Project Summary
The proposed project focusses on better understanding the interactions between the bacteria which inhabit the cystic fibrosis lung. Cystic fibrosis is a genetic disease that causes increased mucus production in the lungs and, as a consequence, colonisation of various types of microorganisms which affect disease progression. One of those organisms is Staphylococcus aureus which is hard to irradiate and has been linked with worse disease prognosis. In this placement, the student will investigate how S. aureus interacts with other bacteria on solid agar; specifically, we will ask whether there are common members of the cystic fibrosis lung microbiome which inhabit or enhance the growth of this pathogen. This will be accomplished by spotting colonies of S. aureus next to colonies of other members of the microbiome and observing changes in the growth diameter of the bacteria when compared to its growth alone.
Full Project Description
Overview: Cystic fibrosis (CF) is a genetic disease that, due to increased mucus production in the lungs, allow communities of microorganisms to inhabit the respiratory tract. Traditionally, the majority of these organisms have been ignored as common “commensal” organisms, and the focus instead has been on principal pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, and Staphylococcus aureus. Staphylococcus aureus is one of the first pathogenic organisms to occupy the lungs of children with CF. This organism is often hard to irradiate and has been linked with decreased morbidity and mortality in individuals with CF. Recently, research has shown that the interactions between commensals and pathogens can result in increased virulence of the pathogen in the lung. For example, in a Drosophila infection model, P. aeruginosa is more pathogenic when it is co-cultivated with various Streptococcus sp. In this summer research placement, we will use in vitro investigations to determine whether S. aureus is similarly affected by various commensal organisms.
Methodology: In this summer placement, the student will work as part of a team to collect and culture a sputum sample collected from an individual with CF. Our previous research has shown that using 13 different selective agars, we can cultivate >82% of the organisms present in these samples. These agars include Mannitol Salt Agar (MSA) which is selective for the growth of S. aureus. After incubation, we will collect individual isolates of (a) S. aureus from the MSA plates, and (b) a variety of morphologically distinct commensal organisms grown on the other agar types. After ensuring the isolates are pure (i.e., only one strain is present) we will conduct a high-throughput competition assay in which we will use a pin replicator to spot 96 commensal strains directly next to S. aureus strains on a non-selective solid agar. After incubation, we will use computational tools such as ImageJ to determine the radius of each spotted colony, and to group commensal-pathogen pairs into virulence categories based on these radii. These categories will include: (a) pairs in which the growth of S. aureus is increased in the presence of the commensal (compared to growth alone; potentiators); (b) pairs in which the growth of S. aureus is decreased in the presence of the commensal (attenuators); (c) pairs in which the growth of S. aureus is unaffected (neutrals). We will then select a small number of commensal-pathogen pairs of interest from each category to investigate in more detail using a variety of microbiological assays (for e.g., assessing the importance of secreted proteins via growth of S. aureus in the sterile spent medium of the commensal; determining the importance of physical proximity by spotting the isolates at varying distances; assessing the importance of the growth environment by testing the strain’s interactions in liquid broth and on a variety of agar types, including minimal medias and agars with varying carbon sources).
From this project, the student will gain experience and familiarity with microbiological techniques, data analysis, and literature review.