Abstract: Background and Objective: Recently, there has been evidence for the accumulation of steroid hormones in the water environment with negative consequences on fish and humans. However, there is paucity of information on how the steroid hormones influence the microbial community in environmental waters. The objective of this study was to determine the occurrence of 17β-estradiol (E2) and its potential influence on the diversity of Aeromonas spp. Materials and Methods: Wastewater samples were obtained from sewage treatment plants in northern South Africa and fish samples were collected from the Nandoni dam. Aeromonas spp. were isolated using microbiological methods and PCR protocols were used for their identification. A commercial Elisa kit was used for measuring the concentration of 17β-estradiol (E2) from the wastewater samples as well as the fish samples. Results: 17β-estradiol (E2) was found in high concentration in sewage samples varying from 0.32-348.6 pg mL–1 while in fish samples, it ranged from 1.1-73.6 pg mL–1. There was a tendency of samples with high E2 concentrations to have higher diversity of Aeromonas spp., implying that steroid hormones may serve as nutrient for Aeromonas spp. Aeromonas hydrophila was the most prevalent species (71%), followed by A. sobria with (68%). Conclusion: The presence of Aeromonas spp. in environmental waters and fish that is consumed by the local community poses a serious health concern. The high content of E2 in treated wastewater is of serious concern as well. For the first time, the present study showed a positive impact of E2 on Aeromonas growth.
INTRODUCTION
For over a decade now, there has been a rising concern about the effects of endocrine disrupting compounds in human and animal health1,2. Estrogens are among those compounds that are classified as endocrine disruptors. Natural and synthetic steroid hormones are known to interfere with growth, reproduction and development in fish and humans3. Natural Estrogens include estrone (E1), 17β-estradiol (E2) and estriol (E3), while 17α-ethinylestradiol (EE2) is a synthetic estrogen among others4. Steroid hormones have been reported to cause adverse effects on fish organisms in concentration5 as low as 1 ng L–1. Some of the effects of hormones in fish include feminization of male fish, induction of vitellogenin, intersex and interference with growth6. These chemicals find their way to environmental waters through sewage discharge and animal wastes, also industries that synthesize these steroid hormones play a role in contaminating the environmental waters through the disposal of their industrial waste materials that includes active forms of these endocrine disruptors7. However, the distribution of these substances in the environment in South Africa is not well researched.
In the aquatic environment, there is a variety of microorganisms, which form part of the normal microflora in water as well as in fish. Such are Aeromonas spp. which are also known to cause a wide spectrum of diseases in man and animals8. The genus Aeromonas comprises of Gram-negative rod-shaped bacilli which are autochthonous in the aquatic environment, i.e., fresh and brackish water, sewage and wastewater, untreated and treated drinking water9. Aeromonas spp. are known as contaminants in fish and a variety of raw meat, milk and milk products and other raw foods10,11. The genus Aeromonas is very diverse with 31 species and 12 subspecies and a few of those are known to be primary pathogens of fish and warm-blooded animals12. These include Aeromonas hydrophila, Aeromonas sobria, A. caviae, A. veronii, A. jandei, A. trota, A. salmonicida, A. media, A. popoffi and A. schubertii13.
Some of the Aeromonas spp., such as Aeromonas hydrophila cause gastroenteritis, septicemia, chronic diarrhea, wound infections, urinary tract infections, peritonitis and respiratory tract infections in humans14. Some are isolated from environmental samples such as fresh water and wastewater as well as from fish tissues and in fish, A. hydrophila causes motile Aeromonas septicaemia15-17. These motile Aeromonas cause such infections in numerous species of cultured and wild freshwater fish such as carp, rainbow trout, brown trout, salmon, eel, carp, channel catfish, tilapia and goldfish18,19. Aeromonas tends to cause infections in fish when their normal environment is compromised, for example, due to poor water quality, nutritional deficiencies and a general poor environment in the aquarium20. Therefore, contamination of aquatic environments by steroid hormones is thought to compromise water quality which may lead to microorganisms causing infections to aquatic organisms. However, there is very limited data on the distribution of steroid hormones in environmental water and wastewater in most African regions including South Africa. Although the impact of steroid hormones on human and fish has been studied, there is no data on the impact of these steroid hormones on bacterial organisms including Aeromonas spp. Therefore, the aim of this study was to determine the distribution of 17β-estradiol (E2) and Aeromonas spp. in wastewater and fish samples and to evaluate the effects of this hormone on Aeromonas diversity.
