Identification and expression of small multidrug resistance transporters in early-branching anaerobic fungi.

Membrane-embedded transporters impart essential functions to cells as they mediate sensing and the uptake and extrusion of nutrients, waste products, and effector molecules. Promiscuous multidrug exporters are implicated in resistance to drugs and antibiotics and are highly relevant for microbial engineers who seek to enhance the tolerance of cell factory strains to hydrophobic bioproducts. Here, we report on the identification of small multidrug resistance (SMR) transporters in early-branching anaerobic fungi (Neocallimastigomycetes). The SMR class of transporters is commonly found in bacteria but has not previously been reported in eukaryotes. In this study, we show that SMR transporters from anaerobic fungi can be produced heterologously in the model yeast Saccharomyces cerevisiae, demonstrating the potential of these proteins as targets for further characterization. The discovery of these novel anaerobic fungal SMR transporters offers a promising path forward to enhance bioproduction from engineered microbial strains.

Unveiling the mechanism of action of acylated temporin L analogues against multidrug-resistant Candida albicans.

The increasing resistance of fungi to conventional antifungal drugs has prompted worldwide the search for new compounds. In this work, we investigated the antifungal properties of acylated Temporin L derivatives, Pent-1B and Dec-1B, against Candida albicans, including the multidrug-resistant strains. Acylated peptides resulted to be active both on reference and clinical strains with MIC values ranging from 6.5 to 26 µM, and they did not show cytotoxicity on human keratinocytes. In addition, we also observed a synergistic or additive effect with voriconazole for peptides Dec-1B and Pent-1B through the checkerboard assay on voriconazole-resistant Candida strains. Moreover, fluorescence-based assays, NMR spectroscopy, and confocal microscopy elucidated a potential membrane-active mechanism, consisting of an initial electrostatic interaction of acylated peptides with fungal membrane, followed by aggregation and insertion into the lipid bilayer and causing membrane perturbation probably through a carpeting effect.

Investigation of a Candida auris outbreak in a skilled nursing facility - Virginia, United States, October 2020-June 2021.

Candida auris, an emerging multi-drug resistant organism, is an urgent public health threat. We report on a C. auris outbreak investigation at a Virginia ventilator skilled nursing facility. During October 2020-June 2021, we identified 28 cases among residents in the ventilator unit. Genomic evidence suggested ≥2 distinct C. auris introductions to the facility. We identified multiple infection and prevention control challenges, highlighting the importance of strengthening multi-drug resistant organism prevention efforts at ventilator skilled nursing facilities.

New Technologies to Diagnose and Treat a Multidrug-Resistant Candida auris: A Patent Review.

Candida auris is responsible for hospital outbreaks worldwide. Some C. auris isolates may show concomitant resistance to azoles, echinocandins, and polyenes, thereby possibly leaving clinicians with few therapeutic options. In addition, this multi-drug-resistant yeast is difficult to identify with conventional methods and has the ability to persist on environmental surfaces causing hospital-acquired infections. The development of new treatment options and tools for identification is critical to control, prevent, and establish an early diagnosis of this emerging pathogen. The aim of this study was to perform a critical patent review to explore and identify the latest advances in therapeutic strategies as well as diagnostic methods for C. auris. A total of 19 patents were identified for a preliminary assessment from the Espacenet database. Three patents were excluded as they were out of focus for this review according to their abstract and/or description. The final selection covered 16 patents, which were surveyed by country, year and classified as treatment or diagnostic methods for C. auris. As noted in the patent reading, in recent years, the interest of academic, government and industry sectors have shown an increasing tendency focused on research and development of new therapeutic molecules and diagnostic methods to combat this emerging pathogen.

