Characterization of Aspergillus section Flavi associated with stored grains.

Increased frequencies of Aspergillus section Flavi and aflatoxins in cereal grains have been seen in recent years due to changes in climate circumstances, such as high temperatures and drought. To assess the microbiological risks of contamination, it is critical to have a reliable and accurate means of identifying the fungi. The main goal of this study was to characterize Aspergillus species from section Flavi obtained from twenty-three samples of barley and maize grains, gathered from different markets in Qena, Egypt, using morphological and molecular techniques. Twenty-three isolates were chosen, one isolate from each sample; they were identified as A. aflatoxiformans (4 isolates), A. flavus (18), and A. parasiticus (1). The existence of four aflatoxin biosynthesis genes was also investigated in relation to the strains' ability to produce total aflatoxins and aflatoxin B1, focusing on the regulatory gene aflR and the structural genes aflD and aflM. All strains producing aflatoxins were linked to the presence of aflR1 and/or aflR2, except two isolates that exhibited aflatoxins but from which aflR1 or aflR2 were not detected, which may be due to one or more missing or unstudied additional genes involved in aflatoxin production. AflD and aflM genes were amplified by 10 and 9 isolates, respectively. Five samples of barley and maize were contaminated by aflatoxins. Fifteen isolates were positive for producing total aflatoxins in the range of 0.1-240 ppm. Antagonistic activity of Trichoderma viride against A. flavus (F5) was assessed at 31.3%. Trichoderma reduced total aflatoxins in all treated seeds, particularly those subjected to Trichoderma formulation.

Molecular identification of keratinophilic fungi associated with hair scalp and antifungal activity of green-synthesis zinc oxide nanoparticles.

A diverse population of fungi colonizes human hair and skin due to millions of years of functional integration and mutual adaptation. The human body, with its fungal communities, forms a complex entity. Microbial imbalance is promoted by instabilities in the host-mycobiota interaction system, which can be related to the development of various diseases. By morphological and molecular identification, 15 genera comprising 24 species were isolated from 18 scalp samples collected from girls. Yeast-like structures were the most common species in this study; they were recovered from six samples (33.3%). They were represented by five species: Arachniotus ruber; Cosmospora aurantiicola; Cutaneotrichosporon oleaginosum; Geotrichum candidum and Suhomyces tanzawaensis. For the synthesis of zinc oxide nanoparticles (ZnO NPs), an aqueous extract of Zingiber officinale was utilized as a reducing and capping agent. The prepared NPs tested by X-ray diffraction, they had a hexagonal wurtzite structure. Most of the ZnO NPs were spherical, and their diameter was about 38.9 nm using a transmission electron microscope. ZnO NPs of the Fourier-transform infrared spectroscopy spectra were recorded in the range of 400-4000 cm-1 . UV-visible diffuse reflectance spectroscopy showed the 200-800 nm wavelength range. ZnO NPs showed the highest activity against Ambrosiella hartigii Ambh2; Cladosporium cladosporioides Cladcl12; C. cf. cucumerinum Cladcu13; S. tanzawaensis Suht34, with minimal inhibitory concentrations 1.25 × 103 µg/mL on the four isolates.

Phylogeny and pathogenicity of Fusarium solani species complex (FSSC) associated with potato tubers.

Potato (Solanum tuberosum L.) is one of the known five crops cultivated throughout the world after corn, barley, cereals, rice, and wheat, due to its content of high carbohydrates. In developing countries, potatoes are especially had valuable contents as a rich source of starch, vitamins C and B6, and essential amino acids. Fusarium solani species complex (FSSC) is one of the prevalent pathogens of potato, causing dry rot in Upper Egypt. In this study, FSSC were isolated and identified from potato tubers based on the morphological and molecular characteristics. F. solani isolates (187) were isolated from infected and noninfected potato tubers collected from various markets in Upper Egypt. Based on the morphology observations, sequence data from amplifying ß-tubulin, and specific translation elongation factor (TEF-1α) genes, all of the chosen 88 FSSC isolates were grouped into three major groups (F. keratoplasticum, F. falciforme, and F. solani). All the tested FSSC were able to produce amylases. The selected isolates were examined for their pathogenic ability on healthy potato tubers, which exhibited pathogenic effects; with lesions sizes were quite variable. F. solani SVUFs73 showed a highly virulent effect.

New morphological criteria and molecular characterization of black aspergilli aggregate from corn, sorghum and wheat grains.

