Giles Holt

Microbiology: lessons from a first attempt at Lake Ellsworth.

During the attempt to directly access, measure and sample Subglacial Lake Ellsworth in 2012–2013, we conducted microbiological analyses of the drilling equipment, scientific instrumentation, field camp and natural surroundings. From these studies, a number of lessons can be learned about the cleanliness of deep Antarctic subglacial lake access leading to, in particular, knowledge of the limitations of some of the most basic relevant microbiological principles. Here, we focus on five of the core challenges faced and describe how cleanliness and sterilization were implemented in the field. In the light of our field experiences, we consider how effective these actions were, and what can be learnt for future subglacial exploration missions. The five areas covered are: (i) field camp environment and activities, (ii) the engineering processes surrounding the hot water drilling, (iii) sample handling, including recovery, stability and preservation, (iv) clean access methodologies and removal of sample material, and (v) the biodiversity and distribution of bacteria around the Antarctic. Comparisons are made between the microbiology of the Lake Ellsworth field site and other Antarctic systems, including the lakes on Signy Island, and on the Antarctic Peninsula at Lake Hodgson. Ongoing research to better define and characterize the behaviour of natural and introduced microbial populations in response to deep-ice drilling is also discussed. We recommend that future access programmes: (i) assess each specific local environment in enhanced detail due to the potential for local contamination, (ii) consider the sterility of the access in more detail, specifically focusing on single cell colonization and the introduction of new species through contamination of pre-existing microbial communities, (iii) consider experimental bias in methodological approaches, (iv) undertake in situ biodiversity detection to mitigate risk of non-sample return and post-sample contamination, and (v) address the critical question of how important these microbes are in the functioning of Antarctic ecosystems.

A metagenomic approach to characterize temperate bacteriophage populations from Cystic Fibrosis and non-Cystic Fibrosis bronchiectasis patients

Pseudomonas aeruginosa (Pa), normally a soil commensal, is an important opportunistic pathogen in Cystic Fibrosis (CF) and non-Cystic Fibrosis Bronchiectasis (nCFBR). Persistent infection correlates with accelerated decline in lung function and early mortality. The horizontal transfer of DNA by temperate bacteriophages can add gene function and selective advantages to their bacterial host within the constrained environment of the lower lung. In this study, we chemically induce temperate bacteriophages from clonal cultures of Pa and identify their mixed viral communities employing metagenomic approaches. We compared 92 temperate phage metagenomes stratified from these clinical backgrounds (47 CF and 45 nCFBR Pa isolates) using MG-RAST and GeneWise2. KEGG analysis shows the complexity of temperate phage accessory gene carriage increases with duration and severity of the disease. Furthermore, we identify the presence of Ig-like motifs within phage structural genes linked to bacterial adhesion and carbohydrate binding including Big_2, He_Pig, and Fn3. This study provides the first clinical support to the proposed bacteriophage adherence to mucus (BAM) model and the evolution of phages interacting at these mucosal surfaces over time.

Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.