Clinical Fungal Isolates Research Articles

Micronutrient availability alters Candida albicans growth and farnesol accumulation: implications for studies using RPMI-1640.

The dimorphic fungus Candida albicans is a major opportunistic pathogen of humans. RPMI-1640 is a chemically defined growth medium commonly used with C. albicans. We identified over 32,000 publications with keywords RPMI and C. albicans. Additionally, Antifungal Susceptibility Testing (AFST) protocols in the United States (CLSI) and Europe (EUCAST) utilize RPMI as a base media to assess drug efficacy against clinical fungal isolates. RPMI contains many nutrients but no added trace metals. We found that the growth characteristics with RPMI were dependent on which MOPS buffer was chosen and the contamination of that buffer by trace levels of Mn(II) and Zn(II). Added Mn(II) was most needed for cell growth while added Zn(II) was most needed for secretion of farnesol and other signaling molecules.

Evaluation of the Combination Effects of Extracts of Jatropha tanjorensis and Adansonia digitata on Clinical Fungal Isolates

Introduction: The proliferation of fake drugs and the multiple resistance seen in conventional fungicidal drugs has led to the use of medicinal plants to treat fungal infections. There are claims that Jatropha tanjorensis and Adansonia digitata have fungicidal potentials, hence this study. Aim: To evaluate the combination effects of the extracts of Jatropha tanjorensis and Adansonia digitata on clinical fungal isolates. Study Design: Randomized sampling of the plants was done by harvesting the plants from different farm in Nsukka Enugu State. Place and Duration of Study: This study was conducted in Enugu State, South East of Nigeria, from November 2023 to March 2024. Methodology: Phytochemical screening was done on the two plants. The methanolic and aqueous (cold and hot) extracts of these plants bark and stem were assessed for their antifungal activities against six fungal isolates using agar diffusion method. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were done according to standard methods. Results: The plants contain alkaloids, terpenoids, saponins, flavonoids, tannin, glycoside and phenol. The hot water extracts of J. tanjorensis stem gave higher zones of inhibition diameter (IZD) than the methanolic extracts and the methanolic extracts of A. digitata bark gave higher IZD than the aqueous extracts. The combination of these two potent extracts showed greatest inhibition on Aspergillus niger, Candida albican and then least inhibition on Trichophyton shoenleinii, this was found to be statistically significant at (P<.05), the P value was (P=0.001). The lowest (MIC) and (MFC) were recorded in A. niger and Candida albican at (1.5625mg/ml) while the highest (MIC) and (MFC) were recorded in T. shoenleinii, at (25mg/ml). Conclusion: This study revealed that the combined plant extracts were more effective against opportunistic fungi than the dermatophytes.

Cold atmospheric plasma inactivates Aspergillus flavus and Fusarium keratoplasticum biofilms and conidia in vitro.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment.Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored.Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro.Methodology. Power parameters (22-27 kVpp, 300-400 Hz and 20-80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml-1) and biofilm formation.Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml-1 within 300 s or less, and complete inhibition after 600 s of treatment.Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.

Evaluation of MMV Pandemic Response Box compounds to identify potent compounds against clinically relevant bacterial and fungal clinical isolates in vitro

