Doctoral Degrees (Microbiology)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3627

Browse

Browsing Doctoral Degrees (Microbiology) by browse.metadata.advisor "Cloete, Thomas Eugene"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Legionella species persistence mechanisms in treated harvested rainwater
    (Stellenbosch : Stellenbosch University, 2017-03) Dobrowsky, Penelope Heather; Khan, Wesaal; Khan, Sehaam; Cloete, Thomas Eugene; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The persistence of Legionella spp. at high pasteurization temperatures poses a threat to human health as a number of Legionella spp. are known to cause Legionnaires’ disease. Research has then indicated that the primary factors that allow Legionella to proliferate and persist in water distribution systems are: the accessibility to nutrients in a water source, water temperature, the presence of free-living amoebae (FLA) and other aquatic bacteria. The focus of the current study was thus to investigate and functionalise selected persistence mechanisms displayed by Legionella spp. that aid in their survival in pasteurized and unpasteurized harvested rainwater. The overall aim of Chapter two was to isolate and identify the dominant Legionella spp. persisting in a domestic rainwater harvesting tank and a solar pasteurization (SOPAS) system and to identify possible FLA vectors of Legionella that remain viable at high pasteurization temperatures (>60°C). For this, pasteurized and unpasteurized tank water samples were screened for the dominant Legionella spp. using culture based techniques. In addition, as FLAs including Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis are the most frequently isolated from hot water systems, ethidium monoazide polymerase chain reaction (EMA-qPCR) was utilised for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri. Eighty-two Legionella spp. were isolated from the unpasteurized tank water samples, where L. longbeachae (35 %) was the most frequently isolated, followed by L. norrlandica (27 %) and L. rowbothamii (4 %). This information provides pertinent knowledge on the occurrence and dominant species of Legionella present in the South African environment. In addition, the SOPAS system was effective in reducing the gene copies of viable N. fowleri (5-log) and V. vermiformis (3-log) to below the lower limit of detection at temperatures of 68–93°C and 74–93°C, respectively. In contrast, as gene copies of viable Legionella and Acanthamoeba were still detected after pasteurization at 68–93°C, it could be concluded that Acanthamoeba spp. primarily act as vectors for Legionella spp. in solar pasteurized rainwater. The primary objective of Chapter three was to determine the resistance of three Legionella species isolated from unpasteurized rainwater [L. longbeachae (env.), L. norrlandica (env.) and L. rowbothamii (env.)], two Legionella reference strains (L. pneumophila ATCC 33152 and L. longbeachae ATCC 33462) and Acanthamoeba mauritaniensis ATCC 50676 to heat treatment (50–90°C). In addition, the resistance of L. pneumophila ATCC 33152 and L. longbeachae (env.) in co-culture with A. mauritaniensis ATCC 50676, respectively, to heat treatment (50–90°C) was determined using EMA-qPCR. The interaction mechanisms exhibited between Legionella and Acanthamoeba during heat treatment (50–90°C) were also elucidated by monitoring the relative expression of genes associated with metabolism and virulence of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L. longbeachae (env.) (lolA) in co-culture with A. mauritaniensis ATCC 50676, respectively. Legionella longbeachae (env.) and L. pneumophila ATCC 33152 were the most resistant to heat treatment as both organisms were still culturable (CFU/mL) following treatment at 50 and 60°C. However, the sensitivity of detection of viable cells was increased when using EMA-qPCR as all Legionella spp. and A. mauritaniensis ATCC 50676 were detected following heat treatment (50–90°C). In addition, while the heat resistance of L. pneumophila ATCC 33152 in co-culture with A. mauritaniensis ATCC 50676 improved, it is postulated that L. longbeachae (env.) is unable to replicate in A. mauritaniensis ATCC 50676 as L. longbeachae (env.) in co-culture was not detected following heat treatment at 80°C and 90°C. Results also showed a clear trend between genes with related function and differential expression during heat treatment (50-90°C). For example, relative to the untreated samples, the expression of lolA remained constant while the expression of sidF increased and the expression of csrA decreased significantly during L. pneumophila ATCC 33152 co-culture with A. mauritaniensis ATCC 50676. Results thus confirm that while heat treatment may reduce the number of viable Legionella spp., L. pneumophila is able to interact with A. mauritaniensis and persist during heat treatment. The overall aim of Chapter four was to elucidate other microbial and physico-chemical characteristics that may be associated with the incidence of Legionella spp. and Acanthamoeba spp. in rainwater harvested from different roofing materials. Overall results indicated that the roofing materials did not influence the incidence of Legionella and Acanthamoeba spp. as these organisms were detected in all tank water samples collected from the Chromadek®, galvanized zinc and asbestos roofing materials. However, significant (p < 0.05) positive Spearman (ρ) correlations were noted between Legionella spp. vs. nitrites and nitrates and between Acanthamoeba spp. vs. barium, magnesium, sodium, silicon, arsenic and phosphate, respectively. In addition, while no significant correlations were observed between Legionella spp. vs. the indicator bacteria (p > 0.05), positive correlations were established between Acanthamoeba spp. vs. total coliforms and Escherichia coli, respectively. Results thus indicated that the incidence of Legionella and Acanthamoeba spp. in harvested rainwater may primarily be due to external pollutants such as dust and animal faecal matter present on the catchment system.