Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Subject "Acinetobacter baumannii -- Molecular genetics"

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    Chemical and biological control of acinetobacter baumannii
    (Stellenbosch : Stellenbosch University, 2023-03) Havevenga, Benjamin; Khan, Wesaal; Ndlovu, Thando; Reyneke, Brandon; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Multidrug (MDR) and extensively drug resistant (XDR) Acinetobacter baumannii (A. baumannii) have emerged as the leading causes of nosocomial infections worldwide and are characterised as critical- priority pathogens by the World Health Organisation. Chapter one (abbreviated version published in Microorganisms) thus focused on the extensive antibiotic resistance and virulence mechanisms employed by A. baumannii as well as potential chemical (i.e., biosurfactants) and biological (i.e., predatory bacteria and bacteriophages) control agents which could be applied to target this opportunistic pathogen. Additionally, limitations associated with these alternative methods, and potential mitigation strategies, including curtailing resistance development using combination therapies, product stabilisation, and large-scale (up-scaling) production, were outlined. The primary focus of this study was thus to determine the efficacy of chemical and biological control strategies to combat or control reference, clinical and environmental MDR and XDR A. baumannii. While the research and development of alternative or novel strategies to combat infections caused by MDR and XDR A. baumannii is crucial, genotypic, and phenotypic characteristics, which may influence therapeutic potential, are often overlooked. The primary aim of Chapter two (published in Microbial Pathogenesis) was then to characterise and compare the genotypic and phenotypic characteristics of clinical (n = 13) and environmental (n = 7) A. baumannii isolates. Genotyping using Repetitive Extragenic Palindromic Sequence-based polymerase chain reaction (REP-PCR) analysis, indicated a low genetic relatedness between the clinical and environmental A. baumannii. Multilocus sequence typing (MLST, Oxford scheme) assigned the clinical A. baumannii isolates to three sequence types (i.e., ST231, ST945 and ST848), while the environmental A. baumannii were assigned to the novel ST2520 (except AB 14 which was assigned to ST945). While the majority of the clinical and environmental A. baumannii isolates were capable of phase variation, with both the translucent and opaque colony phenotypes detected, the clinical isolates exhibited significantly higher biofilm formation capabilities. Moreover, the clinical isolates exhibited significantly higher antibiotic resistance (first line and last resort) profiles, with 92.3% (12/13) classified as XDR, and five clinical A. baumannii isolates exhibiting colistin resistance (38.5%; 5/13). In contrast, while the environmental A. baumannii were predominantly characterised as MDR (57.1%; 4/7), the AB 14 isolate was characterised as XDR. Thus, while the differences in the genotypic, phenotypic, and antibiotic resistance profiles of clinical and environmental A. baumannii were highlighted, the environmental strains were assigned to the novel ST2520, which confirms the existence of this opportunistic pathogen in extra-hospital reservoirs. The primary aim of Chapter three was then to investigate the efficacy of lipopeptides and glycolipids against MDR and XDR strains of A. baumannii. Lipopeptides produced by Bacillus amyloliquefaciens (B. amyloliquefaciens) ST34 and Serratia marcescens (S. marcescens) NP1, and glycolipids produced by Pseudomonas aeruginosa (P. aeruginosa) SB24, were solvent extracted and partially characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS). The presence of surfactin (C13 to C16) and bacillomycin-L were detected in the B. amyloliquefaciens ST34 crude extract; serratamolides A, B, C, and glucosamine derivatives A, B, C, E and K in the S. marcescens NP1 crude extract, and di- and mono- rhamnolipids in the P. aeruginosa SB24 crude extract. Overall, in comparison to the NP1 and SB24 crude extracts, the ST34 crude extract exhibited increased efficacy against the MDR and XDR clinical and environmental A. baumannii, based on the disc diffusion results. Broth microdilutions in combination with the redox dye resazurin, then confirmed that the ST34 crude extract exhibited antimicrobial activity, with a minimum inhibitory concentration ranging from 5 to 20 mg/mL recorded. Using reverse-phase high-performance liquid chromatography (RP-HPLC), the surfactin fractions (Srf1 to Srf4) were purified with results indicating that specifically the Srf2 and Srf4 fractions exhibited potent antimicrobial activity of 60% and 80%, respectively, against representative A. baumannii STs, including the reference MDR AB 1 (ST931), clinical XDR AB 3 (ST231), environmental XDR AB 14 (ST945), and environmental MDR AB 16 (ST2520). In addition, the Srf1 to Srf4 fractions did not exhibit haemolytic activity and in vivo toxicity was not recorded for the ST34 crude extract and Srf2 and Srf4 fractions using the Galleria mellonella model. The ST34 crude extract, Srf2 and Srf4 fractions could thus be employed as novel or alternative strategies to control MDR and XDR A. baumannii. The primary aim of Chapter four was then to investigate the predation capabilities of a Bdellovibrio bacteriovorus (B. bacteriovorus) strain HW 1 (isolated from hospital wastewater) against the reference MDR AB 1 (ST931), clinical XDR AB 3 (ST231), clinical XDR CAC 8 (ST848), environmental XDR AB 14 (ST945), and environmental MDR AB 16 (ST2520). Culture-dependant and ethidium monoazide bromide quantitative polymerase chain reaction (EMA-qPCR) analysis of the co-culture assays indicated that the B. bacteriovorus HW 1 predated on all the MDR and XDR A. baumannii isolates, with log reductions ranging from 2.47 to 3.98 [colony forming units (CFU)/mL] and 2.42 to 4.54 [gene copies (GC)/mL] recorded for the first 24 hours. However, the cell counts and GC for all the A. baumannii strains in co-culture with B. bacteriovorus HW 1, increased at 48 hours and continued to increase until the end of the co-culture trial (i.e., 96 hours). Correspondingly, B. bacteriovorus HW 1 cell counts [plaque forming units (PFU)/mL] increased by 1.92 to 3.24 log at 24 hours, which was further confirmed with EMA-qPCR (GC/mL log increased 2.92 to 4.05). Subsequently, three methods [i.e., filtration, sodium dodecyl sulphate (SDS) treatment and kanamycin treatment] were employed to eliminate the B. bacteriovorus HW 1 from the co-cultures, to determine whether the A. baumannii strains initiated plastic phenotypic resistance to survive predation and persist in the co-culture trials. However, the triple filtration steps, SDS treatment and kanamycin treatment only reduced the cell count (PFU/mL) of B. bacteriovorus HW 1 by 2.93 log, 1.28 log and 0.16 log, respectively, in comparison to the unfiltered and untreated controls. Consequently, as the predatory population was not removed, reverse transcription quantitative polymerase chain reaction analysis (RT-qPCR) was applied to investigate the response of the surviving A. baumannii population in terms of virulence gene expression [i.e., lipopolysaccharide biosynthesis (lpsB) gene and the biofilm associated protein (bap) gene]. While variable results were obtained, overall, the expression analysis for the lpsB and bap genes indicated that the XDR CAC 8 (lpsB gene at 48 hours only) XDR AB 3, XDR AB 14 and MDR 16 (lpsB gene at 24 and 48 hours; bap gene at 48 hours only), during co-culture with B. bacteriovorus HW 1, was significantly lower compared to the expression recorded in the predator free controls at both 24 and 48 hours. These results indicated that although A. baumannii persists and survives predation by B. bacteriovorus HW 1, overall, the surviving prey population exhibited decreased virulence, which could be exploited in combination treatment strategies i.e., combining predatory bacteria pre-treatment (24 hours) with solar disinfection or solar pasteurization, to eliminate or eradicate the target pathogen from water sources.