Species Identification and Mycotoxigenic Potential of Aspergillus Section Flavi Isolated from Maize Marketed in the Metropolitan Region of Asunción, Paraguay.

Zea mays var. amylacea and Zea mays var. indurata are maize ecotypes from Paraguay. Aspergillus section Flavi is the main spoilage fungus of maize under storage conditions. Due to its large intraspecific genetic variability, the accurate identification of this fungal taxonomic group is difficult. In the present study, potential mycotoxigenic strains of Aspergillus section Flavi isolated from Z. mays var. indurata and Z. mays var. amylacea that are marketed in the metropolitan region of Asunción were identified by a polyphasic approach. Based on morphological characters, 211 isolates were confirmed to belong to Aspergillus section Flavi. A subset of 92 strains was identified as Aspergillus flavus by mass spectrometry MALDI-TOF and the strains were classified by MALDI-TOF MS into chemotypes based on their aflatoxins and cyclopiazonic acid production. According to the partial sequencing of ITS and CaM genes, a representative subset of 38 A. flavus strains was confirmed. Overall, 75 A. flavus strains (86%) were characterized as producers of aflatoxins. The co-occurrence of at least two mycotoxins (AF/ZEA, FUM/ZEA, and AF/ZEA/FUM) was detected for five of the Z. mays samples (63%). Considering the high mycological bioburden and mycotoxin contamination, maize marketed in the metropolitan region of Asunción constitutes a potential risk to food safety and public health and requires control measures.

EVALUACION DEL POTENCIAL AFLATOXIGENICO DE AISLADOS DE Aspergillus flavus EN MODELO DE MAÍZ IN VITRO / Evaluation of aflatoxigenic potential of Aspergillus flavus isolates in maize in vitro

ABSTRACT Aflatoxins are secondary metabolites toxic to human and animal health produced by Aspergillus flavus, and which contaminate food throughout the production chain. Knowing and characterizing the fungal population present in food helps us to estimate risk and design measures to mitigate fungal contamination. Thus, the objective of this work was characterizing the toxigenic potential of Aspergillus isolates from corn in in vitro models. For this purpose, two isolates of Aspergillus flavus (CCM-AS02, CCM-AS29) and one of Aspergillus luchuensis (CCM-AS04) from the CCM-UNA culture collection were used. To evaluate the production of aflatoxins in synthetic culture medium, the isolates were plated on coconut agar and yeast extract agar, and the presence of fluorescence was evaluated under UV light (A= 360 nm). To determine the type of aflatoxin, a thin layer chromatography was performed. Finally, an in vitro infection was carried out with the isolates under study, from avatí-morotí maize, acquired commercially, and the aflatoxin concentration was determined with the Afla-V®-VICAM® lateral fluid rapid immunoassay kit. With the results obtained, it can be concluded that the isolates of A. flavus CCM-AS02 and CCM-AS29 from corn are aflatoxigenic under environmental conditions that simulate natural ones and that coincide with the predominant ones in our country, therefore, it is necessary to create awareness of the risk posed by the contamination of corn grains with Aspergillus and aflatoxins in our region and the need to take preventive measures to control this fungus.

RESUMEN Las aflatoxinas son metabolitos secundarios tóxicos para salud humana y animal producidos por Aspergillus flavus, y que contaminan a los alimentos a lo largo de la cadena productiva. Conocer y caracterizar la población fúngica presente en los alimentos nos sirve para estimar riesgo y diseñar medidas para mitigarlo. Siendo así, el objetivo de este trabajo es caracterizar el potencial toxigénico de aislados de Aspergillus provenientes de maíz en modelos in vitro. Para tal fin, se utilizaron dos aislados de A. flavus (CCM-AS02, CCM-AS29) y uno de Aspergillus luchuensis (CCM-AS04) de la colección de cultivos CCM-UNA. Para evaluar la producción de aflatoxinas en medio de cultivo sintético, se sembraron los aislados en agar coco y agar extracto de levadura y se evaluó la presencia de fluorescencia bajo luz UV (A= 360 nm). Para determinar el tipo de aflatoxina, se realizó cromatografía en capa delgada. Por último, se realizó la infección in vitro con los aislados en estudio, de maíz avatí-morotí, adquirido comercialmente y se determinó la concentración de aflatoxinas con la prueba de inmunoensayo rápido de flujo lateral Afla - V®-VICAM®. Con los resultados obtenidos se puede concluir que los aislados de A. flavus CCM-AS02 y CCM-AS29 provenientes de maíz son aflatoxigénicos en las condiciones ambientales que simulan las naturales y que coinciden con las predominantes en nuestro país, por ello, es necesario crear conciencia del riesgo que representa la contaminación de los granos de maíz con Aspergillus y aflatoxinas y la necesidad de tomar medidas preventivas de control de este hongo.

