Browsing by Author "Khan, Wesaal"
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- ItemCharacterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant(SpringerOpen, 2017) Ndlovu, Thando; Rautenbach, Marina; Vosloo, Johann Arnold; Khan, Sehaam; Khan, WesaalBiosurfactants are unique secondary metabolites, synthesised non-ribosomally by certain bacteria, fungi and yeast, with their most promising applications as antimicrobial agents and surfactants in the medical and food industries. Naturally produced glycolipids and lipopeptides are found as a mixture of congeners, which increases their antimicrobial potency. Sensitive analysis techniques, such as liquid chromatography coupled to mass spectrometry, enable the fingerprinting of different biosurfactant congeners within a naturally produced crude extract. Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, isolated from wastewater, were screened for biosurfactant production. Biosurfactant compounds were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI–MS). Results indicated that B. amyloliquefaciens ST34 produced C13–16 surfactin analogues and their identity were confirmed by high resolution ESI–MS and UPLC–MS. In the crude extract obtained from P. aeruginosa ST5, high resolution ESI–MS linked to UPLC–MS confirmed the presence of di- and monorhamnolipid congeners, specifically Rha–Rha–C10–C10 and Rha–C10–C10, Rha–Rha–C8–C10/Rha–Rha–C10–C8 and Rha–C8–C10/Rha–C10–C8, as well as Rha–Rha–C12–C10/Rha–Rha–C10–C12 and Rha–C12–C10/Rha–C10–C12. The crude surfactin and rhamnolipid extracts also retained pronounced antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains and the pathogenic yeast Candida albicans. In addition, the rapid solvent extraction combined with UPLC–MS of the crude samples is a simple and powerful technique to provide fast, sensitive and highly specific data on the characterisation of biosurfactant compounds.
- ItemCo-detection of virulent escherichia coli genes in surface water sources(Public Library of Science, 2015) Ndlovu, Thando; Le Roux, Marcellous; Khan, Wesaal; Khan, SehaamMcNemar’s test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR) assays confirmed the presence of the aggR gene (69%), ipaH gene (23%) and the stx gene (15%) carried by Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Enterohermorrhagic E. coli (EHEC), respectively, in river water samples collected from the Berg River (Paarl, South Africa). Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa). In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC) in 69%, stx (EHEC) in 15%, ipaH (EIEC) in 31% and eae (EPEC) in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC) was detected in 46% of the samples, while eae (EPEC) was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05) between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar’s test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health.
- ItemComparative analysis of solar pasteurization versus solar disinfection for the treatment of harvested rainwater(BioMed Central, 2016-12-09) Strauss, Andre; Dobrowsky, Penelope Heather; Ndlovu, Thando; Reyneke, Brandon; Khan, WesaalBackground: Numerous pathogens and opportunistic pathogens have been detected in harvested rainwater. Developing countries, in particular, require time- and cost-effective treatment strategies to improve the quality of this water source. The primary aim of the current study was thus to compare solar pasteurization (SOPAS; 70 to 79 °C; 80 to 89 °C; and ≥90 °C) to solar disinfection (SODIS; 6 and 8 hrs) for their efficiency in reducing the level of microbial contamination in harvested rainwater. The chemical quality (anions and cations) of the SOPAS and SODIS treated and untreated rainwater samples were also monitored. Results: While the anion concentrations in all the samples were within drinking water guidelines, the concentrations of lead (Pb) and nickel (Ni) exceeded the guidelines in all the SOPAS samples. Additionally, the iron (Fe) concentrations in both the SODIS 6 and 8 hr samples were above the drinking water guidelines. A >99% reduction in Escherichia coli and heterotrophic bacteria counts was then obtained in the SOPAS and SODIS samples. Ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR) analysis revealed a 94.70% reduction in viable Legionella copy numbers in the SOPAS samples, while SODIS after 6 and 8 hrs yielded a 50.60% and 75.22% decrease, respectively. Similarly, a 99.61% reduction in viable Pseudomonas copy numbers was observed after SOPAS treatment, while SODIS after 6 and 8 hrs yielded a 47.27% and 58.31% decrease, respectively. Conclusion: While both the SOPAS and SODIS systems reduced the indicator counts to below the detection limit, EMA-qPCR analysis indicated that SOPAS treatment yielded a 2- and 3-log reduction in viable Legionella and Pseudomonas copy numbers, respectively. Additionally, SODIS after 8 hrs yielded a 2-log and 1-log reduction in Legionella and Pseudomonas copy numbers, respectively and could be considered as an alternative, cost-effective treatment method for harvested rainwater.