MATERIALS AND METHODS
Study area: The study was conducted in the northern part of the Limpopo Province, South Africa from February to November, 2015. Nandoni dam is situated about 20 km from Thohoyandou town along the Luvuvhu River and is surrounded by Ha-Mutoti and Ha-Budeli villages in the Vhembe district. The dam serves primarily for water supply while local populations also catch fishes from the dam as a mean to provide a supplemental protein ration to their meals. Wastewater treatment plants (WWTP), where samples were collected include Muledane, Louis Trichardt, Malamulele, Waterval as well as Elim . The treated wastewater from these plants is sent directly to surrounding rivers.
Sample collection: A total of 30 fishes which weighed between 41.8 and 183.4 g were collected from Nandoni dam at different occasions. About 2 L of water samples was collected from the dam and from the sewage treatment plants at 3 different sections of the plant including the influent, the sedimentation tank and the effluent. All the samples were put in a cooler box with ice to maintain temperature of 4°C and were transported immediately to the Parasitology Laboratory at the University of Venda.
Bacterial isolation
From fish: Aeromonas selective agar from Oxoid (England), was used for the primary isolation of Aeromonas from the samples after a preliminary enrichment in peptone water. Briefly, different parts of the fish were aseptically dissected and inoculated into 9 mL peptone water in sterile bottles. The bottles were closed and shaken thoroughly and were allowed to stand for 30 min, after which a 3-fold serial dilution was carried out in duplicates. About 50 μL Aliquots of each sample was inoculated onto Aeromonas selective agar and incubated at 37°C. After incubation, colony forming units were counted from each plate and recorded and one colony from each plate was taken and inoculated in a 2 mL nutrient broth and incubated for 24 h. After incubation, DNA was extracted from each sample.
From water: Following collection of water samples from different WWTP, samples were transported to the University of Venda, Parasitology Lab. Within 3 h of arrival at the laboratory Aeromonas was isolated using Aeromonas selective agar enriched with ampicillin as follows: 50 μL of each sample was transferred to agar plates and the sample was spread using the glass rod spreader.
Detection of Aeromonas spp. by PCR: Three different PCR protocols were used as previously described using the primers indicated19 in Table 1. All PCR protocols were performed in a total volume of 25 μL. The reaction mixtures contained 12.5 μL dream tag, 0.6 μL of each primer (Aer.hyF, Aer.verF and reverse), 0.25 μL BSA and 5.45 μL PCR water and 5 μL DNA. PCR was performed using the following programs:
| • | For genus specific detection, genus specific primers A16SF and A16SR were used with an initial denaturation at 95°C for 5 min, 50 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec and extension at 72°C for 30 sec and 72°C for 7 min |
| • | For Aeromonas hydrophila and Aeromonas veronii, initial denaturation at 98°C for 150 sec, 5 cycles of denaturation at 93°C for 30 sec, annealing 64°C for 30 sec, extension at 72°C for 30 sec and 28 cycles of denaturation at 93°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 45 sec and final extension at 72°C for 5 min |
| • | For Aeromonas trota and Aeromonas caviae, the cycling conditions were as follows: initial denaturation at 95°C for 2 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 68°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 3 min |
The detection of Aeromonas sobria and Aeromonas salmonicida was performed as per a protocol previously described by Wang et al.21. The primers ASA1F and ASA1R were used for the detection of A. sobria with the following conditions: initial denaturation at 95°C for 5 min, 50 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec and extension at 72°C for 30 sec and 72°C for 7 min. The amplification parameters for Aeromonas salmonicida included an initial denaturation at 95°C for 30 sec, followed by 30 cycles of denaturation at 94°C for 2 min, primer annealing at 57°C for 30 sec, extension at 72°C for 1 min 30 sec. A final extension was performed at 72°C for 3 min. Five microliters of each reaction mixture were then analyzed by gel electrophoresis in 1% agarose at 100 V and the reaction products were visualized with a Bio-Rad gel doc machine after staining with 3% ethidium bromide.
RESULTS
Characteristics of samples used in the study: A total of 103 samples were collected and used in the study. Of these 90 (87.3%) were fish samples, 12 (11.7%) were wastewater samples and 1 was dam sample. All fish samples were collected from Nandoni dam while wastewater and treated wastewater were collected from sewage treatment plants including Malamulele, Muledane, Louis Trichardt, Elim and Waterval.
| Table 1: | List of primers that were used in detecting Aeromonas spp. in fish samples |
| Fig. 1(a-c): | Representative agarose gels of PCR amplification products for (a) A. caviae (260 bp) lane 1-5 are flesh, intestine, gills and intestine from fish and negative control respectively, (b) A. hydrophila (685 bp) lane 1-5 are flesh, intestine, gills, gills from fish and negative control respectively and (c) A. veronii (658 bp) lane 1-9 are influent, influent, effluent, effluent, sediment, effluent and sediment and negative control respectively from wastewater samples |
| Table 2: | Characteristics of the samples used in the study |
The samples were grouped according to the section of the plant from which the original samples were collected. Of the 103 samples 44 samples were tested for the presence of estradiol (E2) and 13 (29.5%) were negative and 31(70.5%) were positive. The results are shown in Table 2.
