SOURCE:

Faculty: Biosences
Department: Applied Microbiology And Brewing

CONTRIBUTORS:

Ezeadila, J.O;
Oyeka, C.A;
Okoli, I;

ABSTRACT:

Candida species live in small numbers in a healthy vagina as normal flora. About 75% of women generally harbour these yeasts without their causing harm to their hosts. However, sometimes these microorganisms can become pathogenic causing vaginal candidiasis especially in immunocompromised individuals. Also, non-albicans Candida species resistant to many antifungal drugs have been identified. This study was therefore undertaken to evaluate the antifungal resistance pattern among Candida species from high vaginal swabs (HVS) of women attending the Obstetrics and Gynecology Unit of the University of Nigeria teaching Hospital, Ituku/Ozalla, Enugu State and to also evaluate their susceptibility to some medicinal plants. A total of 340 high vaginal swab (HVS) specimens were collected from the women and cultured on Sabouraud Dextrose Agar supplemented with Chloramphenicol (0.05mg/ml.) and incubated at 25°C for 24 hours. Identification was done by Colony Characteristics on SDA, Morphology and colour on Chromogenic Candida Agar, Germ Tube Test, Microscopic Morphology on Dalmau plate culture and matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Fresh leaves of Moringa oleifera, Vernonia amygdalina and Ocimum gratissimum were respectively collected from around the Faculty of Biosciences, Nnamdi Azikiwe University, Awka, Umukwa village in Awka, Anambra State and Nsukka, Enugu state. After, 300g of pulverized leaves of each plant was extracted using ethanol in a Soxhlet extractor. Antifungal susceptibility testing of some drugs (Fluconazole (25µg), Ketoconazole (10µg), Voriconazole (1µg), Nystatin (100Units), Amphotericin B (20µg), Flucytosine (1µg), Clotrimazole (10µg) and Itraconazole (50µg) and the plant extracts was respectively performed by disc diffusion Method and agar well diffusion methods. The combined effect of Fluconazole and each of the plant extract against some of the resistant isolates was carried out by the continuous variation Checkerboard method. Extraction and identification of the resistant genes of the isolates was done by using kits and sequencing. Statistical analysis was done using Chi-square test and analysis of variance (p<0.05). Eighty-eight (88) Candida species were isolated with Candida tropicalis (34) being the most predominant (38.63%) followed by Candida parapsilosis 21 (23.86%), Candida albicans 20 (22.73), Candida krusei (Issatchenkia orientalis) 7 (7.95%) and Candida glabrata 6 (6.82%). The prevalence of vaginal Candida colonization was 24.7%. Women ˃50 years had the highest prevalence of Candida colonization (50%) followed by the age group 18 – 25 years, though the difference was not statistically significant (p = 0.067). While the prevalence was higher amongst the non-pregnant women (28.6%) than the pregnant women (16.0%), women in their first trimester had the highest prevalence of 25.0% followed by those in the second trimester (11.1%) and third trimester (5.0%). The percentages of Candida species resistant to Fluconazole, Ketoconazole, Voriconazole, Nystatin, Amphotericin B, Flucytosine, Clotrimazole and Itraconazole were respectively 52.3, 61.9, 35.2, 44.3, 19.3, 86.4, 34.1 and 45.5%. Candida krusei was the most resistant species with 100% resistance to each of Fluconazole, Ketoconazole and Flucytosine. Candida tropicalis was most susceptible showing 79.4% to Amphotericin B. Extract of Moringa oleifera had the highest inhibition zone diameter (19mm) at 200mg/ml against resistant Candida albicans1, followed by extract of Vernonia amygdalina and Ocimum gratissimum with inhibition zone diameters of 18.67mm and 18mm at 100mg/ml and 200mg/ml respectively. Sixty-two (38.27%) out of the 162 combination ratios of Fluconazole with either of the plant extracts was synergistic. The highest number of synergistic combination ratios (48.15%) was observed in the combination of Fluconazole with M. oleifera while 53.7% of the combination ratios with Ocimum gratissimum had no activity. Analysis of the sequencing results showed six (6) amino acid substitutions (I162T, R163H, E164G, V272D, F273I and K276Q) and (Y105F, L112V, A116D, T119K, S120H and P121L), respectively in Candida albicans1 and Candida tropicalis1 that occurred within the three hot spot regions in ERG11 gene. Based on the findings of the present study, Voriconazole is recommended for vaginal candidiasis especially for infections caused by Fluconazole-resistant Candida species. The ethanolic extracts of the three plants used in this study showed varying degrees of antifungal activities, and should thus, be investigated further for possible use in the formulation of antifungal drugs. This study also suggests further research into the functional consequence of the non synonymous mutations observed in this study as these mutations may serve as markers of resistance to azoles. Such knowledge could be used in the prescription and design of new antifungal drugs which are less prone to resistance.