BackgroundMultidrug resistant bacterial and fungal pathogens are resistant to a number of significant front-line drugs, hence, identification of new inhibitory agents to combat them is crucial. In this study, we aim to evaluate the activity of Pandemic Box compounds from Malaria Medicines Venture (MMV) against A. baumannii and P. aeruginosa bacterial, C. auris, C. albicans and A. niger fungal clinical isolates. MethodsIsolates were initially screened with 201 antibacterial and 46 antifungal compounds (10 μM) using a microbroth dilution in triplicates to determine MIC. A persister assay was performed for bacterial pathogens. ResultsOut of 201 antibacterial compounds, twenty-nine and seven compounds inhibited the growth of A. baumannii and P. aeruginosa at 10 μM, respectively. MMV1580854, MMV1579788, eravacycline and epetraborole inhibited both the bacterial test isolates. In a persister assay, MMV1634390 showed complete bactericidal effect against A. baumannii. With antifungal activity compounds, C. auris responded to15 compounds, Six compounds inhibited C. albicans and one was effective against A. niger at 10 μM. The ratio of Minimum Fungicidal Concentration (MFC): Minimum Inhibitory Concentration (MIC) of MMV1782110 was 2 against C. auris. Eberconazole, amorolfine and luliconazole are fungicidal targeting C. albicans at a MFC: MIC ratio of 2. ConclusionFive compounds from MMV Pandemic Box were found to be inhibiting colistin and ceftazidime resistant A. baumannii clinical isolate, also against colistin and β-lactam resistant P. aeruginosa clinical isolate. MMV1634390 showed complete bactericidal effect against A. baumannii in a persister assay. MMV1782110, Eberconazole, amorolfine and luliconazole exhibited potent anti-fungal activity. Further investigations are warranted to identify the targets and mechanism.

Novel automated antifungal susceptibility testing system for yeasts based on dual-detection algorithm of turbidimetry and colorimetry.

Introduction. The increasing prevalence and growing resistance of fungi present a significant peril to public health. There are only four classes of antifungal medicines available today, and few candidates are in clinical trials.Hypothesis/Gap Statement. Rapid and sensitive diagnostic techniques are lacking for most fungal pathogens, and those that do exist are expensive or hard to obtain.Aim. This study aimed to evaluate the feasibility of a novel automated antifungal susceptibility testing system, Fungus AST, in comparison to the broth microdilution method (BMD) recommended by the Clinical and Laboratory Standards Institute (CLSI).Methodology. A total of 101 clinical Candida spp. isolates were collected from the Zengcheng Branch of Nanfang Hospital and subjected to antifungal susceptibility testing. Antifungal susceptibility was assessed using the Fungus AST method and the BMD.Results. In this study, we introduce a novel automated antifungal susceptibility testing system, Fungus AST, which detects the turbidity and/or colour intensity of microdilution wells using a four-wavelength detection technology in real time and is designed to match the growth characteristics of strains over time. Based on our analysis, all reportable ranges of Fungus AST were suitable for clinical fungal isolates in PR China. Within ±twofold dilutions, reproducibility was 100 %. Considering the BMD as a referenced method, ten antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B, 5-flucytosine and nystatin) showed an essential agreement of >95 %. The category agreement of five antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and voriconazole) was excellent at >90 %. One Candida albicans isolate and voriconazole showed a major error (ME) (1.7 %), and no other ME or very ME agents were found.Conclusion. Given the above, it can be argued that the utilization of Fungus AST is a discretionary automated approach. More improvements are needed in Fungus AST compared to the BMD system for a wider range of clinical isolates, including different types of fungi.

The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the β-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial β-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

A Rapid and Efficient Method for the Extraction of Histone Proteins.

Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.

Rapid automated antifungal susceptibility testing system for yeasts based on growth characteristics.

Fungal pathogens are a major threat to public health, as they are becoming increasingly common and resistant to treatment, with only four classes of antifungal medicines currently available and few candidates in the clinical development pipeline. Most fungal pathogens lack rapid and sensitive diagnostic techniques, and those that exist are not widely available or affordable. In this study, we introduce a novel automated antifungal susceptibility testing system, Droplet 48, which detects the fluorescence of microdilution wells in real time and fits growth characteristics using fluorescence intensity over time. We concluded that all reportable ranges of Droplet 48 were appropriate for clinical fungal isolates in China. Reproducibility within ±2 two-fold dilutions was 100%. Considering the Sensititre YeastOne Colorimetric Broth method as a comparator method, eight antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin, amphotericin B, and 5-flucytosine) showed an essential agreement of >90%, except for posaconazole (86.62%). Category agreement of four antifungal agents (fluconazole, caspofungin, micafungin, and anidulafungin) was >90%, except for voriconazole (87.93% agreement). Two Candida albicans isolates and anidulafungin showed a major discrepancy (MD) (2.60%), and no other MD or very MD agents were found. Therefore, Droplet 48 can be considered as an optional method that is more automated and can obtain results and interpretations faster than previous methods. However, the optimization of the detection performance of posaconazole and voriconazole and promotion of Droplet 48 in clinical microbiology laboratories still require further research involving more clinical isolates in the future.

