AUTHOR: EKWEALOR CHITO CLARE
DEPARTMENT: APPLIED MICROBIOLOGY & BREWING
AFFILIATION: NNAMDI AZIKIWE UNIVERSITY, AWKA
The objectives of this study was to isolate and identify organisms involved in skin infections among rice farmers in Ayamelum, Anambra East and Anambra West Local Government Areas of Anambra State, Nigeria, identify some local medicinal plants used by villagers against fungal infections, carry out their phyto-chemical analysis, test the in vitro antifungal activities of these medicinal plants and some selected conventional antifungal drugs against the isolated organisms, screen the isolates for production of some extracellular enzymes and partially characterize the enzymes produced. A total of 2,580 rice farmers were screened for fungal skin infections and clinical specimens of 201 of them with infections were collected and observed by direct microscopy using 20% KOH. The specimens were also cultured on duplicate Sabouraud Dextrose Agar (SDA) plates, supplemented with 0.05mg/ml chloramphenicol and on another set of duplicate SDA plates containing a mixture of 0.05mg/ml chloramphenicol and 0.5mg/ml cyclohexamide. Fungal isolates were studied for their morphological and physiological characteristics. Ten of the isolates were identified using sequencing of the internal transcribed spacer region of the ribosomal DNA by Nationales Konsiliarlabour fur Dermatophyten, Charite Institute fur Microbiologie und Hygiene, Universitatsmedizin, Berlin, Germany. Epidermiological studies were carried out using questionnaires, and statistically analyzed. Isolates were studied for their in vitro susceptibility to four selected conventional antifungal drugs (Ketoconazole, Nystatin, Griseofulvin, Lamisil) at 0.5 – 4mg/disc concentrations. Seven medicinal plants parts (Cassia alata leaf, Cassia alata flower, Solanum nigrum leaf, Solanum nigrum seed, Mitracarpus villosus, Lawsonia inermis, Viscum album) were extracted by soxhlet method using methanol and hexane as solvents. Cold water extraction was also carried out. These extracts were tested at 10-80mg/disc concentrations against the isolated organisms using disc diffusion method. The ability of these organisms to produce carboxymethylcellulase, amylase and xylanase was studied and the effects of temperature and pH on enzyme activity and stability determined. The organisms were also studied for keratinase production. Sixteen species of fungal organisms were isolated in this study, comprising of five species of dermatophytes- Microsporun audouinii five (2.22%), Microsporun ferrugineum two (0.89%), Trichophyton megninii six (2.67%), Trichophyton tonsurans ten (4.44%), Trichophyton rubrum 25 (11.11%), and 11 species of non-dermatophyte molds- Aspergillus terrus 24 (10.67%), Aspergillus candidus 37 (16.44%), Aspergillus sclerotiorum 26 (11.56%), Aspergillus niger 11 (4.89%), Aspergillus flavus 26 (11.56%), Scopulariopsis sp. 18 (8%), Chrysosporium sp. 12 (5.33%), Eupenicillum javanicum two (0.89%), Fusarium sp. 11 (4.89%), Penicillium aculeatum four (1.78%) and Penicillium pinophilum six (2.67%). Infection was found to be non-significant among the studied population. Though there was a correlation between body site and infection, it neither showed any gender relationship nor was it age dependent. Lamisil antifungal drug and methanolic extract of Lawsonia inermis plant inhibited all test organisms at varied concentrations. Aspergillus flavus and Aspergillus terrus, produced the enzymes in very high yields. Effect of temperature on carboxymethylcellulase produced by A. flavus and A. terrus showed maximum activity at ≥ 70 oC and maintained over 70% stability at ≥ 60oC. Maximum activity at 90oC was recorded for xylanase produced by A. terrus. Keratin was degraded, and the significant activity of the enzymes produced, supports the keratin content of the nails and the ability of the fungal isolates to cause infection.
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