Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype–genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.
Salmonella Typhimurium is a major cause of human diarrhoeal infections, usually acquired from chickens, pigs, cattle, or their products. To understand the basis of persistence and pathogenesis in these reservoir hosts, and to inform the design of novel vaccines and treatments, we generated a library of 7,702 S. Typhimurium mutants, each bearing an insertion at a random position in the genome. Using DNA sequencing, we identified the disrupted gene in each mutant and determined its relative abundance in a laboratory culture and after experimental infection of mice, chickens, pigs, and cattle. The method allowed large numbers of mutants to be investigated simultaneously, drastically reducing the number of animals required to perform a comprehensive screen. We identified mutants that grow in culture but do not survive in one or more of the animals. The genes disrupted in these mutants are inferred to be important for the infection process. Most of these genes were required in all three food-producing animals, but smaller subsets of genes may mediate persistence in a specific host species. The data provide the most comprehensive map of virulence-associated genes for any bacterial pathogen in natural hosts and are highly relevant for the design of control strategies.
Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells); cytokeratin 7 (ductal cells); and smooth muscle α-actin (myoepithelial cells) and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.
Salivary gland; Development; Progenitor cells; Multiplex analysis
State-scale and premises-scale gravity models for the spread of highly pathogenic avian influenza (H5N1) in Nigeria and Ghana were used to provide a basis for risk maps for future epidemics and to compare and rank plausible culling and vaccination strategies for control. Maximum likelihood methods were used to fit the models to the 2006–2007 outbreaks. The sensitivity and specificity of the state-scale model-generated probabilities that any given state would be involved in an epidemic were each 57 %. The premises-based model indicated that reactive, countrywide vaccination strategies, in which the order in which flocks are vaccinated was strictly determined by known risk factors for infection, were more effective in reducing the final size of the epidemic and the epidemic impact than vaccinating flocks at random or ring vaccination. The model suggests that an introduction of highly pathogenic avian influenza (H5N1) into Ghana had a high chance (84 %) of causing a major outbreak. That this did not happen was most probably a result of the very swift Ghanaian response to news of the first introductions.
Avian influenza; Nigeria; Ghana; Mathematical model; Control
Obesity, an increasing problem worldwide, is a leading cause of morbidity and mortality. Management principally requires lifestyle (i.e. behavioural) changes. An evidence-base exists of behaviour change techniques for weight loss; however, in routine practice doctors are often unsure about effective treatments and commonly use theoretically-unfounded communication strategies (e.g. information-giving). It is not known if communication skills teaching during undergraduate training adequately prepares future doctors to engage in effective behaviour change talk with patients. The aim of the study was to examine which behaviour change techniques medical undergraduates use to facilitate lifestyle adjustments in obese patients.
Forty-eight medical trainees in their clinical years of a UK medical school conducted two simulated consultations each. Both consultations involved an obese patient scenario where weight loss was indicated. Use of simulated patients (SPs) ensured standardisation of key variables (e.g. barriers to behaviour change). Presentation of scenario order was counterbalanced. Following each consultation, students assessed the techniques they perceived themselves to have used. SPs rated the extent to which they intended to make behavioural changes and why. Anonymised transcripts of the audiotaped consultations were coded by independent assessors, blind to student and SP ratings, using a validated behaviour change taxonomy.
Students reported using a wide range of evidence-based techniques. In contrast, codings of observed communication behaviours were limited. SPs behavioural intention varied and a range of helpful elements of student’s communication were revealed.
Current skills-based communication programmes do not adequately prepare future doctors for the growing task of facilitating weight management. Students are able to generalise some communication skills to these encounters, but are over confident and have limited ability to use evidence-based theoretically informed techniques. They recognise this as a learning need. Educators will need to tackle the challenges of integrating theoretically informed and evidence based behaviour change talk within medical training.
Behaviour change; Obesity; Undergraduate
Nurse led self-help treatments for people with chronic fatigue syndrome/myalgic encephalitis (CFS/ME) have been shown to be effective in reducing fatigue but their cost-effectiveness is unknown.
Cost-effectiveness analysis conducted alongside a single blind randomised controlled trial comparing pragmatic rehabilitation (PR) and supportive listening (SL) delivered by primary care nurses, and treatment as usual (TAU) delivered by the general practitioner (GP) in North West England. A within trial analysis was conducted comparing the costs and quality adjusted life years (QALYs) measured within the time frame of the trial. 296 patients aged 18 and over with CFS/ME diagnosed using the Oxford criteria were included in the cost-effectiveness analysis.
