Browsing by Author "Waso, Monique"

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    Exploring the antimicrobial resistance profiles of WHO critical priority list bacterial strains
    (BMC (part of Springer Nature), 2019-12-23) Havenga, Benjamin; Ndlovu, Thando; Clements, Tanya; Reyneke, Brandon; Waso, Monique; Khan, Wesaal
    Background: The antimicrobial resistance of clinical, environmental and control strains of the WHO “Priority 1: Critical group” organisms, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa to various classes of antibiotics, colistin and surfactin (biosurfactant) was determined. Methods: Acinetobacter baumannii was isolated from environmental samples and antibiotic resistance profiling was performed to classify the test organisms [A. baumannii (n = 6), P. aeruginosa (n = 5), E. coli (n = 7) and K. pneumoniae (n = 7)] as multidrug resistant (MDR) or extreme drug resistant (XDR). All the bacterial isolates (n = 25) were screened for colistin resistance and the mobilised colistin resistance (mcr) genes. Biosurfactants produced by Bacillus amyloliquefaciens ST34 were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI–MS). The susceptibility of strains, exhibiting antibiotic and colistin resistance, to the crude surfactin extract (cell-free supernatant) was then determined. Results: Antibiotic resistance profiling classified four A. baumannii (67%), one K. pneumoniae (15%) and one P. aeruginosa (20%) isolate as XDR, with one E. coli (15%) and three K. pneumoniae (43%) strains classified as MDR. Many of the isolates [A. baumannii (25%), E. coli (80%), K. pneumoniae (100%) and P. aeruginosa (100%)] exhibited colistin resistance [minimum inhibitory concentrations (MICs) ≥ 4mg/L]; however, only one E. coli strain isolated from a clinical environment harboured the mcr-1 gene. UPLC-MS analysis then indicated that the B. amyloliquefaciens ST34 produced C13–16 surfactin analogues, which were identified as Srf1 to Srf5. The crude surfactin extract (10.00 mg/mL) retained antimicrobial activity (100%) against the MDR, XDR and colistin resistant A. baumannii, P. aeruginosa, E. coli and K. pneumoniae strains. Conclusion: Clinical, environmental and control strains of A. baumannii, P. aeruginosa, E. coli and K. pneumoniae exhibiting MDR and XDR profiles and colistin resistance, were susceptible to surfactin analogues, confirming that this lipopeptide shows promise for application in clinical settings.
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    A global review of the microbiological quality and potential health risks associated with roof-harvested rainwater tanks
    (Nature Research, 2019) Hamilton, Kerry; Reyneke, Brandon; Waso, Monique; Clements, Tanya; Ndlovu, Thando; Khan, Wesaal; DiGiovanni, Kimberly; Rakestraw, Emma; Montalto, Franco; Haas, Charles N.; Ahmed, Warish
    A broad body of literature has been published regarding roof-harvested rainwater quality around the world. In particular, the presence of fecal indicator bacteria and pathogenic microorganisms has raised concerns regarding the acceptability of rainwater for potable and non-potable uses. As the use of molecular assays has improved understanding of the diverse microbial communities present in rainwater tanks and their role in providing benefits or harm to human health, a comprehensive review is needed to summarize the state of the science in this area. To provide a summary of microbial contaminants in rainwater tanks and contextual factors, a comprehensive review was conducted here to elucidate the uses of rainwater, factors affecting water quality, concentrations of fecal indicators and pathogens, the attribution of pathogens to host sources using microbial source tracking, microbial ecology, human health risks determined using epidemiological approaches and quantitative microbial risk assessment, and treatment approaches for mitigating risks. Research gaps were identified for pathogen concentration data, microbial source tracking approaches for identifying the sources of microbial contamination, limitations to current approaches for assessing viability, treatment, and maintenance practices. Frameworks should be developed to assess and prioritize these factors in order to optimize public health promotion for roof-harvested rainwater.
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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.
