Characterization of Yeast Isolated from the Gut Microbiota of Tunisian Children with Autism Spectrum Disorder

Citation: Chamtouri, M.; Merghni, A.; Miranda-Cadena, K.; Sakly, N.; Gaddour, N.; de Los Reyes-Gavilán, C.G.; Mastouri, M.; Eraso, E.; Quindós, G. Characterization of Yeast Isolated from the Gut Microbiota of Tunisian Children with Autism Spectrum Disorder. J. Fungi 2024,10, 730. https://doi.org/10.3390/jof10110730 Academic Editor: Antonella Lupetti Received: 16 September 2024 Revised: 9 October 2024 Accepted: 15 October 2024 Published: 22 October 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Fungi Journal of Article Characterization of Yeast Isolated from the Gut Microbiota of Tunisian Children with Autism Spectrum Disorder Mariem Chamtouri 1,2 , Abderrahmen Merghni 3, Katherine Miranda-Cadena 4, Nabil Sakly 5, Naoufel Gaddour 6, Clara G. de Los Reyes-Gavilán1,7 , Maha Mastouri 2, Elena Eraso 4,* and Guillermo Quindós4 1Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), 33300 Villaviciosa, Spain; [email protected] (M.C.); [email protected] (C.G.d.L.R.-G.) 2Laboratory of Transmissible Diseases and Biologically Active Substances LR99ES27, Faculty of Pharmacy, University of Monastir, Monastir 5000, Tunisia; [email protected] 3 Laboratory of Antimicrobial Resistance LR99ES09, Faculty of Medicine of Tunis, University of Tunis El Manar, Tunis 1068, Tunisia; [email protected] 4Department of Immunology, Microbiology and Parasitology, Faculty of Medicine and Nursing, University of the Basque Country, UPV/EHU, 48080 Bilbao, Spain; [email protected] (K.M.-C.); [email protected] (G.Q.) 5Laboratory of Medical and Molecular Parasitology-Mycology (code LR12ES08), Department of Clinical Biology B, Faculty of Pharmacy, University of Monastir, Monastir 5000, Tunisia; [email protected] 6 Unit of Child Psychiatry, Monastir University Hospital, Monastir 5000, Tunisia; [email protected] 7Diet, Microbiota and Health Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain *Correspondence: [email protected]; Tel.: +34-946-01-83-71 Abstract: Research on the microbiota–gut–brain axis in autism has primarily focused on bacteria, with limited attention to fungi. There is a growing interest in understanding the involvement of fungi, particularly Candida, in patients with autism spectrum disorder. The aim of this study was to assess the prevalence, antifungal susceptibility profiles and virulence factors of Candida isolates from the guts of Tunisian children with autism. Twenty-eight children with autism and forty-six controls were enrolled. Candida isolates from the faecal samples were identified using biochemical and molecular methods; antifungal susceptibility testing was determined by the EUCAST broth microdilution method and virulence factors, including biofilm formation, cell surface hydrophobicity and phospholipase and proteinase activities, were assessed in vitro . As a result, Candida was detected in 13 children with autism (46.4%) and 14 control children (30.4%). Candida albicans was found to be the most common species isolate in the faeces of both groups of children. Antifungal susceptibility profiles showed that one Candida isolate was resistant to amphotericin B and anidulafungin (3.7%), six were resistant to micafungin (22.2%) and five were resistant to fluconazole (18.5%). All Candida isolates were biofilm producers. Of the twenty-seven isolates, only four showed phospholipase activity (14.8%), eight showed aspartyl-proteinase activity (29.6%) and nine were hydrophobic (33.3%). These results highlight the presence of Candida in the guts of children with autism, as well as the ability to express multiple virulence factors and the antifungal resistance, and they emphasize the need for further studies to confirm intestinal Candida colonization and its potential role in autism. Keywords: autism; Candida; gut microbiota; antifungal susceptibility; virulence 1. Introduction Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction and communication, as well as restrictive and repetitive behaviours or interests. ASD is one of the most common childhood mental disorders, J. Fungi 2024,10, 730. https://doi.org/10.3390/jof10110730 https://www.mdpi.com/journal/jof J. Fungi 2024,10, 730 2 of 18 with varying degrees of severity. The most recent global prevalence of autism is estimated at 0.76%, which accounts for around 