Bactéries sporulantes dans quelques biotopes aquatiques à Yaoundé (Région du centre, Cameroun) : diversité, impact des facteurs abiotiques et susceptibilité à des antibiotiques/extraits de Moringa oleifera

Abstract

The present study aimed to evaluate the diversity, abundance dynamics, impact of abiotic factors and susceptibility to some antimicrobials of spore-forming bacteria isolated from surface water, groundwater and rainwater in Yaoundé (Cameroon, Central Africa). Water samples were collected at a monthly frequency for 13 months, from March 2021 to March 2022. A total of 15 sampling points were selected and distributed as follows: 6 surface water points, 6 groundwater points (well water) and 3 rainwater harvesting stations. The analyses focused on the isolation of spore forming bacteria by the classic method of surface spreading on specific culture media, followed by their identification by conventional biochemical tests and on API 20E galleries. The abiotic parameters of the water analysed were temperature, pH, electrical conductivity, nitrate and orthophosphate contents and TDS, among others. Antibiotic susceptibility testing (Amoxicillin, Ampicillin, Imipenem, Gentamicin, Amikacin, Ciprofloxacin, Ofloxacin, Sulfamethoxazole, Tetracycline) was then performed by the disc diffusion methods on Müller-Hinton agar, as well as the susceptibility of these spore-forming bacteria to aqueous and hydroethanolic extracts of Moringa oleifera leaves. The concentrations of extracts were 0.5; 1; 1.5 and 2g/L. Incubation temperatures were 23°C and 37°C, and incubation times were 3 hours, 6 hours, 9 hours and 24 hours. Chemical analyses of these M. oleifera leaves were carried out by HPLC and mass spectrometry. The major chemical compounds revealed by the mass spectrum profile have been determined. Bacteriological analyses show that these waters host a spore-forming bacterial microflora consisting of species of the genera Bacillus and Clostridium. The species Bacillus thuringiensis, B. cereus, B. subtilis, B. licheniformis and B. lentus have been found in the different biotopes. Concerning the genus Clostridium, only the species Clostridium perfringens has been identified in the three aquatic biotopes studied. The calculated average surface water abundances of the identified sprouts were 4260, 3450, 130 and 130 CFU/100mL respectively for Bacillus thuringiensis, B. cereus, B. subtilis and Clostridium perfringens. In groundwater they were 930, 320, 690, 20, 50 and 10 CFU/100mL for B. thuringiensis, B. cereus, B. subtilis, B. licheniformis, B. lentus and C. perfringens respectively. In the rainy waters, these calculated mean abundances were 2640, 3080, 90 and 20 CFU/100mL for B. thuringiensis, B. cereus, B. subtilis and C. perfringens, respectively. Physicochemical analyses indicated that temperature, pH, electrical conductivity, TDS, dissolved oxygen, colour, suspended solids, nitrate, phosphate and ammoniacal nitrogen concentrations in the three aquatic environments were subject to spatio-temporal variation. The correlations between these abiotic parameters and the dynamics of bacterial abundance varied between sampling sites, and according to the given abiotic parameter and the bacterial species considered. Similarly, there are one-month lag correlations between the properties of the previous month's rainwater and the microflora abundance dynamics in the surface water and well water sampled in the current month, sometimes referred to as the delayed xxvi impact. The variability of these degrees of binding suggests that the properties of the sampled surface and groundwater would not depend only on those of the region's rainwater, but would result from the interactions of confounding factors. The Kruskal-Wallis "H" comparison test showed that there are significant differences (P<0.05) between the abundances of the germ species in the 3 types of aquatic biotopes. The antibiograms showed that the strains of the different bacterial species express resistance to the majority of the antibiotics tested. The inhibition diameters of most antibiotics with respect to each of the bacterial species varied significantly (P<0.05) between the different aquatic biotopes. In the presence of aqueous and hydroethanolic extracts of M. oleifera leaves, the abundance of culturable spore-forming bacteria isolated from the 3 types of biotopes decreased. Bacteria of the genus Bacillus were subject to relative inhibitions at different concentrations of the two types of extracts after all incubation periods and at temperatures 23ºC and 37ºC. The species Clostridium perfringens appeared to be sensitive only to hydroethanolic extracts with concentrations of 1.5 and 2 g/L after 6 and 24 hours of incubation. Overall, the decreases in the abundances of the spore-forming bacteria identified appeared to increase with increasing concentrations of the extracts. Chemical analyses of extracts from M. oleifera leaves showed the presence of numerous chemical compounds including sucrose (heteroside), glucomoringinin (glucosinolate), neochlorogenic acid (phenolic acid), 0 acetylshanzhiside methyl ester (iridoid), isoquercetin (flavonoid), Kaempferol-3-O-glucoside and Kaempferol acetylglucoside (flavonoid heteroside). Some of these compounds have bactericidal or bacteriostatic properties. These results show the potential use of M. oleifera extracts in the microbiological treatment of water intended for human consumption, in particular the treatment of these waters against B. thuringiensis, B. cereus and C. perfringens.