Browsing by Author "Claassen-Weitz, Shantelle"
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- ItemCurrent knowledge and future research directions on fecal bacterial patterns and their association with asthma(Frontiers Media, 2016) Claassen-Weitz, Shantelle; Wiysonge, Charles S.; Machingaidze, Shingai; Thabane, Lehana; Horsnell, William G. C.; Zar, Heather J.; Nicol, Mark P.; Kaba, MamadouENGLISH SUMMARY : Asthma is a complex respiratory condition that involves interplay between genetic predisposition, environmental, and immunological factors (Edwards et al., 2012). It is considered to be one of the most common chronic diseases, affecting ~300 million people (Masoli et al., 2004), and causing an estimated 250,000 deaths annually (Bateman et al., 2008). Furthermore, because of an increased Westernized lifestyle and urbanization in developing countries, it is estimated that by 2025 the global burden of asthma will increase by 100 million people (Masoli et al., 2004).
- ItemHIV-exposure, early life feeding practices and delivery mode impacts on faecal bacterial profiles in a South African birth cohort(Nature Research, 2018) Claassen-Weitz, Shantelle; Gardner-Lubbe, Sugnet; Nicol, Paul; Botha, Gerrit; Mounaud, Stephanie; Shankar, Jyoti; Nierman, William C.; Mulder, Nicola; Budree, Shrish; Zar, Heather J.; Nicol, Mark P.; Kaba, MamadouThere are limited data on meconium and faecal bacterial profiles from African infants and their mothers. We characterized faecal bacterial communities of infants and mothers participating in a South African birth cohort. Stool and meconium specimens were collected from 90 mothers and 107 infants at birth, and from a subset of 72 and 36 infants at 4–12 and 20–28 weeks of age, respectively. HIV-unexposed infants were primarily exclusively breastfed at 4–12 (49%, 26/53) and 20–28 weeks (62%, 16/26). In contrast, HIV-exposed infants were primarily exclusively formula fed at 4–12 (53%; 10/19) and 20–28 weeks (70%, 7/10). Analysis (of the bacterial 16S rRNA gene sequences of the V4 hypervariable region) of the 90 mother-infant pairs showed that meconium bacterial profiles [dominated by Proteobacteria (89%)] were distinct from those of maternal faeces [dominated by Firmicutes (66%) and Actinobacteria (15%)]. Actinobacteria predominated at 4–12 (65%) and 20–28 (50%) weeks. HIV-exposed infants had significantly higher faecal bacterial diversities at both 4–12 (p = 0.026) and 20–28 weeks (p = 0.002). HIV-exposed infants had lower proportions of Bifidobacterium (p = 0.010) at 4–12 weeks. Maternal faecal bacterial profiles were influenced by HIV status, feeding practices and mode of delivery. Further longitudinal studies are required to better understand how these variables influence infant and maternal faecal bacterial composition.
- ItemOptimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens(BMC (part of Springer Nature), 2020-05-12) Claassen-Weitz, Shantelle; Gardner-Lubbe, Sugnet; Mwaikono, Kilaza S.; Du Toit, Elloise; Zar, Heather J.; Nicol, Mark P.Background: Careful consideration of experimental artefacts is required in order to successfully apply highthroughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and “denoising” approaches for sequencing low biomass specimens. Results: We found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = − 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination. Conclusions: We provide insight into experimental design, quality control steps and “denoising” approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results.