α-Pinene: Docking Study, Cytotoxicity, Mechanism of Action, and Anti-Biofilm Effect against Candida albicans.

Candida albicans is associated with serious infections in immunocompromised patients. Terpenes are natural-product derivatives, widely studied as antifungal alternatives. In a previous study reported by our group, the antifungal activity of α-pinene against C. albicans was verified; α-pinene presented an MIC between 128-512 µg/mL. In this study, we evaluate time-kill, a mechanism of action using in silico and in vitro tests, anti-biofilm activity against the Candida albicans, and toxicity against human cells (HaCaT). Results from the molecular-docking simulation demonstrated that thymidylate synthase (-52 kcal mol-1), and δ-14-sterol reductase (-44 kcal mol-1) presented the best interactions. Our in vitro results suggest that α-pinene's antifungal activity involves binding to ergosterol in the cellular membrane. In the time-kill assay, the antifungal activity was not time-dependent, and also inhibited biofilm formation, while rupturing up to 88% of existing biofilm. It was non-cytotoxic to human keratinocytes. Our study supports α-pinene as a candidate to treat fungal infections caused by C. albicans.

Antifungal and Antibiofilm Activity of Riparin III against Dermatophytes.

The ability of dermatophytes to develop biofilms is possibly involved in therapeutic failure because biofilms impair drug effectiveness in the infected tissues. Research to find new drugs with antibiofilm activity against dermatophytes is crucial. In this way, riparins, a class of alkaloids that contain an amide group, are promising antifungal compounds. In this study, we evaluated the antifungal and antibiofilm activity of riparin III (RIP3) against Trichophyton rubrum, Microsporum canis, and Nannizzia gypsea strains. We used ciclopirox (CPX) as a positive control. The effects of RIP3 on fungal growth were evaluated by the microdilution technique. The quantification of the biofilm biomass in vitro was assessed by crystal violet, and the biofilm viability was assessed by quantifying the CFU number. The ex vivo model was performed on human nail fragments, which were evaluated by visualization under light microscopy and by quantifying the CFU number (viability). Finally, we evaluated whether RIP3 inhibits sulfite production in T. rubrum. RIP3 inhibited the growth of T. rubrum and M. canis from 128 mg/L and N. gypsea from 256 mg/L. The results showed that RIP3 is a fungicide. Regarding antibiofilm activity, RIP3 inhibited biofilm formation and viability in vitro and ex vivo. Moreover, RIP3 inhibited the secretion of sulfite significantly and was more potent than CPX. In conclusion, the results indicate that RIP3 is a promising antifungal agent against biofilms of dermatophytes and might inhibit sulfite secretion, one relevant virulence factor.

In vitro and ex vivo antibiofilm activity of riparin 1, and its nor and dinor homologs, against dermatophytes.

Dermatophytosis is one of the most frequent superficial mycoses in the world. They are mainly caused by the dermatophytes Trichophyton rubrum and Microsporum canis. Biofilm production is an essential factor in the pathogenesis of dermatophytes; it confers drug resistance and significantly impairs antifungal effectiveness. Therefore, we evaluated the antibiofilm activity of an alkamide-type alkaloid called riparin 1 (RIP1) against clinically relevant dermatophytes. We also produced synthetic nor (NOR1) and dinor (DINOR1) homologs for pharmacological evaluation, with a 61-70% yield. We used in vitro (96-well polystyrene plates) and ex vivo (hair fragments) models to verify the effects of these compounds on the formation and viability of biofilms. RIP1 and NOR1 showed antifungal activity against strains of T. rubrum and M. canis, but DINOR1 showed no significant antifungal activity against the dermatophytes. Furthermore, RIP1 and NOR1 significantly reduced the viability of biofilms in vitro and ex vivo (P < 0.05). RIP1 was more potent than NOR1, possibly due to the distance between the p-methoxyphenyl and the phenylamide moieties in these compounds. Due to the significant antifungal and antibiofilm activities observed for RIP1 and NOR1, we suggest that they could be useful in the treatment of dermatophytosis.