16% of the global child population [ 1 – 3 ]. The etiopathogenesis of autism remains poorly understood, as this disorder involves genetic abnormalities, dysregulation of the immune system, inflammation, environmental factors and modifications of gut microbiota. Gastrointestinal (GI) symptoms, such as constipation, abdominal pain, flatulence and diarrhoea are quite common in patients with ASD and are strongly associated with the severity of ASD [ 4 – 6 ]. The frequently reported GI problems may be associated with an altered gut microbiota, highlighting a close connection between gut and brain, the so-called “microbiota–gut–brain axis”, a physiological bidirectional network of communication between the gut and the brain [ 7 , 8 ]. Indeed, the gut microbiota could influence the central nervous system through multiple mechanisms, including neural, immune and endocrine ones, and through the production of microbial toxins [ 4 ]. Consistent evidence of microbiota dysbiosis in ASD has been shown in recent years. Despite the heterogeneity of results among studies, most of them observed an increase in the presence of genera such as Clostridium,Sutterella and Faecalibacterium and a lower proportion of Bifidobacterium [ 9 – 11 ]. Furthermore, although several studies have assessed the gut microbiota profile in ASD children, to our knowledge, few studies have analysed the gut mycobiota and its potential role in ASD [4,12]. Some reports showed an increased abundance of Candida species in the faecal samples of children with ASD [ 13 – 15 ]. Candida colonization can cause malabsorption of carbohydrates and minerals, and can release ammonia and toxins, which have been reported to be associated with some autistic behaviour [ 16 , 17 ]. These findings suggest that Candida may be involved in the ASD pathogenesis. Therefore, an extensive understanding of the role of intestinal Candida colonization in ASD development is needed. The present study aimed to determine the prevalence of Candida colonization in the guts of Tunisian children with ASD. In addition, the distribution of Candida species, in vitro antifungal susceptibility of the collected isolates and the production of several virulence factors, such as biofilm formation, cell surface hydrophobicity and proteinase and phospholipase activities, were evaluated. We also compared the clinical data and investigated the association between ASD severity, GI disorders and Candida intestinal colonization among children with ASD and control (CC) groups. 2. Subjects and Methods 2.1. Subjects A total of 28 children with ASD aged between 4 and 10 years old were recruited at the Unit of Child and Adolescent Psychiatry, Department of Psychiatry, Fattouma Bourguiba University Hospital, Monastir, Tunisia between 2019 and 2020. During the same period, 46 age-matched children were enrolled from siblings and children of the general population as a control group. ASD patients were diagnosed according to the diagnostic and statistical manual of Mental Disorders (DSM-5) criteria [ 1 ], the Autism Diagnostic Inventory-Revised (ADI-R) [ 18 ] and the Autism Diagnostic Observation Schedule-2 (ADOS-2) [ 19 ]. The severity of autism was assessed by the childhood autism rating scale (CARS) [ 20 ]. Exclusion criteria included associated pathologies that can lead to risks: neurological disorders not strictly associated with autism, type 1 diabetes, genetic syndromes, coeliac disease, food intolerance or inflammatory bowel disease. Subjects in this study were not treated with antibiotics, antifungal drugs, probiotics and/or prebiotics for at least one month before sampling. The clinical data of children with ASD and control children were collected in Table 1. J. Fungi 2024,10, 730 3 of 18 Table 1. Demographic characteristics and clinical manifestations of Tunisian children from the region of Monastir with ASD (n= 28) and CC group (n= 46). Characteristics ASD Group n(%) CC Group n(%) p-Value Gender 0.08 Male 22 (78.6%) 27 (58.7%) Female 6 (21.4%) 19 (41.3%) Age (mean ±SD) 7.93 ±2.09 7.39 ±2.02 0.119 4–7 years 10 (35.7%) 25 (54.3%) 8–10 years 18 (64.3%) 21 (45.7%) GI disorders 18 (64.3%) 21 (45.7%) 0.119 Constipation 17 (60.7%) 8 (17.4%) 0.000 * Diarrhoea 1 (3.6%) 1 (2.2%) 1.000 Vomiting 1 (3.6%) 0 (0.0%) 0.378 Oesophageal reflux 1 (3.6%) 0 (0.0%) 0.378 Abdominal pain 7 (25.0%) 14 (30.4%) 0.615 CARS score 30–36 (mild to moderate ASD) 11 (39.3%) 37–60 (severe ASD) 17 (60.7%) ASD, autism spectrum disorder; CC, control children; CARS, childhood autism rating scale; (*) indicates significant difference between ASD and CC groups. 