Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains

Understanding how micro-organisms respond to acid pH is central to their control and successful exploitation

Microbes from the three domains of life, bacteria, archaea and eukarya, share the need to sense and respond to changes in the external and internal concentrations of protons. When the proton concentration is high, acidic conditions prevail and cells must respond appropriately to ensure that macromolecules and metabolic processes are sufficiently protected to sustain life. While we have learned much in recent decades about the mechanisms that microbes use to cope with acid, including the unique challenges presented by organic acids, there is still much to learn and much to be gained from developing a deeper understanding of the effects and responses to acid in microbes. In this perspective article, we survey the key molecular mechanisms that are known to be important for microbial survival during acid stress. We discuss the research approaches that have been taken to investigate the problem and highlight promising new avenues. We highlight the importance of understanding acid stress in controlling spoilage and pathogenic microbes in the food chain. We discuss the influence of acid on pathogens during the course of infections and highlight the potential of using organic acids in treatments for some types of infection. We explore the influence of acid stress on photosynthetic microbes, and on biotechnological and industrial processes, including those needed to produce organic acids. Finally, we invite colleagues with an interest in microbial responses to low pH to participate in the EU-funded COST Action project called EuroMicropH and contribute to a comprehensive database of literature on this topic that we are making publicly available

Evaluation of a co-culture of rapidly isolated chondrocytes and stem cells seeded on tri-layered collagen-based scaffolds in a caprine osteochondral defect model

Cartilage has poor regenerative capacity and thus damage to the joint surfaces presents a major clinical challenge. Recent research has focussed on the development of tissue-engineered and cell-based approaches for the treatment of cartilage and osteochondral injuries, with current clinically available cell-based approaches including autologous chondrocyte implantation and matrix-assisted autologous chondrocyte implantation. However, these approaches have significant disadvantages due to the requirement for a two-stage surgical procedure and an in vitro chondrocyte expansion phase which increases logistical challenges, hospital times and costs. In this study, we hypothesized that seeding biomimetic tri-layered scaffolds, with proven regenerative potential, with chondrocyte/infrapatellar fat pad stromal cell co-cultures would improve their regenerative capacity compared to scaffolds implanted cell-free. Rapid cell isolation techniques, without the requirement for long term in vitro culture, were utilised to achieve co-cultures of chondrocytes and stromal cells and thus overcome the limitations of existing cell-based techniques. Cell-free and cell-seeded scaffolds were implanted in osteochondral defects, created within the femoral condyle and trochlear ridge, in a translational large animal goat model. While analysis showed trends towards delayed subchondral bone healing in the cell-seeded scaffold group, by the 12 month timepoint the cell-free and cell-seeded groups yield cartilage and bone tissue with comparable quality and quantity. The results of the study reinforce the potential of the biomimetic tri-layered scaffold to repair joint defects but failed to demonstrate a clear benefit from the addition of the CC/FPMSC co-culture to this scaffold. Taking into consideration the additional cost and complexity associated with the cell-seeded scaffold approach, this study demonstrates that the treatment of osteochondral defects using cell-free tri-layered scaffolds may represent a more prudent clinical approach

The role of stress and stress adaptations in determining the fate of the bacterial pathogen listeria monocytogenes in the food chain

The foodborne pathogen Listeria monocytogenes is a highly adaptable organism that can persist in a wide range of environmental and food-related niches. The consumption of contaminated ready-to-eat foods can cause infections, termed listeriosis, in vulnerable humans, particularly those with weakened immune systems. Although these infections are comparatively rare they are associated with high mortality rates and therefore this pathogen has a significant impact on food safety. L. monocytogenes can adapt to and survive a wide range of stress conditions including low pH, low water activity, and low temperature, which makes it problematic for food producers who rely on these stresses for preservation. Stress tolerance in L. monocytogenes can be explained partially by the presence of the general stress response (GSR), a transcriptional response under the control of the alternative sigma factor sigma B (GB) that reconfigures gene transcription to provide homeostatic and protective functions to cope with the stress. Within the host GB also plays a key role in surviving the harsh conditions found in the gastrointestinal tract. As the infection progresses beyond the GI tract L, monocytogenes uses an intracellular infectious cycle to propagate, spread and remain protected from the host\u27s humoral immunity. Many of the virulence genes that facilitate this infectious cycle are under the control of a master transcriptional regulator called PrIA. In this review we consider the environmental reservoirs that enable L. monocytogenes to gain access to the food chain and discuss the stresses that the pathogen must overcome to survive and grow in these environments. The overlap that exists between stress tolerance and virulence is described. We review the principal measures that are used to control the pathogen and point to exciting new approaches that might provide improved means of control in the future

The role of stress and stress adaptations in determining the fate of the bacterial pathogen listeria monocytogenes in the food chain

