Detection of aeromonas species in relation to the occurrence of estrogens and testosterone in various water resources in Limpopo Province, South Africa and Lusaka, Zambia

Abstract

Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds.