Development of a real-time PCR and multiplex PCR assay for the detection and identification of mycotoxigenic fungi in stored maize grains.

This study aimed to identify important mycotoxigenic fungi and accurate detection of mycotoxin in stored maize grains using molecular methods. The current study also optimised the real-time PCR (RT-PCR) assay. The melting curve was established to identify isolated fungal species of Aspergillus (4), Fusarium (3), Penicillium (3), and Alternaria (one). A multiplex polymerase chain reaction (mPCR) technique was developed for the detection and characterisation of mycotoxin producing fungi, mycotoxin metabolic pathway genes, and the determination of eleven mycotoxins in stored maize grains using high-performance liquid chromatography (HPLC). The mPCR results indicated positive signals for potentially mycotoxigenic fungal species tested of Aspergillus, Fusarium, Penicillium, and Alternaria. A protocol for multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was tested to distinguish between free and contaminated, stored maize with aflatoxin B1 (AFB1). The expression pattern of four aflatoxin biosynthetic pathway genes, AFB1 (aflQ, aflP, aflO, and aflD), was a good marker for contaminated, stored maize grains. HPLC analysis showed that maize grain samples were contaminated with mycotoxins, and the concentration was above the detection level. The results indicate that the polyphasic approach might provide a sensitive, rapid, and accurate method for detecting and identifying mycotoxigenic fungal species and mycotoxins in stored maize grains.

An Eco-Friendly Approach Utilizing Green Synthesized Titanium Dioxide Nanoparticles for Leather Conservation against a Fungal Strain, Penicillium expansum AL1, Involved in the Biodeterioration of a Historical Manuscript.

The main hypothesis of the present research is investigating the efficacy of titanium oxide nanoparticles (TiO2-NPs) to prevent the growth of fungal strains when applied on leather under an experimental study. Therefore, fifteen fungal strains were isolated from a deteriorated historical manuscript (papers and leathers) and identified by traditional methods and ITS sequence analysis, including Aspergillus chevalieri (one isolate), A. nidulans (two strains), A. flavus (four strains), A. cristatus (one strain), A. niger (one strain), Paecilomyces fulvus (two strains), Penicillium expansum (two strains), and P. citrinum (two strains). The enzymes cellulase, amylase, pectinase, and gelatinase, which play a crucial role in biodegradation, were highly active in these fungal strains. TiO2-NPs were formed using the cell-free filtrate of the probiotic bacterial strain, Lactobacillus plantarum, and characterized. Data showed that the TiO2-NPs were successfully formed with a spherical shape and anatase phase with sizes of 2-8 nm. Moreover, the EDX analysis revealed that the Ti and O ions occupied the main component with weight percentages of 41.66 and 31.76%, respectively. The in vitro cytotoxicity of TiO2-NPs toward two normal cell lines, WI38 and HFB4, showed a low toxicity effect against normal cells (IC50 = 114.1 ± 8.1µg mL-1 for Wi38, and 237.5 ± 3.5µg mL-1 for HFB4). Therefore, concentrations of 100 µg mL-1 were used to load on prepared leather samples before inoculation with fungal strain P. expansum AL1. The experimental study revealed that the loaded TiO2-NPs have the efficacy to inhibit fungal growth with percentages of 73.2 ± 2.5%, 84.2 ± 1.8%, and 88.8 ± 0.6% after 7, 14, and 21 days, respectively. Also, the analyses including SEM, FTIR-ART, color change, and mechanical properties for leather inoculated with fungal strain AL1 in the absence of NPs showed high damage aspects compared to those inoculated with fungal strains in the presence of TiO2-NPs.

Prevalence, antibiotic profile, virulence determinants, ESBLs, and non-ß-lactam encoding genes of MDR Proteus spp. isolated from infected dogs.

This study investigated the prevalence, antibiogram, virulence, extended-spectrum ß-lactamases (ESBLs), and non-ß-lactam encoding genes of Proteus species isolated from infected dogs in Ismailia province, Egypt. The study was conducted on 70 fecal swabs collected from dogs with diarrhea for bacteriological identification of Proteus spp. The positive isolates were evaluated for antibiotic susceptibility, molecular tests of virulence, ESBLs, and non-ß-lactam encoding genes. Prevalence of Proteus spp. was 35.7% (25/70), including Proteus mirabilis (n = 23) and Proteus vulgaris (n = 2). The Proteus spp. prevalence revealed diversity, higher in males than females, in ages < 12 weeks. Investigation of antimicrobial resistance was found against penicillin and amoxicillin (100%), amoxicillin-clavulanic acid (32%), cephalosporins: cefotaxime and ceftazidime (36%), and monobactam: aztreonam (28%) as ESBLs, in addition to tetracycline (32%) and trimethoprim sulfamethoxazole (100%). The strains retrieved by PCR revealed ureC, zapA, and rsbA virulence genes with variant prevalence as 92%, 60%, and 52%, respectively. In addition, the recovered strains contained ESBL genes with a dramatic variable prevalence of 100%, 92%, 36%, and 32%, to bla TEM, bla SHV, bla CTX-M, and bla OXA-1, respectively, and non ß-lactam encoding genes with a prevalence of 100%, 48%, 44%, 20%, and 12%, to sul1, tetA, intI1, qnrA, and aadA1. Moreover, 28% (7/25) of recovering strains were MDR (multidrug-resistant) up to four classes of antimicrobials, and 48% (12/25) of the examined strains were MDR up to three antimicrobial classes. In conclusion, to the best of our knowledge, our study could be the first report recording MDR Proteus spp. in dogs in Egypt.

