Siouxsie Wiles | The University of Auckland (original) (raw)
Papers by Siouxsie Wiles
Objectives Tuberculosis drug development is hampered by the slow growth of Mycobacterium tubercul... more Objectives Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.
Objectives The current method for testing new drugs against tuberculosis in vivo is the enumerati... more Objectives The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.
Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesion... more Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen.
Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We con... more Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable.
Abstract Bioluminescence is an excellent reporter system for analysing bacterial colonization and... more Abstract Bioluminescence is an excellent reporter system for analysing bacterial colonization and clearance dynamics in vivo. Many bacterial species have been rendered bioluminescent, allowing the sensitive detection of bacterial burden and metabolic activity in real-time and in situ in living animals.
Despite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 ... more Despite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 million new cases and 1.7 million deaths worldwide in 2009 (World Health Organisation, 2010). Furthermore, an estimated one third of the worlds' population is infected with the bacterium responsible, Mycobacterium tuberculosis.
Abstract Dosing of experimental animals and the removal of blood are two of the most frequent pro... more Abstract Dosing of experimental animals and the removal of blood are two of the most frequent procedures performed in biomedical research using live animals. Despite the apparently simple nature of these procedures, they can, if not correctly carried out, have significant effects on the welfare of the animals and the scientific value of the results. There are several methods by which research staff may obtain training in the administration of substances.
Photodermatology, …, Jan 1, 2008
Asian immigrants to the United Kingdom demonstrate much higher tuberculosis rates than the indige... more Asian immigrants to the United Kingdom demonstrate much higher tuberculosis rates than the indigenous population. This is postulated to be because of their low vitamin D levels, consequent upon a combination of diet and their reduced ultraviolet (UV) exposure in the United Kingdom, because vitamin D enhances antimycobacterial activity in in vitro systems. The aim of this study was to examine the relationship between UVB exposure, vitamin D levels and tuberculo-immunity in Asian immigrants in the United Kingdom. Suberythemal UVB treatments were given to eight subjects on 3 consecutive days, using broadband UVB fluorescent lamps. Blood was sampled for 25-hydroxyvitamin D (25-OH D) and whole blood functional assays were performed for antimycobacterial immunity. The mean 25-OH D level increased from a baseline of 11.23 ng/ml (95% CI 6.7-20.39) to 20.39 ng/ml (95% CI 16.6-20) following UVB treatment, P o0.01. However, no significant change in antimycobacterial immunity occurred following UVB exposure. This pilot study in Asian subjects with good baseline tuberculo-immunity has not supported a role for UVB-induced 25-OH D in the immune response to Mycobacterium tuberculosis.
Biomaterials, Jan 1, 2008
Imaging of in vivo gene expression using luciferase expression in various organs has been used fo... more Imaging of in vivo gene expression using luciferase expression in various organs has been used for several years. In contrast to other organs, in vivo imaging of the lung, particularly after non-viral gene transfer has not been extensively studied. The aim of this study was to address several questions:(1) Does in vivo light emission correlate with standard tissue homogenate-based luciferase detection in a dose-dependent manner? Recombinant Sendai virus (SeV) transduces airway epithelial cells very efficiently and was used to ...
FEMS microbiology reviews, Jan 1, 2011
According to World Health Organization estimates, infectious organisms are responsible for approx... more According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo.
Infection and …, Jan 1, 2008
Vaccine, Jan 1, 2009
Currently there is no licensed vaccine against the human pathogen Streptococcus pyogenes. The hig... more Currently there is no licensed vaccine against the human pathogen Streptococcus pyogenes. The highly conserved IL-8 cleaving S. pyogenes cell envelope proteinase SpyCEP is surface expressed and is a potential vaccine candidate. A recombinant N-terminal part of SpyCEP (CEP) was expressed and purified. AntiCEP antibodies were found to neutralize the IL-8 cleaving activity of SpyCEP. CEP-immunized mice had reduced bacterial dissemination from focal S. pyogenes intramuscular infection and intranasal infection. We also identified a functional SpyCEP-homolog protease SeCEP, expressed by the equine pathogen Streptococcus equi, which was able to cleave both human and equine IL-8. CEP-immunized mice also demonstrated reduced bacterial dissemination from S. equi intramuscular infection. Therefore immunization against SpyCEP may provide protection against other streptococci species with homologous proteases.
PLoS One, Jan 1, 2010
Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a m... more Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
Infection and …, Jan 1, 2008
The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal g... more The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.
