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    Isolation and characterization of bacteriophages from wastewater as potential biocontrol agents for Escherichia coli.
    (2022) Ntuli, Nontando Percevierance.; Schmidt, Stefan.
    Host-specific lytic bacteriophages have regained momentum as an alternative treatment option to control and eliminate pathogenic bacteria. This study aimed to isolate, characterize and evaluate the potential application of Escherichia coli phages as a biocontrol agent in wastewater. In this study, four lytic Escherichia coli phages were isolated from wastewater for biocontrol purposes, using the double-layer method with E. coli (ATCC-25922) as a host. The phage morphology was characterized using transmission electron microscopy, with further parameters such as host range, phage stability at different temperatures, and pH values analyzed additionally. The genome of two selected phages (NPS and NPM) was sequenced, and the capacity of the phage isolate NPM to eliminate E. coli from artificial wastewater was evaluated and compared to conventional chlorination. All the four phage isolates showed typical T4 phage appearance with isometric capsids and contractile tails of different sizes, matching the family Myoviridae in the order Caudovirales. They exhibited a narrow host range limited to E. coli isolates, with two exceptions: phage NPS and NPM additionally lysed Salmonella Typhimurium (ATCC-14028). The four phage isolates were even able to lyse MDR (multidrug-resistant) E. coli isolates, such as the strain FP29. The four phages had burst sizes ranging from 70-115 per host cell and a latency period of 10- 20 minutes. All the four bacteriophages were stable at pH 5-9 but completely inactivated at pH 12. Exposure to 60°C for 10 minutes reduced phage titers by 1.5- log, while exposure to 80°C for 10 minutes completely inactivated all four phage isolates. The two genomes (NPS and NPM) were 99% identical and had similar sizes (169 536 bp), but phage NPS differed from phage NPM in view of its host range and plaque morphology. Another difference observed at the genome level was a shift of coding sequences between phage NPS and NPM. Phage isolate NPM achieved a 3.5- log reduction of E. coli cells present in artificial wastewater at an MOI of 0.1 in 120 minutes. A 90-minute chlorine treatment achieved a log reduction in the same range, highlighting that phages have the potential as environmentally friendly biocontrol agents in wastewater treatment.
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    The use of zebrafish to assess water quality and remediation efforts.
    (2023) Zondi, Thandolwethu Beauty.; Hewer, Raymond.
    Although wastewater effluents continue to be significant polluters of aquatic ecosystems in developing countries with limited water resources, little is known about the ecotoxicity induced by these effluents on fish throughout their early life stages. Several wastewater treatment plants (WWTPs) in South Africa (SA) do not adequately meet the minimal wastewater treatment requirements established by the country's Department of Water and Sanitation (DWS). Moreover, contaminants of emerging concern (CECs) originating from synthetic or natural sources, are widely distributed in aquatic environments of SA. This includes a broad range of natural and chemical compounds, such as aspirin (44243 ng/L), Fluoroquinolones (27100 ng/L), Atenolol (25900 ng/L), Nalidixic acid (25234 ng/L) and Ciprofloxacin (20514 ng/L). In addition to chemical compounds, endocrine disrupting chemicals, pharmaceuticals and personal care products are also distributed in the water systems. In the process of wastewater treatment, agents such as flocculants, coagulants, chemical precipitants (e.g., calcium hydroxide or sodium hydroxide) and chlorine disinfectants are utilized in wastewater treatment settings. However, research to understand the adverse effects that can be caused by these agents on aquatic organisms is still ongoing in SA. In order to bridge this knowledge gap, advanced techniques could be employed to help reveal adverse effects of wastewater as well as any shortcomings of current water remediation techniques. Using an appropriate aquatic model organism with highly conserved physiological pathways present in higher vertebrates (including humans), a rich behavioural repertoire, and occurrence in a variety of habitats would be a novel approach. To this effect, this study employed zebrafish with the aim to monitor six distinct wastewater samples from various regions of SA and to assess the effectiveness of currently used water remediation techniques such as chlorination. Two wastewater effluents, namely, Southern Works Final Effluents (SWFE) and Jacob’s Incoming (JB) alerted potential toxicity during chemical characterization with suboptimal pH (SWFE = 9.02 ± 0.16 and JB = 5.65 ± 0.02) and total alkalinity of zero (0 mg/L) detected for both effluents. The lethal toxicity of these effluents was seen by the elevation of mortality rate up to 77 ± 2.89 % and 100 ± 0.00 %, respectively for SWFE and JB at 40 %, with corresponding LC50 values of 17.77 % and 16.46 %. The zebrafish jaw and face, heart, brain, fins, notochord, somite and tail were significantly deformed (p < 0.05) post-exposure to these effluents, as revealed by morphological scores upon the analysis of the zebrafish’s body structure. Moreover, there was a delay in development due to the aforementioned effluents, unsuccessful hatching, craniofacial abnormalities, pericardial and yolk sac oedema, notochord abnormality somite defects and spinal cord curvature. In addition, locomotor activity of zebrafish was inhibited following observation of distance travelled, frozen moments, acceleration rates, swimming trajectories and exploration rate. Surprisingly, safety of these wastewaters was restored by chemical precipitation revealing non-lethal pH ranges of 6.02 - 8.02 and 6.65 - 7.65 for SWFE and JB, reducing the mortality rate to non-significant levels (p > 0.05) compared to the control. Also, sodium bicarbonate (NaHCO3) at 120 mg/L was found effective at supplementing the wastewater total alkalinity. In contrast, Amanzimtoti water before and after chlorination (TB and TA), Incoming Badulla (IB) and Chatsworth Incoming (CI) exhibited no consistent lethality effects on zebrafish and induced no apparent stress as demonstrated by insignificant expression (p > 0.05) of the stress protein: heat shock protein 70 (HSP70). However, the insignificant mortality v rate (p > 0.05) in the water tested before (TB) and after (TA) chlorination appeared to be the same (~25 %) indicating that chlorination is not enough at completely remediating wastewater. Our study is a pioneer in evaluating the ecotoxicological impact of wastewater effluents from localized regions of a developing country like South Africa in relation to the adjustment of water quality parameters for the neutralization of contaminants. To better understand emerging contaminants released as effluents in SA's water bodies and their interactions with aquatic organisms at the adult stage, more studies needs to be developed.