Discovery a novel of thiazolo[3,2-a]pyridine and pyrazolo[3,4-d]thiazole derivatives as DNA gyrase inhibitors; design, synthesis, antimicrobial activity, and some in-silico ADMET with molecular docking study

In our continuous efforts to develop novel and effectual antimicrobial agents, we herein synthesized a novel thiazolo[3,2-a]pyridine and pyrazolo[3,4-d]thiazole derivatives hybrid with pyrazole scaffold. The structure of seventeen synthesized derivatives was confirmed using IR, NMR, Mass spectra, and elemental analysis. The desired compounds have undergone an appraisal for their antimicrobial activity in vitro against six clinical MDR bacterial and fungal isolates through the agar well-diffusion, demonstrating promising results. Additionally, five thiazolo[3,2-a]pyridine derivatives 2a, 2e, 2g, 3c, and 3d revealed significant potent activity against the tested pathogens, with compound 2e as the most active compound with values ranging MIC = 0.125–0.25 µg/mL and MBC = 0.0625–0.125 µg/mL against bacterial isolate and MIC = 0.25 µg/mL and MFC = 0.125 µg/mL against two fungal isolates. In the MBC, MFC, and time-kill, the active target compounds showed -cidal activities towards the pathogenic isolates at different concentrations, and the isolates were not recovered from the treated groups with 1XMIC after 6-24 h of bacterial incubation and after 12 h of fungal incubation, confirming -cidal activity. Moreover, compound 2e exhibited DNA gyrase inhibitor with IC50 = 7.35 µM compared with Ciprofloxacin (IC50 = 47.68 µM). Finally, the docking simulation was performed to determine the mode of molecular interaction. The results exhibited that compound 2e revealed the lowest binding energy S = - 20.84 Kcal/mol compared with Ciprofloxacin S = -13.67 Kcal/mol. In silico, ADMET displayed that compound 2e obeys the Lipinski rule and, therefore, is predicted to be oral bioavailability with a non-toxic and skin sensitization profile.

Colonization and azole resistance of oropharyngeal <i>Candida</i> fungi in intensive care patients with COVID-19