Prevalence of Aeromonas spp. according to type of samples: Following PCR amplification, the correct bands were obtained for the different Aeromonas spp. as shown in Fig. 1 from fish samples and wastewater samples. Aeromonas sobria occurred in all the wastewater samples while A. hydrophila occurred in 75.0% of the wastewater samples. However, A. caviae was most prevalent in fish samples occurring in 67.8% of the samples, but the difference was not statistically significant p = 0.358 (p>0.05). Aeromonas salmonicida and Aeromonas trota were not detected in any of the samples. The results are shown in Table 3.
Prevalence of Aeromonas spp. in the presence of estradiol (E2): Table 4 shows the prevalence of Aeromonas spp. in relation to the presence of estradiol (E2). The prevalence of A. sobria was much higher in samples that had estradiol (E2) 90.3% compared to the samples that did not (53.8%). The difference was statistically significant with p = 0.006 (p<0.006). Aeromonas caviae had the prevalence of 71.0% in samples that had estradiol (E2), however the difference with samples that did not have estradiol (E2) was not significant. On the contrary, A. hydrophila was more common in samples that did not have the hormone although the difference was not significant.
Variation of estradiol (E2) concentration from wastewater and fish samples: Estradiol (E2) concentrations varied from 0.32-348.64 pg mL–1. E2 concentrations were determined by ELISA which had been proven to be a very sensitive method. In the present study, raw samples had high concentration with the highest being 348.64 pg mL–1 although one effluent sample had high concentration of 279.31 pg mL–1 suggesting that, that wastewater treatment plant was not working well.
| Fig. 2: | A graph showing variation of concentration of estradiol (E2) from wastewater samples |
| Table 3: | Prevalence of Aeromonas spp./type of samples |
| Table 4: | Prevalence of Aeromonas spp. in the presence of estradiol (E2) |
Having such large amount of hormones in the effluent may lead to their accumulation in environmental waters (streams, rivers as well as dams); which also results in aquatic organisms being affected by these compounds (Fig. 2).
The concentration of estradiol (E2) varied also according to the part of the fishes tested and ranged from 0.001-27.82 pg mL–1 with flesh samples having the highest concentration (Fig. 3). Further analysis showed that there is a correlation between hormone concentration and multiple infections. It was observed that the higher the hormone concentration, the higher the number of multiple infections by Aeromonas spp. (Fig. 4). Regression analysis also revealed that there is correlation between estradiol (E2) and multiple infection with p = 0.0048 (p<0.05) and R2 with a value of 0.1822. This suggested that hormones do influence the members of Aeromonas spp. and particularly their occurrence in fish.
DISCUSSION
The present study found high concentrations of 17β-estradiol in wastewater as well as treated waste water with potential influence on the distribution of Aeromonas spp. in these samples. Aeromonas hydrophila was the most prevalent species isolated from fish and water samples.
| Fig. 3: | A graph showing varying concentrations of estradiol (E2) from fish samples |
| Fig. 4: | Scatter graph shows correlation between hormone concentration and multiple infection |
White dots on the graph represent the number of Aeromonas species |
|
In fact, A. hydrophila is the most common Aeromonas spp. that is also involved in human disease. Similar findings have been described by other authors such as Janda and Abbot, who also found that A. hydrophila was the most prevalent in fish samples and they suggested that it may be because it is known to be the most common pathogen linked to fish kills all over the world, which results in economic losses13. In the current study, the prevalence of A. hydrophila was followed by that of A. sobria, this is close to the results obtained by Beaz-Hidalgo et al.24 and Austin and Austin25 who showed that A. sobria was the commonly isolated species in diseased fish in Spain. These differences may be due to geographic difference. This study also shares the results with a study done by Hu26 in 2012 who found A. hydrophila to be the most prevalent species isolated from diseased fish.
In the presence of estradiol (E2), our results showed that A. sobria was the most common species with (90.3%) and p-value of 0.006. This is an indication that A. sobria might be capable of metabolizing estradiol much better compared to the other Aeromonas spp. To our knowledge no studies have investigated the metabolism of this hormone by microorganism, this do not match with the occurrence of Aeromonas spp. in general, because the most prevalent was A. hydrophila. But now in the presence of estradiol (E2) the most common species is A. sobria. This may be due to that not all samples were tested for estradiol (E2) and many of the tested samples were positive for A. sobria, or it may be that A. sobria strives most in the presence of estradiol (E2). This was followed by A. hydrophila, but the difference was not significant. Most of the flesh samples from fish showed high concentration of Estradiol, followed by gills samples and then intestines samples; this suggests the possibility that hormones accumulate in the flesh of aquatic organisms. A previous study showed that Aeromonas hydrophila was not able to degrade estrone in a monoculture27. The increased growth of Aeromonas spp. in the present study is a proof that these organisms could be able to degrade estradiol. Further studies should investigate the metabolism of this hormone by Aeromonas spp.
