Metabolomics as a tool to explore the staphylococcal biofilm

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.

The effect of the proton pump inhibitor pantoprazole on the biology of Campylobacter jejuni

Campylobacter is a major cause of acute bacterial gastroenteritis worldwide, with the highest number of infections being attributed to Campylobacter jejuni. C. jejuni is a Gram negative, spiral, motile bacterium that belongs to the campylobacterales order and is related to both Helicobacter spp. and Wolinella sp.. It has long been established that proton pump inhibitors (PPIs) and other benzimidazole derivatives display anti-Helicobacter activity in vitro. PPIs have in the past been shown to affect Helicobacter pylori growth, survival, motility, morphology, adhesion/invasion potential and susceptibility to conventional antibiotics. PPIs are highly effective drugs that are well tolerated, safe for prolonged daily use and are therefore in high demand. Both the PPIs omeprazole and lansoprazole featured in the top ten drugs prescribed in England in 2014. In 2014 Campylobacter was also the most commonly diagnosed gastrointestinal infection in Scotland, in England and Wales and also in Europe. It has previously been generally accepted that patients who are being treated with PPIs are more susceptible to enteric infections such as Campylobacter than people not taking PPIs. The effect of PPI exposure on H. pylori has been investigated rigorously in the past. A single previous study has hinted that PPIs may also be capable of affecting the related organism C. jejuni,but investigations have been extremely limited in comparison to those investigating the effect of PPIs on H. pylori. This study has investigated the in vitro effects of direct contact with PPIs on the biology ofC. jejuni. Exposure to the PPI pantoprazole was found to affect C. jejuni growth/survival, motility, morphology, biofilm formation, invasion potential and susceptibility to some conventional antibiotics. Microarray studies showed that the cmeA and Cj0561c genes were significantly up-regulated in response to pantoprazole exposure and a CmeABC deficient mutant was found to be significantly more susceptible to killing by pantoprazole than was the parent strain. Proteomic analysis indicated that the oxidative stress response of C. jejuni was induced following exposure to sub-lethal concentrations of pantoprazole. C. jejuni gene expression was assessed using qRT-PCR and the genes encoding for thiol peroxidase and GroEL co-chaperonin (both involved in the C. jejuni oxidative stress response) were found to be around four times higher in response to exposure to sub-lethal concentrations of pantoprazole. Experiments using the oxidative stress inhibitors thiourea (a hydroxyl radical quencher) and bipyridyl (a ferrous iron chelator) showed that killing by pantoprazole was not mediated by hydroxyl radical production.