Patients with COVID-19 are susceptible to developing oropharyngeal candidiasis and invasive candidiasis. Invasive fungal infections can complicate the clinical course of COVID-19 and are associated with a substantially increased mortality. An important reason for the successful treatment of candidiasis is to determine the sensitivity of clinical fungal isolates to antimycotics. A microbiological study of oropharyngeal swabs was performed in 54 patients aged 33 to 94 years (mean age 67.4 years) with severe and extremely severe COVID-19, who were treated in the intensive care unit at the height of clinical manifestations. Most patients (95%) had comorbidities: hypertension (68.5%), diabetes mellitus (24%), coronary artery disease (22.2%), chronic heart failure (38.9%), obesity (23.8%), cardiac arrhythmias (20.4%), chronic cerebral ischemia (56.1%), varicose veins of the lower extremities (5.5%), chronic kidney disease (20.4%), liver cirrhosis (5.5%), HIV infection (5.5%). A comparative analysis of oropharyngeal microbial biocenosis showed differences in the frequency of occurrence of opportunistic microbial species, as well as in the number of members in microbial associations. Polyspecific fungal-bacterial associations were more common and dominated polyspecific bacterial associations. According to the results, a high frequency of oropharyngeal colonization in patients with severe COVID-19 by strains of fungi of the genus Candida was established. The vast majority of strains of fungi of the genus Candida (85.2%) were resistant to antimycotics of the azole group. A high frequency (85.2%) of oropharyngeal colonization in resuscitation patients with azole-resistant strains of fungi of the genus Candida was revealed. C. albicans dominated among Candida. C. albicans was isolated in 37 (68.5%) patients, C. pararapsilosis in 6 (11.1%), C. tropicalis in 3 (5.5%), C. krusei in 2 (3.7%)%), C. kefyr in 1 (1.9%), C. glabrata in 1 (1.9%) patient. In 11 (20.4%) patients, associations of C. albicans and other Candida species were identified. The level of fungi in the material was etiologically significant ( 104105 CFU/tamp.) in 80% of patients. The vast majority of strains (more than 70%) were resistant to fluconazole and voriconazole, which should be taken into account when prescribing candidiasis therapy in patients with COVID-19. The MIC for fluconazole against C. albicans strains was 1024 g/ml for 4 isolates, 512 g/ml for 7 isolates, and 128 g/ml for 15 isolates. The maximum MIC values for voriconazole against C. albicans was 256 g/ml.

Identification and susceptibility testing of oral candidiasis in advanced cancer patients

BackgroundPatients with advanced cancer are prone to develop different opportunistic oral infection due to anti-cancer treatment or the malignancies themselves. Studies of oral fungal samples show an increased prevalence of non-Candida albicans species in mixed oral infections with Candida albicans. Non-C. albicans and C. albicans are associated with varying degrees of resistance to azoles, which may have implications for treatment. This study aimed to assess the diversity and antifungal susceptibility of Candida species detected in the oral cavity.MethodsAn observational study with microbiological analysis was conducted. Clinical fungal isolates were collected from patients in a hospice unit in 2014–2016. Isolates were re-grown on chromID® Candida plates in 2020. Single colony of each species was re-cultivated and prepared for biochemical identification with a VITEK2® system and verified by gene sequencing. Etest was performed on RPMI agar, and the antifungals fluconazole, amphotericin B, anidulafungin and nystatin were applied.ResultsFifty-six isolates from 45 patients were identified. Seven different Candida species and one Saccharomyces species were detected. The results of biochemical identification were confirmed with sequencing analysis. Thirty-six patients had mono infection, and nine out of 45 patients had 2–3 different species detected. Of C. albicans strains, 39 out of 40 were susceptible to fluconazole. Two non-C. albicans species were resistant to fluconazole, one to amphotericin B and three to anidulafungin.ConclusionC. albicans was the predominant species, with a high susceptibility to antifungal agents. Different Candida species occur in both mono and mixed infections. Identification and susceptibility testing may therefore lead to more effective treatment and may prevent the development of resistance among patients with advanced cancer.Trail registrationThe study Oral Health in Advanced Cancer was registered at ClinicalTrials.gov (#NCT02067572) in 20/02/2014.

Artificial intelligence-aided rapid and accurate identification of clinical fungal infections by single-cell Raman spectroscopy.

Integrating artificial intelligence and new diagnostic platforms into routine clinical microbiology laboratory procedures has grown increasingly intriguing, holding promises of reducing turnaround time and cost and maximizing efficiency. At least one billion people are suffering from fungal infections, leading to over 1.6 million mortality every year. Despite the increasing demand for fungal diagnosis, current approaches suffer from manual bias, long cultivation time (from days to months), and low sensitivity (only 50% produce positive fungal cultures). Delayed and inaccurate treatments consequently lead to higher hospital costs, mobility and mortality rates. Here, we developed single-cell Raman spectroscopy and artificial intelligence to achieve rapid identification of infectious fungi. The classification between fungi and bacteria infections was initially achieved with 100% sensitivity and specificity using single-cell Raman spectra (SCRS). Then, we constructed a Raman dataset from clinical fungal isolates obtained from 94 patients, consisting of 115,129 SCRS. By training a classification model with an optimized clinical feedback loop, just 5 cells per patient (acquisition time 2 s per cell) made the most accurate classification. This protocol has achieved 100% accuracies for fungal identification at the species level. This protocol was transformed to assessing clinical samples of urinary tract infection, obtaining the correct diagnosis from raw sample-to-result within 1 h.