Treatment as usual is less expensive and leads to better patient outcomes compared with Supportive Listening. Treatment as usual is also less expensive than Pragmatic Rehabilitation. PR was effective at reducing fatigue in the short term, but the impact of the intervention on QALYs was uncertain. However, based on the results of this trial, PR is unlikely to be cost-effective in this patient population.
This analysis does not support the introduction of SL. Any benefits generated by PR are unlikely to be of sufficient magnitude to warrant recommending PR for this patient group on cost-effectiveness grounds alone. However, dissatisfaction with current treatment options means simply continuing with ‘treatment as usual’ in primary care is unlikely to be acceptable to patients and practitioners.
The trial registration number is IRCTN74156610
Cost-effectiveness; Primary care; CFS/ME; Self-management; Supportive listening; Pragmatic rehabilitation
NICE guidelines emphasise the need for a confident, early diagnosis of Chronic Fatigue Syndrome/ Myalgic Encephalitis (CFS/ME) in Primary Care with management tailored to the needs of the patient. Research suggests that GPs are reluctant to make the diagnosis and resources for management are currently inadequate. This study aimed to develop resources for practitioners and patients to support the diagnosis and management of CFS/ME in primary care.
Semi structured interviews were conducted with patients, carers, GPs, practice nurses and CFS/ME specialists in North West England. All interviews were audio recorded, transcribed and analysed qualitatively using open explorative thematic coding. Two patient involvement groups were consulted at each stage of the development of resources to ensure that the resources reflect everyday issues faced by people living with CFS/ME.
Patients and carers stressed the importance of recognising CFS/ME as a legitimate condition, and the need to be believed by health care professionals. GPs and practice nurses stated that they do not always have the knowledge or skills to diagnose and manage the condition. They expressed a preference for an online training package. For patients, information on getting the most out of a consultation and the role of carers was thought to be important. Patients did not want to be overloaded with information at diagnosis, and suggested information should be given in steps. A DVD was suggested, to enable information sharing with carers and family, and also for those whose symptoms act as a barrier to reading.
Rather than use a top-down approach to the development of training for health care practitioners and information for patients and carers, we have used data from key stakeholders to develop a patient DVD, patient leaflets to guide symptom management and a modular e-learning resource which should equip GPs to diagnose and manage CFS/ME effectively, meet NICE guidelines and give patients acceptable, evidence-based information.
Chronic fatigue syndrome; ME; Resources; Patient; Practitioner; Qualitative research; Primary health care
Despite respiratory tract infections usually being viral and self-limiting, most primary care consultations still result in an antibiotic prescription. The National Institute for Health and Clinical Excellence (NICE) introduced the ‘delayed prescribing’ (DP) strategy. It remains unknown how useful UK clinicians find this approach
To investigate how DP is used within UK primary care, and the benefits and challenges associated with this strategy
Design and setting
Qualitative interview and focus group study in UK scheduled and unscheduled care primary care settings
Data were gathered through semi-structured interviews (n = 49) and six focus groups with GPs, trainee GPs, and nurse prescribers (NPs). An iterative analysis approach, using grounded theory principles, was used to generate themes from the dataset.
Prescribers were familiarwith DP but used it infrequently. DP was often used to manage diagnostic uncertainty, although NPs, trainee GPs, and GPs working in unscheduled care services preferred patients to reconsult under these circumstances. Prescribers used DP to avoid conflict, although some had found more effective strategies to achieve this. Prescribers were generally uncomfortable giving clinical responsibility to patients, and DP was perceived to communicate a conflicting message to patients about antibiotic efficacy.
DP was not considered to be a helpful strategy for managing patients with self-limiting respiratory tract infections within primary care and the findings do not support the centrality of DP in NICE guidelines as a primary means of reducing antibiotic prescribing. Future training and guidelines should encourage alternative ways of communicating empathy, addressing patient beliefs, and encouraging self-management.
antibacterial agents; drug prescriptions; health communication; primary care; qualitative research
Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.
The evidence base for a range of psychosocial and behavioural interventions in managing and supporting patients with long-term conditions (LTCs) is now well-established. With increasing numbers of such patients being managed in primary care, and a shortage of specialists in psychology and behavioural management to deliver interventions, therapeutic interventions are increasingly being delivered by general nurses with limited training in psychological interventions. It is unknown what issues this raises for the nurses or their patients. The purpose of the study was to examine the challenges faced by non-specialist nurses when delivering psychological interventions for an LTC (chronic fatigue syndrome/myalgic encephalomyelitis [CFS/ME]) within a primary care setting.
