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Klebsiella pneumoniae is an urgent health threat which has been identified by world medical agencies as a major risk to public health. The lack of efficient medicines against infectious diseases is becoming one of the leading causes of mortality worldwide. Gram-negative pathogens are becoming increasingly resistant to current medical innovations, whereby if no prompt action is taken will result in no treatments being available. One way that multiple pathogens including K. pneumoniae have evolved to become increasingly resilient to current human medicine is by existing in biofilms. Living within a highly-organised community has become an important survival strategy due to the many advantages that it has versus existing in a planktonic state. Living within a biofilm results in increased drug-resistance of bacteria due to the gradient of both nutrients and oxygen which are linked to an increase in doubling times of bacterial cells and a decrease in metabolism. Biofilms are also linked with an upregulation in efflux pumps, mutations in antibiotic targets (Høiby et al., 2010) . It is therefore important that research is performed to fully understand and begin tackling them. Biofilm formation is important for K. pneumoniae dissemination and virulence. This research aimed to investigate which genes are involved in biofilm formation in K. pneumoniae 52145 using an extensive random mutagenesis approach in optimal screening conditions. These genes were then characterised to identify their role in both the biofilm and within the host. A total of eighty-five hits were found to be involved in biofilm formation in K. pneumoniae. There were hits in multiple previously identified biofilm-related genes involved in fimbriae, sugar transporters, transcriptional regulators, capsule synthesis, metabolism, and O-antigen. However, there were many hits that were in found in this study that have not been previously identified in other K. pneumoniae transposon screens. These included hits in polysaccharides, siderophores, Type VI secretion system and peptidoglycan. Fimbriae and cellulose were also found for the first time to play a role in virulence in G. mellonella. There were several hits in T6SS secretion system related genes which is a novel area regarding biofilm formation and has not been reported in K. pneumoniae previously. Overall, this work is the first time that a transposon screen investigating biofilm formation has been performed in K. pneumoniae 52145, which has revealed that there are a multitude of genes involved in biofilm formation. This study has provided a foundation in which further research can be performed to further characterise the role that these hits play in both biofilm formation and in host pathogen interactions. Due to the increase in antibiotic resistance, it is vital that alternative therapies are investigated. The hits identified in this study could be used towards potential therapeutic targets in the future to tackle K. pneumoniae 52145 living in biofilms in the respiratory tract of patients.

Klebsiella pneumoniae is an emerging pathogen for which there are limited therapeutic options. K. pneumoniae survives in macrophages within specialised Klebsiella containing vacuoles (KCV) by subverting phagosome maturation. Here, we have investigated the metabolic adaptation of K. pneumoniae to its intracellular lifestyle. Isogenic mutants were constructed for key enzymes from each metabolic pathway and assessed for intracellular survival. We found that the glyoxylate shunt was dispensable for K. pneumoniae survival within the KCV, but disruption of glycolysis, gluconeogenesis and pentose phosphate pathway impaired survival within macrophages. From the tricarboxylic acid cycle, fumarate reductase and isocitrate and malate dehydrogenases play a role in intracellular survival whereas neither aconitase nor citrate synthase do. Klebsiella-controlled blockage of phagolysosome maturation was compromised for most that showed an intracellular survival phenotype, but 6-phosphofructokinase and 6-phosphogluconate dehydrogenase did not affect this pathway, possibly due to a nutritional deficit. Disruption of these enzymes also resulted in recruitment of Rab14 to the KCV, unlike the mutants that were unable to prevent phagolysosomal fusion. The ability, or lack thereof, to recruit Rab14 to the KCV was reflected in Kp52145 and the isogenic mutants ability to activate AKT in macrophages. Klebsiella mutants of fructose bisphosphate aldolase, malate and 6-phosphogluconate dehydrogenases induced host inflammatory response upon infection above that of wild type Kp52145. The remaining mutants with intracellular survival deficits did not elicit immune responses beyond that seen with Kp52145 infection. Interestingly, alterations in the metabolic profile of Kp52145 did not affect the induction of IL-10 in macrophages. We determined macrophage metabolism upon Klebsiella infection using a pharmacologic approach. Inhibition of glycolysis negatively impacted Klebsiella intracellular survival, whereas fatty acid oxidation or oxidative phosphorylation inhibition did not. We have also demonstrated that macrophage glycolysis is required by the host to illicit an immune response to infection. Mechanistically, we determined that the STAT6 and IL-10 are exploited to aid intracellular survival in macrophages. In conclusion, we have uncovered the metabolic profile of intracellular K. pneumoniae and how the macrophage is rewired upon infection.

