. Despite their widespread use, they are rare opportunistic pathogens responsible for a high proportion of Saccharomyces mycosis cases. The potential virulence attributes of S. 'boulardii' as well as its interactions with the human immune system have been studied, however, no information is available on how these yeasts may change due to in-host evolution. To fill this gap, we compared the general phenotypic characteristics, cell morphology, virulence factors, epithelial and immunological interactions, and pathogenicity of four probiotic product samples, two mycosis, and eight non-mycosis samples of S. 'boulardii'. We assessed the characteristics related to major steps of yeast infections. Mycosis and non-mycosis isolates both displayed novel characters when compared to the product isolates, but in the case of most virulence factors and in pathogenicity, differences were negligible or, surprisingly, the yeasts from products showed elevated levels. No isolates inflicted considerable damage to the epithelial model or bore the hallmarks of immune evasion. Our results show that strains in probiotic products possess characteristics that enable them to act as pathogens upon permissive conditions, and their entry into the bloodstream is not due to active mechanisms but depends on the host. Survival in the host is dependent on yeast phenotypic characteristics which may change in many ways once they start evolving in the host. These facts call attention to the shortcomings of virulence phenotyping in yeast research, and the need for a more thorough assessment of probiotic use.

Unpredictable In Vitro Killing Activity of Amphotericin B against Four Candida auris Clades.

Candida auris is an emerging multiresistant yeast against which amphotericin B (AMB) is still the first therapeutic choice in certain clinical situations (i.e., meningitis, endophthalmitis, and urinary tract infections). As data about the in vitro killing activity of AMB against C. auris clades are lacking, we determined MICs, minimum fungicidal concentrations (MFCs), and killing activity of AMB against 22 isolates representing the 4 major C. auris clades (South Asian n = 6; East Asian n = 4; South African n = 6, and South American n = 6). MIC values were ≤1 mg/L regardless of clades; MFC ranges were, 1-4 mg/L, 2-4 mg/L, 2 mg/L, and 2-8 mg/L for South Asian, East Asian, South African, and South American clades, respectively. AMB showed concentration-, clade-, and isolate-dependent killing activity. AMB was fungicidal at 1 mg/L against two of six, two of four, three of six, and one of six isolates from the South Asian, East Asian, South African, and South American clades, respectively. Widefield fluorescence microscopy showed cell number decreases at 1 mg/L AMB in cases of the South Asian, East Asian, and South African clades. These data draw attention to the weak killing activity of AMB against C. auris regardless of clades, even when MICs are low (≤1 mg/L). Thus, AMB efficacy is unpredictable in treatment of invasive C. auris infections.

Evolution of the Gram-Negative Antibiotic Resistance Spiral over Time: A Time-Series Analysis.

We followed up the interplay between antibiotic use and resistance over time in a tertiary-care hospital in Hungary. Dynamic relationships between monthly time-series of antibiotic consumption data (defined daily doses per 100 bed-days) and of incidence densities of Gram-negative bacteria (Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, and Acinetobacter baumannii) resistant to cephalosporins or carbapenems were followed using vector autoregressive models sequentially built of time-series ending in 2015, 2016, 2017, 2018, and 2019. Relationships with Gram-negative bacteria as a group were fairly stable across years. At species level, association of cephalosporin use and cephalosporin resistance of E. coli was shown in 2015-2017, leading to increased carbapenem use in these years. Association of carbapenem use and carbapenem resistance, as well as of carbapenem resistance and colistin use in case of A. baumannii, were consistent throughout; associations in case of Klebsiella spp. were rarely found; associations in case of P. aeruginosa varied highly across years. This highlights the importance of temporal variations in the interplay between changes in selection pressure and occurrence of competing resistant species.

In vitro and in vivo interaction of caspofungin with isavuconazole against Candida auris planktonic cells and biofilms.

