In recent years, there is a growing concern about the alarming spread of antimicrobial resistance... more In recent years, there is a growing concern about the alarming spread of antimicrobial resistance (AMR) in different environments. Increasingly, many species of bacteria, fungi and viruses are becoming immune to the most commonly used pharmaceuticals. One of the causes of the development of the resistance is the persistence of these drugs, excreted by humans, in municipal and hospital wastewater (WW). Consequently, wastewater treatment plants (WWTPs) are a primary source of antimicrobial resistance genes as novel pollutants. This systematic review sought to examine the relevant literature on pharmaceutical residues (PRs) responsible for AMR in municipal and hospital WW in order to propose a classification of the PRs of greatest concern and provide an updated source for AMR management in WWTPs. Among 546 studies collected from four databases, 18 were included in the present review. The internal and external validity of each study was assessed, and the risk of bias was evaluated on a ...
Strains of enteroaggregative Escherichia coli (EAggEC), characterised by their pattern of adhesio... more Strains of enteroaggregative Escherichia coli (EAggEC), characterised by their pattern of adhesion to HEp-2 cells known as the `stacked brick' formation, are a significant cause of chronic diarrhoea in certain under-developed countries. Strains of EAggEC are detected either by a HEp-2 adhesion cell test or by an `aggregative adhesion' gene probe. The pathogenic mechanisms expressed by EAggEC are only poorly understood and the aim of the research described was to obtain a better understanding of how these bacteria cause disease. The adhesion of EAggEC to HEp-2 cells was shown in the majority of strains not to involve fimbriae and was thought to result from physical properties of strains such as charge, since EAggEC adhered to `fixed' HEp-2 cells and readily agglutinated a range of different erythrocytes. Certain strains of EAggEC, which also hybridised with a probe for diffuse adhesion, expressed membrane-associated proteins (MAPs) of 18 or 20 kDa responsible for HEp-2 ad...
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable f... more The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment. Clostridium difficile is a pathogen of major significance worldwide, being recognised as the leading cause of antibiotic associated diarrhoea in the nosocomial setting. In the U.S. increased rates of C. difficile infection (CDI) and relapse, seen in up to 20.9% of CDI these cases 1 , incur annual health care costs in excess of $4.8 billion 2. Consequently, there is an urgent need to understand the pathogenic traits of this organism and the influence of expression of specific genes during infection. To date, most research has centred on the study of the two major toxins A and B (TcdA & TcdB) whose production and activity are responsible for the majority of symptoms observed during CDI 3,4. Whilst experimental vaccines based on these toxins reduce or eliminate symptoms 5 they do not prevent germination, colonisation and sporulation of the bacteria, which are all essential steps in transmission from host to host. Greater understanding of these processes is essential if we are to identify new targets for future prophylactic or therapeutic treatments. One factor limiting insight into the molecular analysis of this pathogen is the inability to visualise and monitor ongoing cellular processes. Fluorophores, derived from green fluorescent protein (GFP) have been used successfully in other species 6 , but require molecular oxygen for fluorescence maturation, thus preventing their use in anaerobic systems. In addition, many Clostridium species generate a green autofluorescence when excited with blue/ultraviolet (UV) light 7 making visualisation with green fluorescent reporters problematic. However, advances in C. difficile bioimaging are beginning to occur, with the oxygen-dependent fluorescent proteins [Cyan Fluorescent Protein (CFP) and mCherry] used to characterise the cellular localisation of MdlA/B/C & ZapA in dividing bacteria 7,8. In addition, tags based on O-6-methylguanine-DNA methyltransferase (SNAP-tags) have been used successfully to investigate protein localisation 9,10. These elegant studies have significantly extended our understanding of the temporal and spatial interplay between the RNA polymerase sigma subunits during sporulation. However, due to oxygen requirements or cell toxicity of substrates used, they are unsuited for monitoring real time protein dynamics in anaerobic environments. In contrast, small flavin-based photosensory modules
Various studies have suggested that the gut microbiome interacts with the host and may have a sig... more Various studies have suggested that the gut microbiome interacts with the host and may have a significant role in the aetiology of obesity and Type 2 Diabetes (T2D). It was hypothesised that bacterial communities in obesity and T2D differ from control and compromise normal interactions between host and microbiota. Obesity and T2D were developed in rats by feeding a high-fat diet or a high-fat diet plus a single low-dose streptozotocin administration, respectively. The microbiome profiles and their metabolic potentials were established by metagenomic 16S rRNA sequencing and bioinformatics. Taxonomy and predicted metabolism-related genes in obesity and T2D were markedly different from controls and indeed from each other. Diversity was reduced in T2D but not in Obese rats. Factors likely to compromise host intestinal, barrier integrity were found in Obese and T2D rats including predicted, decreased bacterial butyrate production. Capacity to increase energy extraction via ABC-transporters and carbohydrate metabolism were enhanced in Obese and T2D rats. T2D was characterized by increased proinflammatory molecules. While obesity and T2D show distinct differences, results suggest that in both conditions Bacteroides and Blautia species were increased indicating a possible mechanistic link.
C lostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have show... more C lostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have shown that effective protection can be mediated in hamsters through the inclusion of specific recombinant fragments from toxin A and B in a systemically delivered vaccine. Interestingly while neutralizing antibodies to the binding domains of both toxin A and B are moderately protective, enhanced survival is observed when fragments from the glucosyltransferase region of toxin B replace those from the binding domain of this toxin. In this addendum, we discuss additional information that has been derived from such vaccination studies. This includes observations on efficacy and cross-protection against different ribotypes mediated by these vaccines and the challenges that remain for a vaccine which prevents clinical symptoms but not colonization. The use and value of vaccination both in the prevention of infection and for treatment of disease relapse will be discussed. Vaccination against Clostridium difficile using toxin fragments
Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated... more Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated with specific changes to gut microbiome composition. In particular, loss of microbes that modify bile salt acids (BSA) play a central role; primary bile acids stimulate spore germination whilst secondary bile acids limit C. difficile vegetative growth. To determine the relative contribution of bile salt (BS) metabolism on C. difficile disease severity, we treated mice with three combinations of antibiotics prior to infection. Mice given clindamycin alone became colonized but displayed no tissue pathology while severe disease, exemplified by weight loss and inflammatory tissue damage occurred in animals given a combination of five antibiotics and clindamycin. Animals given only the five antibiotic cocktails showed only transient colonization and no disease. C. difficile colonization was associated with a reduction in bacterial diversity, an inability to amplify bile salt hydrolase (BSH) sequences from fecal DNA and a relative increase in primary bile acids (pBA) in cecal lavages from infected mice. Further, the link between BSA modification and the microbiome was confirmed by the isolation of strains of Lactobacillus murinus that modified primary bile acids in vitro, thus preventing C. difficile germination. Interestingly, BSH activity did not correlate with disease severity which appeared linked to alternations in mucin, which may indirectly lead to increased exposure of the epithelial surface to inflammatory signals. These data confirm the role of microbial metabolic activity in protection of the gut and highlights the need for greater understanding the function of bacterial communities in disease prevention.
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable f... more The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. diff... more Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
Antimicrobial agents and chemotherapy, Jan 7, 2015
Microbiome dysbiosis caused by antibiotic treatment has been associated with both the susceptibil... more Microbiome dysbiosis caused by antibiotic treatment has been associated with both the susceptibility and relapsing of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplification in situ, but few studies have focused on their use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. Whilst it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage; lysing 18 and 62 of the ribotypes and strains tested respectively. Single-phage treatments of ribotypes 076, 014/020 and 027 strains showed an initial reduct...
Clostridium difficile is the main cause of antibiotic-associated disease, a disease of high socio... more Clostridium difficile is the main cause of antibiotic-associated disease, a disease of high socio-economical importance that has recently been compounded by the global spread of the 027 (BI/NAP1/027) ribotype. C. difficile cases attributed to ribotype 027 strains have high recurrence rates (up to 36 %) and increased disease severity. The hamster model of infection is widely accepted as an appropriate model for studying aspects of C. difficile host-pathogen interactions. Using this model we characterized the infection kinetics of the UK 2006 outbreak strain, R20291. Hamsters were orally given a dose of clindamycin, followed 5 days later with 10 000 C. difficile spores. All 100 % of the hamsters succumbed to infection with a mean time to the clinical end point of 46.7 h. Colonization of the caecum and colon were observed 12 h postinfection reaching a maximum of approximately 3¾10 4 c.f.u. per organ, but spores were not detected until 24 h post-infection. At 36 h post-infection C. difficile numbers increased significantly to approximately 6¾10 7 c.f.u. per organ where numbers remained high until the clinical end point. Increasing levels of in vivo toxin production coincided with increases in C. difficile numbers in organs reaching a maximum at 36 h post-infection in the caecum. Epithelial destruction and polymorphonuclear leukocyte (PMN) recruitment occurred early on during infection (24 h) accumulating as gross microvilli damage, luminal PMN influx, and blood associated with mucosal muscle and microvilli. These data describe the fatal infection kinetics of the clinical UK epidemic C. difficile strain R20291 in the hamster infection model. Abbreviations: CDI, Clostridium difficile infection; MLVA, multilocus variable-number tandem-repeat analysis; PMN, polymorphonuclear leukocyte; SEM, scanning electron microscopy.
Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a 'stacked brick' pattern... more Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a 'stacked brick' pattern (EAggEC), and four nonadherent control strains, were examined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA) HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outermembrane protein (MAP), to agglutinate erythrocytes, and to produce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outer membrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did not express an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cells with a stacked brick pattern, these strains exhibit a diverse range of physical and biochemical properties. From the results of this study, it was concluded that currently, the possession of EAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain the only reliable means of identifying EAggEC.
European Journal of Pharmaceutics and Biopharmaceutics, 2009
With the emergence of widespread antibiotic resistance, there has been renewed interest in the us... more With the emergence of widespread antibiotic resistance, there has been renewed interest in the use of bacteriophages. While their potency, safety and specificity have underpinned their clinical potential, to date, little work has been focussed on their formulation with respect to controlled release and/or passive targeting. Here, we show that bacteriophages selective for Staphylococcus aureus or Pseudomonas aeruginosa can be encapsulated into biodegradable polyester microspheres via a modified w/o/w double emulsion-solvent extraction protocol with only a partial loss of lytic activity. Loss of lytic activity could be attributed to the exposure of the bacteriophages to the water-dichloromethane interface, with the lyophilization process itself having little effect. The microspheres were engineered to have an appropriate size and density to facilitate inhalation via a dry-powder inhaler and fluorescently labeled bacteriophages were distributed entirely within the internal porous matrix. The release profile showed a burst release phase (55-63% release within 30 min), followed by a sustained release till around 6 h, as appropriate for pulmonary delivery. Despite the poor shelf-life of the formulation, the work is proof-of-concept for the formulation and controlled delivery of bacteriophages, as suitable for the treatment of bacterial lung infections.
Twenty-two strains of enteroaggregative Escherichia coli (EAggEC), isolated from four outbreaks o... more Twenty-two strains of enteroaggregative Escherichia coli (EAggEC), isolated from four outbreaks of diarrhoeal disease in England, were examined for a range of phenotypic attributes including the ability to produce fimbriae, haemolysins and siderophores, and cell-surface properties such as surface charge and hydrophobicity. Strains of EAggEC isolated from two of these outbreaks belonged to a diverse range of serotypes and were heterogeneous in phenotype. Strains of EAggEC isolated from the other two outbreaks belonged predominantly to serotypes O86 : H34 and O98 : H-, respectively. Only two strains expressed fimbriae and two strains produced an 18 kDa membrane associated protein (MAP), suggesting that EAggEC express a range of adhesion mechanisms to produce the cell arrangement recognized as the ' stacked brick ' formation. The possible explanation for the diversity of EAggEC serotypes is discussed.
Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is nec... more Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture-and PCR detection, preenrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only.
articLe addenduM C lostridium difficile is a major cause of antibiotic associated diarrhea. Recen... more articLe addenduM C lostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have shown that effective protection can be mediated in hamsters through the inclusion of specific recombinant fragments from toxin A and B in a systemically delivered vaccine. Interestingly while neutralizing antibodies to the binding domains of both toxin A and B are moderately protective, enhanced survival is observed when fragments from the glucosyltransferase region of toxin B replace those from the binding domain of this toxin. In this addendum, we discuss additional information that has been derived from such vaccination studies. This includes observations on efficacy and cross-protection against different ribotypes mediated by these vaccines and the challenges that remain for a vaccine which prevents clinical symptoms but not colonization. The use and value of vaccination both in the prevention of infection and for treatment of disease relapse will be discussed.
