Biofilm metabolomics: the development of mass spectrometry and chromatographic methodology for the analysis of dual-species pathogenic biofilms

QH345 Biochemistry;QR Microbiology

Haggarty, Jennifer ; Engineering and Physical Sciences Research Council (EPSRC) ; Burgess, Karl E. V.

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: Metabolomics, 2D-LC, mass spectrometry, ion chromatography, liquid chromatography, IC-MS, LC-MS, dual-species biofilms, Staphylococcus aureus, Candida albicans, microbial interactions, energy metabolism, purine biosynthesis, pyrimidine biosynthesis, methionine metabolism, microbial metabolism, serially combined columns, polymicrobial infection.;   

Others  :  http://theses.gla.ac.uk/8616/4/2017haggartyphd.pdf

来源: University of Glasgow

【 摘 要 】

Polymicrobial diseases arise when multiple microorganisms colonize a host and form multi-species biofilms. Within polymicrobial communities bacteria, fungi, viruses and/or parasites directly and indirectly interact with one another in a multitude of ways. The composition and the interactions between organisms within polymicrobial biofilms govern disease severity and patient outcomes. Polymicrobial infections are of significant interest because of the escalating development of antimicrobial resistance and the increasing involvement polymicrobial biofilms in chronic and systemic infections.The Gram-positive bacteria Staphylococcus aureus and dimorphic fungi Candida albicans have been shown to coexist within the human host in polymicrobial biofilm communities which often result in increased disease severity and mortality. Both of these commensals are opportunistic human pathogens that cause a plethora of infections ranging from relatively non-lethal local infections to life-threatening systemic infections in immunocompromised individuals.S. aureus and C. albicans have been co-associated with a number of polymicrobial diseases including cystic fibrosis and polymicrobial sepsis. Furthermore, S. aureus and C. albicans dual-infections have been associated with increased virulence and antimicrobial resistance. Although an effort has been made to unravel the relationship between S. aureus and C. albicans, metabolomics offers a powerful analytical tool to gain a better understanding of the interactions between this bacteria and fungus. To gain a better understanding of these interactions novel methods must be developed to modulate biofilm growth.Metabolomics is intended to analyse the complete small molecule component of a biological system. Analytically, the diversity present in these compounds presents huge opportunities for improvement. The overall aim of this research was to develop novel metabolomics methods and to apply these methods to the analysis of a S. aureus/C. albicans dual species biofilm to aid in the understanding of the relationship between this bacteria and fungi.Characterisation of the S. aureus/C. albicans biofilm in comparison in to the mono-species was carried out using a number of techniques, including fluorescence microscopy, SEM imaging, qPCR and transcriptional analysis, which indicated that these two organisms interact with each other on a physical and molecular lever. Although the presence of C. albicans facilitates S. aureus biofilm formation in sera, the presence of the bacteria reduced the number of C. albicans within the dual-species biofilm compared to the fungal mono-species and caused ‘crinkled’ hyphae which suggested possible antagonistic behaviour towards the fungi. An untargeted liquid chromatography-mass spectrometry separation method was developed that effectively retained both polar and nonpolar compounds by serially coupling a reversed-phase liquid chromatography (RPLC) column to a hydrophilic interaction liquid chromatography (HILIC) column via a T-piece. Two independent pumps were incorporated into the system to allow independent gradient control of the two columns. The high dilution between the columns, achieved by the difference in flow rates, enabled the retention and separation of both polar and nonpolar standards and numerous polar and non-polar metabolites extracted from beer. Good peak shapes and retention time reproducibility was achieved across all compound classes analysed.Next, a targeted ion-chromatography mass-spectrometry method was developed for the analysis of central carbon metabolism intermediates, specifically those involved in glycolysis, the tricarboxylic acid (TCA) cycle and the Electron Transport Chain (ETC). A total mix of all of the energy metabolites standards analysed were able to be separated and detected using IC-MS, with the exception of DHAP, G3P, oxaloacetate, acetyl-CoA, succinyl-CoA, NAD and NADP. The method displayed good reproducibility and limits of detection.The complexity of the extracted biofilms proved challenging to the IC-MS. Sample variation and low intensities in some sample groups (particularly the S. aureus samples) resulted in lower detection than expected. The RPLC/HILIC method provided hundreds of metabolite detections, but suffered in comparison to the conventional HILIC method, likely due to far greater optimisation of the original technique, leading to the utilisation of the routine pHILIC method in place of the serially combined method. Untargeted metabolomics analysis highlighted significant changes in a number of metabolic pathways including purine, pyrimidine, methionine and cysteine metabolism between the S. aureus and C. albicans mono- species and the dual-species biofilms. The differences detected within individual pathways suggest a difference in behaviour when the microorganisms are cultured with one another. The dramatic downregulation of a large portion of essential metabolites within purine, pyrimidine, cysteine and methionine pathways is indicative of the bacteria struggling to proliferate and form strong biofilms in sera. Down-regulation of many of the pathways in the dual-species biofilm compared to the C. albicans mono-species biofilm suggests that the presence of S. aureus within the biofilm could be having an adverse effect on the C. albicans. The results and conclusions herein provide greater understanding of the clinically important interaction between S. aureus and C. albicans. Microscopic and molecular characterisation enabled visualisation of the dual-species biofilm. The development and application of metabolomics techniques highlighted changes in metabolism between the mono- and dual-species biofilms, indicating that the relationship between S. aureus & C. albicans may not be completely synergistic, as previously suggested.Although the metabolomics methods developed during this study performed well, with regards to the separation of simple standard mixes and the complex beer sample, were not suitable for biofilm analysis. Through continued sample preparation and chromatographic optimisation these novel methods could offer relatively simple alternatives to more time consuming and complex chromatographic procedures.