2.2. Clinical Diagnosis The DSM-5 is a reference guide for mental health professionals to diagnose, classify and identify mental health disorders. It was published in May 2013 by the American Psychiatric Association (APA) [ 1 ]. The ADI-R is a structured interview administered to parents and/or caregivers of children with suspected ASD. It contains 93 items relating to social interaction, communication and restricted, repetitive and stereotyped behaviours. This exam usually takes about 2 or 3 h to complete [ 18 ]. The ADOS is a standardized protocol for diagnosing ASD. It consists of a series of activities that evaluate communication, reciprocal social interaction, imagination and creativity. The ADOS exam lasts approximately 40 min [ 19 ]. The CARS is a 15-item behavioural rating scale used for assessing the severity of ASD. The CARS score can range between 15 and 60. Children who have a score of less than 30 are in the non-autistic range. Mild to moderate autism is indicated by a score of 30 to 36, whereas severe autism is indicated by a score of 37 to 60 [20]. 2.3. Faecal Sample Collection and Yeast Isolation A total of 74 faecal samples were collected from children in sterile containers by the first investigator and transported to the laboratory for processing on the same day. From each sample, 1 g of faeces was resuspended in 10 mL of 0.9% sterile saline solution and homogenized by vortexing. Ten µ L of this mixture was inoculated on Sabouraud dextrose agar medium with chloramphenicol and incubated aerobically at 37 ◦C for 24–48 h. 2.4. Candida Identification Candida identification was performed by morphologic, biochemical and molecular methods [ 21 ]. Isolated colonies with various morphologies were Gram stained and observed under the microscope. Each colony type was subcultured for purity and stored at − 20 ◦ C for further analysis. Thawed isolates were cultured for 48 h at 37 ◦ C on Candida chromogenic (Condalab, Madrid, Spain) and ChromID Candida (bioMérieux, Craponne, France) agars and identification was carried out considering colony colour and morphology. Colonies that appeared with colours other than green and blue on the selective media used were respectively identified using API ID32C (bioMérieux). All the isolates that grew as green colonies on Candida chromogenic agar and blue colonies on ChromID Candida were tested to distinguish between Candida albicans,Candida dubliniensis and Candida africana by amplification of the hyphal wall protein 1 gene J. Fungi 2024,10, 730 4 of 18 (HWP1) based on the different amplicon sizes—700 bp for C. africana, 569 bp for C. dubliniensis and approximately 941 bp for C. albicans (Figure S1) [ 21 ]. Briefly, the isolates were plated on Sabouraud dextrose agar (Difco, Becton Dickinson, Franklin Lakes, NJ, USA) and incubated overnight at 37 ◦ C. Then, a single colony was cultured in yeast extract peptone dextrose broth (YEPD, Panreac, Barcelona, Spain) and incubated for 48 h at 30 ◦ C in shaking conditions. DNA was extracted from Candida isolates using a DNeasy Ultraclean Microbial Kit (QIAGEN, Germantown, MD, USA) following the manufacturer’s instructions. A Nano Drop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure DNA concentration and purity. Extracted DNA was stored at − 20 ◦ C until use. The HWP1 gene was amplified using the primers CRR-f: 5 ′ -GTTTTTGCAACTTCTCTTTGTA-3 ′ and CRR-r: 5 ′ -ACAGTTGTATCATG TTCAGT-3 ′ and the PCR was performed according to the protocol described by Romeo et al. [ 21 ]. PCR reaction conditions were as follows: denaturation at 95 ◦ C for 5 min, 30 cycles of denaturation at 94 ◦ C for 45 s, primer annealing at 58 ◦ C for 40 s and extension at 72 ◦ C for 55 s, followed by a final extension at 72 ◦ C for 10 min in a C 1000TM Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR products were separated by electrophoresis on a 1.5% agarose gel stained with Gel Red (Biotium, Fremont, CA, USA) and then visualized on a UV transilluminator. A DNA ladder (HyperladderTM 50 bp [50–2000 bp]; Bioline, London, UK) was used as a molecular weight standard. DNA of C. albicans NCPF 3153, C. dubliniensis NCPF 3949 and C. africana ATCC MYA-2669 were included as positive controls. 