Browsing by Author "Klopper, Kyle Brent"

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    Biofilm dynamics : linking in situ biofilm biomass and metabolic activity measurements in real-time under continuous flow conditions
    (Nature Research, 2020) Klopper, Kyle Brent; De Witt, Riaan N.; Bester, Elanna; Dicks, Leon Milner Theodore, 1961-; Wolfaardt, Gideon M.
    The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass–function relationships and aid in the development of biofilm control strategies.
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    Elucidating the duality of biofilms as both a proliferation and survival strategy using novel IoT technology
    (Stellenbosch : Stellenbosch University, 2023-12) Klopper, Kyle Brent; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Microbes rarely exist and proliferate as individual cells, but rather form complex community structures known as biofilms. Biofilm formation ensures that microbial communities are ubiquitous throughout both natural and man-made environments, where the presence of microbial biofilms can be advantageous or detrimental, depending on the context. Biofilm formation is traditionally viewed as a mechanism for microbial survival, since it provides protection from adverse environmental challenges, including starvation, desiccation, predation and antimicrobials. However, a more contemporary perspective also takes the role of biofilms in microbial proliferation into consideration. The reductionist approach, which classifies a biofilm as either a survival or a proliferation mechanism often leads to poor outcomes when the aim is to transition fundamental biofilm research to the real-world and its associated problems. Owing to the inherently complex and dynamic nature of biofilms, a multi-parameter approach is required to better understand its form-function relationship. The development of the novel BioSpec system provides a simple, reliable, and cost-effective tool to monitor biofilm biomass at high- resolution and in real-time, without disturbing the biofilm structure. Coupling of the BioSpec with the carbon dioxide evolution monitoring system (CEMS), facilitates the simultaneous measurement of two important interrelated biofilm metrics; namely biofilm biomass and metabolic activity. The ability to monitor these parameters non-destructively in real-time provides an unprecedented level of insight into biofilm dynamics, as demonstrated here when challenged with biocides at sub-inhibitory and inhibitory concentrations. The inherent complexity of biofilms requires biofouling mitigation strategies that are based on well-informed chemical treatment dosing regimens and the right combination of biocides and dispersants. In industrial applications, the rapid response of microbial respiration rates to chemical treatment offers a strong utility to monitor the success of antimicrobial application, whereas biofilm biomass measurement shows promise as a direct measure of dispersal or removal efficacy. The role of biofilms as ‘planktonic cell production factories’, even when challenged with antimicrobials, was again highlighted in this thesis and emphasises the need for a change in perspective. Microbial proliferation is of critical importance to multiple facets of society ranging from the food, to the medical and industrial sectors. For too long, the emphasis has been placed on the survival attributes of biofilms, thereby neglecting the equally important role of biofilms as mechanism of proliferation and dissemination. The latter has important implications for sanitation regimes in food and beverage processing facilities, where planktonic cell dissemination and secondary biofilm formation at distant locations within a facility, pose serious challenges and risks.
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    Isolation and characterization of novel Lactobacillus spp. with promising gastro-intestinal survival and adhesion properties
    (Stellenbosch : Stellenbosch University, 2017-12) Klopper, Kyle Brent; Dicks, Leon Milner Theodore; Deane, Shelly M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: The human gastrointestinal tract (GIT) is a complex organ system, and is closely associated with immunological and hormonal functions. A delicate balance needs to be maintained between the selective and beneficial colonization of allochthonous and autochthonous microorganisms, which contribute to the preservation of gut homeostasis and protect the host against infections. Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2, isolated from the feces of healthy humans, formed biofilms on a hydrophobic abiotic surface (polystyrene) under static conditions and were selected for further studies. Meaningful differences in cell surface properties were observed between the isolates, with strain HFI-K2 exhibiting a significantly greater basic surface property, in addition to a significantly higher surface hydrophobicity (37.71 %, p˂ 0.05) compared to that recorded for strain HFI-LD5 (8.82 %, p˂ 0.05). The hydrophobic nature of L. rhamnosus HFI-K2 in conjunction with better biofilm formation, may contribute to a greater GIT colonization ability. Neither of the two strains isolated degraded mucus, and their growth was not irreversibly inhibited when exposed to acidic conditions (pH 2.5) and bile salts (0.5 % and 1.0 % w/v), suggesting that they may survive conditions in the GIT. To confirm planktonic and sessile survival of L. rhamnosus HFI-K2 and L. reuteri HFI-LD5 in the human GIT, the effect of three simulated, fasting-state gastrointestinal fluids (gastric fluid, pH 2, 2 h exposure; intestinal fluid, pH 7.5, 6 h exposure and colonic fluid pH 7, 24h exposure) on both free-living and attached cell viability of the strains was assessed. Exposure to simulated gastric juice had the greatest effect on both planktonic cell viability and biofilm metabolic activity. The sequential introduction of the simulated gastrointestinal fluids initiated the detachment of biofilm biomass, accompanied by a decrease in the metabolic activity, as recorded by changes in CO2 production, by the use of the carbon dioxide measurement system (CEMS). However, as soon as the exposure was halted and sterile culture medium was reintroduced, the remaining biofilm biomass responded by producing CO2, followed by the recovery of biofilm biomass and re-establishment of pre-exposure activity within 24 h. In contrast to the complete loss of planktonic L. rhamnosus HF1-K2 viability after exposure to gastric juice, biofilms of this strain not only recovered within 24 h after exposure, but also exhibited increased metabolic activity after recovery. To our knowledge, this is the first study to assess the effect of simulated, fasting-state gastrointestinal fluids on lactobacilli biofilms. Monitoring of CO2 production as a real-time indicator of metabolic activity in a biofilm provided insight to the differential survival responses of lactic acid bacteria under fasting-state gastrointestinal conditions. The ability of L. reuteri HFI-LD5 and L. rhamnosus HFI-K2 to survive acid, bile and simulated gastrointestinal fluid induced stresses, coupled with biofilm formation under dynamic flow conditions, may contribute to improved survival and persistence of these strains within the human GIT. These characteristics, especially those exhibited by L. rhamnosus HFI-K2, are promising indicators for the application of these isolates as probiotic supplements.