- ItemExploring 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, WesaalBackground: 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.
- ItemGeographical distribution, characterisation and evaluation of Saccharomyces cerevisiae strains isolated from South African vineyards in the warmer, inland regions of the Western Cape(Stellenbosch : Stellenbosch University, 1999-12) Khan, Wesaal; Pretorius, I. S.; Lambrechts, M. G.; Van Der Westhuizen, T. J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH SUMMARY: The natural or spontaneous fermentation of grape must sugars, by yeast that originate from the grapes and winery equipment, to ethanol, carbon dioxide and other minor, but important metabolites, produces wine. The early stages of fermentation are usually carried out by the yeast genera with low fermentative activity, such as Hanseniaspora, Kloeckera, Candida and Pichia. An increase in the alcohol content of the wine causes the suppression of these genera, leaving Saccharomyces cerevisiae, which plays a significant and dominating role, to complete the fermentation process. For this reason S. cerevisiae is then generally accepted and known as the "wine yeast" in the wine industry. Wines made in different areas from the same grape variety with apparently similar fruit composition and processing, often taste noticeably different. It may be that differences in the microflora, naturally present in the two areas, play an important role in producing flavour differences in the wines. The genetic diversity of the yeast flora in nature was proven, by a number of microbiological and oenological studies, to be greatly influenced by a series of parameters, which among others includes the age of the vineyard, grape variety, viticultural and oenological practices, geographic location and climatic conditions. This project, as part of a larger research programme described by Pretorius, Van der Westhuizen & Augustyn (1999), was directly aimed at isolating and characterising indigenous S. cerevisiae yeast strains from vineyards based in the warmer, inland wine regions of the Western Cape in South Africa. Research by Van der Westhuizen, Augustyn & Pretorius (1999a) and Van der Westhuizen et al. (1999b) on the wine yeast strains present on grapes in the cooler, coastal areas of the same province enabled us to compare these strains isolated from these two contrasting climatic zones on a molecular, physiological and biochemical level. The ideal or ultimate aim of any such study would be to determine the entire sequence of the bases of the yeast nucleic acid, for each of these isolates. Molecular biological, physiological and biochemical techniques can, however, be used as indicators of the homology of the chromosomes of strains or isolates obtained. The specific aims and approaches to this study were as follows: Firstly approximately 1 kg of grapes were sampled from each of the 19 sites in the warmer, inland wine regions. Areas sampled included: Villiersdorp, Robertson (2 sites), Nuy, Du Toits Kloof, Bonnievale, Ashton, Montagu (2 sites), Rawsonville, Riebeeck-Kasteel (2 sites), Porterville (2 sites), Wolseley, Malmesbury (2 sites), Sianghoek and Tulbagh. These grapes were aseptically crushed and the must allowed to spontaneously ferment at 15°C. Thirty single colonies per fermentation were isolated when the residual sugar was less than 4 g/l. Characterisation of these S. cerevisiae isolates was done using molecular biological fingerprinting techniques such as electrophoretic karyotyping by pulsed field gel electrophoresis [Contour-Clamped Homogenous Electric Field, (CHEF)] and Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) using five specific Operon Kit C (OPC) primers. Comparison of chromosomal banding patterns per sampling site revealed the presence of 30 S. cerevisiae strains hereafter designated W1-W30. Initially it was thought a total of 21 yeast strains (based on CHEF banding pattern results only) were isolated from the warmer, inland regions. However, by comparing the CHEF-DNA analysis and RAOP-PCR data it was possible to distinguish between all of the strains isolated, except W1 and W5. Comparing these chromosomal banding patterns to the banding patterns collected in the Nietvoorbij database revealed that no single strain recovered was indigenous to both the warmer areas of this study and the cooler areas sampled by Van der Westhuizen at al. (1999a, 1999b). Further comparison and characterisation of these strains by means of various physiological and biochemical techniques such as the ability to flocculate; ferment various carbon sources; survive stress resistance at 55°C and -20°C by monitoring cell growth after 24 and 48 