The foodborne pathogen Listeria monocytogenes is a highly adaptable organism that can persist in a wide range of environmental and food-related niches. The consumption of contaminated ready-to-eat foods can cause infections, termed listeriosis, in vulnerable humans, particularly those with weakened immune systems. Although these infections are comparatively rare they are associated with high mortality rates and therefore this pathogen has a significant impact on food safety. L. monocytogenes can adapt to and survive a wide range of stress conditions including low pH, low water activity, and low temperature, which makes it problematic for food producers who rely on these stresses for preservation. Stress tolerance in L. monocytogenes can be explained partially by the presence of the general stress response (GSR), a transcriptional response under the control of the alternative sigma factor sigma B (GB) that reconfigures gene transcription to provide homeostatic and protective functions to cope with the stress. Within the host GB also plays a key role in surviving the harsh conditions found in the gastrointestinal tract. As the infection progresses beyond the GI tract L, monocytogenes uses an intracellular infectious cycle to propagate, spread and remain protected from the host's humoral immunity. Many of the virulence genes that facilitate this infectious cycle are under the control of a master transcriptional regulator called PrIA. In this review we consider the environmental reservoirs that enable L. monocytogenes to gain access to the food chain and discuss the stresses that the pathogen must overcome to survive and grow in these environments. The overlap that exists between stress tolerance and virulence is described. We review the principal measures that are used to control the pathogen and point to exciting new approaches that might provide improved means of control in the future

International partnerships in telehealth: healthcare, industry and education

PROJECT BACKGROUND: In 2010 an internationally renowned American healthcare organisation partnered with Irish industry and higher education in Waterford with the goal to expand their telehealth services. Combining the skills and expertise of Nurse Consultants, Nurse Educators, IT Specialists and Healthcare Executives, these collaborative partnerships led to the delivery of telehealth services to North America from an Irish base, and to the development of new European telehealth programmes and telehealth training in Ireland. The telehealth service includes the provision of telephone triage, health information and advice, disease management and hospital discharge programmes to clients in Ireland, the UK and the USA. Telehealth nursing is an evolving specialty that requires the development of competence in key areas of information and communication technologies, assessment, triage and critical thinking in clinical decision-making within an environment where distance separates the nurse from the client. AIMS AND OBJECTIVES: The aim of this paper is to report on the development, implementation and evaluation of the telehealth service with a focus on the telephone triage and advice service and the hospital discharge programmes. Objectives of this paper include describing this telehealth initiative with reference to the changing nature of global healthcare provision; discuss the educational strategy and accredited programme for training competent telehealth nurses; report the results of the evaluation of nurse performance in telephone triage and present the data relating to the impact of hospital discharge programmes on patient satisfaction and readmission rates. METHODS AND RESULTS: The evaluation of the telehealth training programme was undertaken six months post initial training and service commencement. One hundred triage and health information calls were reviewed, against best practice standards and programme learning outcomes, during a four-month period. Quantitative and qualitative data that demonstrates evidence of learning transfer from training to practice and the development of nurse competence from advanced beginner to levels of proficiency will be presented. The hospital discharge programmes have undergone continuous monitoring and reporting since commencement. This has enabled the collection of evidence that supports this brief telephone intervention as a method of reducing hopsital re-admissions and increasing patient satisfcation. Quantitative results will be presented and analysed in relation to the impact on patient satisfaction and readmission rates for patients discharged from cardiovascular, renal and digestive disease services. CONCLUSIONS: This project has demonstrated the effectiveness of partnerships in healthcare, industry and education in achieving the development, implementation and evaluation of international telehealth services. Initial education, training and ongoing support and development of nurses is essential for quality telehealth provision. Weekly call review, constructive feedback and reflection on practice are effective strategies for performance assurance and improvement. As a growing element of integrated healthcare, telehealth modules should be included in pre and post-registration nursing education curricula. Further collaboration between industry, healthcare and education are necessary in moving the telehealth agenda forward for the benefit of integrating services that impact positively on service users

Development and optimization of an egfp-based reporter for measuring the general stress response inlisteria monocytogenes

A characteristic of the food-borne pathogen Listeria monocytogenes is its tolerance to the harsh conditions found both in minimally processed foods and the human gastrointestinal tract. This trait is partly under the control of the alternative sigma factor sigma B (sigma(B)). To study the mechanisms that trigger the activation of sB, and hence the development of stress tolerance, we have developed a fluorescent reporter fusion that allows the real-time activity of sB to be monitored. The reporter, designated P-lmo2230::egfp, fuses the strong sigma(B)-dependent promoter from the lmo2230 gene (which encodes a putative arsenate reductase) to a gene encoding enhanced green fluorescence protein (EGFP). The reporter was integrated into the genomes of the wild-type strain L. monocytogenes EGD-e as well as two mutant derivatives lacking either sigB or rsbV. The resulting strains were used to study sigma(B) activation in response to growth phase and hyperosmotic stress. The wild-type was strongly fluorescent in stationary phase or in cultures with added NaCl and this fluorescence was abolished in both the sigB and rsbV backgrounds, consistent with the sigma(B)-dependency of the lmo2230 promoter. During sudden osmotic upshock (addition of 0.5 M NaCl during growth) a real-time increase in fluorescence was observed microscopically, reaching maximal activation after 30 min. Flow cytometry was used to study the activation of sigma(B) at a population level by hyperosmotic stress during exponential growth. A strong and proportional increase in fluorescence was observed as the salt concentration increased from 0 to 0.9 M NaCl. Interestingly, there was considerable heterogeneity within the population and a significant proportion of cells failed to induce a high level of fluorescence, suggesting that sB activation occurs stochastically in response to hyperosmotic stress. Thus the Plmo2230::egfp is a powerful tool that will allow the stress response to be better studied in this important human pathogen