Biochemical Analysis of CaurSOD4, a Potential Therapeutic Target for the Emerging Fungal Pathogen Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen. With high mortality rates, there is an urgent need for new antifungals to combat C. auris. Possible antifungal targets include Cu-only superoxide dismutases (SODs), extracellular SODs that are unique to fungi and effectively combat the superoxide burst of host immunity. Cu-only SODs are essential for the virulence of diverse fungal pathogens; however, little is understood about these enzymes in C. auris. We show here that C. auris secretes an enzymatically active Cu-only SOD (CaurSOD4) when cells are starved for Fe, a condition mimicking host environments. Although predicted to attach to cell walls, CaurSOD4 is detected as a soluble extracellular enzyme and can act at a distance to remove superoxide. CaurSOD4 selectively binds Cu and not Zn, and Cu binding is labile compared to bimetallic Cu/Zn SODs. Moreover, CaurSOD4 is susceptible to inhibition by various metal-binding drugs that are without effect on mammalian Cu/Zn SODs. Our studies highlight CaurSOD4 as a potential antifungal target worthy of consideration.

In Vitro Antifungal Activity of Manogepix and Other Antifungal Agents against South African Candida auris Isolates from Bloodstream Infections.

We determined the susceptibility of South African Candida auris bloodstream surveillance isolates to manogepix, a novel antifungal, and several registered antifungal agents. C. auris isolates were submitted to a reference laboratory between 2016 and 2017. Species identification was confirmed by phenotypic methods. We determined MICs for amphotericin B, anidulafungin, caspofungin, micafungin, itraconazole, posaconazole, voriconazole, fluconazole, and flucytosine using Sensititre YeastOne and manogepix using a modified Clinical and Laboratory Standards Institute broth microdilution method. Clade distribution was determined for a subset of isolates using whole-genome sequencing. Of 394 tested isolates, 357 were resistant to at least 1 antifungal class. The manogepix MIC range was 0.002 to 0.06 µg/mL for 335 isolates with fluconazole monoresistance. Nineteen isolates were resistant to both fluconazole and amphotericin B yet still had low manogepix MICs (range, 0.004 to 0.03 µg/mL). Two isolates from the same patient were panresistant but had manogepix MICs of 0.004 µg/mL and 0.008 µg/mL. Comparing MIC50 values, manogepix was >3-fold more potent than azoles, 4-fold more potent than echinocandins, and 9-fold more potent than amphotericin B. Of 84 sequenced isolates, the manogepix MIC range for 70 clade III isolates was 0.002 to 0.031 µg/mL, for 13 clade I isolates was 0.008 to 0.031 µg/mL, and for one clade IV isolate, 0.016 µg/mL. Manogepix exhibited potent activity against all isolates, including those resistant to more than one antifungal agent and in three different clades. These data support manogepix as a promising candidate for treatment of C. auris infections. IMPORTANCE Since C. auris was first detected in South Africa in 2012, health care-associated transmission events and large outbreaks have led to this pathogen accounting for more than 1 in 10 cases of candidemia. A large proportion of South African C. auris isolates are highly resistant to fluconazole but variably resistant to amphotericin B and echinocandins. There is also an emergence of pandrug-resistant C. auris isolates, limiting treatment options. Therefore, the development of new antifungal agents such as fosmanogepix or the use of new combinations of antifungal agents is imperative to the continued effective treatment of C. auris infections. Manogepix, the active moiety of fosmanogepix, has shown excellent activity against C. auris isolates. With the emergence of C. auris isolates that are pandrug-resistant in South Africa, our in vitro susceptibility data support manogepix as a promising new drug candidate for treatment of C. auris and difficult-to-treat C. auris infections.

Reduction of susceptibility to azoles and 5-fluorocytosine and growth acceleration in Candida albicans in glucosuria.

Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia and glucosuria, and is a risk factor for Candida infections. To reveal the potential effects of glucosuria on Candida spp., we investigated their growth and antifungal susceptibilities in normal human urine to which glucose was added. The viable cell numbers of Candida spp. were more than 10 fold higher in the urine added 3000 mg/dL glucose than in plain urine. In antifungal susceptibility, more than 80% of Candida albicans clinical isolates increased minimum inhibitory concentrations of azoles and 5-fluorocytosine with the addition of glucose, and exceeded their breakpoints. In most of the C. albicans clinical isolates, the mRNA expression of the azole resistance genes ERG11, CDR1, CDR2, and MDR1 in the presence of glucose in urine. These observations provide valuable information about the clinical course and therapeutic effects of azoles against C. albicans infections in patients with diabetes mellitus and hyperglucosuria.