A qualitative study nested within a randomised controlled trial [ISRCTN 74156610] explored the experiences and acceptability of two different psychological interventions (pragmatic rehabilitation and supportive listening) from the perspectives of nurses, their supervisors, and patients. Semi structured in-depth interviews were conducted with three nurse therapists, three supervisors, and 46 patients. An iterative approach was used to develop conceptual categories from the dataset.
Analyses identified four sets of challenges that were common to both interventions: (i) being a novice therapist, (ii) engaging patients in the therapeutic model, (iii) dealing with emotions, and (iv) the complexity of primary care. Each challenge had the potential to cause tension between therapist and patient. A number of strategies were developed by participants to manage the tensions.
Tensions existed for nurses when attempting to deliver psychological interventions for patients with CFS/ME in this primary care trial. Such tensions should be addressed before implementing psychological interventions within routine clinical practice. Similar tensions may be found for other LTCs. Our findings have implications for developing therapeutic alliances and highlight the need for regular supervision.
Managing early warning signs is an effective approach to preventing relapse in bipolar disorder. Involving relatives in relapse prevention has been shown to maximize the effectiveness of this approach. However, family-focused intervention research has typically used expert therapists, who are rarely available within routine clinical services. It remains unknown what issues exist when involving relatives in relapse prevention planning delivered by community mental health case managers. This study explored the value and barriers of involving relatives in relapse prevention from the perspectives of service users, relatives and care-coordinators.
Qualitative interview study nested within a randomized controlled trial of relapse prevention for individuals with bipolar disorder. The purposive sample of 52 participants comprised service users (n = 21), care coordinators (n = 21) and relatives (n = 10). Data were analyzed using a grounded theory approach.
All parties identified benefits of involving relatives in relapse prevention: improved understanding of bipolar disorder; relatives gaining a role in illness management; and improved relationships between each party. Nevertheless, relatives were often discouraged from becoming involved. Some staff perceived involving relatives increased the complexity of their own role and workload, and some service users valued the exclusivity of their relationship with their care-coordinator and prioritized taking individual responsibility for their illness over the benefits of involving their relatives. Barriers were heightened when family relationships were poor.
Whilst involving relatives in relapse prevention has perceived value, it can increase the complexity of managing bipolar disorder for each party. In order to fully realize the benefits of involving relatives in relapse prevention, additional training and support for community care coordinators is needed.
Non-didactically delivered curriculum based group psychoeducation has been shown to be more effective than both group support in a specialist mood disorder centre in Spain (with effects lasting up to five years), and treatment as usual in Australia. It is unclear whether the specific content and form of group psychoeducation is effective or the chance to meet and work collaboratively with other peers. The main objective of this trial is to determine whether curriculum based group psychoeducation is more clinically and cost effective than unstructured peer group support.
Single blind two centre cluster randomised controlled trial of 21 sessions group psychoeducation versus 21 sessions group peer support in adults with bipolar 1 or 2 disorder, not in current episode but relapsed in the previous two years. Individual randomisation is to either group at each site. The groups are carefully matched for the number and type of therapists, length and frequency of the interventions and overall aim of the groups but differ in content and style of delivery. The primary outcome is time to next bipolar episode with measures of the therapeutic process, barriers and drivers to the effective delivery of the interventions and economic analysis. Follow up is for 96 weeks after randomisation.
The trial has features of both an efficacy and an effectiveness trial design. For generalisability in England it is set in routine public mental health practice with a high degree of expert patient involvement.
National Institute for Health Research, England.
Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.
Submandibular gland; AQP5; Development; Prenatal; Postnatal; Sexual dimorphism
Objective To evaluate the effectiveness of home delivered pragmatic rehabilitation—a programme of gradually increasing activity designed collaboratively by the patient and the therapist—and supportive listening—an approach based on non-directive counselling—for patients in primary care with chronic fatigue syndrome/myalgic encephalomyelitis or encephalitis (CFS/ME).
Design Single blind, randomised, controlled trial.
Setting 186 general practices across the north west of England between February 2005 and May 2007.
Participants 296 patients aged 18 or over with CFS/ME (median illness duration seven years) diagnosed using the Oxford criteria.
Interventions Participants were randomly allocated to pragmatic rehabilitation, supportive listening, or general practitioner treatment as usual. Both therapies were delivered at home in 10 sessions over 18 weeks by one of three adult specialty general nurses who had received four months’ training, including supervised practice, in each of the interventions. GP treatment as usual was unconstrained except that patients were not to be referred for systematic psychological therapies during the treatment period.
