Browsing by Author "Kode, Megan Ann"

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    Predatory bacteria: alternative antimicrobial and biocontrol agents
    (Stellenbosch : Stellenbosch University, 2023-03) Kode, Megan Ann; Khan, Wesaal; Waso-Reyneke, Monique; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: While Bdellovibrio-and-like organisms (BALOs) are ubiquitous in nature, their diversity in environmental niches is underexplored. The isolation and characterisation of novel strains should thus be investigated, as these strains may exhibit an increased predation efficiency on pathogenic bacteria prevalent in the environment. The primary aim of the current study was thus to isolate and characterise BALOs from various water sources, and to investigate the efficacy of Bdellovibrio spp. in combination with solar disinfection (SODIS) and flocculation, for the removal of multi-drug resistant (MDR) human pathogens from artificial rainwater. Chapter one (abbreviated version published in Frontiers in Chemistry, 2022) thus focussed on elucidating the diversity of the BALO families, as well as the potential application of predatory bacteria and their natural products (including BALO and non-BALO strains) as therapeutic, biocontrol, and preservative agents. In Chapter two, BALOs were isolated from various water sources, including rivers, streams, surface runoff, clinical wastewater, and marine water. The isolation of several Bdellovibrio spp. and one Halobacteriovorax sp. was confirmed by polymerase chain reaction (PCR) analysis and sequencing, with phylogenetic analysis indicating that these isolates clustered with other strains in the Bdellovibrionaceae and Halobacteriovoraceae families, respectively. However, it was hypothesised that the isolated Halobacteriovorax sp. strain may belong to a novel species, as it branched independently from the Halobacteriovorax spp. reference strains. Two BALOs, namely Bdellovibrio bacteriovorus (B. bacteriovorus) TWPF3 and Halobacteriovorax sp. GBVP3, were selected for further characterisation, as B. bacteriovorus TWPF3 formed numerous and large plaques, and the Halobacteriovorax sp. GBVP3 was the only isolate from the Halobacteriovoraceae family. The prey range of both predatory bacteria was assessed using twenty-three Gram-negative bacterial strains (i.e., four clinical; seven environmental; twelve laboratory strains), with results indicating that B. bacteriovorus TWPF3 formed plaques when exposed to the clinical strains Escherichia coli (E. coli) MCC2, and Klebsiella pneumoniae (K. pneumoniae) KP3, and the laboratory strains K. pneumoniae ATCC 13883, K. pneumoniae PF, Salmonella typhimurium (S. typhimurium) ATCC 14028, and Shigella sonnei (S. sonnei) ATCC 25931. Halobacteriovorax sp. GBVP3 then formed plaques using four laboratory strains [K. pneumoniae ATCC 13883, Serratia marcescens (S. marcescens) ATCC 13880, S. sonnei ATCC 25931, and Vibrio cholerae (V. cholerae)], and one environmental strain [Vibrio parahaemolyticus (V. parahaemolyticus) GB6] as prey. The antibiogram for all predation-sensitive bacteria was assessed using Kirby-Bauer analysis, where only E. coli MCC2 and K. pneumoniae KP3 were classified as MDR, and K. pneumoniae PF was classified as extensively drug-resistant (XDR). The B. bacteriovorus TWPF3 and Halobacteriovorax sp. GBVP3 were then co-cultured with each of the respective predation-sensitive bacterial strains, with samples collected after 0, 24, 48, 72, and 96 h assessed using culture-based methods. Results indicated that B. bacteriovorus TWPF3 reduced K. pneumoniae ATCC 13883, S. typhimurium ATCC 14028 and S. sonnei ATCC 25931 by a maximum of 1.63 logs, 0.50 logs and 1.31 logs, respectively, while Halobacteriovorax sp. GBVP3 reduced the cell counts of K. pneumoniae ATCC 13883 and V. cholerae by 1.21 logs and 0.98 logs, respectively. Ethidium monoazide bromide quantitative PCR (EMA-qPCR) analysis was subsequently used to assess the co-culture assays where the prey cell counts were reduced by ≥ 2 logs. Overall, significant reductions in the cell counts and gene copies of MDR E. coli MCC2 [2.05 (72 h) and 1.50 logs (72 h), respectively], MDR K. pneumoniae KP3 [2.25 (72 h) and 3.59 logs (48 h), respectively], and XDR K. pneumoniae PF [3.79 (72 h) and 2.29 logs (96 h), respectively], were recorded after co-culture with B. bacteriovorus TWPF3. Similarly, significant reductions in the cell counts and gene copies of S. marcescens ATCC 13880 [2.38 (48 h) and 2.47 logs (96 h), respectively], S. sonnei ATCC 25931 [3.40 (48 h) and 6.19 logs (24 h), respectively] and V. parahaemolyticus GB6 [2.34 (48 h) and 2.61 logs (96 h), respectively], were recorded after co-culture with Halobacteriovorax sp. GBVP3. Based on the results obtained, B. bacteriovorus TWPF3 and Halobacteriovorax sp. GBVP3 could potentially be applied as biocontrol agents or for therapeutic uses, to target MDR and human pathogenic bacteria. Communities that do not have access to piped water supplies often rely on alternative water sources such as harvested rainwater, for potable and domestic uses. Various microbial pathogens have however, been detected in these water sources and while treatment methods such as chlorination, filtration, and SODIS, have been implemented for their removal, K. pneumoniae and Pseudomonas aeruginosa (P. aeruginosa), amongst many other microorganisms, have been found to persist. The aim of Chapter three (published in the Journal of Environmental Chemical Engineering, 2022) was thus to investigate the efficacy of combination treatments for the eradication of MDR K. pneumoniae and XDR P. aeruginosa from artificial rainwater. The combination treatments included a 72-h predatory bacteria pre-treatment using B. bacteriovorus PF13 (isolated by a member of the Water Resource Laboratory) or B. bacteriovorus TWPF3, or a dual-predatory pre-treatment using both B. bacteriovorus PF13 and TWPF3. The different predatory bacteria pre-treatments were followed by SODIS (for 6 h), and flocculation (for 1 h) using Moringa oleifera (M. oleifera) seed extract. Overall, the use of the dual predatory pre-treatment, followed by SODIS, significantly reduced the MDR K. pneumoniae cell counts and gene copies (as determined by EMA-qPCR) by 8.46 logs and 4.40 logs, respectively, to below the detection limit. Contrastingly, while none of the predatory pre- treatments were found to reduce the XDR P. aeruginosa cell concentration, SODIS with no pre- treatment was able to significantly reduce the cell counts and gene copies of XDR P. aeruginosa by 6.40 and 3.81 logs, respectively. The M. oleifera flocculation treatment (following SODIS) did not however, significantly reduce the cell concentrations of the prey strains. Analysis of the antibiogram of the MDR K. pneumoniae and XDR P. aeruginosa using the VITEK® 2 Compact System, then showed that no difference in the organisms’ antibiotic-resistance profiles was observed after the implementation of the various treatment stages. Results obtained in the current study thus indicated that MDR K. pneumoniae was significantly reduced to below the detection limit by the dual-predatory bacteria pre-treatment followed by SODIS, and future research should elucidate the interaction and predation kinetics when multiple predatory bacteria are used to target bacterial pathogens.