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Access Microbiology, 2019
Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigat... more Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigate the molecular mechanisms by which acetic acid acts as an antibacterial agent. To do this, we grew a high-density transposon library in uropathogenic E. coli EO499 serotype 131 in M9 media at pH 7 and pH 5.5 with acetic acid concentrations of 40 mM and 4 mM, respectively, or without added acetic acid. Sequencing libraries were generated from total bacterial populations after growth, and sequenced using a transposon-specific primer to generate positions and frequencies for each transposon. By comparing numbers of reads before and after the stress, we identified candidate genes where transposon inserts led to a decrease of fitness under acetic acid stress. Eight of these were chosen for further study: nuoM, nuoG, sucA, sthA, pitA, apaH, rssB and ytfP. Because of the difficulties of constructing gene deletions in the uropathogenic strain for validating the TraDIS results, we tested the relative fitness of the corresponding gene deletion mutants from the Keio library (in strain BW25113), with the growth conditions used for EO499. Interestingly, only a few knockouts showed a reduction in relative fitness in time course competitions at pH 5.5 with acetic acid. This may due to the differences between strains used in TraDIS and competition. To overcome this issue, we have also isolated transposon mutants from E. coli EO499 transposon library for the determination of relative fitness. The results will be presented.
Frontiers in microbiology, 2017
Bile salts are potent antimicrobial agents and are an important component of innate defenses in t... more Bile salts are potent antimicrobial agents and are an important component of innate defenses in the intestine, giving protection against invasive organisms. They play an important role in determining microbial ecology of the intestine and alterations in their levels can lead to increased colonization by pathogens. We have previously demonstrated survival of the opportunistic pathogen Staphylococcus aureus in the human colonic model. Thus investigating the interaction between S. aureus and bile salts is an important factor in understanding its ability to colonize in the host intestine. Harnessing bile salts may also give a new avenue to explore in the development of therapeutic strategies to control drug resistant bacteria. Despite this importance, the antibacterial activity of bile salts on S. aureus is poorly understood. In this study, we investigated the antibacterial effects of the major unconjugated and conjugated bile salts on S. aureus. Several concentration-dependent antibact...
The analysis of circulating tumour DNA (ctDNA) promises to extend current tissue-specific cancer ... more The analysis of circulating tumour DNA (ctDNA) promises to extend current tissue-specific cancer screening programmes to multi-cancer early detection and measurable disease monitoring to solid tumours using minimally invasive blood draws (liquid biopsies). Most studies so far have focused on using targeted deep sequencing to detect the low-abundance, fragmented ctDNA. Few studies have integrated information from multiple modalities using shallow 1x WGS. Here, we developed an integrated bioinformatics pipeline for ctDNA detection based on whole genome TET-Assisted Pyridine Borane Sequencing (TAPS) of plasma samples sequenced at 80x or higher. We conducted a diagnostic accuracy study in a case-control cohort of patients presenting to the UK National Health Service's (NHS) primary care pathway with non-specific symptoms of cancer, who either did not have cancer or who were subsequently diagnosed with cancer and referred to surgery with curative intent. TAPS is a base-level-resoluti...
Bile salts are potent antimicrobial agents and are an important component of innate defenses in t... more Bile salts are potent antimicrobial agents and are an important component of innate defenses in the intestine, giving protection against invasive organisms. They play an important role in determining microbial ecology of the intestine and alterations in their levels can lead to increased colonization by pathogens. We have previously demonstrated survival of the opportunistic pathogen Staphylococcus aureus in the human colonic model. Thus investigating the interaction between S. aureus and bile salts is an important factor in understanding its ability to colonize in the host intestine. Harnessing bile salts may also give a new avenue to explore in the development of therapeutic strategies to control drug resistant bacteria. Despite this importance, the antibacterial activity of bile salts on S. aureus is poorly understood. In this study, we investigated the antibacterial effects of the major unconjugated and conjugated bile salts on S. aureus. Several concentration-dependent antibacterial mechanisms were found. Unconjugated bile salts at their minimum inhibitory concentration (cholic and deoxycholic acid at 20 and 1 mM, respectively) killed S. aureus, and this was associated with increased membrane disruption and leakage of cellular contents. Unconjugated bile salts (cholic and deoxycholic acid at 8 and 0.4 mM, respectively) and conjugated bile salts (glycocholic and taurocholic acid at 20 mM) at their sub inhibitory concentrations were still able to inhibit growth through disruption of the proton motive force and increased membrane permeability. We also demonstrated that unconjugated bile salts possess more potent antibacterial action on S. aureus than conjugated bile salts.