Main outcome measures The primary clinical outcomes were fatigue and physical functioning at the end of treatment (20 weeks) and 70 weeks from recruitment compared with GP treatment as usual. Lower fatigue scores and higher physical functioning scores denote better outcomes.
Results A total of 257 (87%) of the 296 patients who entered the trial were assessed at 70 weeks, the primary outcome point. Analysis was on an intention to treat basis, with robust treatment effects estimated after adjustment for missing data using probability weights. Immediately after treatment (at 20 weeks), patients allocated to pragmatic rehabilitation (n=95) had significantly improved fatigue (effect estimate -1.18, 95% confidence interval -2.18 to -0.18; P=0.021) but not physical functioning (-0.18, 95% CI -5.88 to +5.52; P=0.950) compared with patients allocated to treatment as usual (n=100). At one year after finishing treatment (70 weeks), there were no statistically significant differences in fatigue or physical functioning between patients allocated to pragmatic rehabilitation and those on treatment as usual (-1.00, 95% CI -2.10 to +0.11; P=0.076 and +2.57, 95% CI 3.90 to +9.03; P=0.435). At 20 weeks, patients allocated to supportive listening (n=101) had poorer physical functioning than those allocated to treatment as usual (-7.54, 95% CI -12.76 to -2.33; P=0.005) and no difference in fatigue. At 70 weeks, patients allocated to supportive listening did not differ significantly from those allocated to treatment as usual on either primary outcome.
Conclusions For patients with CFS/ME in primary care, pragmatic rehabilitation delivered by trained nurse therapists improves fatigue in the short term compared with unconstrained GP treatment as usual, but the effect is small and not statistically significant at one year follow-up. Supportive listening delivered by trained nurse therapists is not an effective treatment for CFS/ME.
Trial registration International Standard Randomised Controlled Trial Number IRCTN74156610.
Despite both parties often expressing dissatisfaction with consultations, patients with medically unexplained symptoms (MUS) prefer to consult their general practitioners (GPs) rather than any other health professional. Training GPs to explain how symptoms can relate to psychosocial problems (reattribution) improves the quality of doctor–patient communication, though not necessarily patient health.
To examine patient experiences of GPs’ attempts to reattribute MUS in order to identify potential barriers to primary care management of MUS and improvement in outcome.
Patients consulting with MUS whose GPs had been trained in reattribution. A secondary sample of patients of control GPs was also interviewed to ascertain if barriers identified were specific to reattribution or common to consultations about MUS in general.
Thematic analysis of in-depth interviews.
Potential barriers include the complexity of patients’ problems and patients’ judgements about how to manage their presentation of this complexity. Many did not trust doctors with discussion of emotional aspects of their problems and chose not to present them. The same barriers were seen amongst patients whose GPs were not trained, suggesting the barriers are not particular to reattribution.
Improving GP explanation of unexplained symptoms is insufficient to reduce patients’ concerns. GPs need to (1) help patients to make sense of the complex nature of their presenting problems, (2) communicate that attention to psychosocial factors will not preclude vigilance to physical disease and (3) ensure a quality of doctor–patient relationship in which patients can perceive psychosocial enquiry as appropriate.
doctor–patient communication; medically unexplained symptoms; reattribution
NICE guidelines emphasise the role of the primary care team in the management of patients with Chronic Fatigue Syndrome/Myalgic Encephalitis (CFS/ME). A key stage in effective management is making an accurate early diagnosis, supported by appropriate referral.
A nested qualitative study within a multi-centre randomised controlled trial which aimed to explore GPs' views on their role in making the diagnosis of CFS/ME and subsequent management of patients in primary care. Semi-structured interviews with 22 GPs. Interviews were transcribed verbatim and an iterative approach used to develop themes from the dataset.
GPs described difficulties in defining CFS/ME and suggested that their role in making a diagnosis was to exclude physical causes for the patient's symptoms, but they reported little confidence in positively attributing the label of CFS/ME to a patient and their symptoms. GPs suggested that the label of CFS/ME could be potentially harmful for the patient. The role of referral to secondary care was debated and GPs struggled defining their own role in management of this group of patients.
Until GPs feel comfortable making the diagnosis of CFS/ME and facilitating initial management, and have appropriate services to refer patients to, there will continue to be delays in confirming the diagnosis and patients presenting in primary care with fatigue may not receive appropriate care.
Salmonella enterica serovar Enteritidis (S. Enteritidis) has caused major epidemics of gastrointestinal infection in many different countries. In this study we investigate genome divergence and pathogenic potential in S. Enteritidis isolated before, during and after an epidemic in Uruguay.