A Markerless CRISPR-Mediated System for Genome Editing in Candida auris Reveals a Conserved Role for Cas5 in the Caspofungin Response.

Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide. It can cause life-threatening disseminated infections in humans, with mortality rates upwards of 50%. The molecular mechanisms underlying its multidrug resistance and pathogenic properties are largely unknown. Few methods exist for genome editing in C. auris, all of which rely on selectable markers that limit the number of modifications that can be made. Here, we present a markerless CRISPR/Cas9-mediated genome editing system in C. auris. Using this system, we successfully deleted genes of interest and subsequently reconstituted them at their native loci in isolates across all five C. auris clades. This system also enabled us to introduce precision genome edits to create translational fusions and single point mutations. Using Cas5 as a test case for this system, we discovered a conserved role for Cas5 in the caspofungin response between Candida albicans and C. auris. Overall, the development of a system for precise and facile genome editing in C. auris that can allow edits to be made in a high-throughput manner is a major step forward in improving our understanding of this important human fungal pathogen. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris. Here, we present a markerless genome editing system for C. auris that relies on CRISPR/Cas9 technology and works to modify the genomes of all known C. auris clades. Using this system, we discovered a conserved role for Cas5 in the caspofungin response between C. albicans and C. auris. Overall, the development of a system for facile genome editing in C. auris is a major step forward in improving our understanding of this important human fungal pathogen.

In vitro study on the potential fungicidal effects of atorvastatin in combination with some azole drugs against multidrug resistant Candida albicans.

The resistance of Candida albicans to azole drugs represents a great global challenge. This study investigates the potential fungicidal effects of atorvastatin (ATO) combinations with fluconazole (FLU), itraconazole (ITR), ketoconazole (KET) and voriconazole (VOR) against thirty-four multidrug-resistant (MDR) C. albicans using checkerboard and time-kill methods. Results showed that 94.12% of these isolates were MDR to ≥ two azole drugs, whereas 5.88% of them were susceptible to azole drugs. The tested isolates exhibited high resistance rates to FLU (58.82%), ITR (52.94%), VOR (47.06%) and KET (35.29%), whereas only three representative (8.82%) isolates were resistant to all tested azoles. Remarkably, the inhibition zones of these isolates were increased at least twofold with the presence of ATO, which interacted in a synergistic (FIC index ≤ 0.5) manner with tested azoles. In silico docking study of ATO and the four azole drugs were performed against the Lanosterol 14-alpha demethylase enzyme (ERG11) of C. albicans. Results showed that the mechanism of action of ATO against C. albicans is similar to that of azole compounds, with a docking score (-4.901) lower than azole drugs (≥5.0) due to the formation a single H-bond with Asp 225 and a pi-pi interaction with Thr 229. Importantly, ATO combinations with ITR, VOR and KET achieved fungicidal effects (≥ 3 Log10 cfu/ml reduction) against the representative isolates, whereas a fungistatic effect (≤ 3 Log10 cfu/ml reduction) was observed with FLU combination. Thus, the combination of ATO with azole drugs could be promising options for treating C. albicans infection.

Appraisal of Chitosan-Gum Arabic-Coated Bipolymeric Nanocarriers for Efficient Dye Removal and Eradication of the Plant Pathogen Botrytis cinerea.