Analysis of the synergistic antifungal activity of everolimus and antifungal drugs against dematiaceous fungi.

Chromoblastomycosis (CBM) is a form of chronic mycosis that affects the skin and mucous membranes and is caused by species of dematiaceous fungi including Exophiala spp., Phialophora spp., and Fonsecaea spp. The persistence of this disease and limitations associated with single-drug treatment have complicated efforts to adequately manage this condition. In this study, a microdilution assay was used to explore the synergistic antifungal activity of everolimus (EVL) in combination with itraconazole (ITC), voriconazole (VRC), posaconazole (POS), and amphotericin B (AMB) against a range of clinical dematiaceous fungal isolates. These analyses revealed that the EVL+POS and EVL+ITC exhibited superior in vitro synergistic efficacy, respectively inhibiting the growth of 64% (14/22) and 59% (13/22) of tested strains. In contrast, the growth of just 9% (2/22) of tested strains was inhibited by a combination of EVL+AMB, and no synergistic efficacy was observed for the combination of EVL+VRC. Overall, these findings indicate that EVL holds promise as a novel drug that can be synergistically combined with extant antifungal drugs to improve their efficacy, thereby aiding in the treatment of CBM.

358. Surveillance of Activity of Antifungals: Data from the Analysis of Resistance in Antifungals (ARIA) 2020 Study

Abstract Background ARIA is a new annual global surveillance initiative collecting yeast and fungal isolates from worldwide designed to determine resistance to antifungal agents and trends over time. The ARIA program was developed in 2020 to provide a repository of recent clinical fungal isolates with known susceptibility profiles and to monitor resistance trends over time. ARIA reports the susceptibility patterns of its earliest data concerning echinocandins, second-generation triazoles, and fluconazole against clinical Candida albicans, non-albicans strains including C. auris, Aspergillus and Fusarium isolates from worldwide sources. Methods Isolates were collected from hospital worldwide during 2020, shipped to a central laboratory and re-identified by MALDI-TOF or molecular methods. MIC tests were performed by broth microdilution method in line with CLSI susceptibility testing standards. Percentage of susceptibility was calculated according to CLSI breakpoints. Antifungals tested were amphotericin B (AMB), anidulafungin (ANID), fluconazole (FLU), isavuconazole (ISA), micafungin (MIC), posaconazole (POS), and voriconazole (VOR). All testing was performed according to CLSI M27-A4 and M38-A2 methodologies. Results See table. Conclusion Ongoing antifungal resistance surveillance like the ARIA program is of utmost importance in order to monitor the efficacy of traditional empirical therapy and for the development of novel antifungal agents. This repository and ongoing ARIA program will provide a resource to better support the biopharmaceutical industry’s goals to develop new and more potent antifungal agents. Disclosures All Authors: No reported disclosures.

Elucidating the antifungal activity and mechanism of action of bioactive phytochemicals against fungal dermatitis isolates.