Corn, sorghum and wheat grains are used as livestock feed in the world. Identification of black aspergilli associated with these grains is necessary to make sure of the safety of the grains because its occurrence is an indicator of mycotoxin production. Forty-five isolates were isolated from the samples collected from Upper Egypt's markets and identified morphologically based on colony color, conidia, stipe and vesicle size and molecularly by using ß-tubulin and calmodulin genes. Isolates were divided into 30 strains of Aspergillus welwitschiae and 15 strains of A. niger. We have found new criteria in the morphological identification of A. welwitschiae as its colony color was black to brown with yellow edge, but in A. niger was black with white edge, also A. welwitschiae sometimes produced finely-to-distinctly roughened brownish conidia on malt extract agar (MEA) media. Thirteen isolates of A. welwitschiae and six of A. niger were recognized as potential producers for ochratoxin A.

Molecular characterization of ochratoxigenic fungi associated with poultry feedstuffs in Saudi Arabia.

Fungal and mycotoxins contamination of food and poultry feeds can occur at each step along the chain from grain production, storage, and processing. A total of 200 samples comprising of mixed poultry feedstuffs (n = 100) and their ingredients (n = 100) were collected from Riyadh, Alhassa, Qassium, and Jeddah cities in Saudi Arabia. These samples were screened for contamination by fungi. Penicillium chrysogenum was the predominant species taking into its account and frequency, respectively, in both mixed poultry feedstuff and barley samples (4,561.9 and 687 fungal colony-forming units (CFU)/g) and (66% and 17%). Moisture content was an important indicator for the count of fungi and ochratoxin A. Ochratoxin analysis of plate cultures was performed by a HPLC technique. Sample of mixed poultry feedstuff which was collected from Jeddah displayed the highest level of ochratoxin (14.8 µg/kg) and moisture content (11.5%). Corn grains samples were highly contaminated by ochratoxin A (450 and 423 µg/kg) and recorded the highest moisture contents (14.1 and 14.5%). Ochratoxin A production in fungal species isolated from mixed poultry feedstuff samples were high with P. verrucosum (5.5 µg/kg) and A. niger (1.1 µg/kg). In sorghum and corn grains, the highest ochratoxins producing species were P. viridicatum (5.9 µg/kg) and A. niger (1.3 µg/kg), respectively. Sixty-three isolates of A. niger were ochratoxigenic, and all of them showed the presence of pks genes using PKS15C-MeT and PKS15KS primer pairs. The detection technique of A. niger in poultry feedstuff samples described in the present study was successfully used as a rapid and specific protocol for early detection of A. niger without cultivation on specific media.

Conventional detection and quantification real-time PCR of the pks-1 gene of Chaetomium globosum.

Chaetomium globosum is known as a potential biocontrol indicator against various soil and seedborne pathogens. Precise data are necessary for population monitoring of C. globosum for its effective use in agriculture. A sequence-characterized amplified region marker has been applied for the detection of this biocontrol agent, which will help to detect C. globosum at the site of its application. Out of 17 isolates of C. globosum, only 8 isolates of C. globosum amplified a monomorphic band of 1,900 bp. C. globosum is known for producing chaetoglobosin A. The pks-1 gene is unique in C. globosum in that it is involved in chaetoglobosin A production, melanin formation, and sporulation. Real-time PCR of pks-1 was used to compare the expressions of the pks-1 gene and chaetoglobosin A biosynthesis and sporulation. It was found that the sporulation of C. globosum was associated with the levels of pks-1 gene expression; Cg2 isolate showed high expression of the pks-1 gene, 41.21%, and also produced a great number of spores and perithecia. The association between the pks-1 gene expression and chaetoglobosin A production was estimated. The Pks-1 gene was expressed by all C. globosum isolates except one isolate, C1, which is another species of Chaetomium. In addition, all C. globosum isolates produced chaetoglobosin A with different concentrations and did not express the same levels of pks-1. This finding may be a result of the solvent type used in the extraction.

The anti-fasciolasis properties of silver nanoparticles produced by Trichoderma harzianum and their improvement of the anti-fasciolasis drug triclabendazole.

Recently, new strains of Fasciola demonstrated drug resistance, which increased the need for new drugs or improvement of the present drugs. Nanotechnology is expected to open some new opportunities to fight and prevent diseases using an atomic scale tailoring of materials. The ability to uncover the structure and function of biosystems at the nanoscale, stimulates research leading to improvement in biology, biotechnology, medicine and healthcare. The size of nanomaterials is similar to that of most biological molecules and structures; therefore, nanomaterials can be useful for both in vivo and in vitro biomedical research and applications. Therefore, this work aimed to isolate fungal strains from Taif soil samples, which have the ability to synthesize silver nanoparticles. The fungus Trichoderma harzianum, when challenged with silver nitrate solution, accumulated silver nanoparticles (AgNBs) on the surface of its cell wall in 72 h. These nanoparticles, dislodged by ultrasonication, showed an absorption peak at 420 nm in a UV-visible spectrum, corresponding to the plasmon resonance of silver nanoparticles. The transmission electron micrographs of dislodged nanoparticles in aqueous solution showed the production of reasonably monodisperse silver nanoparticles (average particle size: 4.66 nm) by the fungus. The percentage of non hatching eggs treated with the Triclabendazole drug was 69.67%, while this percentage increased to 89.67% in combination with drug and AgNPs.