The in vitro and in vivo efficacy of caspofungin was determined in combination with isavuconazole against Candida auris. Drug-drug interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss independence model and an immunocompromised mouse model. Median planktonic minimum inhibitory concentrations (pMICs) of 23 C. auris isolates were between 0.5 and 2 mg/l and between 0.015 and 4 mg/l for caspofungin and isavuconazole, respectively. Median pMICs for caspofungin and isavuconazole in combination showed 2-128-fold and 2-256-fold decreases, respectively. Caspofungin and isavuconazole showed synergism in 14 out of 23 planktonic isolates (FICI range 0.03-0.5; Bliss cumulative synergy volume range 0-4.83). Median sessile MICs (sMIC) of 14 biofilm-forming isolates were between 32 and >32 mg/l and between 0.5 and >2 mg/l for caspofungin and isavuconazole, respectively. Median sMICs for caspofungin and isavuconazole in combination showed 0-128-fold and 0-512-fold decreases, respectively. Caspofungin and isavuconazole showed synergistic interaction in 12 out of 14 sessile isolates (FICI range 0.023-0.5; Bliss cumulative synergy volume range 0.13-234.32). In line with the in vitro findings, synergistic interactions were confirmed by in vivo experiments. The fungal kidney burden decreases were more than three log volumes in mice treated with combination of 1 mg/kg caspofungin and 20 mg/kg isavuconazole daily; this difference was statistically significant compared with control mice (P < 0.001). Despite the favorable effect of isavuconazole in combination with caspofungin, further studies are needed to confirm the therapeutic advantage of this combination when treating an infection caused by C. auris.

Candida auris poses a continuous therapeutic challenge. We demonstrate an approach where the combination of caspofungin and isavuconazole showed a potent activity against planktonic cells and biofilms. This synergism helps to expand the therapeutic options against C. auris.

Comparison of In Vitro Killing Activity of Rezafungin, Anidulafungin, Caspofungin, and Micafungin against Four Candida auris Clades in RPMI-1640 in the Absence and Presence of Human Serum.

Candida auris is an emerging and frequently multidrug-resistant pathogen against which the echinocandins are the preferred therapeutic option. We compared killing activities of anidulafungin, caspofungin, micafungin, and rezafungin against 13 isolates representing four C. auris clades (South Asian n = 3; East Asian n = 3; South African n = 3; South American n = 4, of which two were of environmental origin). Minimum inhibitory concentration MICs and killing kinetics in RPMI-1640 and RPMI-1640 plus 50% serum (50% serum) were determined. The four echinocandins were never fungicidal and induced large aggregates in RPMI-1640 and, less markedly, in 50% serum. Colony forming unit CFU decreases were found more consistently in 50% serum than in RPMI-1640. Isolates from the East Asian clade were killed at ≥1-≥ 4 mg/L with all echinocandins regardless of media. Anidulafungin and micafungin produced killing at peak drug serum concentration (8 mg/L) against environmental but not clinical isolates from the South American and the South African clades. Micafungin at ≥8 mg/L but not anidulafungin produced CFU decreases against the South Asian clade as well. In 50% serum, rezafungin at ≥1-≥ 8 mg/L produced killing against all four clades. The next generation echinocandin, rezafungin, showed the same or better activity at clinically attainable trough concentration regardless of media, compared with anidulafungin, caspofungin, and micafungin against all four tested C. auris clades.

The Role of Uniform Meropenem Usage in Acinetobacter baumannii Clone Replacement.

The dominant carbapenem resistant Acinetobacter baumannii harboring blaOXA-23-like carbapenemase was replaced by blaOXA-40-like carriers in a Hungarian tertiary-care center with high meropenem but relatively low imipenem use. We hypothesized that alterations in antibiotic consumption may have contributed to this switch. Our workgroup previous study examined the relation between resistance spiral and the antibiotic consumption, and the results suggest that the antibiotic usage provoked the increasing resistance in case of A. baumannii. We aimed at measuring the activity of imipenem and meropenem to compare the selection pressure exerted by the different carbapenems in time-kill assays. Strain replacement was confirmed by whole genome sequencing, core-genome multilocus sequence typing (cgMLST), and resistome analysis. Based on results of the time-kill assays, we found a significant difference between two different sequence-types (STs) in case of meropenem, but not in case of imipenem susceptibility. The newly emerged ST636 and ST492 had increased resistance level against meropenem compared to the previously dominant ST2 and ST49. On the other hand, the imipenem and colistin resistance profiles were similar. These results suggest, that the uniform meropenem usage may have contributed to A. baumannii strain replacement in our setting.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.