Multilocus Sequence Typing and Antifungal Susceptibility of Candida albicans Isolates From Milk and Genital Tract of Dromedary Camel.

Multilocus sequence typing (MLST) was used to study the genetic diversity and population structure of 48 Candida albicans (C. albicans) isolates from the udder or genital tract of apparently healthy or diseased camels. This study aimed also to determine the frequency of C. albicans isolates in the genital tract and udder of healthy or diseased female dromedary camels. A total of 240 mature dromedary camels (230 females and 10 males) were categorized based on the clinical examination of gentile tract and udder into five groups [fertile females (n = 70), infertile females (n = 115), healthy udder (n = 15), mastitis (n = 30), and fertile males (n = 10)]. Swabs were collected from male and female genital tracts of dromedary camels and milk samples were collected from healthy and diseased udders. C. albicans was isolated from 20% of the samples. The frequency of isolation was significantly higher (p < 0.00001) in disease camels (75%) compared with apparently healthy camels (25%). Most of C. albicans was isolated from infertile female genitalia (62.50%) which was significantly higher than that isolated from fertile female genitalia (16.67%). Multilocus sequence (MLS) analysis identified seven different diploid sequence types (DSTs) including DST2, DST50, DST62, DST69, DST124, DST142, and DST144. The most frequently identified DTS was DST69 (13/48) which significantly higher (p ≤ 0.05) than DST2, DST62, and DST124. The frequency of identification of DST50, DST142, and DST 144 was significantly higher (p ≤ 0.05) than DST62. DST62 and DST124 were isolated only from diseased camels. DST62 was isolated only from mastitic milk. DST124 was isolated only from infertile female genitalia. The percentage of DST50 and DST 142 was significantly higher in diseased camels (infertile females) than in the apparently healthy ones (fertile females). DST2 and DST50 were isolated only from female genitalia of apparent health and diseased camels. The C. albicans isolated from diseased camels had significantly higher biofilm formation, hydrophobicity, phospholipase, proteinase, and hemolysin activities compared with the isolates from apparent healthy camels. All isolates were sensitive to amphotericin B, itraconazole, micafungin, posaconazole and voriconazole. In conclusion, the present study represents the first molecular typing of C. albicans in samples isolated from milk and the genital tract of the dromedary camel. MLST is a useful tool for studying the epidemiology and evolution of C. albicans. Early identification of Candida species and attention to Candida virulence factors and their antifungal susceptibility patterns is very important for establishing strategies to control and/or prevent candidiasis by novel therapeutic management. Amphotericin B, itraconazole, micafungin, posaconazole, or voriconazole can be efficient in treatment of candidiasis.

Transcriptome Profiling Reveals Differential Gene Expression of Laccase Genes in Aspergillus terreus KC462061 during Biodegradation of Crude Oil.

Fungal laccases have high catalytic efficiency and are utilized for the removal of crude oil because they oxidize various aliphatic and aromatic hydrocarbons and convert them into harmless compounds or less toxic compounds, thus accelerating the biodegradation potential of crude oil. Laccases are important gene families and the function of laccases genes varied widely based on transcription and function. Biodegradation of crude oil using Aspergillus terreus KC462061 was studied in the current study beside the transcription level of eight laccase (Lcc) genes have participated in biodegradation in the presence of aromatic compounds, and metal ions. Time-course profiles of laccase activity in the presence of crude oil indicated that the five inducers individual or combined have a very positive on laccase activity. In the status of the existence of crude oil, the synergistic effect of Cu-ABTS compound caused an increase in laccase yields up to 22-fold after 10 days than control. The biodegradation efficiencies of A. terreus KC462061 for aliphatic and aromatic hydrocarbons of crude oil were 82.1 ± 0.2% and 77.4 ± 0.6%, respectively. The crude oil biodegradation efficiency was improved by the supplemented Cu-ABTS compound in A. terreus KC462061. Gas chromatography-mass spectrometry was a very accurate tool to demonstrate the biodegradation efficiencies of A. terreus KC462061 for crude oil. Significant differences were observed in the SDS-PAGE of A. terreus KC462061 band intensities of laccase proteins after the addition of five inducers, but the Cu-ABTS compound highly affects very particular laccase electrophoresis. Quantitative real-time polymerase chain reaction (qPCR) was used for the analysis of transcription profile of eight laccase genes in A. terreus KC462061 with a verified reference gene. Cu2+ ions and Cu-ABTS were highly effective for efficient laccase expression profiling, mainly via Lcc11 and 12 transcription induction. The current study will explain the theoretical foundation for laccase transcription in A. terreus KC462061, paving the road for commercialization and usage.