PLoS ONE, 2011
An anaerobic three-stage continuous culture model of the human colon (gut model), which represent... more An anaerobic three-stage continuous culture model of the human colon (gut model), which represent different anatomical areas of the large intestine, was used to study the effect of S. aureus infection of the gut on the resident faecal microbiota. Studies on the development of the microbiota in the three vessels were performed and bacteria identified by culture independent fluorescence in situ hybridization (FISH). Furtheremore, short chain fatty acids (SCFA), as principal end products of gut bacterial metabolism, were measured along with a quantitative assessment of the predominant microbiota. During steady state conditions, numbers of S. aureus cells stabilised until they were washed out, but populations of indigenous bacteria were transiently altered; thus S. aureus was able to compromise colonisation resistance by the colonic microbiota. Furthermore, the concentration of butyric acid in the vessel representing the proximal colon was significantly decreased by infection. Thus infection by S. aureus appears to be able to alter the overall structure of the human colonic microbiota and the microbial metabolic profiles. This work provides an initial in vitro model to analyse interactions with pathogens.
Indian Journal of Microbiology, 2011
Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaper... more Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaperones in processes like protein folding and protein trafficking between different intracellular compartments. They are induced during stress conditions like oxidative stress, nutritional deficiencies and radiation. They are released into extracellular compartment during necrosis. However, recent research findings highlights that, they are not solely present in cytoplasm, but also released into extracellular compartment during normal conditions and even in the absence of necrosis. When present in extracellular compartment, they have been shown to perform various functions like antigen presentation, intercellular signaling and induction of pro-inflammatory cytokines. Heat shock proteins represents as dominant microbial antigens during infection. The phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins has led to proposition that, microbial heat shock proteins can induce self reactivity to host heat shock proteins and result in autoimmune diseases. The self-reactivity of heat shock proteins protects host against disease by controlling induction and release of pro-inflammatory cytokines. However, antibodies to self heat shock proteins haven been implicated in pathogenesis of autoimmune diseases like arthritis and atherosclerosis. Some heat shock proteins are potent inducers of innate and adaptive immunity. They activate dendritic cells and natural killer cells through toll-like receptors, CD14 and CD91. They play an important role in MHC-antigen processing and presentation. These immune effector functions of heat shock proteins are being exploited them as therapeutic agents as well as therapeutic targets for various infectious diseases and cancers.
Annals of Microbiology, 2009
The aim of this study was to understand development of resistance to alamethicin (a model barrel ... more The aim of this study was to understand development of resistance to alamethicin (a model barrel stave pore forming antimicrobial peptide) by investigating changes in phospholipid profile, fatty acid side chain analysis and extent of alamethicin insertion in biomimetic membrane prepared form wild type strains and five folds alamethicin resistant variants of Staphylococcus aureus NCDC 110, Enterococcus faecalis NCDC 114 and Bacillus cereus NCDC 66. The wild type strains NCDC 110, 114, 66, were sensitive to alamethicin with IC 50 5.5, 3.25 and 2.0 μg/ml respectively. Wild type strains were cultured in the presence of alamethicin to select resistant variants with IC 50 29.0, 17.0 and 9.5 μg/ml respectively. The phospholipid profile analysis revealed increase in amino-group containing phospholipids to amino-group lacking phospholipids ratio between wild-type and resistant variant in S. aureus and B. cereus but decreased in E. faecalis. Predominant fatty acids in all strains were composed of even number of carbons. Linoleic acid was detected only in resistant strain of B. cereus. As indicated by saturated-to-unsaturated fatty acids ratio, the membrane from S. aureus and E. faecalis became more rigid, whereas, in B. cereus it became more fluid. Using a colorimetric in vitro assay, a decrease in alamethicin insertion in the biomimetic membrane could be observed upon acquisition of resistance. The membranes of five-fold alamethicin-resistant S. aureus, E. faecalis and B. cereus revealed changes in membrane fluidity and surface charge upon acquisition of resistance to alamethicin.