Dermatophytosis has been the most common cause of superficial fungal infections which invade the keratinized tissues of body such as nail, hair, and skin, respectively. Although these infections are treatable and many commercial drugs are available that can be applied topically (clotrimazole, fluconazole, itraconazole, miconazole, voriconazole) on the infected areas but they have very low efficacy and has high probability of relapse. To increase the efficacy of treatment, the patient receives supplementary oral medicines for prolong duration that leads to hepatotoxicity. Previously, it has been reported that some wild medicinal plants possess antifungal capacity due to the presence of bioactive molecules. In present study, these phytochemicals (viz. tannins, saponins, alkaloids, flavonoids) derived from three test plants [Acacia nilotica (babul), Catharanthus roseus (sadabahar) and Ricinus communis (Arandi)] are used as sources of direct medicinal agents to develop an antidermatophytic drug formulation against the clinical fungal isolates associated with affected population. The mechanism of their antifungal potential of partially purified phytochemicals were analyzed using agar well diffusion method, food inhibition assessment and DNA cleavage analysis. The data revealed that the alkaloids are the most potent component possessing antifungal property that is recommended to be used to formulate topical ointment for the dermatophytic infection after competent regulatory approvals. This can be used as promising source of alternative treatment approach and as a competent substitute for chemically synthesized hepatotoxic drugs that are available in market.

Performance Evaluation of Bruker Biotyper, ASTA MicroIDSys, and VITEK-MS and Three Extraction Methods for Filamentous Fungal Identification in Clinical Laboratories.

Filamentous fungi are a major cause of life-threatening infections in immunocompromised patients; thus, rapid and accurate identification is critical. Filamentous fungal identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been demonstrated with high sensitivity and reproducibility; however, its wider application has been limited in clinical laboratories because of practical challenges such as database availability or lack of standardization. In this study, we compared the performance of the Bruker Biotyper, ASTA MicroIDSys, and Vitek MS for 84 clinical filamentous fungal isolates. Moreover, the sensitivity of three independent sample preparation methods (direct, on plate, in tube) was compared. Bruker Biotyper identified 71.43% (60/84) of isolates correctly (species, genus, or complex/group level). ASTA MicroIDSys and Vitek MS showed accuracy rates of 70.24% (59/84) and 55.95% (47/84), respectively. We found that any difference in sensitivity may be attributed to the database of the systems. In addition, the "in tube" method showed the highest sensitivity among the three methods; however, there was no statistical difference among them. For the broader application of MALDI-TOF MS for filamentous fungal identification, further studies from multiple perspectives are required.

The Antimicrobial Effects of Saudi Sumra Honey against Drug Resistant Pathogens: Phytochemical Analysis, Antibiofilm, Anti-Quorum Sensing, and Antioxidant Activities.

Honey exhibited potential antimicrobial activity against multidrug resistant (MDR) bacteria that continues to be a serious health problem. We reported the in-vitro activity of Saudi Sumra honey against clinical pathogenic bacteria and fungi, antibiofilm, anti-quorum-sensing (QS) and antioxidant activities in relation to its phytochemical composition assessed by gas chromatography-mass spectrometry (GC-MS). Broth dilution method and scavenging activities against 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and β-carotene bleaching assays were performed. The GC-MS analysis of Sumra honey showed that 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one 1-methylcyclopropanemethanol were the major identified phytoconstituents. Sumra honey showed a minimum inhibitory concentration (MIC) to clinical isolates of Staphylococcus aureus including methicillin-resistant Staphylococcus aureus (MRSA) at 300 mg/mL, Pseudomonas aeruginosa (250 mg/mL), Escherichia coli (350 mg/mL) and Acinetobacter baumannii (250 mg/mL); clinical fungal isolates—Candida auris (600 mg/mL) and Cryptococcus neoformans (>1000 mg/mL); wild type fungal isolates—Candida krusei (>1000 mg/mL) and Candida albicans (700 mg/mL). In addition, Sumra honey demonstrated promising inhibition targeting biofilm formation by 59% for Bacillus subtilis, 48% for S. aureus, 38% for E. coli, and 33.63% for P. aeruginosa. The violacein production in Chromobacterium violaceum was reduced to 68%, whereas pyocyanin production in P. aeruginosa was reduced to 54.86% at ½ MIC. Furthermore, Sumra honey exhibited strong antioxidant activities (DPPH − IC50 = 7.7 mg/mL; ABTS − IC50 = 5.4 mg/mL; β-carotene − IC50 = >20 mg/mL). Overall, obtained data highlighted the promising potential therapeutic use of Sumra honey treating infections caused by MDR bacteria and fungi. Moreover, Sumra honey can be a good candidate as an inhibitor agent for bacterial cellular communication in strains of P. aeruginosa and C. violaceum.

