Browsing by Author "Van Zyl, Kristien Nel"

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    The effect of storage conditions on microbial communities in stool
    (Public Library of Science, 2020) Van Zyl, Kristien Nel; Whitelaw, Andrew C.; Newton-Foot, Mae
    Microbiome research has experienced a surge of interest in recent years due to the advances and reduced cost of next-generation sequencing technology. The production of high quality and comparable data is dependent on proper sample collection and storage and should be standardized as far as possible. However, this becomes challenging when samples are collected in the field, especially in resource-limited settings. We investigated the impact of different stool storage methods common to the TB-CHAMP clinical trial on the microbial communities in stool. Ten stool samples were subjected to DNA extraction after 48-hour storage at -80˚C, room temperature and in a cooler-box, as well as immediate DNA extraction. Three stool DNA extraction kits were evaluated based on DNA yield and quality. Quantitative PCR was performed to determine the relative abundance of the two major gut phyla Bacteroidetes and Firmicutes, and other representative microbial groups. The bacterial populations in the frozen group closely resembled the immediate extraction group, supporting previous findings that storage at -80˚C is equivalent to the gold standard of immediate DNA extraction. More variation was seen in the room temperature and coolerbox groups, which may be due to the growth temperature preferences of certain bacterial populations. However, for most bacterial populations, no significant differences were found between the storage groups. As seen in other microbiome studies, the variation between participant samples was greater than that related to differences in storage. We determined that the risk of introducing bias to microbial community profiling through differences in storage will likely be minimal in our setting.
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    Exploring the gut microbiome of children from Cape Town communities
    (Stellenbosch : Stellenbosch University, 2021-12) Van Zyl, Kristien Nel; Newton-Foot, Mae; Whitelaw, Andrew Christopher; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.
    ENGLISH ABSTRACT: Despite the increase in microbiome investigations in the last decade, descriptions of the microbiota remain limited in developing countries, particularly in children. Little is known about the gut microbiota of young children in South Africa and there is a need for investigations of the composition and diversity of microbiota and the influence of demographic, clinical, and environmental factors on the microbiota in this setting. This study formed part of the ongoing Tuberculosis child multidrug-resistant preventive therapy (TB-CHAMP) clinical trial, which aims to determine the efficacy and safety of levofloxacin preventive therapy in a cohort of children <5 years exposed to multidrug-resistant tuberculosis in the household. This sub-study explored the composition, diversity, and factors influencing the baseline bacterial and fungal gut microbiota of these children, prior to randomisation into treatment or placebo arms. A pilot study was performed to assess the effect of stool sample storage and transport conditions on the microbiota and to develop a practical DNA extraction standard operating procedure for the remainder of the sub-study and future microbiome studies in this setting. Quantitative PCR showed that the gut microbiota was not affected by storage and transport conditions common to this setting. Targeted 16S rRNA and internal transcribed spacer 1 gene sequencing was performed on the Illumina platform to characterise the bacterial and fungal microbiota. The bacterial microbiota of young children from Cape Town communities were shown to be similar to those in children from developing countries and age was the main driver of differences in both bacterial and fungal communities. While the risk for dysbiosis was shown to be low overall, recent antibiotic use and household pollution were associated with reduced bacterial diversity. The inconsistent detection and abundance of fungi even in participants of the same age, highlighted the need for expanded research, including dietary and longitudinal analysis. The need for longitudinal analysis in paediatric populations was further emphasised by a systematic review on the effects of antibiotics on the microbiome, particularly in populations who are at risk for increased antibiotic exposure. The findings also demonstrated the population and target site-specific responses of the microbiome to antibiotics, which underlined the need for individual evaluation of the effect of antibiotics on the gut microbiome. This study set the groundwork for future investigations in the TB-CHAMP cohort, including the temporal development and stability of the gut microbiome in these children, and the impact of levofioxacin preventive therapy on the microbiota and resistance reservoirs in the gut.
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    Genotypic and epidemiological characterization of Methicillin resistant Staphylococcus aureus (MRSA) and Coagulase Negative Staphylococcal (CoNS) strains isolated at Tygerberg Hospital
    (Stellenbosch : Stellenbosch University, 2016-12) Van Zyl, Kristien Nel; Hoek, Kim Gilberte Pauline; Whitelaw, Andrew Christopher; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology: Medical Microbiology
    Background The global burden of methicillin resistant Staphylococcus aureus (MRSA) can be largely attributed to S. aureus’ ability to acquire the resistance element, SCCmec (Staphylococcal chromosome complex mec). Classification of SCCmec types is an essential component of staphylococcal epidemiology and is based on the arrangement and classes of the mec and ccr gene complexes and Open Reading Frames (ORFs) in the joining (J) regions. At least twelve SCCmec types and numerous subtypes have been described to date. We identified potentially novel and novel variant SCCmec types in MRSA isolates from a tertiary hospital in Cape Town, South Africa. This study aimed to describe the molecular structure and possible origin of these novel elements in our setting, and to determine the prevalence of these SCCmec types. Methods We screened 87 clinical MRSA and 100 methicillin resistant coagulase negative Staphylococci (MR-CoNS) isolates using a multiplex PCR for SCCmec typing. Additional typing employed a combination of six multiplex PCRs on 3 MRSA isolates each from the novel and novel variant types. Whole genome sequencing (WGS) was performed on representative isolates using the Illumina next-generation sequencing platform. Results Among the MRSA isolates, 36% contained the novel SCCmec type (ccrC/Class A mec), followed by SCCmec IV (24%), the novel variant SCCmec type (ccrA1B1, ccrC/Class B mec) (17%) and SCCmec III (11%). Only one MR-CoNS isolate contained the novel type. Preliminary genomic analysis support the PCR findings, and show the presence of a possible truncated SCCHg element in the novel, novel variant and SCCmec III isolates. Discussion We have successfully optimized and implemented two SCCmec typing assays on MRSA and MR-CoNS isolates in our setting and in doing so, identified possible novel and novel variant SCCmec elements. The novel SCCmec type is common among local MRSA isolates, and may reflect clonal spread within the hospital. Preliminary WGS analysis showed that these isolates are composites of SCCHg and SCCmec elements, however further phylogenetic analysis is required to provide insights into how these elements emerged.