hours, respectively; and produce extracellular enzymes, was determined. Even though no distinct differences could be observed in the physiological and biochemical behaviour of strains isolated from the warmer, inland and cooler, coastal wine regions, it was shown that strains W1 and W5, which had the same DNA banding patterns, were in fact different as W1 was galactose negative and W5 was galactose positive. Strain W1 could also grow invasively into the agar medium whereas W5 could not. This comparison revealed that 30 unique strains were in fact isolated from the warmer, inland wine regions of the Western Cape. Investigations into possible survival mechanisms of S. cerevisiae was done by evaluating their ability to form pseudohyphae/grow invasively. Results indicated that these factors could contribute to the organisms over-wintering ability, but clearly other mechanisms must also be involved. Molecular biological, physiological and biochemical characterisation techniques should then be used in collaboration with each other to obtain an overall, positive and extensive profile of any yeast species or strain. These results also then prove invaluable in not only providing information, but also elucidating any possible differences between strains isolated from different climatic zones. The fermentation potential of these 30 unique strains was evaluated and of these strains three are included in the breeding programme at the ARC-Institute for Fruit, Vine and Wine, Nietvoorbij, aimed at improving wine yeast.
- ItemA 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, WarishA 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.
- ItemMicrobial interactions in drinking water systems(Stellenbosch : Stellenbosch University, 2004-03) Khan, Wesaal; Wolfaardt, Gideon M.; Saftic, S.; Rohns, H-P.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Microorganisms show a tendency to accumulate on surfaces in aqueous environments to form biofilms. Microbial biofilms represent a significant problem in public health microbiology as the development of these microbial communities, especially in water distribution systems, may lead to (i) the enhanced growth of opportunistic pathogens, (ii) the development of organoleptic problems, (iii) the reduction in the flow rate and (iv) the regrowth of microorganisms. In this project, biofilm monitors were installed in a large water distribution system to study biofilm phenomena in drinking water systems, and to deduce the biological stability and quality of the potable water. Measurements of biofilm formation potential showed that biofilms did not reach a steady state after 100 to 150 days. The microbial cells in these biofilms were mostly non-culturable. The contribution of the heterotrophic colony count to active biomass, as determined with cell numbers based on ATP measurements were often < 1%, while the ratio of heterotrophic plate counts and direct acridine orange counts were also <1%. The ratio between cell numbers based on ATP measurements and direct acridine orange counts were often < 100%. Results also showed that under certain conditions, such as those investigated in the present study, 1 pg of ATP may not be equal to approximately 104 active bacteria/cells, as stipulated by previous investigations, and that the average ATP content per active bacterial cell is indeed less than 10-16 - 10-15 g. It was calculated that threshold values for assimilable, and dissolved organic carbon below -5 IJg Gil and -0.5 mg Gil, respectively, should be target values for the control of biofilm formation in this system. It was shown that polyethylene, polyvinylchloride, teflon, plexiglass, copper, zinc-coated steel and aluminium provide favourable attachment surfaces that allowed primary colonisation and subsequent biofilm formation. Significant (p < 0.05) differences in surface colonisation on the materials were observed, indicating that the composition of the material has a direct influence on microbial colonisation. The two grades of stainless steel evaluated in this study were the least favourable materials for biofilm formation. It was further demonstrated that the nature of the surface of these materials, flow conditions and water type all had a direct influence on biofilm formation. While modification of the attachment surface did not result in significant differences (p > 0.05) in disinfection efficiency of two commonly used biocides, the concentration of the biocide, as well as the material to which the biofilm is attached, greatly influenced biocidal efficiency. The results show that biofilm monitoring needs to be implemented at the water treatment plants in addition to common biostability measurements.