S5.4b Genomic variation structure in clinical isolate collections alters benefits of association screening: a study of Cryptococcus neoformans var. grubii in Colombia

S5.4 Free oral paper session, September 22, 2022, 3:00 PM - 4:30 PMObjectiveRecent genome-wide genotype-phenotype association studies (GWAS) of sets of clinical fungal isolates have reported problems resulting from strong phylogenetic clustering (apparent quasi-clonal population structure, difficulties in localizing association signals on chromosomes). We wished to directly investigate such difficulties, for whole-genome sequence data, and corresponding phenotypic data we had generated.MethodsOur recently released Illumina read sequences (Bioproject PRJNA669191) for 29 clinical Colombian isolate genomes of Cryptococcus neoformans var. grubii (28 VNI and 1 VNII as outgroup)12 were assembled and aligned to the annotated H99 VNI reference genome. Single nucleotide polymorphisms (SNPs) were called for the VNI alignment. MLST types, phenotypes, and source metadata of the same isolates12 were integrated with the genomic data. Analyses were interpreted in the light of published literature.ResultsThe 28 VNI isolates were assigned an order considering MLST classification and whole-genome phylogenetic distances. Most SNPs were biallelic, allowing straightforward barcoding: each of the 28 isolates was given a bar if it had the minor allele. Barcode profiles (SNP state vectors) were found repeated numerous times within genes and across entire chromosomes, indicating that neither fine nor ‘rough’ mapping of associations would succeed, except where flagged genes had already been characterized by relevant molecular biology experiments. The same repetitiveness can allow amplicons of individual MLST loci to tag and inform on variation present in other loci in the genome. The gene for the capsule-associated protein CAP59 and its promoter region illustrate such implications at a small (genic) scale.ConclusionsMost associations of phenotypes with fungal genomes from modestly sampled clinical isolate populations cannot be localized to one or a few regions in a genome. This problem is less restrictive in human GWAS studies or (possibly) in studies of recombining fungi sampled from the environment3. It limits the benefits for molecular etiologic understanding that one might expect from GWAS of isolates obtained in clinical contexts, where key fungal phenotypes can be under stronger selection pressure. Explanatory power may be low if one compares just MLST types or phylogenetic clusters4 or examines only variation within individual MLST types. More informative results may be gained by restricting the genomic search space a priori to a subset of genes that are likely to be related to a phenotype, and then zooming in to individual SNPs that are known/suspected to affect protein structure/function1 or influence transcription/translation of the gene. Where the modes of regulation of a key gene are still not completely characterized despite decades of research, such as in CAP59, the presence of a phenotypically monitored, lone SNP in a strategic position within an almost SNP-less region can give potentially precious hints where otherwise none exist; we illustrate this strategy of SNP-focused local exploration for the promoter/enhancer and currently used MLST amplicon of CAP59.Sources:Vélez N, Vega-Vela N, et al. (2022), J Fungi 8(3):245.Vélez N, Monteoliva L, et al. (2022). J Fungi 8(1):57.Whiston E, Taylor JW (2015). G3 6(2):235-244.Felsenstein J (2008). Am Nat. 171(6):713-725.