Access Microbiology, 2019
Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigat... more Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigate the molecular mechanisms by which acetic acid acts as an antibacterial agent. To do this, we grew a high-density transposon library in uropathogenic E. coli EO499 serotype 131 in M9 media at pH 7 and pH 5.5 with acetic acid concentrations of 40 mM and 4 mM, respectively, or without added acetic acid. Sequencing libraries were generated from total bacterial populations after growth, and sequenced using a transposon-specific primer to generate positions and frequencies for each transposon. By comparing numbers of reads before and after the stress, we identified candidate genes where transposon inserts led to a decrease of fitness under acetic acid stress. Eight of these were chosen for further study: nuoM, nuoG, sucA, sthA, pitA, apaH, rssB and ytfP. Because of the difficulties of constructing gene deletions in the uropathogenic strain for validating the TraDIS results, we tested the relative fitness of the corresponding gene deletion mutants from the Keio library (in strain BW25113), with the growth conditions used for EO499. Interestingly, only a few knockouts showed a reduction in relative fitness in time course competitions at pH 5.5 with acetic acid. This may due to the differences between strains used in TraDIS and competition. To overcome this issue, we have also isolated transposon mutants from E. coli EO499 transposon library for the determination of relative fitness. The results will be presented.
Frontiers in microbiology, 2017
Bile salts are potent antimicrobial agents and are an important component of innate defenses in t... more Bile salts are potent antimicrobial agents and are an important component of innate defenses in the intestine, giving protection against invasive organisms. They play an important role in determining microbial ecology of the intestine and alterations in their levels can lead to increased colonization by pathogens. We have previously demonstrated survival of the opportunistic pathogen Staphylococcus aureus in the human colonic model. Thus investigating the interaction between S. aureus and bile salts is an important factor in understanding its ability to colonize in the host intestine. Harnessing bile salts may also give a new avenue to explore in the development of therapeutic strategies to control drug resistant bacteria. Despite this importance, the antibacterial activity of bile salts on S. aureus is poorly understood. In this study, we investigated the antibacterial effects of the major unconjugated and conjugated bile salts on S. aureus. Several concentration-dependent antibact...
The analysis of circulating tumour DNA (ctDNA) promises to extend current tissue-specific cancer ... more The analysis of circulating tumour DNA (ctDNA) promises to extend current tissue-specific cancer screening programmes to multi-cancer early detection and measurable disease monitoring to solid tumours using minimally invasive blood draws (liquid biopsies). Most studies so far have focused on using targeted deep sequencing to detect the low-abundance, fragmented ctDNA. Few studies have integrated information from multiple modalities using shallow 1x WGS. Here, we developed an integrated bioinformatics pipeline for ctDNA detection based on whole genome TET-Assisted Pyridine Borane Sequencing (TAPS) of plasma samples sequenced at 80x or higher. We conducted a diagnostic accuracy study in a case-control cohort of patients presenting to the UK National Health Service's (NHS) primary care pathway with non-specific symptoms of cancer, who either did not have cancer or who were subsequently diagnosed with cancer and referred to surgery with curative intent. TAPS is a base-level-resoluti...
Bile salts are potent antimicrobial agents and are an important component of innate defenses in t... more Bile salts are potent antimicrobial agents and are an important component of innate defenses in the intestine, giving protection against invasive organisms. They play an important role in determining microbial ecology of the intestine and alterations in their levels can lead to increased colonization by pathogens. We have previously demonstrated survival of the opportunistic pathogen Staphylococcus aureus in the human colonic model. Thus investigating the interaction between S. aureus and bile salts is an important factor in understanding its ability to colonize in the host intestine. Harnessing bile salts may also give a new avenue to explore in the development of therapeutic strategies to control drug resistant bacteria. Despite this importance, the antibacterial activity of bile salts on S. aureus is poorly understood. In this study, we investigated the antibacterial effects of the major unconjugated and conjugated bile salts on S. aureus. Several concentration-dependent antibacterial mechanisms were found. Unconjugated bile salts at their minimum inhibitory concentration (cholic and deoxycholic acid at 20 and 1 mM, respectively) killed S. aureus, and this was associated with increased membrane disruption and leakage of cellular contents. Unconjugated bile salts (cholic and deoxycholic acid at 8 and 0.4 mM, respectively) and conjugated bile salts (glycocholic and taurocholic acid at 20 mM) at their sub inhibitory concentrations were still able to inhibit growth through disruption of the proton motive force and increased membrane permeability. We also demonstrated that unconjugated bile salts possess more potent antibacterial action on S. aureus than conjugated bile salts.