2.5. In Vitro Antifungal Susceptibility Testing The activity of six antifungal drugs against all Candida isolates were tested: amphotericin B (Sigma-Aldrich, Madrid, Spain), micafungin (Astellas Pharma Inc., Tokyo, Japan), anidulafungin (Pfizer SA, Madrid, Spain), fluconazole (Pfizer SA), isavuconazole (Basilea Pharmaceutica International, Allschwil, Switzerland) and ibrexafungerp (Scynexis Inc., Jersey City, NJ, USA). Stock solutions of each drug were prepared in dimethyl sulfoxide (DMSO) and stored at − 80 ◦ C until use. The final drug concentration ranged from 0.008 to 4 mg/L for amphotericin B, anidulafungin, micafungin and isavuconazole, from 0.125 to 64 mg/L for fluconazole and from 0.016 to 16 mg/L for ibrexafungerp. The minimal inhibitory concentrations (MICs) of the antifungal drugs were determined by broth microdilution method in 96-well flat-bottom microtiter plates, in RPMI 1640 medium, according to EUCAST guidelines [ 22 ]. Prior to the experiment, an inoculum was prepared in sterile distilled water for each isolate obtained from an overnight culture at 37 ◦ C. The final inoculum, between 0.5–2.5 × 10 5 CFU/mL, was added to the microtiter plates. The plates were then incubated at 37 ◦ C for 24 h and the absorbance was measured at a wavelength of 450 nm using an Infinite F50 spectrophotometer (Tecan, Männedorf, Switzerland ). Two reference strains Candida krusei ATCC 6258 (currently Pichia kudriavzevii) and Candida parapsilosis ATCC 22019 were used as quality controls. MICs were read at 24 h and defined as the antifungal concentration that inhibited at least 50% of Candida growth, except for amphotericin B, for which the MIC evaluated 90% of growth inhibition. For each species and antifungal, descriptive statistics, including MIC ranges and geometric mean MICs (GM), were calculated. The interpretation of susceptibility was performed according the EUCAST clinical breakpoints [ 23 ]. No interpretive breakpoints are available for isavuconazole and ibrexafungerp. 2.6. Biofilm Development Prior to the experiment, Candida strains were inoculated in YEPD broth and were incubated overnight under orbital shaking 120 rpm at 30 ◦ C. Cells were harvested and washed three times with sterile phosphate buffered saline solution (PBS, Sigma-Aldric). After cell counting by microscopy using a Burker haemocytometer, cell suspensions of each Candida isolate were adjusted to a final concentration of 10 6 cells/mL, with RPMI 1640 medium supplemented with L-glutamine and buffered with morpholine propanesulfonic acid (MOPS, Sigma-Aldrich) to pH 7. Candida biofilm formation was performed on J. Fungi 2024,10, 730 5 of 18 sterile, flat-bottomed honeycomb 100-well microtiter plates (Labsystems, Vantaa, Finland). One hundred µ L of each adjusted cell suspension was transferred into the wells of the plate. The microtiter plates were then incubated at 37 ◦ C. After 24 h and 48 h, the wells were washed with 100 µL of sterile PBS to remove unattached and weakly attached cells [24]. Biofilm biomass was quantified using crystal violet (CV) staining [ 25 ]. After washing, the biofilms were dried at room temperature for 30 min, then a volume of 100 µ L of 0.4% CV solution (Merck, Darmstadt, Germany) was added to each well and stained for 20 min. Afterwards, the excess CV was removed by rinsing the plates twice with 250 µ L of sterile distilled water. Finally, the bound CV was released by adding 150 µ L of 33% acetic acid. Biofilm metabolic activity was measured using a colorimetric method based on the reduction of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2Htetrazolium hydroxide (XTT, Sigma-Aldrich) [ 26 ]. Briefly, XTT was prepared as a saturated solution at a concentration of 0.5 g/L in Ringer’s lactate. The solution was filter-sterilized using a 0.22 µ m-pore-size filter (Sarstedt, Nümbrecht, Germany) and aliquots were stored at − 70 ◦ C until use. Before each assay, 100 µ L of an aliquot of XTT mixed with 1 µ M menadione was added to each well and the microplates were incubated in the dark at 37 ◦ C for 2 h. Absorbances were measured at two time points, 24 and 48 h, using a BioScreen C MBR microplate reader (Growth Curves Ltd., Turku, Finland) at a wavelength of 600 nm for biofilm biomass and 492 nm for biofilm metabolic activity. Two independent experiments were performed with five replicates for each condition, and the reference strains C. albicans SC5314 and C. albicans Ca2 hypha-defective mutant (graciously donated by Professor Antonio Cassone, Instituto Superiore di Sanità, Rome, Italy) were included as positive and negative controls, respectively. 