P083 Susceptibility pattern of fungal isolated from patients with otomycosis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesAntifungal resistance is posing several new concerns to clinicians. Increased rates of azole and echinocandin resistance in various non-albicans Candida species and azole resistance in A. fumigatus may arise due to clinical or environmental exposure to these drugs. The study evaluated the antifungal susceptibility for clinical fungal isolates causing otomycosis.MethodsA total of 89 Aspergillus isolates containing A. niger (58 isolates), A. flavus (19 isolates), A. fumigatus (12 isolates), and 25 Candida isolates containing C. parapsilosis (14 isolates), C. orthopsilosis (6 isolates), and C. albicans (5 isolates) collected from individuals with confirmed otomycosis during October 2020-November 2021 were tested for antifungal susceptibility testing (AFST). AFST of ketoconazole, voriconazole, tioconazole, amphotericin B, miconazole, fluconazole, nystatin, and itraconazole was conducted using the broth microdilution method based on CLSI (M38-A2, M27-A3) protocols. Conidia of molds and colonies of yeasts were harvested from fungal cultures on SDA incubated at 35°C; the turbidity of the suspension was then adjusted to OD630 nm = 80%-82%T for molds and 75%-77%T for yeasts.ResultsMainly, all antifungals examined were effective against most Aspergillus isolates, aside from tioconazole (GM = 5.54767 μg/ml) and nystatin (GM = 2.10151 μg/ml). Terbinafine (GM = 1.69824 μg/ml) had minimal in vitro effects (Table 1). Nystatin (GM = 2.94853 μg/ml) and itraconazole (GM = 1.08673 μg/ml) showed higher GM MICs against all Candida species isolates. Conversely, amphotericin B (GM = 0.07129 μg/ml) in Aspergillus, ketoconazole (GM = 0.02570), and voriconazole (GM = 0.03686 μg/ml) in Candida showed the highest antifungal activity (Table 2). Regarding the CLSI-M59 document for ECV, one A. niger (MIC 8 μg/ml), A. flavus (MIC 2 μg/ml), and A. fumigatus (MIC 2 μg/ml) isolates were non-wild type against itraconazole. A total of 3 A. niger non-wild type isolates with MIC 4 μg/ml against voriconazole were inspected (Table 1). Three C. albicans isolates with high itraconazole MICs (two 8 μg/ml and one 16 μg/ml) were observed (Table 2). Even though the MIC50 of Aspergillus niger for tolnaftate was 0.37 μg/ml, 9 isolates with high MICs (16 μg/ml) were found.ConclusionThe foremost commonest yeast isolates in this study, C. parapsilosis, exhibit significant sensitivity to various antifungals, including ketoconazole, voriconazole, tioconazole, amphotericin B, miconazole, fluconazole, and itraconazole. However, contrary to other studies, nystatin had high MICs and is not recommended as an effective drug. Since the pattern of antifungal susceptibility is varied among the cryptic species of Aspergillus sections, we recommend that physicians request a drug susceptibility testing before antibiotic therapy to prevent the development of resistance.

Occidiofungin: Actin Binding as a Novel Mechanism of Action in an Antifungal Agent.

The identification and development of natural products into novel antimicrobial agents is crucial to combat the rise of multidrug-resistant microorganisms. Clinical fungal isolates have been identified, which have shown resistance to all current clinical antifungals, highlighting a significant need to develop a novel antifungal agent. One of the natural products produced by the bacterium Burkholderia contaminans MS14 is the glycolipopeptide occidiofungin. Occidiofungin has demonstrated in vitro activity against a multitude of fungal species, including multidrug-resistant Candida auris strains, and in vivo effectiveness in treating vulvovaginal candidiasis. Characterization of occidiofungin revealed the mechanism of action as binding to actin to disrupt higher-order actin-mediated functions leading to the induction of apoptosis in fungal cells. Occidiofungin is the first small molecule capable of disrupting higher-order actin functions and is a first-in-class compound that is able to circumvent current antifungal resistant mechanisms by fungal species. Anticancer properties and antiparasitic activities, against Cryptosporidium parvum, have also been demonstrated in vitro. The novel mechanism of action and wide spectrum of activity highlights the potential of occidiofungin to be developed for clinical use.