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    Identifying the primary microbial and chemical source tracking markers in harvested rainwater for the detection of faecal contamination
    (Stellenbosch : Stellenbosch University, 2017-03) Waso, Monique; Khan, Wesaal; Khan, Sehaam; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Rainwater harvesting has been earmarked as an additional source of fresh water. However, research has indicated that the microbiological quality is substandard as pathogens have been detected in this water source. As it is impractical to monitor for the presence of all pathogens in a water source, indicator organisms are routinely utilised to monitor water quality and predict the presence of pathogens in contaminated environmental waters. Various research groups have however indicated that the analysis of indicator organisms in a water source may not be sufficient to accurately identify the source of contamination. Supplementary indicators are therefore required to accurately identify contamination sources, with chemical and microbial source tracking markers currently being investigated and applied to various water sources. The primary focus of the current study was thus to identify a toolbox of microbial source tracking (MST) and chemical source tracking (CST) markers that could be utilised to supplement indicator organism analysis of domestic rainwater harvesting (DRWH) systems. To achieve this aim, harvested rainwater (n = 60) and rooftop debris (n = 60) samples were screened for a range of MST (conventional PCR) and CST (high-performance liquid chromatography tandem mass spectrometry) markers previously utilised in literature to analyse various water sources (Chapter two). All the tank water samples collected at the Kleinmond Housing Scheme site (Kleinmond, Western Cape), were also screened for traditional indicator organisms using culture based techniques. Additionally, Escherichia coli (E. coli) and enterococci were screened for in all tank water and rooftop debris samples using quantitative PCR (qPCR) analysis. Based on the conventional PCR results, Bacteroides HF183, adenovirus, Lachnospiraceae and human mitochondrial DNA (mtDNA) were the most prevalent MST markers. These markers were subsequently quantified in the tank water and rooftop debris samples by qPCR. The HF183 marker was then detected at a mean concentration of 5.1 × 103 and 4.7 × 103 gene copies/μL in the tank water and rooftop debris, respectively. Adenovirus was detected at 3.2 × 102 and 6.4 × 103 gene copies/μL; human mtDNA was detected at 1.1 × 106 and 3.0 × 105 gene copies/μL and Lachnospiraceae was detected at 3.0 × 104 and 6.9 × 103 gene copies/μL in the tank water and rooftop debris samples, respectively. Additionally, E. coli and enterococci were quantifiable in all tank water and rooftop debris samples by qPCR analysis. The CST markers caffeine, salicylic acid, acetaminophen, triclosan, triclocarban and methylparaben were then detected at μg/L levels in all the tank water [except salicylic acid (98%)] and rooftop debris samples. A secondary aim was to establish correlations between the MST and CST markers as well as indicator organisms to ascertain which markers may be employed to supplement indicator organism analysis of DRWH systems. In the tank water samples, significant positive correlations were observed for adenovirus versus E. coli (enumerated with the culturing techniques) (p = 0.000), the HF183 marker versus E. coli (quantified by qPCR) (p = 0.023), Lachnospiraceae versus heterotrophic bacteria (p = 0.000) and human mtDNA versus enterococci (enumerated with the culturing techniques) (p = 0.026). In addition, significant positive correlations were observed for caffeine versus enterococci (quantified by qPCR) (p = 0.000); faecal coliforms (p = 0.001); total coliforms (p = 0.000) and enterococci (enumerated with culturing techniques) (p = 0.002). Salicylic acid also positively correlated with total coliforms (p = 0.024) in the tank water samples. For the rooftop debris samples, significant positive correlations were observed for E. coli (quantified by qPCR) versus methylparaben (p = 0.000) and salicylic acid (p = 0.042), respectively. Based on the results obtained, it is thus evident that faecal contamination and anthropogenic activities may be the primary sources of contamination in the DRWH systems. Moreover, the markers Bacteroides HF183, Lachnospiraceae, human mtDNA, adenovirus, caffeine, salicylic acid and methylparaben may be utilised to supplement traditional indicator organism analysis for the monitoring of harvested rainwater. It is however recommended that future studies focus on correlation analysis of the source tracking markers with pathogens frequently detected in harvested rainwater, in order to determine which source tracking markers may be utilised as surrogates for these pathogens and subsequently as supplementary indicators. Avian species are vectors of microorganisms in the environment and have been identified as major sources of faecal contamination of DRWH systems. The focus of Chapter three was thus to design and validate (on a small-scale) novel MST markers for the detection of avian faecal contamination in the DRWH systems. Three primer sets [AVF1 and AVR (designated AV1); AVF2 and AVR (designated AV2); and ND5F and ND5R (designated ND5)] were subsequently designed to target regions of the NADH dehydrogenase subunit 5 mitochondrial DNA gene of avian species. Mitochondrial DNA is abundant in animal faecal matter and may thus be readily detected. Conventional PCR assays were optimised for each of the three primer sets. Avian and non-avian faecal samples were then screened to validate the host-specificity and host-sensitivity of the mtDNA markers. The mtDNA markers AV1, AV2 and ND5 displayed a host-sensitivity of 1.00, 0.892 and 0.622, respectively. While the host-specificity of each assay was equal to 0.316, 0.0526 and 0.237 for AV1, AV2 and ND5, respectively. Tank water samples (n = 60) and rooftop debris (n = 60) were then screened for the prevalence of the three markers. Overall, AV1 was the dominant marker detected in the tank water (85%) and rooftop debris (90%) samples. Bayes’ theorem then indicated that there was an 89.2% and 92.9% probability that the AV1 marker detected true avian faecal contamination in the tank water and rooftop debris samples, respectively. The AV1 marker thus exhibited the greatest potential as an avian mtDNA marker for the detection of avian faecal contamination in DRWH systems. However, based on the low host-specificity obtained for all three primer sets (AV1, AV2 and ND5), further optimisation should include the use of a Taqman™ probe to increase the specificity of this marker.