2.7. Cell Surface Hydrophobicity Assay Cell surface hydrophobicity (CSH) was determined using the microbial adhesion to hydrocarbon (MATH) test [ 27 , 28 ]. Briefly, the yeast cells grown overnight at 30 ◦ C in YEPD broth were washed twice with sterile PBS and resuspended in the same buffer to adjust an absorbance between 0.4 and 0.5 at 600 nm (A0). Three mL of this yeast cell suspension was overlaid with 0.4 mL of n-hexadecane (Sigma-Aldrich). After vigorous vortexing, aqueous and organic phases were allowed to separate for 10 min at 30 ◦ C and the absorbance of the aqueous phase was measured at 600 nm (A1). The percentage of hydrophobicity was calculated according to the following equation: percentage of CSH = [1 −(A1/A0)] ×100 . The highly hydrophobic strains exhibited CSH values of more than 50%, and the moderately hydrophobic strains had CSH values ranging between 20 and 50%. Hydrophilic strains had CSH values of less than 20% [29]. 2.8. Determination of Phospholipase and Proteinase Activity Isolates were grown on Sabouraud dextrose agar plates overnight at 37 ◦ C and cells were then suspended in sterile saline solution to a final suspension of 10 7 cells/mL. Ten µ L of this suspension was inoculated on each specific medium. Phospholipase activity was evaluated following the method described by Polak [ 30 ], using malt agar plates supplemented with 1 M NaCl, 5 mM CaCl 2 and 8% sterile egg yolk emulsion [ 31 ]. The strain C. albicans UPV/EHU 04-125 was included as positive control. Aspartyl proteinase activity was assessed using bovine serum albumin (BSA) agar plates [ 32 ]. Briefly, the BSA medium consisted of 1.17% yeast carbon base (Difco), 0.01% yeast extract (Condalab) and 0.2% BSA (Sigma-Aldrich). The medium was sterilized by filtration and mixed with a stock solution of autoclaved 2% bacto-agar (Difco). C. dubliniensis UPV/EHU 00-134 was used as positive control [ 33 ]. After inoculation, the plates were incubated at 37 ◦ C for 6 days. The phospholipase activity (Pz) value was determined as the ratio of the diameter of the colony to the total diameter of the colony plus precipitation zone and was classified as follows: 0.35–0.5 (high producers); 0.51–0.74 (moderate producers); 0.75–0.9 (low producers) and 1 (non-producers). The aspartyl-proteinase activity was es- J. Fungi 2024,10, 730 6 of 18 tablished as the diameter of a transparent halo around growing colonies. Candida isolates were classified as non-producers (when no visible halo was present), moderate producers (when the diameter of the halo was 1–2 mm) and high producers (when the diameter of the halo was >2 mm) [33]. Each isolate was assayed in triplicate. 2.9. Statistical Analysis Statistical analysis of quantitative and qualitative data, including descriptive statistics, was performed. Frequencies comparison of categorical data was performed with Chisquare test or Fisher’s exact test. The intergroup differences of continuous data were conducted by Student’s test when data showed a normal distribution and Mann–Whitney nonparametric test when data did not show a normal distribution. All tests were two sided and a p-value < 0.05 was considered as statistically significant. Data were analysed using SPSS software (version 26) and figures were constructed using GraphPad Prism (version 8.0.2). 3. Results 3.1. Clinical Characteristics of the Study Population As shown in Table 1, there were no statistically significant differences in age and gender between the ASD and CC groups. Moreover, GI disorders did not significantly differ when comparing both groups, except for constipation: constipation was more frequent in the ASD group than the CC group (60.7% vs. 17.4%, respectively, p= 0.000). 