Pathogenicity of some Fusarium species associated with superficial blemishes of potato tubers.

As an organ for reserve and propagation, the tuber grows underground and is in contact with soil-borne microorganisms, making it potentially exposed to blemishes. Therefore, the objective of this study was the possibility of using some modern methods of molecular diagnostics and detection of the presence of fungal contaminants in potato blemishes in Al-Qasim (Saudi Arabia). Polygonal lesions were the most observed blemish type in the collected samples. One hundred and sixty isolates were recovered from different types of blemishes obtained in this study. Fusarium, Penicillium, Ilyonectria, Alternaria and Rhizoctonia were the most common genera collected from different blemish types. Using ITS region sequencing, all collected fungi were identified at the species level. All Fusarium strains collected during this study were used to detect their pathogenicity against potato tubers. This is the first comprehensive report on the identification of major pathogenic fungi isolated from potato tuber blemishes in Saudi Arabia.

Mycobiota and mycotoxins (aflatoxins and ochratoxin) associated with some Saudi date palm fruits.

This study aimed to determine the mycological profile of the retail date fruits distributed in different markets at Taif, Saudi Arabia. The presence of aflatoxins and ochratoxin A was also measured. Twenty-two fungal species belonging to 12 genera were isolated from 50 different date samples. Aspergillus flavus, A. niger, Penicillium chrysogenum, and Rhizopus stolonifer were the most prevalent species among isolated fungi. Eighty isolates of A. flavus and 36 of A. niger were detected for their aflatoxins and ochratoxin production potentials using thin layer chromatography. Toxicity test using Artimia larvae indicated that seven out of 18 A. flavus isolates had aflatoxins potentials, while nine out of 36 isolates of A. niger were ochratoxigenic. The quadruplex polymerase chain reaction using specific primers demonstrated the presence of four genes: nor A, ver 1, omt A, and avf A in seven A. flavus toxigenic isolates. Nine A. niger toxigenic isolates showed positive results for the presence of the PKS gene. In conclusion, the present study highlighted the potential hazards of mycotoxins on human health from consuming raw dates. Rapid molecular detection methods described here might help the food authorities to assure the safety of raw dates distributed in local markets.

Molecular characterization of ochratoxigenic fungi associated with raisins.

Dried grapes (raisin) may carry a significant mycological load contaminated via cultivation, postharvest processing practices, and drying processing. The contamination of raisin with fungi can accelerate spoilage or illness, if pathogens are present. Since raisins are used as food additives in many dishes in Saudi kitchen, there are healthy concerns on the safety of raisins consumed. In this article, the mycological profile of raisins sold in different markets at Jeddah, Kingdom of Saudi Arabia, was studied. The black raisin samples showed high fungal load compared with the white samples. Aspergillus, Alternaria, Cladosporium, Epicoccum Fusarium, and Rhizopus were the most prevalent genera isolated from raisin samples. Among six Aspergillus species isolated in this study, As. carbonarius and As. niger were the most frequently isolated species. Ochratoxin A (OTA) production in raisins was investigated using a combination of chromatographical (thin layer) and molecular (random amplified polymorphic DNA and Multiplex polymerase chain reaction) techniques. The OTA was detected in 70% of the raisin samples. Also, As. carbonarius (14 out of 19 isolates) and As. niger (2 isolates out of 9) were recognized as potential producers for OTA. Also, some molecular markers for detecting the contamination of raisin samples with OTA directly without isolating the producers were tested.

Bacterial load of fresh vegetables and their resistance to the currently used antibiotics in Saudi Arabia.