The treatment of textile wastewater comprising many dyes as contaminants endures an essential task for environmental remediation. In addition, combating antifungal multidrug resistance (MDR) is an intimidating task, specifically owing to the limited options of alternative drugs with multitarget drug mechanisms. Incorporating natural polymeric biomaterials for drug delivery provides desirable properties for drug molecules, effectively eradicating MDR fungal growth. The current study fabricated the bipolymeric drug delivery system using chitosan-gum arabic-coated liposome 5ID nanoparticles (CS-GA-5ID-LP-NPs). This study focused on improving the solubility and sustained release profile of 5I-1H-indole (5ID). These NPs were characterized and tested mechanically as a dye adsorbent as well as their antifungal potencies against the plant pathogen, Botrytis cinerea. CS-GA-5ID-LP-NPs showed 71.23% congo red dye removal compared to crystal violet and phenol red from water and effectively had an antifungal effect on B. cinerea at 25 µg/mL MIC concentrations. The mechanism of the inhibition of B. cinerea via CS-GA-5ID-LP-NPs was attributed to stabilized microtubule polymerization in silico and in vitro. This study opens a new avenue for designing polymeric NPs as adsorbents and antifungal agents for environmental and agriculture remediation.

Bacterial and fungal growth in sputum cultures from 165 COVID-19 pneumonia patients requiring intubation: evidence for antimicrobial resistance development and analysis of risk factors.

BACKGROUND: Coronavirus SARS-CoV-2 causes COVID-19 illness which can progress to severe pneumonia. Empiric antibacterials are often employed though frequency of bacterial coinfection superinfection is debated and concerns raised about selection of bacterial antimicrobial resistance. We evaluated sputum bacterial and fungal growth from 165 intubated COVID-19 pneumonia patients. Objectives were to determine frequency of culture positivity, risk factors for and outcomes of positive cultures, and timing of antimicrobial resistance development. METHODS: Retrospective reviews were conducted of COVID-19 pneumonia patients requiring intubation admitted to a 1058-bed four community hospital system on the east coast United States, March 1 to May 1, 2020. Length of stay (LOS) was expressed as mean (standard deviation); 95% confidence interval (95% CI) was computed for overall mortality rate using the exact binomial method, and overall mortality was compared across each level of a potential risk factor using a Chi-Square Test of Independence. All tests were two-sided, and significance level was set to 0.05. RESULTS: Average patient age was 68.7 years and LOS 19.9 days. Eighty-three patients (50.3% of total) originated from home, 10 from group homes (6.1% of total), and 72 from nursing facilities (43.6% of total). Mortality was 62.4%, highest for nursing home residents (80.6%). Findings from 253 sputum cultures overall did not suggest acute bacterial or fungal infection in 73 (45%) of 165 individuals sampled within 24 h of intubation. Cultures ≥ 1 week following intubation did grow potential pathogens in 72 (64.9%) of 111 cases with 70.8% consistent with late pneumonia and 29.2% suggesting colonization. Twelve (10.8% of total) of these late post-intubation cultures revealed worsened antimicrobial resistance predominantly in Pseudomonas, Enterobacter, or Staphylococcus aureus. CONCLUSIONS: In severe COVID-19 pneumonia, a radiographic ground glass interstitial pattern and lack of purulent sputum prior to/around the time of intubation correlated with no culture growth or recovery of normal oral flora ± yeast. Discontinuation of empiric antibacterials should be considered in these patients aided by other clinical findings, history of prior antimicrobials, laboratory testing, and overall clinical course. Continuing longterm hospitalisation and antibiotics are associated with sputum cultures reflective of hospital-acquired microbes and increasing antimicrobial resistance. TRIAL REGISTRATION: Not applicable as this was a retrospective chart review study without interventional arm.

Schiff bases of sulphonamides as a new class of antifungal agent against multidrug-resistant Candida auris.

Invasive Candida infections in hospitalized and immunocompromised or critically ill patients have become an important cause of morbidity and mortality. There are increasing reports of multidrug resistance in several Candida species that cause Candidemia, including C. glabrata and C. auris, with limited numbers of antifungal agents available to treat patients with invasive Candida infections. Therefore, there is an urgent need to discover new antifungal agents that work against multidrug-resistant Candida species, particularly C. auris, which has been identified as an emerging global pathogen. In this article, we report a new class of antifungal agents, the Schiff bases of sulphonamides, that show activity against all Candida species tested, with an MIC range of 4-32 µg/ml. Compound 2b showed activity against C. glabrata and a panel of fluconazole-resistant C. auris strains, with MICs of 4-16 µg/ml. The drug-like nature of these Schiff bases offers opportunities to optimize these compounds with medicinal chemistry techniques to obtain more potent analogs that can be progressed toward pre-clinical evaluation.

