Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Subject "Antimicrobial -- Testing"

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    Exploring the antimicrobial and antifouling properties of secondary metabolites produced by serratia marcescens
    (Stellenbosch : Stellenbosch University, 2021-04) Clements, Tanya Lee; Khan, Wesaal; Ndlovu, Thando; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: The emergence of multi- (MDR) and extensive drug-resistant (XDR) bacterial and fungal pathogens constitutes a major public health concern and has led to the prioritisation of research into the discovery of novel bioactive compounds. Microbial secondary metabolites serve as promising alternative antimicrobial and antifouling agents, with Serratia species representing a potential untapped source of novel and structurally diverse bioactive compounds. Chapter one (abbreviated version published in Applied Microbiology and Biotechnology) focused on the classification, biosynthesis, production and application of secondary metabolites produced by Serratia species. The primary focus of this dissertation was subsequently to identify secondary metabolites produced by environmental Serratia species that display antimicrobial and antifouling activity, and elucidate the secondary metabolic profiles and chemical structures of these compounds. In Chapter two (published in Microbiological Research), various environmental sources were screened for Serratia isolates capable of biosurfactant production during secondary metabolism. A total of 569 presumptive Serratia strains were subsequently isolated from wastewater treatment plants, an oil refinery, winery and olive oil estates, river water and rainwater samples. Preliminary screening methods (i.e. oil spreading method, emulsification assay and surface tension measurements) and molecular typing identified twenty-two pigmented (n = 11; P1 to P11) and non- pigmented (n = 11; NP1 to NP11) Serratia marcescens (S. marcescens) presumptive biosurfactant producers. Based on the physico-chemical analysis, molecular analysis and preliminary antimicrobial testing, ultra-performance liquid chromatography (UPLC) linked to electrospray ionisation mass spectrometry (ESI-MS) was used to identify the secondary metabolites produced by S. marcescens strains P1, NP1 and NP2. Strains P1 and NP1 produced serrawettin W1 homologues (also known as serratamolides) as well as prodigiosin (P1) and glucosamine derivative A (NP1). In contrast, serrawettin W2 analogues were predominantly identified in the NP2 extract. Antimicrobial analysis then indicated that the P1 and NP1 crude extracts exhibited broad-spectrum antimicrobial activity against opportunistic pathogens, such as MDR Pseudomonas aeruginosa (P. aeruginosa), methicillin-resistant Staphylococcus aureus and a clinical Cryptococcus neoformans strain. While an XDR Acinetobacter baumannii strain was susceptible to the NP2 extract, a narrower spectrum of antimicrobial activity was observed in comparison to the other two strains. The compounds produced by the P1 (pigmented) and NP1 (non-pigmented) S. marcescens strains could thus serve as a promising source of antimicrobial agents for therapeutic application. An integrated approach involving the use of reverse-phase high-performance liquid chromatography (RP-HPLC), ESI-MS, UPLC linked to tandem mass spectrometry (UPLC-MSᵉ) and molecular networking (using the Global Natural Products Social molecular network platform), was applied in Chapter three to unravel the secondary metabolic profiles and structures of the bioactive compounds produced by S. marcescens P1 and NP1. The mass spectrometry-based molecular networking guided the structural elucidation of 18 compounds for the P1 strain (including 6 serratamolides, 10 glucosamine derivatives, prodigiosin and serratiochelin A) and 15 compounds for the NP1 strain (including 8 serratamolides, 6 glucosamine derivatives and serratiochelin A) using the UPLC-MSᵉ fragmentation profiles. It was proposed that the serratamolide homologues consisted of two L-serine residues (cyclic or open-ring) linked to two fatty acyl chains (lengths of C₁₀, C₁₂ or C₁₂:₁). The glucosamine derivative homologues consisted of four residues, including glucose / hexose, valine, butyric acid (or oxo-hexanoic acid for derivative at m/z 627.4192) and a saturated or unsaturated fatty acyl chain (lengths of C₁₃ to C₁₇). The putative structures of a novel open-ring serratamolide homologue and eight novel glucosamine derivative congeners were described. The minimum inhibitory and bactericidal concentrations revealed that prodigiosin exhibited potent activity against Enterococcus faecalis (E. faecalis), followed by glucosamine derivative A and serratamolides A, B and C. The integrated approach thus provided insight into the secondary metabolic profile and structures of novel congeners produced by the S. marcescens strains. In Chapter four, the biofilm disruption and antiadhesive potential of the P1 and NP1 crude extracts was evaluated using the Minimum Biofilm Eradication Concentration (MBEC) Assay® against single- and dual-species biofilms. Plate count and viability-quantitative polymerase chain reaction indicated that the P1 and NP1 extracts significantly reduced (≥ 2 logs) biofilms formed by E. faecalis, while the single-species P. aeruginosa biofilm was more susceptible (≥ 2 logs) to the P1 extract. The P1 and NP1 extracts significantly reduced the dual-species P. aeruginosa and E. faecalis biofilm; however, in comparison to the single-species E. faecalis biofilm, increased concentrations of both extracts were required to reduce E. faecalis by ≥ 2 logs. Moreover, pre- absorption of the P1 and NP1 extracts (at 50 mg/mL) onto the pegs of the MBEC Assay® reduced the adhesion of mono-culture P. aeruginosa and E. faecalis cells by ≥ 80% based on cell counts and gene copies. In contrast, for the co-culture experiments, significant reductions (≥ 90% based on cell counts and gene copies) in the adhesion of only E. faecalis to the P1 and NP1 coated pegs were observed. Serratamolides and glucosamine derivatives present in the P1 and NP1 extracts were subsequently covalently immobilised onto high-density polyethylene (HDPE) and polyvinyl chloride (PVC) discs. The P1 and NP1 coated HDPE reduced the adhesion of P. aeruginosa cells by ≥ 87% based on plate counts and ≥ 64% based on gene copies, while the E. faecalis cells were reduced by ≥ 96% based on plate counts and ≥ 87% based on gene copies. The P1 and NP1 coated PVC also effectively reduced the adhesion of P. aeruginosa cells by ≥ 81% based on plate counts and ≥ 99% based on gene copies however, minor reductions in E. faecalis adhesion were observed. While it is recommended that the antifouling potential of the biomaterials be tested against mixed microbial communities and that the serratamolides and glucosamine derivatives be immobilised onto various other piping materials frequently used in the water, food and medical industries; this preliminary analysis indicates that the P1 and NP1 extracts could potentially be applied as a preventative strategy to delay the onset of biofilm formation on polymeric materials.