From the different sewage treatment plants, samples from the influent (raw sewage) had higher concentration of estradiol which is not surprising considering that the influent contains lot of chemical pollutants from different sewage drainage such as from households and institutions. The concentration of Estradiol in effluents is what is of concern because it is not expected to be high, yet from some treatment plants the effluents showed rather high concentration of Estradiol, which means that the plants were not effective in removing this kind of pollutants which might end up accumulating in environmental waters where the effluents are released. The accumulation of the hormones in the rivers will also lead to their accumulation in the bodies of aquatic organisms; affecting their health28. The concentration of estradiol (E2) from Nandoni dam in the current study was high with a value of 43.9 pg mL–1. This was in comparison to the concentration of estradiol (E2) found in dams built on ephemeral rivers in a study done by Faul and colleagues who found the value of estradiol29 (E2) to be 7.2 pg mL–1. This may be because Nandoni dam receives large amounts of inflow from rivers that are catchments of effluents from different wastewater treatment plants and the estrogenic pollutants that are in the water accumulates in the dam over time.
Estradiol (E2) seemed to be an important factor on the diversity of Aeromonas spp., because the higher the estradiol (E2) concentration, the higher the number of Aeromonas spp. These results are in agreement with a study done by Zhang and colleagues who found that, the presence of Estrone and estradiol (E2) in soil increased soil microbial community, suggesting that microorganisms may be using hormones as nutrients for their growth30. This could also imply that the same could happen to microorganisms in the aquatic environment, which could lead to overflow of pathogenic microorganisms that may end up infecting aquatic organisms causing diseases.
Aeromonas salmonicida and Aeromonas trota were absent from the samples tested. The failure of the primer set to detect A. salmonicida may be related to the primer target site or that they did not grow at 37°C21,31. The failure to detect Aeromonas trota may also be related to the primer used. Or it could be that these 2 species were not available in all the samples. Of the samples that were sequenced the sequence information obtained covered approximately 600 nucleotides and allowed for identification of members of the genus Aeromonas. Based on the constructed phylogenetic tree using the KT371350 reference sequence, isolates showed clustering with the reference strains of respective species and were identified as Aeromonas caviae/veronii species and a cluster of Aeromonas sobria. Joseph et al.32 also found that different strains of Aeromonas spp. that are closely related cluster together when a phylogenetic tree is constructed. Aeromonas veronii was found to be more prevalent in environmental samples. This is in alignment with a study done by Khor who also found that Aeromonas veronii was more common in environmental samples (freshwater)6. Aeromonas spp. as well as other bacterial species living in natural water sources are able to cause a wide range of diseases to both humans and animals. Therefore, it is essential to keep their levels down by implementing measures that discourage their growth. The presence of hormones and other endocrine disrupting chemicals (EDCs) in environmental niches may affect not only on the organisms living in these spaces, but also on microorganisms by enhancing their overgrowth in these environments. The development of methods for the improved elimination of these substances in the environment is of paramount importance. Further studies should include more hormones including the artificial 17 alpha ethinyl estradiol which is part of most birth control pills used by women throughout the world. Communities should be educated on the disposal of such material that may contain human body fluids to avoid the contamination of natural water sources.
CONCLUSION
In conclusion, this study demonstrated that Aeromonas spp. are prevalent in wastewater and in fish from the Nandoni Dam. There was a high concentration of estradiol (E2) in the sewage samples. Two STPs were able to reduce the concentration of estradiol in the wastewater while 2 others did not. This could lead to high concentrations of estradiol (E2) in environmental waters that come in contact with the partially treated wastewater. Although there was no straight association between estradiol (E2) and Aeromonas diversity there was a tendency of samples with high estradiol (E2) concentrations to have high numbers of Aeromonas spp. as well. However, further studies are needed to confirm this hypothesis.
SIGNIFICANCE STATEMENT
This study discovered the possible potential effect of estradiol (E2) on Aeromonas spp. which will be beneficial in serving as preliminary data for more research in order to explore such findings. These findings will help researchers to uncover the critical areas of the interaction between steroid hormones and bacterial organisms. Thus, a new theory on this type of interaction may be arrived at and further research is needed to explore these possibilities.
ACKNOWLEDGMENT
The authors would like to express their sincere gratitude to the National Research Foundation of South Africa (NRF) for funding the study (NRF E384).