Browsing by Author "Ndlovu, Thando"

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    Bioprospecting for novel biosurfactants and biosurfactant producing bacteria in wastewater
    (Stellenbosch : Stellenbosch University, 2017-03) Ndlovu, Thando; Khan, Wesaal; Khan, Sehaam; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Biosurfactants are surface active amphiphilic compounds, synthesised by numerous bacteria, fungi and yeast. They are known to exhibit broad spectrum antimicrobial activity and are currently applied as antimicrobial agents, antiadhesives, foaming agents, emulsifiers etc. in the cosmetic, food, pharmaceutical and biotechnology industries. The primary aim of the study was thus to bioprospect for novel biosurfactants and biosurfactant-producing bacteria in a wastewater treatment plant (WWTP). Wastewater was selected as it is a suitable environment for the growth of diverse microorganisms and the presence of numerous organic and inorganic contaminants were postulated to enable the flourishing of biosurfactant-producing microorganisms. Chapter 1 then outlined literature pertaining to biofurfactants, their characterisation and mode of action, amongst many other topics. Chapter 2 of this study focused on the distribution and diversity of biosurfactant-producing bacteria isolated from wastewater. Wastewater samples were collected from various points of the Stellenbosch WWTP and culturable isolates were screened for possible biosurfactant production using the oil spreading and drop collapse methods. Surface tension and emulsification activities were then used for the partial characterisation of the produced biosurfactant compounds. Thirty-two of the 667 bacterial isolates were regarded as biosurfactant producers and were classified into the Aeromonadaceae, Bacillaceae, Enterobacteriaceae, Gordoniaceae and the Pseudomonadaceae families using 16S rRNA analysis. Bacillus and Pseudomonas were among the most dominant genera, which constituted 21.8% (7/32) and 12.5% (4/32) of all isolates, respectively. High surface tension reduction of the growth medium (71.1 mN/m) was also observed for the Bacillus ST34 (34.4 mN/m) and the Pseudomonas ST5 (32.3 mN/m) isolates. In addition, the Bacillus ST34 and Pseudomonas ST5 isolates tested positive for the sfp and rhlB genes involved in the biosynthesis of surfactin and rhamnolipid biosurfactants. While numerous studies have reported on the isolation of biosurfactant-producing bacteria from contaminated soil and terrestrial environments, the current study indicated that municipal wastewater could be exploited for the isolation of diverse biosurfactant-producing bacterial strains. In chapter 3, 32 biosurfactant-producing isolates were then genotypically differentiated utilising repetitive element PCRs (rep PCRs) [targeting the repetitive extragenic palindromic (REP) and the BOX element sequences]. This molecular differentiation was performed as the genetic diversity amongst bacterial species is known to produce different concentrations and proportions of various homologues of biomolecules such as biosurfactants and antibiotics. With the use of the conventional PCR assays, some of the isolates were identified as Bacillus subtilis (n = 4), Aeromonas hydrophila (n = 3) and Bacillus amyloliquefaciens (n = 2), amongst others. These bacterial species were genotypically differentiated into four, three and two sub-species (strains), respectively, utilising rep PCRs. The BOX AIR and REP primers utilised for rep PCR in the current study thus provided a powerful tool to discriminate between biosurfactant-producing bacterial isolates identified as the same species. Chapter 4 focused on the characterisation and antimicrobial activity of the biosurfactant extracts produced by the isolates B. amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5. Crude biosurfactants from ST34 and ST5 culture broth were extracted using solvent extraction based methods. Thereafter, the high resolution ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS) method, developed in the current study, was utilised to characterise the produced compounds. Results indicated that B. amyloliquefaciens ST34 primarily produced the C13, C14, C15 and C16 surfactin analogues when grown on mineral salt medium (MSM) supplemented with glycerol. For P. aeruginosa ST5, high resolution ESI-MS linked to UPLC confirmed the presence of dirhamnolipid congeners, specifically Rha-Rha-C10-C10 as well as monorhamnolipid congeners, specifically Rha-C10-C10. The crude surfactin and rhamnolipid extracts were also assessed for their antimicrobial activities and displayed significant antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains. The quantitative and qualitative effects of various substrates utilised for the surfactin and rhamnolipid production by B. amyloliquefaciens ST34 and P. aeruginosa ST5 strains, respectively, were assessed in chapter 5. For B. amyloliquefaciens ST34, maximum biosurfactant production was observed in the MSM supplemented with fructose (28 mg/L). In addition, four surfactin analogues were produced by B. amyloliquefaciens ST34 using the different substrates, however, the Srf2-4 (C13-15 surfactins) were the most dominant in all the B. amyloliquefaciens ST34 extracts. For P. aeruginosa ST5, maximum biosurfactant production was observed in the MSM supplemented with glucose (307 mg/mL). In addition, six rhamnolipid congeners were produced by P. aeruginosa ST5 using the different substrates, however, similar to results obtained in Chapter four, the dRL2 (Rha-Rha-C10-C10) and mRL2 (Rha-C10-C10) were the most abundant compounds produced in all P. aeruginosa ST5 extracts.
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    Characterisation 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, Wesaal
    Biosurfactants 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.
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    Co-detection of virulent escherichia coli genes in surface water sources
    (Public Library of Science, 2015) Ndlovu, Thando; Le Roux, Marcellous; Khan, Wesaal; Khan, Sehaam
    McNemar’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.
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    Comparative 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, Wesaal
    Background: 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.
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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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    Variants 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, Wesaal
    The 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.