3.2. Association between ASD Severity and Clinical Data in the ASD Group According to the CARS score, children with ASD were divided into two groups: children with mild–moderate ASD (n= 11) and children with severe ASD (n= 17). We found significant associations between GI disorders and ASD severity (p= 0.020): a higher percentage of children suffering from severe ASD (82.4%) had GI disorders. In contrast, only 36.4% of children with mild to moderate autism presented GI disorders (Figure 1). Constipation was also associated with ASD severity (p= 0.006). Children with severe ASD were more likely to present constipation than children with mild to moderate ASD (82.4% vs. 27.3%) (Figure 1). No significant differences were observed between ASD severity and other clinical parameters. J. Fungi 2024, 10, x FOR PEER REVIEW 6 of 19 extract (Condalab) and 0.2% BSA (Sigma-Aldrich). The medium was sterilized by filtration and mixed with a stock solution of autoclaved 2% bacto-agar (Difco). C. dubliniensis UPV/EHU 00-134 was used as positive control [33]. After inoculation, the plates were incubated at 37 °C for 6 days. The phospholipase activity (Pz) value was determined as the ratio of the diameter of the colony to the total diameter of the colony plus precipitation zone and was classified as follows: 0.35–0.5 (high producers); 0.51–0.74 (moderate producers); 0.75–0.9 (low producers) and 1 (non-producers). The aspartyl-proteinase activity was established as the diameter of a transparent halo around growing colonies. Candida isolates were classified as non-producers (when no visible halo was present), moderate producers (when the diameter of the halo was 1–2 mm) and high producers (when the diameter of the halo was >2 mm) [33]. Each isolate was assayed in triplicate. 2.9. Statistical Analysis Statistical analysis of quantitative and qualitative data, including descriptive statistics, was performed. Frequencies comparison of categorical data was performed with Chisquare test or Fisher’s exact test. The intergroup differences of continuous data were conducted by Student’s test when data showed a normal distribution and Mann–Whitney nonparametric test when data did not show a normal distribution. All tests were two sided and a p-value < 0.05 was considered as statistically significant. Data were analysed using SPSS software (version 26) and figures were constructed using GraphPad Prism (version 8.0.2). 3. Results 3.1. Clinical Characteristics of the Study Population As shown in Table 1, there were no statistically significant differences in age and gender between the ASD and CC groups. Moreover, GI disorders did not significantly differ when comparing both groups, except for constipation: constipation was more frequent in the ASD group than the CC group (60.7% vs. 17.4%, respectively, p = 0.000). 3.2. Association between ASD Severity and Clinical Data in the ASD Group According to the CARS score, children with ASD were divided into two groups: children with mild–moderate ASD (n = 11) and children with severe ASD (n = 17). We found significant associations between GI disorders and ASD severity (p = 0.020): a higher percentage of children suffering from severe ASD (82.4%) had GI disorders. In contrast, only 36.4% of children with mild to moderate autism presented GI disorders (Figure 1). Constipation was also associated with ASD severity (p = 0.006). Children with severe ASD were more likely to present constipation than children with mild to moderate ASD (82.4% vs. 27.3%) (Figure 1). No significant differences were observed between ASD severity and other clinical parameters. Figure 1. Presence of GI disorders and constipation in children suffering from mild to moderate ASD and severe ASD. GI, gastrointestinal; ASD, autism spectrum disorder. Asterisks (*) indicate significant differences between the two ASD subgroups: mild to moderate and severe. Figure 1. Presence of GI disorders and constipation in children suffering from mild to moderate ASD and severe ASD. GI, gastrointestinal; ASD, autism spectrum disorder. Asterisks (*) indicate significant differences between the two ASD subgroups: mild to moderate and severe. 