Activity and Mechanism of Action of Antifungal Peptides from Microorganisms: A Review.

Harmful fungi in nature not only cause diseases in plants, but also fungal infection and poisoning when people and animals eat food derived from crops contaminated with them. Unfortunately, such fungi are becoming increasingly more resistant to traditional synthetic antifungal drugs, which can make prevention and control work increasingly more difficult to achieve. This means they are potentially very harmful to human health and lifestyle. Antifungal peptides are natural substances produced by organisms to defend themselves against harmful fungi. As a result, they have become an important research object to help deal with harmful fungi and overcome their drug resistance. Moreover, they are expected to be developed into new therapeutic drugs against drug-resistant fungi in clinical application. This review focuses on antifungal peptides that have been isolated from bacteria, fungi, and other microorganisms to date. Their antifungal activity and factors affecting it are outlined in terms of their antibacterial spectra and effects. The toxic effects of the antifungal peptides and their common solutions are mentioned. The mechanisms of action of the antifungal peptides are described according to their action pathways. The work provides a useful reference for further clinical research and the development of safe antifungal drugs that have high efficiencies and broad application spectra.

Investigation of culturable human gut mycobiota from the segamat community in Johor, Malaysia.

Although several studies have already been carried out in investigating the general profile of the gut mycobiome across several countries, there has yet to be an officially established baseline of a healthy human gut mycobiome, to the best of our knowledge. Microbial composition within the gastrointestinal tract differ across individuals worldwide, and most human gut fungi studies concentrate specifically on individuals from developed countries or diseased cohorts. The present study is the first culture-dependent community study assessing the prevalence and diversity of gut fungi among different ethnic groups from South East Asia. Samples were obtained from a multi-ethnic semi-rural community from Segamat in southern Malaysia. Faecal samples were screened for culturable fungi and questionnaire data analysis was performed. Culturable fungi were present in 45% of the participants' stool samples. Ethnicity had an impact on fungal prevalence and density in stool samples. The prevalence of resistance to fluconazole, itraconazole, voriconazole and 5-fluorocytosine, from the Segamat community, were 14%, 14%, 11% and 7% respectively. It was found that Jakun individuals had lower levels of antifungal resistance irrespective of the drug tested, and male participants had more fluconazole resistant yeast in their stool samples. Two novel point mutations were identified in the ERG11 gene from one azole resistant Candida glabrata, suggesting a possible cause of the occurrence of antifungal resistant isolates in the participant's faecal sample.

Multidrug Resistance in Mammals and Fungi-From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms.

Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure-function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings.

Genome-Wide Analysis of Experimentally Evolved Candida auris Reveals Multiple Novel Mechanisms of Multidrug Resistance.

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome-wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes, and echinocandins. We show the stepwise accumulation of copy number variations and novel mutations in genes both known and unknown in antifungal drug resistance. Echinocandin resistance was accompanied by a codon deletion in FKS1 hot spot 1 and a substitution in FKS1 "novel" hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1, a segmental duplication of chromosome 1 containing ERG11, and a whole chromosome 5 duplication, which contains TAC1b The latter was associated with increased expression of ERG11, TAC1b, and CDR2 but not CDR1 The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross-resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential for and diversity of MDR development in C. auris, even in a clade until now not associated with MDR (clade II), stressing its clinical importance and the urge for future research.IMPORTANCECandida auris is a recently discovered human fungal pathogen and has shown an alarming potential for developing multi- and pan-resistance toward all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of multidrug resistance (MDR) in C. auris is monitored in vitro Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.

Clinical implications of multidrug-resistant microorganisms and fungi isolated from patients with intra-abdominal infections in the Republic of Korea: a multicenter study.