Journal of microbiological …, Jan 1, 2003
A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutage... more A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutagenesis of microorganisms isolated from a polluted phenolic-remediating wastewater treatment system. Four Pseudomonad species were engineered to carry a stable chromosomal copy of the lux operon (luxCDABE) derived from Photorhabdus luminescens. These recombinant reporter microorganisms were tested for bioluminescence response to relevant phenol concentrations in the laboratory and to phenolic-containing effluents generated by an industrial wastewater treatment plant. The reporters displayed proportional responses of bioluminescence decay with increasing phenol concentrations up to 800 mg l À 1 of phenol. When deployed against samples from the treatment system, they showed superior operational range and sensing capabilities to that observed for industry standard microorganisms such as Vibrio fischeri. Specifically, the engineered strains accurately predicted toxicity shifts in all the treatment compartments under study (with phenolic concentrations ranging from approximately 10 to 600 mg l À 1 ) with a low coefficient of variation of replicate determinations (between 1.16% and 8.32%). This work highlights the utility of genetic modification of native microorganisms from sites of interest to provide robust and ecologically relevant organism-based reagents for toxicity monitoring with the potential for in situ deployment. D
Nature Reviews …, Jan 1, 2006
| Models occupy an essential position in the study of infectious disease as a result of the ethic... more | Models occupy an essential position in the study of infectious disease as a result of the ethical problems of exposing humans to potentially lethal agents. Deliberately induced infections in well-defined animal models provide much useful information about disease processes in an approximation of their natural context. Despite this, animal models are not the natural disease process, and recent experimental advances show, perhaps not unsurprisingly, that there are large differences between natural infections and animal models. Focusing on mouse models of bacterial pathogens, we discuss some of these discrepancies and suggest ways of improving model systems in the future.
Cellular microbiology, Jan 1, 2005
Applied and …, Jan 1, 2005
The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has le... more The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.
Infection and …, Jan 1, 2006
Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhag... more Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal E. coli during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that C. rodentium colonizes the rectum, a site previously unreported for this pathogen.
Journal of microbiological …, Jan 1, 2001
A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial... more A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy. q
Objectives Tuberculosis drug development is hampered by the slow growth of Mycobacterium tubercul... more Objectives Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.
Objectives The current method for testing new drugs against tuberculosis in vivo is the enumerati... more Objectives The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.
Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesion... more Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen.
Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We con... more Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable.
Abstract Bioluminescence is an excellent reporter system for analysing bacterial colonization and... more Abstract Bioluminescence is an excellent reporter system for analysing bacterial colonization and clearance dynamics in vivo. Many bacterial species have been rendered bioluminescent, allowing the sensitive detection of bacterial burden and metabolic activity in real-time and in situ in living animals.
Despite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 ... more Despite all efforts, tuberculosis (TB) still constitutes a serious global health threat with 9.4 million new cases and 1.7 million deaths worldwide in 2009 (World Health Organisation, 2010). Furthermore, an estimated one third of the worlds' population is infected with the bacterium responsible, Mycobacterium tuberculosis.
Abstract Dosing of experimental animals and the removal of blood are two of the most frequent pro... more Abstract Dosing of experimental animals and the removal of blood are two of the most frequent procedures performed in biomedical research using live animals. Despite the apparently simple nature of these procedures, they can, if not correctly carried out, have significant effects on the welfare of the animals and the scientific value of the results. There are several methods by which research staff may obtain training in the administration of substances.
Photodermatology, …, Jan 1, 2008
Asian immigrants to the United Kingdom demonstrate much higher tuberculosis rates than the indige... more Asian immigrants to the United Kingdom demonstrate much higher tuberculosis rates than the indigenous population. This is postulated to be because of their low vitamin D levels, consequent upon a combination of diet and their reduced ultraviolet (UV) exposure in the United Kingdom, because vitamin D enhances antimycobacterial activity in in vitro systems. The aim of this study was to examine the relationship between UVB exposure, vitamin D levels and tuberculo-immunity in Asian immigrants in the United Kingdom. Suberythemal UVB treatments were given to eight subjects on 3 consecutive days, using broadband UVB fluorescent lamps. Blood was sampled for 25-hydroxyvitamin D (25-OH D) and whole blood functional assays were performed for antimycobacterial immunity. The mean 25-OH D level increased from a baseline of 11.23 ng/ml (95% CI 6.7-20.39) to 20.39 ng/ml (95% CI 16.6-20) following UVB treatment, P o0.01. However, no significant change in antimycobacterial immunity occurred following UVB exposure. This pilot study in Asian subjects with good baseline tuberculo-immunity has not supported a role for UVB-induced 25-OH D in the immune response to Mycobacterium tuberculosis.