3.3. Prevalence of Candida in the Gut Microbiota of Children with ASD and Control Children Candida was detected in 13 children with ASD (13 out of 28, 46.4%) and in 14 control children (14 out of 46, 30.4%) (Figure 2, Table S1). The difference in Candida prevalence between the two groups of children was not statistically significant (p= 0.166). The species C. albicans was the most frequent in faecal samples of both ASD children (n= 7 out of 13 isolates; 53.8%) and the CC group (n= 10 out of 14 isolates; 71.4%). The most common non-C. albicans species detected in the stools of the group with ASD were Candida glabrata J. Fungi 2024,10, 730 7 of 18 (currently Nakaseomyces glabratus;n= 2; 15.4%), C. parapsilosis (n= 2; 15.4%), C. dubliniensis (n= 1; 7.7%) and Candida guilliermondii (currently Meyerozyma guilliermondii;n= 1; 7.7%). As for the control group, the identification of non-C. albicans species yielded detection of C. glabrata (n= 2; 14.3%), C. dubliniensis (n= 1; 7.1%) and C. krusei (n= 1; 7.1%). There were no significant differences in the distribution of Candida species or in the diversity of these species between children with ASD and controls (p= 0.480). No cultures with more than one species were detected. J. Fungi 2024, 10, x FOR PEER REVIEW 7 of 19 3.3. Prevalence of Candida in the Gut Microbiota of Children with ASD and Control Children Candida was detected in 13 children with ASD (13 out of 28, 46.4%) and in 14 control children (14 out of 46, 30.4%) (Figure 2, Table S1 Online resource 2). The difference in Candida prevalence between the two groups of children was not statistically significant (p = 0.166). The species C. albicans was the most frequent in faecal samples of both ASD children (n = 7 out of 13 isolates; 53.8%) and the CC group (n = 10 out of 14 isolates; 71.4%). The most common non-C. albicans species detected in the stools of the group with ASD were Candida glabrata (currently Nakaseomyces glabratus; n = 2; 15.4%), C. parapsilosis (n = 2; 15.4%), C. dubliniensis (n = 1; 7.7%) and Candida guilliermondii (currently Meyerozyma guilliermondii; n = 1; 7.7%). As for the control group, the identification of non-C. albicans species yielded detection of C. glabrata (n = 2; 14.3%), C. dubliniensis (n = 1; 7.1%) and C. krusei (n = 1; 7.1%). There were no significant differences in the distribution of Candida species or in the diversity of these species between children with ASD and controls (p = 0.480). No cultures with more than one species were detected. Figure 2. Prevalence of Candida in stools of children with ASD and CC group. ASD, autism spectrum disorder; CC, control children. C. glabrata (n = 2), C. parapsilosis (n = 2), C. dubliniensis (n = 1) and C. guilliermondii (n = 1) were non-C. albicans species isolated in stools of children with ASD. C. glabrata (n = 2), C. dubliniensis (n = 1) and C. krusei (n = 1) were non-C. albicans species isolated in stools of the control children group. 3.4. Association Between Candida Gut Colonization and the Clinical Data of the ASD Group In the group of children with ASD, there were no statistically significant differences between faecal specimens with positive or negative Candida cultures regarding their gender or age. Similarly, no statistically significant differences in Candida presence were observed between ASD children with or without GI disorders and with or without severe ASD (Table S2, Online resource 3). 3.5. Antifungal Susceptibility Profile of Candida Isolates The activities of the six antifungal agents tested against the Candida isolates are presented in Table 2. In the group of children suffering from ASD, all C. albicans (n = 7) were susceptible to amphotericin B, one isolate was resistant to anidulafungin, two were resistant to micafungin and four were resistant to fluconazole. C. glabrata isolates (n = 2) were resistant to micafungin, susceptible—dose dependent—to fluconazole and susceptible to amphotericin B and anidulafungin. The two isolates of C. parapsilosis were susceptible to anidulafungin, micafungin and fluconazole and one isolate was found to be amphotericin B resistant. The isolate of C. dubliniensis was susceptible to amphotericin B and fluconazole. Figure 2. Prevalence of Candida in stools of children with ASD and CC group. ASD, autism spectrum disorder; CC, control children. C. glabrata (n= 2), C. parapsilosis (n= 2), C. dubliniensis (n= 1) and C. guilliermondii (n= 1) were non-C. albicans species isolated in stools of children with ASD. C. glabrata ( n= 2 ), C. dubliniensis (n= 1) and C. krusei (n= 1) were non-C. albicans species isolated in stools of the control children group. 3.4. Association Between Candida Gut Colonization and the Clinical Data of the ASD Group In the group of children with ASD, there were no statistically significant differences between faecal specimens with positive or negative Candida cultures regarding their gender or age. Similarly, no statistically significant differences in Candida presence were observed between ASD children with or without GI disorders and with or without severe ASD (Table S2). 