The purpose of this study was to evaluate the clinical significance of fungi and multidrug-resistant organisms (MDROs) isolated from patients with intra-abdominal infections (IAIs). This multicenter study included consecutive patients admitted for microbiologically proven IAIs at 6 university-affiliated hospitals in South Korea between 2016 and 2018. A total of 1571 patients were enrolled. Multivariable logistic regression analysis revealed that the isolation of MDROs, isolation of Candida spp., underlying renal diseases, Charlson comorbidity score ≥ 3, septic shock, failure to receive a required surgery or invasive intervention, secondary bacteremia due to IAIs, and lower body mass index were found to be independent predictors for 28-day mortality. However, the isolation of Enterococcus spp. was not identified as a significant risk factor. MDROs and Candida spp. were found in 42 (2.7%) and 395 (25.1%), patients respectively. The isolation of MDROs or Candida spp. was a surrogate marker of 28-day mortality.

On the emergence, spread and resistance of Candida auris: host, pathogen and environmental tipping points.

Over a decade ago, a multidrug-resistant nosocomial fungus Candida auris emerged worldwide and has since become a significant challenge for clinicians and microbiologists across the globe. A resilient pathogen, C. auris survives harsh disinfectants, desiccation and high-saline environments. It readily colonizes the inanimate environment, susceptible patients and causes invasive infections that exact a high toll. Prone to misidentification by conventional microbiology techniques, C. auris rapidly acquires multiple genetic determinants that confer multidrug resistance. Whole-genome sequencing has identified four distinct clades of C. auris, and possibly a fifth one, in circulation. Even as our understanding of this formidable pathogen grows, the nearly simultaneous emergence of its distinct clades in different parts of the world, followed by their rapid global spread, remains largely unexplained. We contend that certain host-pathogen-environmental factors have been evolving along adverse trajectories for the last few decades, especially in regions where C. auris originally appeared, until these factors possibly reached a tipping point to compel the evolution, emergence and spread of C. auris. Comparative genomics has helped identify several resistance mechanisms in C. auris that are analogous to those seen in other Candida species, but they fail to fully explain how high-level resistance rapidly develops in this yeast. A better understanding of these unresolved aspects is essential not only for the effective management of C. auris patients, hospital outbreaks and its global spread but also for forecasting and tackling novel resistant pathogens that might emerge in the future. In this review, we discuss the emergence, spread and resistance of C. auris, and propose future investigations to tackle this resilient pathogen.

Multifunctional properties and safety evaluation of lactic acid bacteria and yeasts associated with fermented cereal doughs.

Spontaneous cereal fermentations involve diverse lactic acid bacteria (LAB) and yeasts which may include multifunctional and safe or unsafe strains. This study assessed acidification ability, safety, antifungal activity and free amino acids release ability of LAB and yeasts previously isolated from spontaneously fermented cereal doughs in Benin. Fourteen LAB and thirteen yeast strains were studied in liquid media and/or in a model cereal dough prepared in laboratory conditions. Antifungal activity was assessed against Candida glabrata in liquid medium. Amino acids were determined by pre-column derivatization and separation with reversed-phase HPLC. Antimicrobial susceptibility was analysed by minimum inhibitory concentration determination. The acidification ability was higher for LAB compared to yeast strains. All LAB strains retarded the growth of C. glabrata Cg1 with the highest inhibition recorded for Weissella confusa Wc1 and Wc2. The highest free amino acid content was found in the doughs fermented with Pichia kudriavzevii Pk2 and Pk3. All the LAB strains were susceptible to ampicillin, chloramphenicol, erythromycin, but displayed phenotypic resistance to kanamycin, streptomycin and tetracycline. Positive PCR amplicon of resistance genes were detected in the following cases: 2 LAB strains were positive for kanamycin (aph(3)III), 5 strains were positive for streptomycin (aadA and/or strA and/or strB) and 3 strains were positive for tetracycline (tet (L) and/or tet (M)). For yeasts, most of the P. kudriavzevii strains were resistant to amphotericin B, fluconazole and itraconazole opposite to K. marxianus and Saccharomyces cerevisiae strains which were susceptible. The results obtained are valuable for selecting safe and multifunctional strains for cereal fermentation in West Africa.