Biomaterials, Jan 1, 2008
Imaging of in vivo gene expression using luciferase expression in various organs has been used fo... more Imaging of in vivo gene expression using luciferase expression in various organs has been used for several years. In contrast to other organs, in vivo imaging of the lung, particularly after non-viral gene transfer has not been extensively studied. The aim of this study was to address several questions:(1) Does in vivo light emission correlate with standard tissue homogenate-based luciferase detection in a dose-dependent manner? Recombinant Sendai virus (SeV) transduces airway epithelial cells very efficiently and was used to ...
FEMS microbiology reviews, Jan 1, 2011
According to World Health Organization estimates, infectious organisms are responsible for approx... more According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo.
Infection and …, Jan 1, 2008
Vaccine, Jan 1, 2009
Currently there is no licensed vaccine against the human pathogen Streptococcus pyogenes. The hig... more Currently there is no licensed vaccine against the human pathogen Streptococcus pyogenes. The highly conserved IL-8 cleaving S. pyogenes cell envelope proteinase SpyCEP is surface expressed and is a potential vaccine candidate. A recombinant N-terminal part of SpyCEP (CEP) was expressed and purified. AntiCEP antibodies were found to neutralize the IL-8 cleaving activity of SpyCEP. CEP-immunized mice had reduced bacterial dissemination from focal S. pyogenes intramuscular infection and intranasal infection. We also identified a functional SpyCEP-homolog protease SeCEP, expressed by the equine pathogen Streptococcus equi, which was able to cleave both human and equine IL-8. CEP-immunized mice also demonstrated reduced bacterial dissemination from S. equi intramuscular infection. Therefore immunization against SpyCEP may provide protection against other streptococci species with homologous proteases.
PLoS One, Jan 1, 2010
Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a m... more Background: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
Infection and …, Jan 1, 2008
The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal g... more The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-κB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC ΔnleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-κB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-κB response elements, we found that NleH causes an increase in NF-κB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.
Journal of microbiological …, Jan 1, 2003
A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutage... more A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutagenesis of microorganisms isolated from a polluted phenolic-remediating wastewater treatment system. Four Pseudomonad species were engineered to carry a stable chromosomal copy of the lux operon (luxCDABE) derived from Photorhabdus luminescens. These recombinant reporter microorganisms were tested for bioluminescence response to relevant phenol concentrations in the laboratory and to phenolic-containing effluents generated by an industrial wastewater treatment plant. The reporters displayed proportional responses of bioluminescence decay with increasing phenol concentrations up to 800 mg l À 1 of phenol. When deployed against samples from the treatment system, they showed superior operational range and sensing capabilities to that observed for industry standard microorganisms such as Vibrio fischeri. Specifically, the engineered strains accurately predicted toxicity shifts in all the treatment compartments under study (with phenolic concentrations ranging from approximately 10 to 600 mg l À 1 ) with a low coefficient of variation of replicate determinations (between 1.16% and 8.32%). This work highlights the utility of genetic modification of native microorganisms from sites of interest to provide robust and ecologically relevant organism-based reagents for toxicity monitoring with the potential for in situ deployment. D
Nature Reviews …, Jan 1, 2006
| Models occupy an essential position in the study of infectious disease as a result of the ethic... more | Models occupy an essential position in the study of infectious disease as a result of the ethical problems of exposing humans to potentially lethal agents. Deliberately induced infections in well-defined animal models provide much useful information about disease processes in an approximation of their natural context. Despite this, animal models are not the natural disease process, and recent experimental advances show, perhaps not unsurprisingly, that there are large differences between natural infections and animal models. Focusing on mouse models of bacterial pathogens, we discuss some of these discrepancies and suggest ways of improving model systems in the future.
Cellular microbiology, Jan 1, 2005
Applied and …, Jan 1, 2005
The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has le... more The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.
Infection and …, Jan 1, 2006
Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhag... more Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal E. coli during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that C. rodentium colonizes the rectum, a site previously unreported for this pathogen.
Journal of microbiological …, Jan 1, 2001
A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial... more A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy. q