Trichothecene Genotype Profiling of Wheat Fusarium graminearum Species Complex in Paraguay.

Paraguay is a non-traditional wheat-producing country in one of the warmest regions in South America. Fusarium Head Blight (FHB) is a critical disease affecting this crop, caused by the Fusarium graminearum species complex (FGSC). A variety of these species produce trichothecenes, including deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). This study characterized the phylogenetic relationships, and chemotype diversity of 28 strains within FGSC collected from wheat fields across different country regions. Phylogenetic analysis based on the sequence of elongation factor-1α gene (EF-1α) from 28 strains revealed the presence of four species in the FGSC: F. graminearum sensu stricto, F. asiaticum, F. meridionale and F. cortaderiae. Ten strains selected for further analysis revealed that all F. graminearum strains were 15-ADON chemotype, while the two strains of F. meridionale and one strain of F. asiaticum were NIV chemotype. Thus, the 15-ADON chemotype of F. graminearum sensu stricto was predominant within the Fusarium strains isolated in the country. This work is the first report of phylogenetic relationships and chemotype diversity among Fusarium strains which will help understand the population diversity of this pathogen in Paraguay.

Presence of Aflatoxin M1 in Commercial Milk in Paraguay.

ABSTRACT: The presence of aflatoxin M1 (AFM1) in milk is a public health concern because milk is a significant part of human diets worldwide. In economies where AFM1 monitoring is low or nonexistent, the possibility of AFM1 contamination might be increased. Our study was conducted to detect and quantify AFM1 in fluid milk and milk drinks of various brands, fat concentrations, packages, and heat treatments sold in the Metropolitan Area of Asunción, Paraguay. Eighty samples were collected from supermarkets in the Metropolitan Area of Asunción following a nonprobability sampling method. An enzyme-linked immunosorbent assay for AFM1 (25 to 500 ppt) was used to quantify the toxin, and results were analyzed with nonparametric methods. All samples were positive values for AFM1 (above the detection limit of 25 ng/kg); 85% of the samples had 30 to 50 ng/kg, and 15% had >500 ng/kg. No significant difference in AFM1 concentration was found based on fat concentration, heat treatment, or type of packaging of these milk products; however, significant differences were found between brands.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 µm; truncated with protruding hilum and varied in length from 1.0 to 1.5 µm and diameters from 2.0 to 2.2 µm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

Presencia de aflatoxina M1 en fórmulas lácteas infantiles comercializadas en el área metropolitana a Asunción, Paraguay

RESUMEN Introducción: La aflatoxina M1 (AFM1) es un metabolito tóxico derivado de la aflatoxina B. Su ingestión en lactantes se ha relacionado con retraso en el crecimiento, aumento de susceptibilidad a enfermedades infecciosas, reducción de la eficiencia en la inmunización y cirrosis. En Paraguay se recomienda lactancia exclusiva hasta los 6 meses, sin embargo se comercializan diferentes marcas de fórmulas infantiles dirigidas a lactantes menores de 6 meses. Objetivo: Detectar y cuantificar la presencia de AFM1 en fórmulas para lactantes comercializadas en el Área Metropolitana. Materiales y Métodos: Se adquirieron fórmulas fluidas (n=18) y en polvo (n=91) para lactantes de 0 a 12 meses de farmacias y supermercados del Área Metropolitana de Asunción y fueron analizados mediante el ensayo de inmunoafinidad ligado a enzimas (ELISA). Resultados: 9,75% (0 a 6 meses) y 2% (6 a 12 meses) de las fórmulas lácteas en polvo y 100% de las fórmulas fluidas resultaron positivas para AFM1. La mediana de contenido de AFM1 en formulas en polvo fue de 1820 ng/kg y 510 ng/kg en las marcas A y B. En las fórmulas líquidas fue de 31,8 ng/kg y 33,6 ng/kg para las dos marcas analizadas respectivamente, p=0,0001. Conclusiones: Se detectó AFM1 en todas las fórmulas líquidas analizadas, y en el 2 y 9,7% de las fórmulas en polvo de las marcas Ay B respectivamente Los niveles de AFM1 fueron mayores en las fórmulas en polvo.

