Department of Microbiology

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Browsing Department of Microbiology by browse.metadata.advisor "Ahmed, Warish"

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    Human health risks associated with harvested rainwater: implementation of biocontrol strategies
    (Stellenbosch : Stellenbosch University, 2020-04) Waso, Monique; Khan, Wesaal; Khan, Sehaam; Ahmed, Warish; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Rainwater harvesting has been earmarked as an additional fresh water source, which could be utilised to supplement municipal water supplies, especially in water scarce regions. However, various studies have indicated that the microbial quality of this water source is substandard. These microbial contaminants may pose a significant health risk to end-users and it is recommended that treatment systems are implemented to reduce the level of contamination in rainwater. Solar disinfection (SODIS) has been identified as an easy-to-use and cost-effective strategy that could be used to disinfect water. A minimum of 6 hours solar exposure is generally required for effective disinfection of water and photocatalytic nanomaterials such as titanium dioxide (TiO2) have subsequently been employed to improve SODIS efficiency by decreasing the treatment time. Research has however, indicated that while SODIS is effective in significantly reducing the concentration of microbial contaminants in water sources, various pathogens and opportunistic pathogens employ survival strategies and persist after treatment. A combination of physical, chemical and biological treatments, which target these persistent organisms directly, should therefore be investigated. For the purpose of this dissertation, the use of Bdellovibrio bacteriovorus (B. bacteriovorus), a Gram-negative predatory bacterium, was investigated. The primary aim of Chapter 2 (published in Microbiological Research, 2019) was thus to isolate B. bacteriovorus from wastewater and investigate the interaction of this predator with Gram-negative and Gram-positive prey using culture-based (spread plating and double-layer agar overlays) and molecular methods [ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR)]. The predation activity of B. bacteriovorus on the different prey cells was assessed and compared in a nutrient poor [diluted nutrient broth (DNB)] and nutrient deficient medium (HEPES buffer). A B. bacteriovorus isolate (PF13) was subsequently co-cultured with Pseudomonas fluorescens (P. fluorescens), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium). Results indicated that P. fluorescens (maximum log reduction of 4.21) and K. pneumoniae (maximum log reduction of 5.13) were sensitive to predation in DNB and HEPES buffer, while E. faecium (maximum log reduction of 2.71) was sensitive to predation in DNB and S. aureus (maximum log reduction of 1.80) was sensitive to predation in HEPES buffer. Predation of Gram-positive prey by B. bacteriovorus was thus dependent on the specific prey cells used and the media employed to assess these interactions. In contrast, for P. aeruginosa, while the culture-based analysis indicated that the cell counts were reduced, the EMA-qPCR analysis indicated that the concentration of P. aeruginosa was not significantly reduced in DNB or HEPES buffer. The use of EMA-qPCR can thus aid in accurately monitoring and quantifying both predator and prey cells during co-culture experiments in a time-effective manner. The aim of Chapter 3 (published in Water Research, 2020) was to subsequently apply B.bacteriovorus PF13 as a pre-treatment to SODIS and solar photocatalytic disinfection. Thephotocatalyst used was immobilised titanium-dioxide reduced graphene oxide (TiO2-rGO). Synthetic rainwater was seeded with K. pneumoniae and E. faecium, with results indicating that the use of B. bacteriovorus pre-treatment in combination with solar photocatalysis resulted in the greatest reduction in K. pneumoniae concentrations in the shortest treatment time, with the cell counts reduced by 9.30 logs to below the detection limit (BDL) within 120 min. In contrast, for E. faecium the most effective treatment was solar photocatalysis or SODIS without the B. bacteriovorus pre-treatment, as the viable counts of E. faecium were reduced by 8.00 logs to BDL (within 210 min) and the gene copies were reduced by ~3.39 logs after 240 min. It was thus evident that the application of B. bacteriovorusmay specifically enhance the disinfection of Gram-negative bacteria. Additionally, the use of the photocatalyst further enhanced the disinfection of the Gram-negative bacteria, while the same trend was not observed for E. faecium. Recirculating the water in solar photocatalytic reactors may, however, enhance disinfection of Gram-positive bacteria, by exerting mechano-osmotic stress on the cells and should be investigated in future research. As conflicting results regarding the interaction between B. bacteriovorus and Gram-positive bacteria have been reported, the aim of Chapter 4 (published in Microbiological Research, 2020) was to monitor and compare the expression of attack phase (AP) and growth phase (GP) genes of B. bacteriovorus in co-culture with Gram-positive and Gram-negative prey. Bdellovibrio bacteriovorus PF13 was thus co-cultured with Escherichia coli (E. coli; control), K. pneumoniae and E. faecium. Relative qPCR analysis indicated that the AP genes bd0108 (type IVa pili retraction/extrusion) and merRNA (massively expressed riboswitch RNA) were highly expressed in the B. bacteriovorus AP cells, whereafter expression in co-culture with all the prey strains was reduced. The fliC1 gene (flagellar filament) was also expressed at a high level in the AP cells, however, after 240 min of co-culture with E. faecium the expression of fliC1 remained low (at 0.759-fold), while in the presence of the Gram-negative prey, fliC1 expression increased (in comparison to the expression recorded after 30 min) to 4.62 (E. coli) and 2.69-fold (K. pneumoniae). In addition, bd0816 (peptidoglycan-modifying enzyme) and groES1 (chaperone protein) were not induced in the presence of E. faecium, however, after exposure to the Gram-negative prey, bd0816 expression increased during the early GP, while groES1 expression gradually increased during the early GP and GP. It was thus concluded that B. bacteriovorus senses the presence of potential prey when exposed to Gram-positive and Gram-negative prey however, the GP genes were not induced when B. bacteriovorus was co-cultured with E. faecium. This indicates that B. bacteriovorus may not actively grow in the presence of E. faecium and the second predatory cue (which induces active growth of B. bacteriovorus) may be lacking under the conditions employed in this study. Limited information on the expression of predatory-specific genes of B. bacteriovorus in co-culture with Gram-positive prey cells is available. Recent studies have however, indicated that B. bacteriovorus can prey on Gram-positive bacteria and investigating the expression of these predatory-specific genes may elucidate the genetic mechanisms this predator employs to survive in the presence of these atypical prey.