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Staphylococcus aureus is a bacterium found in the milk of up to 38% of healthy lactating mothers; however, S. aureus is isolated with increased frequency from colostrum and mastitis milk. Both of these milk types have lower lactose concentrations compared to mature milk from healthy lactating mothers, which may indicate that lactose has a role in determining whether S. aureus can survive in human milk. The aim of this study was (1) to investigate whether the presence of S. aureus in human milk is associated with the milk's lactose concentration, and (2) to determine whether different lactose concentrations can affect the ability of S. aureus isolates to grow in vitro. Human milk samples were collected at 10 weeks postpartum from mothers participating in the Drakenstein Child Health Study (Cape Town, South Africa) and underwent NMR spectroscopy to determine their metabolome. A subset of these samples (n = 117) was cultured to isolate S. aureus. Milk samples with lactose concentrations of less than 166 mM were more likely to have S. aureus present, compared to samples with lactose concentrations of over 166 mM (p < 0.001). In vitro, the growth of S. aureus was negatively correlated with the lactose concentration of axenic culture. Lactose concentrations associated with human milk appear to have an inhibitory effect on the growth of S. aureus human milk isolates. Therefore, low-lactose human milk could potentially be a risk factor for increased S. aureus growth and the development of S. aureus-associated mastitis. [ABSTRACT FROM AUTHOR]

Lake Magic, located on the Yilgarn Craton in southern Western Australia, is a round lake with ~1 km diameter and harbours a unique environment on Earth. It exhibits co-stressors of pH 1.6-4.5 and salinity of 32% TDS. The lake is known to have the highest concentration of dissolved Al, Fe and Si in the world. Lake Magic undergoes stages of flooding, evapo-concentration and desiccations, and hence, is a home to a diverse population of halophilic and acidophilic microorganisms. Understanding the diversity dynamics and survival strategies of extremophiles enables us to comprehend how they interact to drive key biogeochemical cycles, and has broad applications in biomining, synthetic ecology and biotechnology. Here, we used a temporal approach to understand prokaryotic and eukaryotic diversity in the lake sediment and salt mat using high throughput amplicon sequencing. To further understand the survival strategies of microbes, their microbial interactions were studied using a high throughput co-culture method. We established a bottom up approach by using bacterial species isolated from Lake Magic in a well-controlled synthetic community fed with labelled isotope substrates and analysed them through NanoSIMS. We studied the species metabolic interactions in all mono and pairwise cultures. The results indicated that the microbial diversity and composition are significantly affected by lake conditions, and species become more specialised in buffering the increased acidity in the lake as a strategy to survive. NanoSIMS analysis gave an in-depth view of carbon and nitrogen exchange between isolates, shedding light on the potential tactics to survive in this extreme environment. [ABSTRACT FROM AUTHOR]

Stable isotope probing is a combined molecular and isotopic technique used to probe the identity and function of uncultivated microorganisms within environmental samples. Employing stable isotopes of common elements such as carbon and nitrogen, RNA-SIP exploits an increase in the buoyant density of RNA caused by the active metabolism and incorporation of heavier mass isotopes into the RNA after cellular utilization of labeled substrates pulsed into the community. Labeled RNAs are subsequently separated from unlabeled RNAs by density gradient centrifugation followed by identification of the RNAs by sequencing. Therefore, RNA stable isotope probing is a culture-independent technique that provides simultaneous information about microbiome community, composition and function. This chapter presents the detailed protocol for performing an RNA-SIP experiment, including the formation, ultracentrifugation, and fractional analyses of stable isotope-labeled RNAs extracted from environmental samples.