In recent years, there is a growing concern about the alarming spread of antimicrobial resistance... more In recent years, there is a growing concern about the alarming spread of antimicrobial resistance (AMR) in different environments. Increasingly, many species of bacteria, fungi and viruses are becoming immune to the most commonly used pharmaceuticals. One of the causes of the development of the resistance is the persistence of these drugs, excreted by humans, in municipal and hospital wastewater (WW). Consequently, wastewater treatment plants (WWTPs) are a primary source of antimicrobial resistance genes as novel pollutants. This systematic review sought to examine the relevant literature on pharmaceutical residues (PRs) responsible for AMR in municipal and hospital WW in order to propose a classification of the PRs of greatest concern and provide an updated source for AMR management in WWTPs. Among 546 studies collected from four databases, 18 were included in the present review. The internal and external validity of each study was assessed, and the risk of bias was evaluated on a ...
Strains of enteroaggregative Escherichia coli (EAggEC), characterised by their pattern of adhesio... more Strains of enteroaggregative Escherichia coli (EAggEC), characterised by their pattern of adhesion to HEp-2 cells known as the `stacked brick' formation, are a significant cause of chronic diarrhoea in certain under-developed countries. Strains of EAggEC are detected either by a HEp-2 adhesion cell test or by an `aggregative adhesion' gene probe. The pathogenic mechanisms expressed by EAggEC are only poorly understood and the aim of the research described was to obtain a better understanding of how these bacteria cause disease. The adhesion of EAggEC to HEp-2 cells was shown in the majority of strains not to involve fimbriae and was thought to result from physical properties of strains such as charge, since EAggEC adhered to `fixed' HEp-2 cells and readily agglutinated a range of different erythrocytes. Certain strains of EAggEC, which also hybridised with a probe for diffuse adhesion, expressed membrane-associated proteins (MAPs) of 18 or 20 kDa responsible for HEp-2 ad...
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable f... more The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment. Clostridium difficile is a pathogen of major significance worldwide, being recognised as the leading cause of antibiotic associated diarrhoea in the nosocomial setting. In the U.S. increased rates of C. difficile infection (CDI) and relapse, seen in up to 20.9% of CDI these cases 1 , incur annual health care costs in excess of $4.8 billion 2. Consequently, there is an urgent need to understand the pathogenic traits of this organism and the influence of expression of specific genes during infection. To date, most research has centred on the study of the two major toxins A and B (TcdA & TcdB) whose production and activity are responsible for the majority of symptoms observed during CDI 3,4. Whilst experimental vaccines based on these toxins reduce or eliminate symptoms 5 they do not prevent germination, colonisation and sporulation of the bacteria, which are all essential steps in transmission from host to host. Greater understanding of these processes is essential if we are to identify new targets for future prophylactic or therapeutic treatments. One factor limiting insight into the molecular analysis of this pathogen is the inability to visualise and monitor ongoing cellular processes. Fluorophores, derived from green fluorescent protein (GFP) have been used successfully in other species 6 , but require molecular oxygen for fluorescence maturation, thus preventing their use in anaerobic systems. In addition, many Clostridium species generate a green autofluorescence when excited with blue/ultraviolet (UV) light 7 making visualisation with green fluorescent reporters problematic. However, advances in C. difficile bioimaging are beginning to occur, with the oxygen-dependent fluorescent proteins [Cyan Fluorescent Protein (CFP) and mCherry] used to characterise the cellular localisation of MdlA/B/C & ZapA in dividing bacteria 7,8. In addition, tags based on O-6-methylguanine-DNA methyltransferase (SNAP-tags) have been used successfully to investigate protein localisation 9,10. These elegant studies have significantly extended our understanding of the temporal and spatial interplay between the RNA polymerase sigma subunits during sporulation. However, due to oxygen requirements or cell toxicity of substrates used, they are unsuited for monitoring real time protein dynamics in anaerobic environments. In contrast, small flavin-based photosensory modules
Various studies have suggested that the gut microbiome interacts with the host and may have a sig... more Various studies have suggested that the gut microbiome interacts with the host and may have a significant role in the aetiology of obesity and Type 2 Diabetes (T2D). It was hypothesised that bacterial communities in obesity and T2D differ from control and compromise normal interactions between host and microbiota. Obesity and T2D were developed in rats by feeding a high-fat diet or a high-fat diet plus a single low-dose streptozotocin administration, respectively. The microbiome profiles and their metabolic potentials were established by metagenomic 16S rRNA sequencing and bioinformatics. Taxonomy and predicted metabolism-related genes in obesity and T2D were markedly different from controls and indeed from each other. Diversity was reduced in T2D but not in Obese rats. Factors likely to compromise host intestinal, barrier integrity were found in Obese and T2D rats including predicted, decreased bacterial butyrate production. Capacity to increase energy extraction via ABC-transporters and carbohydrate metabolism were enhanced in Obese and T2D rats. T2D was characterized by increased proinflammatory molecules. While obesity and T2D show distinct differences, results suggest that in both conditions Bacteroides and Blautia species were increased indicating a possible mechanistic link.
C lostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have show... more C lostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have shown that effective protection can be mediated in hamsters through the inclusion of specific recombinant fragments from toxin A and B in a systemically delivered vaccine. Interestingly while neutralizing antibodies to the binding domains of both toxin A and B are moderately protective, enhanced survival is observed when fragments from the glucosyltransferase region of toxin B replace those from the binding domain of this toxin. In this addendum, we discuss additional information that has been derived from such vaccination studies. This includes observations on efficacy and cross-protection against different ribotypes mediated by these vaccines and the challenges that remain for a vaccine which prevents clinical symptoms but not colonization. The use and value of vaccination both in the prevention of infection and for treatment of disease relapse will be discussed. Vaccination against Clostridium difficile using toxin fragments
Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated... more Susceptibility of patients to antibiotic-associated C. difficile disease is intimately associated with specific changes to gut microbiome composition. In particular, loss of microbes that modify bile salt acids (BSA) play a central role; primary bile acids stimulate spore germination whilst secondary bile acids limit C. difficile vegetative growth. To determine the relative contribution of bile salt (BS) metabolism on C. difficile disease severity, we treated mice with three combinations of antibiotics prior to infection. Mice given clindamycin alone became colonized but displayed no tissue pathology while severe disease, exemplified by weight loss and inflammatory tissue damage occurred in animals given a combination of five antibiotics and clindamycin. Animals given only the five antibiotic cocktails showed only transient colonization and no disease. C. difficile colonization was associated with a reduction in bacterial diversity, an inability to amplify bile salt hydrolase (BSH) sequences from fecal DNA and a relative increase in primary bile acids (pBA) in cecal lavages from infected mice. Further, the link between BSA modification and the microbiome was confirmed by the isolation of strains of Lactobacillus murinus that modified primary bile acids in vitro, thus preventing C. difficile germination. Interestingly, BSH activity did not correlate with disease severity which appeared linked to alternations in mucin, which may indirectly lead to increased exposure of the epithelial surface to inflammatory signals. These data confirm the role of microbial metabolic activity in protection of the gut and highlights the need for greater understanding the function of bacterial communities in disease prevention.
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable f... more The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. diff... more Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
Antimicrobial agents and chemotherapy, Jan 7, 2015
Microbiome dysbiosis caused by antibiotic treatment has been associated with both the susceptibil... more Microbiome dysbiosis caused by antibiotic treatment has been associated with both the susceptibility and relapsing of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplification in situ, but few studies have focused on their use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. Whilst it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage; lysing 18 and 62 of the ribotypes and strains tested respectively. Single-phage treatments of ribotypes 076, 014/020 and 027 strains showed an initial reduct...
Clostridium difficile is the main cause of antibiotic-associated disease, a disease of high socio... more Clostridium difficile is the main cause of antibiotic-associated disease, a disease of high socio-economical importance that has recently been compounded by the global spread of the 027 (BI/NAP1/027) ribotype. C. difficile cases attributed to ribotype 027 strains have high recurrence rates (up to 36 %) and increased disease severity. The hamster model of infection is widely accepted as an appropriate model for studying aspects of C. difficile host-pathogen interactions. Using this model we characterized the infection kinetics of the UK 2006 outbreak strain, R20291. Hamsters were orally given a dose of clindamycin, followed 5 days later with 10 000 C. difficile spores. All 100 % of the hamsters succumbed to infection with a mean time to the clinical end point of 46.7 h. Colonization of the caecum and colon were observed 12 h postinfection reaching a maximum of approximately 3¾10 4 c.f.u. per organ, but spores were not detected until 24 h post-infection. At 36 h post-infection C. difficile numbers increased significantly to approximately 6¾10 7 c.f.u. per organ where numbers remained high until the clinical end point. Increasing levels of in vivo toxin production coincided with increases in C. difficile numbers in organs reaching a maximum at 36 h post-infection in the caecum. Epithelial destruction and polymorphonuclear leukocyte (PMN) recruitment occurred early on during infection (24 h) accumulating as gross microvilli damage, luminal PMN influx, and blood associated with mucosal muscle and microvilli. These data describe the fatal infection kinetics of the clinical UK epidemic C. difficile strain R20291 in the hamster infection model. Abbreviations: CDI, Clostridium difficile infection; MLVA, multilocus variable-number tandem-repeat analysis; PMN, polymorphonuclear leukocyte; SEM, scanning electron microscopy.
Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a 'stacked brick' pattern... more Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a 'stacked brick' pattern (EAggEC), and four nonadherent control strains, were examined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA) HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outermembrane protein (MAP), to agglutinate erythrocytes, and to produce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outer membrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did not express an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cells with a stacked brick pattern, these strains exhibit a diverse range of physical and biochemical properties. From the results of this study, it was concluded that currently, the possession of EAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain the only reliable means of identifying EAggEC.
European Journal of Pharmaceutics and Biopharmaceutics, 2009
With the emergence of widespread antibiotic resistance, there has been renewed interest in the us... more With the emergence of widespread antibiotic resistance, there has been renewed interest in the use of bacteriophages. While their potency, safety and specificity have underpinned their clinical potential, to date, little work has been focussed on their formulation with respect to controlled release and/or passive targeting. Here, we show that bacteriophages selective for Staphylococcus aureus or Pseudomonas aeruginosa can be encapsulated into biodegradable polyester microspheres via a modified w/o/w double emulsion-solvent extraction protocol with only a partial loss of lytic activity. Loss of lytic activity could be attributed to the exposure of the bacteriophages to the water-dichloromethane interface, with the lyophilization process itself having little effect. The microspheres were engineered to have an appropriate size and density to facilitate inhalation via a dry-powder inhaler and fluorescently labeled bacteriophages were distributed entirely within the internal porous matrix. The release profile showed a burst release phase (55-63% release within 30 min), followed by a sustained release till around 6 h, as appropriate for pulmonary delivery. Despite the poor shelf-life of the formulation, the work is proof-of-concept for the formulation and controlled delivery of bacteriophages, as suitable for the treatment of bacterial lung infections.
Twenty-two strains of enteroaggregative Escherichia coli (EAggEC), isolated from four outbreaks o... more Twenty-two strains of enteroaggregative Escherichia coli (EAggEC), isolated from four outbreaks of diarrhoeal disease in England, were examined for a range of phenotypic attributes including the ability to produce fimbriae, haemolysins and siderophores, and cell-surface properties such as surface charge and hydrophobicity. Strains of EAggEC isolated from two of these outbreaks belonged to a diverse range of serotypes and were heterogeneous in phenotype. Strains of EAggEC isolated from the other two outbreaks belonged predominantly to serotypes O86 : H34 and O98 : H-, respectively. Only two strains expressed fimbriae and two strains produced an 18 kDa membrane associated protein (MAP), suggesting that EAggEC express a range of adhesion mechanisms to produce the cell arrangement recognized as the ' stacked brick ' formation. The possible explanation for the diversity of EAggEC serotypes is discussed.
Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is nec... more Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture-and PCR detection, preenrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only.
articLe addenduM C lostridium difficile is a major cause of antibiotic associated diarrhea. Recen... more articLe addenduM C lostridium difficile is a major cause of antibiotic associated diarrhea. Recently, we have shown that effective protection can be mediated in hamsters through the inclusion of specific recombinant fragments from toxin A and B in a systemically delivered vaccine. Interestingly while neutralizing antibodies to the binding domains of both toxin A and B are moderately protective, enhanced survival is observed when fragments from the glucosyltransferase region of toxin B replace those from the binding domain of this toxin. In this addendum, we discuss additional information that has been derived from such vaccination studies. This includes observations on efficacy and cross-protection against different ribotypes mediated by these vaccines and the challenges that remain for a vaccine which prevents clinical symptoms but not colonization. The use and value of vaccination both in the prevention of infection and for treatment of disease relapse will be discussed.
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Papers by Janice Spencer