- ItemMolecular detection of acanthamoeba spp., naegleria fowleri and vermamoeba (hartmannella) vermiformis as vectors for legionella spp. in untreated and solar pasteurized harvested rainwater(BioMed Central, 2016) Dobrowsky, Penelope H.; Khan, Sehaam; Cloete, Thomas E.; Khan, WesaalBackground: Legionella spp. employ multiple strategies to adapt to stressful environments including the proliferation in protective biofilms and the ability to form associations with free-living amoeba (FLA). The aim of the current study was to identify Legionella spp., Acanthamoeba spp., Vermamoeba (Hartmannella) vermiformis and Naegleria fowleri that persist in a harvested rainwater and solar pasteurization treatment system. Methods: Pasteurized (45 °C, 65 °C, 68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples were screened for Legionella spp. and the heterotrophic plate count was enumerated. Additionally, ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) was utilized for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri in pasteurized (68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples, respectively. Results: Of the 82 Legionella spp. isolated from unpasteurized tank water samples, Legionella longbeachae (35 %) was the most frequently isolated, followed by Legionella norrlandica (27 %) and Legionella rowbothamii (4 %). Additionally, a positive correlation was recorded between the heterotrophic plate count vs. the number of Legionella spp. detected (ρ = 0.710, P = 0.048) and the heterotrophic plate count vs. the number of Legionella spp. isolated (ρ = 0.779, P = 0.0028) from the tank water samples collected. Solar pasteurization was effective in reducing the gene copies of viable V. vermiformis (3-log) and N. fowleri (5-log) to below the lower limit of detection at temperatures of 68–93 °C and 74–93 °C, respectively. Conversely, while the gene copies of viable Legionella and Acanthamoeba were significantly reduced by 2-logs (P = 0.0024) and 1-log (P = 0.0015) overall, respectively, both organisms were still detected after pasteurization at 93 °C. Conclusions: Results from this study indicate that Acanthamoeba spp. primarily acts as the vector and aids in the survival of Legionella spp. in the solar pasteurized rainwater as both organisms were detected and were viable at high temperatures (68–93 °C).
- ItemVariants of lipopeptides and glycolipids produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa cultured in different carbon substrates(SpringerOpen, 2017) Ndlovu, Thando; Rautenbach, Marina; Khan, Sehaam; Khan, WesaalThe quantitative and qualitative effect of water immiscible and miscible carbon-rich substrates on the production of biosurfactants, surfactin and rhamnolipids, by Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, respectively, was analysed. A small-scale high throughput 96 deep-well micro-culture method was utilised to cultivate the two strains in mineral salt medium (MSM) supplemented with the water miscible (glucose, glycerol, fructose and sucrose) and water immiscible carbon sources (diesel, kerosene and sunflower oil) under the same growth conditions. The biosurfactants produced by the two strains were isolated by acid precipitation followed by an organic solvent extraction. Ultra-performance liquid chromatography coupled to electrospray ionisation mass spectrometry was utilised to analyse yields and characterise the biosurfactant variants. For B. amyloliquefaciens ST34, maximum surfactin production was observed in the MSM supplemented with fructose (28 mg L−1). In addition, four surfactin analogues were produced by ST34 using the different substrates, however, the C13–C15 surfactins were dominant in all extracts. For P. aeruginosa ST5, maximum rhamnolipid production was observed in the MSM supplemented with glucose (307 mg L−1). In addition, six rhamnolipid congeners were produced by ST5 using different substrates, however, Rha–Rha–C10–C10 and Rha–C10–C10 were the most abundant in all extracts. This study highlights that the carbon sources utilised influences the yield and analogues/congeners of surfactin and rhamnolipids produced by B. amyloliquefaciens and P. aeruginosa, respectively. Additionally, glucose and fructose were suitable substrates for rhamnolipid and surfactin, produced by P. aeruginosa ST5 and B. amyloliquefaciens ST34, which can be exploited for bioremediation or as antimicrobial agents.