This study was carried out to describe the bacterial load and the occurrence of some disease-causing enteric bacteria on raw vegetables sold in Saudi markets. The study further aimed to analyze antibiotic resistance rates, production of extended-spectrum beta lactamase, and plasmid carriage among bacterial population of raw vegetables. Results revealed that none of them contained Bacillus cereus, Salmonella, and Escherichia coli O157:H7. However, Staphylococcus aureus and Shigella were detected in 11.8% and 4.4% of the samples, respectively. The bacterial loads ranged from 3 to 8 log(10) CFUg(-1) for aerobic bacteria and 1 to 4 log(10) CFUg(-1) for coliforms as well as Enterobacteriaceae. The isolates exhibited resistance in decreasing order for ampicillin (76.5%), cephalothin (69.5%), trimethoprime-sulfamethoxazole (36.7%), aminoglycosides (21.9%), tetracycline (17.2%), fluoroquinolones (17.2%), amoxycillin-clavulanic acid (13.3%), and chloramphenicol (7.8%). Maximum resistance to extended-spectrum beta-lactam antibiotics occurred in 14.8% of isolates and the production of extended-spectrum beta-lactamase was achieved by 2.3% of isolates. Multiple resistances to four or more antimicrobial agents along with plasmid with varied sizes were documented. These investigations indicate the occurrence of antibiotic resistance and plasmid carriage among bacterial isolates populating raw vegetables.

Molecular characterization of mycobiota and aflatoxin contamination of retail wheat flours from Jeddah markets.

The mycological profile of the retail wheat flour selling in different markets at Jeddah (Kingdom of Saudi Arabia) was studied. The most common genera were Aspergillus (isolated from 70% of the tested samples), Penicillium (30%), Eurotium (14%), and in a lesser extent Fusarium (20%) and Alternaria (18%). Twenty-nine strains of Aspergillus flavus were screened for their ability to produce aflatoxins (AFs). Four strains produced only aflatoxin B1 (AFB1), two strains produced AFB1 and aflatoxin B2, and one strain produced AFB1, aflatoxin G1, and aflatoxin G2. Random amplified polymorphic DNA-polymerase chain reaction technique could not differentiate between toxigenic and nontoxigenic strains of A. flavus. AF regulatory gene was detected in three flour samples and in seven A. flavus isolates.

Antibiotic resistance in Escherichia coli isolated from retail raw chicken meat in Taif, Saudi Arabia.

The present study was carried out to screen and analyze the genetic characteristics of antibiotic resistance in Escherichia coli strains isolated from chicken meat marketed in the local markets of the Taif region in Saudi Arabia. A total of 119 samples were purchased from various supermarkets and examined for bacterial contamination with resistant E. coli. Thirty-seven E. coli isolates were evaluated for their antibiotic susceptibilities and the presence of class 1 integrons and antibiotic resistance genes. Results of antibiograms revealed that E. coli isolates were resistant to one or more of the antibiotics tested. Resistance was most frequently observed against sulphafurazole (89.2%), ampicillin (78.4%), nalidixic acid (70.3%), streptomycin (48.6%), chloramphenicol (32.4%), and gentamicin (24.3%). Fifteen E. coli strains have multidrug resistance phenotypes and harbored at least three antibiotic resistance genes. The bla(TEM) (beta-lactamase) and sul (sulfonamide) resistance encoding genes were detected in all the tested isolates. Polymerase chain reaction screening detected class 1 integrons in all multiresistant E. coli isolates. The present study provides an assessment of the occurrence of multidrug resistance of E. coli from raw chicken meat collected from local markets.

Molecular characterization of Mucor circinelloides and Rhizopus stolonifer strains isolated from some saudi fruits.

Thirteen strains of Mucor circinelloides and nine strains of Rhizopus stolonifer were collected from different Saudi fruits. Also, two strains of M. circinelloides and one strain of R. stolonifer isolated from soil were used in this study to analyze the genetic diversity among these 25 strains. The dendrogram constructed from random amplified polymorphic DNA-polymerase chain reaction results using two primers (V6 and M13) showed no correlation between clustering system and sources of isolation for M. circinelloides strains, while Rhizopus strains clustered according to the sources of isolation. The phylogenetic tree based on the unweighted pair group method with arithmetic average of internal transcribed spacer (ITS) rDNA sequence revealed the variation of the ITS region among Mucor strains as well as Rhizopus strains.

Diversity of keratinophilic fungi on human hairs and nails at four governorates in upper egypt.

The mycobiota of 160 hair and nail samples collected from 4 different governorates in upper Egypt were estimated using soil plate method for isolating keratinophilic and dermatophytic fungi. Twenty-three fungi were recorded on both hair and nail samples collected from the four governorates. Highest fungal diversity (20) was collected from Red Sea samples followed by Qena (18) and Aswan (17) while lowest fungal diversity was recorded from Sohage samples. The common genera were Aphanoascus, Aspergillus, Penicillium, Paecilomyces and Chrysosporium. The most prevalent species belonging to these genera were: A. fulvescens, Aphanoascus sp. A. flavus link, A. flavus var. columnaris, P. chrysogenium. P. lilacinus and C. sulfureum. True dermatophytes such as Nannizzia fulva appeared in 20~30% of the male samples.