Gut microbiota are important for human health; contributing to host metabolism and limiting pathogen infections. Colonic microbiota lives in almost anoxic environment and separated from the host epithelium by mucus layer. Host epithelia adapt to the hypoxic environment in the lumen by stabilising the transcription factor HIF-1α. HIF-1α is critical for expression of certain enteric antimicrobial peptides to maintain intestinal integrity. Mucins, the main glycoprotein component of mucus, are highly glycosylated and specific microbiota forage mucin's glycans. Previous studies showed that HIF-1α conditional knockout mice have a thinner sterile mucus layer as well as altered expression of mucin glycosylation enzymes. Our hypothesis is that HIF-1a expression influences mucus thickness, sterility, and glycan profile. In this study, we used colonic epithelial cells in vitro; Caco2 (enterocyte cell line) and HT29MTX (goblet cell line). Cell lines were cultured either in normoxia, hypoxia or treated with pharmacological modulators of HIF to study the regulation of the mucin glycosyltransferase enzymes, mucus composition, and effect of altered mucus layer on commensals/pathogenesis bacteria growth. Using differentiated Caco2, we showed higher C1GALT1 (the main mucin glycosylation enzyme) expression in hypoxia compared to normoxia over 8-hours of exposure time. We also studied the metabolically incorporation of different labelled sugars in mucin structure using "click chemistry" reaction. Azido labelled N-acetylgalactosamine (GalNAc) shows lower GalNAc incorporation in HIF inhibited mucus produced in goblet cells compare to HIF stabilised one. N-acetylglucosamine (GlcNAc) revealed high incorporation in HMW mucus glycoprotein structures produced by the cells under Normoxia compared with mucus produced under hypoxic conditions. Mucin-2 Immunofluorescent labelling shows the higher MUC2 expression in HIF stabilised cells and not significant changes in normoxic and HIF inhibited cells which shows the role of HIF in adaptive immune respond in hypoxia. The antimicrobial activity of mucus secreted by HT29 and Caco2 was also studied using E. coli and S. Typhimurium as candidate for commensal and pathogenic bacteria, respectively. The E. coli was sensitive to mucus produced by normoxic, hypoxic, and HIF inhibited cells. However, the S. Typhimurium showed not significant sensitivity to all mucus samples (a source of antimicrobial factor). Finally, to study the carbon source availability of mucus layer secreted by goblet cell line, in vitro, we used Akkermansia muciniphila, an anaerobic commensal mucin-degrading bacterial species, and incubated with different mucus samples (collected from differently treated cells) in minimal growth conditions (M9 salts) under anaerobic conditions and analysed its growth after 24 hours. Subsequently, we studied the influence of liberated/residual glycans on the growth of commensal E. coli (K12 MG1655). Overall, the E. coli growth curve showed the bacteria supplemented with all the different mucus samples had more growth than the bacteria treated with only M9 salt media. The mucus sample produced under hypoxia and HIF stabilisation also provided higher growth for commensal bacteria compared with the bacteria treated with mucus sample produced under normoxic/HIF inhibitor conditions. In conclusion, our findings indicate that the gut mucus properties are influenced by epithelial oxygenation state, and this may have a high influence on the composition of the microbiota.

In recent years, e-cigarettes have been marketed as an alternative to cigarette smoking that is less damaging to health. However, there is a lack of scientific evidence to support this assertation that they are a safer alternative to cigarettes. This study aimed to evaluate the effect of e-cigarettes on key respiratory pathogens which included S. aureus (SA), P. aeruginosa (PA), H. influenzae (HI), S. pneumoniae (SP) and M. catarrhalis (MCAT). Results from this study have shown that neat e-liquids can reduce bacterial viability with particular flavours causing significant reductions in bacterial viability. It was found that e-liquids contained numerous chemical compounds some of which had antimicrobial properties. The greater the number of chemical compounds present in the e-liquid the greater the reduction in bacterial viability. E-liquid induced antimicrobial pressure affected HI, MCAT and SP isolates to a greater degree than PA and SA isolates. It was discovered that both vaped and neat e-liquids increased biofilm formation in PA and SA isolates whilst decreasing biofilm formation in HI, SP and MCAT isolates. The vaping of the e-liquids indicated that PGVG and flavours had significant effects on biofilm formation in bacteria. Additionally, e-cigarette vapour extract (ECVE) exposure modified the expression of two PA virulence factors, pyoverdine and pyocyanin. The chemical components of e-liquids components can act as environmental stressors resulting in modification of important bacterial virulence factors. Exposing respiratory bacteria to ECVE or cigarette smoke extract (CSE) before infection was found to result in significant increases and decreases in the release of IL-8 and IL-6 and activation of NF-κB in human cells. The degree of effect on cell inflammation appeared to be dependent on the bacterial isolate and whether it was exposed to ECVE or CSE. The exposure of respiratory isolates to ECVE or CSE can modulate the host immune response to infection. Overall, the study shows that e-cigarette exposure can induce changes in key respiratory pathogens associated with chronic lung diseases. These changes could impact host-bacterial interactions and may increase bacterial persistence and inflammatory potential.