PLoS ONE, 2011
An anaerobic three-stage continuous culture model of the human colon (gut model), which represent... more An anaerobic three-stage continuous culture model of the human colon (gut model), which represent different anatomical areas of the large intestine, was used to study the effect of S. aureus infection of the gut on the resident faecal microbiota. Studies on the development of the microbiota in the three vessels were performed and bacteria identified by culture independent fluorescence in situ hybridization (FISH). Furtheremore, short chain fatty acids (SCFA), as principal end products of gut bacterial metabolism, were measured along with a quantitative assessment of the predominant microbiota. During steady state conditions, numbers of S. aureus cells stabilised until they were washed out, but populations of indigenous bacteria were transiently altered; thus S. aureus was able to compromise colonisation resistance by the colonic microbiota. Furthermore, the concentration of butyric acid in the vessel representing the proximal colon was significantly decreased by infection. Thus infection by S. aureus appears to be able to alter the overall structure of the human colonic microbiota and the microbial metabolic profiles. This work provides an initial in vitro model to analyse interactions with pathogens.
Indian Journal of Microbiology, 2011
Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaper... more Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaperones in processes like protein folding and protein trafficking between different intracellular compartments. They are induced during stress conditions like oxidative stress, nutritional deficiencies and radiation. They are released into extracellular compartment during necrosis. However, recent research findings highlights that, they are not solely present in cytoplasm, but also released into extracellular compartment during normal conditions and even in the absence of necrosis. When present in extracellular compartment, they have been shown to perform various functions like antigen presentation, intercellular signaling and induction of pro-inflammatory cytokines. Heat shock proteins represents as dominant microbial antigens during infection. The phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins has led to proposition that, microbial heat shock proteins can induce self reactivity to host heat shock proteins and result in autoimmune diseases. The self-reactivity of heat shock proteins protects host against disease by controlling induction and release of pro-inflammatory cytokines. However, antibodies to self heat shock proteins haven been implicated in pathogenesis of autoimmune diseases like arthritis and atherosclerosis. Some heat shock proteins are potent inducers of innate and adaptive immunity. They activate dendritic cells and natural killer cells through toll-like receptors, CD14 and CD91. They play an important role in MHC-antigen processing and presentation. These immune effector functions of heat shock proteins are being exploited them as therapeutic agents as well as therapeutic targets for various infectious diseases and cancers.
Annals of Microbiology, 2009
The aim of this study was to understand development of resistance to alamethicin (a model barrel ... more The aim of this study was to understand development of resistance to alamethicin (a model barrel stave pore forming antimicrobial peptide) by investigating changes in phospholipid profile, fatty acid side chain analysis and extent of alamethicin insertion in biomimetic membrane prepared form wild type strains and five folds alamethicin resistant variants of Staphylococcus aureus NCDC 110, Enterococcus faecalis NCDC 114 and Bacillus cereus NCDC 66. The wild type strains NCDC 110, 114, 66, were sensitive to alamethicin with IC 50 5.5, 3.25 and 2.0 μg/ml respectively. Wild type strains were cultured in the presence of alamethicin to select resistant variants with IC 50 29.0, 17.0 and 9.5 μg/ml respectively. The phospholipid profile analysis revealed increase in amino-group containing phospholipids to amino-group lacking phospholipids ratio between wild-type and resistant variant in S. aureus and B. cereus but decreased in E. faecalis. Predominant fatty acids in all strains were composed of even number of carbons. Linoleic acid was detected only in resistant strain of B. cereus. As indicated by saturated-to-unsaturated fatty acids ratio, the membrane from S. aureus and E. faecalis became more rigid, whereas, in B. cereus it became more fluid. Using a colorimetric in vitro assay, a decrease in alamethicin insertion in the biomimetic membrane could be observed upon acquisition of resistance. The membranes of five-fold alamethicin-resistant S. aureus, E. faecalis and B. cereus revealed changes in membrane fluidity and surface charge upon acquisition of resistance to alamethicin.