ABSTRACT Introduction: Aflatoxin M1 (AFM1) is a toxic metabolite derived from aflatoxin B. Its ingestion in infants has been related to growth retardation, increased susceptibility to infectious diseases, reduced immunization efficiency, and cirrhosis. In Paraguay, exclusive breastfeeding is recommended up to 6 months of age, however, different brands of infant formulas targeted at this age range are marketed. Objective: To detect and quantify the presence of AFM1 in infant formulas marketed in the Metropolitan Area. Materials and Methods: Fluid (n = 18) and powder (n = 91) formulas for infants aged 0 to 12 months were purchased from pharmacies and supermarkets in the Metropolitan Area of Asunción and were analyzed using the enzyme-linked immunoaffinity assay (ELISA). Results: 9.75% (0 to 6 months) and 2% (6 to 12 months) of the powdered milk formulas and 100% of the fluid formulas were positive for AFM1. The median content of AFM1 in powder formulas was 1820 ng/kg and 510 ng/kg in brands A and B, while in liquid formulas it was 31.8 ng/kg and 33.6 ng/kg for the two brands analyzed respectively (p = 0.0001). Conclusions: AFM1 was detected in all the liquid formulas analyzed, and in 2 and 9.7% of the powder formulas of brands A and B, respectively. The levels of AFM1 were higher in the powder formulas.

Presencia de micotoxinas en preparados y colados comerciales para lactantes en el área metropolitana de Asunción, Paraguay

Introducción: Las micotoxicosis son enfermedades producidas por micotoxinas, metabolitos secundarios tóxicos producidos por hongos filamentosos. Los lactantes son especialmente susceptibles a este tipo de toxinas debido a la inmadurez anatómica y funcional de sus sistemas digestivo e inmune, lo que se refleja en la relación entre la cantidad de alimento ingerida y su peso. Objetivo: Determinar la presencia de micotoxinas en alimentos para lactantes comercializados en farmacias y supermercados del Área Metropolitana. Materiales y métodos: Se colectaron al azar 66 unidades de productos de seis marcas diferentes de preparados y colados comerciales importados, dulces y salados, de farmacias y supermercados del Área Metropolitana. Posteriormente, fueron analizados mediante el ensayo de inmunoafinidad ligado a enzimas (ELISA). Se realizó el análisis de varianza y la posterior comparación de medias de las concentraciones de micotoxinas mediante la prueba de Tukey (IC= 95%), con el estadístico InfoStat®. Resultados: Las micotoxinas prevalentes fueron aflatoxinas (AF) y Toxina T2, que se presentaron en 39% de las unidades muestrales analizadas, tanto en preparados como en colados. En tercer orden de importancia se encuentra ocratoxina A (OTA), detectada en 18% de las mismas. En cuarto lugar, deoxinivalenol (DON) se detectó en 4% los productos. Se presentaron diferencias significativas entre los preparados y los colados, siendo los colados los que en media presentaron niveles más altos de concentraciones de todas las micotoxinas estudiadas. Conclusiones: Se constataron niveles variables de AF, OTA, T2 y DON en los alimentos para lactantes comercializados en el Área Metropolitana. Los colados presentaron concentraciones más elevadas de micotoxinas en media en todos los productos analizados. Tanto AF como OTA superaron los límites máximos permitidos por las normas internacionales.

Introduction: Mycotoxicoses are diseases caused by mycotoxins, secondary toxic metabolites produced by filamentous fungi. Infants are especially susceptible to this type of toxins due to the anatomical and functional immaturity of their digestive and immune systems, which is related to the amount of food eaten and their weight. Objective: To determine the presence of mycotoxins in foods for infants sold in pharmacies and supermarkets in the Metropolitan Area. Materials and methods: 66 units of products from six different brands of imported commercial sweet and salty preparations and strained foods from pharmacies and supermarkets in the Metropolitan Area were randomly collected. Subsequently, they were analyzed by the enzyme-linked immunoaffinity test (ELISA). The variance analysis and the subsequent comparison of means of mycotoxin concentrations were performed using the Tukey test (95% CI), with the InfoStat® statistic. Results: The prevalent mycotoxins were aflatoxins (AF) and T-2 Toxin, which were present in 39% of the sample units analyzed, both in preparations and in strains. In third order of importance we detected Ochratoxin A (OTA) in 18% of the units. Fourth, deoxinivalenol (DON) products were detected in 4%. There were significant differences between the preparations and the strains, with the strains having the highest concentration levels of all the mycotoxins studied. Conclusions: Variable levels of AF, OTA, T-2 and DON were found in infant foods marketed in the Metropolitan Area. The strains showed a higher average concentrations of mycotoxins in all the products analyzed. Both AF and OTA exceeded the maximum limits allowed by international standards.