3.5. Antifungal Susceptibility Profile of Candida Isolates The activities of the six antifungal agents tested against the Candida isolates are presented in Table 2. In the group of children suffering from ASD, all C. albicans (n= 7) were susceptible to amphotericin B, one isolate was resistant to anidulafungin, two were resistant to micafungin and four were resistant to fluconazole. C. glabrata isolates (n= 2) were resistant to micafungin, susceptible—dose dependent—to fluconazole and susceptible to amphotericin B and anidulafungin. The two isolates of C. parapsilosis were susceptible to anidulafungin, micafungin and fluconazole and one isolate was found to be amphotericin B resistant. The isolate of C. dubliniensis was susceptible to amphotericin B and fluconazole. In the CC group, all C. albicans isolates (n= 10) were susceptible to amphotericin B, anidulafungin, micafungin and fluconazole. Two C. glabrata isolates were susceptible to amphotericin B and anidulafungin and resistant to micafungin; one isolate was fluconazole resistant, and one was susceptible, dose dependent. The C. dubliniensis isolate was susceptible to amphotericin B and fluconazole and the C. krusei isolate was susceptible to amphotericin B and anidulafungin. Furthermore, low MIC values were found for isavuconazole and they ranged from 0.008 to 0.03 mg/L for C. albicans and from 0.008 to 1 mg/L for non-C. albicans. MICs of ibrexafungerp against Candida isolates were also tested in vitro , ranging from 0.016 to 1 mg/L. The lowest ibrexafungerp MICs were observed against C. albicans isolates (GM 0.02 mg/L, MIC range 0.016–0.03 mg/L) and the highest were obtained for the C. guilliermondii isolate (MIC = 1 mg/L). Adopting wild-type upper limits recently proposed by Quindós et al. [ 34 ] for C. albicans (0.5 mg/L), C. glabrata (1 mg/L), C. parapsilosis (2 mg/L), J. Fungi 2024,10, 730 8 of 18 and for C. krusei (4 mg/L), no non-wild-type phenotype for ibrexafungerp was observed, and all isolates were considered susceptible to this antifungal drug. J. Fungi 2024,10, 730 9 of 18 Table 2. In vitro activities of amphotericin B, anidulafungin, micafungin, fluconazole, isavuconazole and ibrexafungerp against Candida isolated from the faecal samples of children with autism and control children. Antifungal Drugs Species (n) Children with Autism Control Children MIC (mg/L) Isolates n(%) MIC (mg/L) Isolates n(%) Range MIC GM Mean S I R Range MIC GM Mean S I R Amphotericin B Candida albicans (17) 0.03–0.125 0.16 7 (41.2) 0 0 0.06–0.5 0.16 10 (58.8) 0 0 Candida glabrata (4) 0.125–0.25 0.18 2 (50) 0 0 0.125–0.25 0.18 2 (50) 0 0 Candida parapsilosis (2) 0.125–4 2.06 1 (50) 0 1 (50) Candida dubliniensis (2) 0.03 0.03 1 (50) 0 0 0.03 0.03 1 (50) 0 0 Candida guilliermondii (1) 0.125 0.12 ND ND ND Candida krusei (1) 0.25 0.25 1 (100) 0 0 Anidulafungin Candida albicans (17) 0.008–4 0.58 6 (35.3) 0 1 (5.8) 0.008–0.03 0.02 10 (58.8) 0 0 Candida glabrata (4) 0.06 0.06 2 (50) 0 0 0.03–0.06 0.05 2 (50) 0 0 Candida parapsilosis (2) 0.5–4 2.25 2 (100) 0 0 Candida dubliniensis (2) 0.03 0.03 ND ND ND 0.03 0.03 ND ND ND Candida guilliermondii (1) 0.06 0.06 ND ND ND Candida krusei (1) 0.03 0.03 1 (100) 0 0 Micafungin Candida albicans (17) 0.03–4 0.59 5 (29.4) 0 2 (11.8) 0.016–0.03 0.03 10 (58.8) 0 0 Candida glabrata (4) 0.06–0.125 0.09 0 0 2 (50) 0.06–0.125 0.09 0 0 2 (50) Candida parapsilosis (2) 0.008–1 0.5 2 (100) Candida dubliniensis (2) 0.06 0.06 ND ND ND 0.06 0.06 ND ND ND Candida guilliermondii (1) 0.06 0.06 ND ND ND Candida krusei (1) 0.06 0.06 ND ND ND Isavuconazole Candida albicans (17) 0.008–0.016 0.01 ND ND ND 0.008–0.03 0.02 ND ND ND Candida glabrata (4) 0.25–1 0.63 ND ND ND 0.5–1 0.75 ND ND ND Candida parapsilosis (2) 0.016–0.03 0.02 ND ND ND Candida dubliniensis (2) 0.008 0.008 ND ND ND 0.016 0.016 ND ND ND Candida guilliermondii (1) 0.125 0.125 ND ND ND Candida krusei (1) 1 1 ND ND ND Fluconazole Candida albicans (17) 0.125–64 25.19 3 (16.6) 0 4 (23.5) 0.125–0.25 0.162 10 (58.9) 0 0 Candida glabrata (4) 4–8 6 0 2 (50) 0 4–64 34 0 1 (25) 1 (25) Candida parapsilosis (2) 0.5 0.5 2 (100) 0 0 Candida dubliniensis (2) 0.125 0.125 1 (50) 0 0 0.25 0.25 1 (50) 0 0 Candida guilliermondii (1) 1 1 ND ND ND Candida krusei (1) 32 32 ND ND ND J. 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