266 S. Enteritidis isolates were genotyped using RAPD-PCR and a selection were subjected to PFGE analysis. From these, 29 isolates spanning different periods, genetic profiles and sources of isolation were assayed for their ability to infect human epithelial cells and subjected to comparative genomic hybridization using a Salmonella pan-array and the sequenced strain S. Enteritidis PT4 P125109 as reference. Six other isolates from distant countries were included as external comparators.
Two hundred and thirty three chromosomal genes as well as the virulence plasmid were found as variable among S. Enteritidis isolates. Ten out of the 16 chromosomal regions that varied between different isolates correspond to phage-like regions. The 2 oldest pre-epidemic isolates lack phage SE20 and harbour other phage encoded genes that are absent in the sequenced strain. Besides variation in prophage, we found variation in genes involved in metabolism and bacterial fitness. Five epidemic strains lack the complete Salmonella virulence plasmid. Significantly, strains with indistinguishable genetic patterns still showed major differences in their ability to infect epithelial cells, indicating that the approach used was insufficient to detect the genetic basis of this differential behaviour.
The recent epidemic of S. Enteritidis infection in Uruguay has been driven by the introduction of closely related strains of phage type 4 lineage. Our results confirm previous reports demonstrating a high degree of genetic homogeneity among S. Enteritidis isolates. However, 10 of the regions of variability described here are for the first time reported as being variable in S. Enteritidis. In particular, the oldest pre-epidemic isolates carry phage-associated genetic regions not previously reported in S. Enteritidis. Overall, our results support the view that phages play a crucial role in the generation of genetic diversity in S. Enteritidis and that phage SE20 may be a key marker for the emergence of particular isolates capable of causing epidemics.
Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.
Salmonella Typhimurium infection of mice is an established model of systemic typhoid fever in humans. Mutations that inactivate genes that are important for virulence produce attenuated S. Typhimurium bacteria that have potential for use as live vaccines. To investigate the infection process we have produced a large pool of random insertion mutants, and developed a novel microarray-based technology, Transposon Mediated Differential Hybridisation (TMDH), that allows us to determine the gene disrupted by each insertion. Comparison of data obtained from the mutant pool grown in laboratory culture (input) with equivalent data produced after passage of the pool through mice (output) enables genes that are important for the infection process to be determined, since they are absent or less prevalent in the output pool. We have constructed defined deletion mutants of several of the candidate genes identified in the TMDH screen, and have shown that they are attenuated for virulence and effective live vaccines.
Salmonella enterica serovar Typhimurium (S. Typhimurium) infection causes an inflammatory response through activation of Toll-like receptor 4 by lipopolysaccharide. Dexamethasone, a glucocorticoid analogue, suppresses inflammatory responses by many mechanisms including inhibition of the lipopolysaccharide-induced production of proinflammatory mediators. There is little information on the effect of glucocorticoids on murine salmonellosis. In this study, we treated susceptible BALB/c mice by subcutaneous implantation of slow-release dexamethasone pellets before infection with S. Typhimurium. Dexamethasone promotes bacterial growth early in infection and induces a dose-dependent increase in bacterial growth within mouse livers and spleens. The bacterial load in organs from infected placebo-treated mice was lower than that in dexamethasone-treated mice. Glucocorticoids inhibit lipopolysaccharide-induced inflammation partially through the steroid-inducible protein annexin-A1 (ANXA1). Infection of wild-type and ANXA1 knock-out mice with S. Typhimurium led to similar organ bacterial loads. ANXA1 also did not affect the bacterial load in organs from infected dexamethasone-treated mice. This suggests that glucocorticoids, independently of ANXA1, accelerate S. Typhimurium growth in vivo in susceptible BALB/c mice.
glucocorticoid; dexamethasone; Salmonella; infection; annexin-A1
In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth.
We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes.
We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.
Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as αVβ5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revealed that an RSD motif in the EHV-1 envelope glycoprotein D (gD) is critical for entry via endocytosis. In addition, we show that EHV-1 enters peripheral blood mononuclear cells predominantly via the endocytic pathway, whereas in equine endothelial cells entry occurs mainly via fusion at the plasma membrane. Taken together, the data in this study provide evidence that EHV-1 entry via endocytosis is triggered by the interaction between cellular integrins and the RSD motif present in gD and, moreover, that EHV-1 uses different cellular entry pathways to infect important target cell populations of its natural host.
Enhanced relapse prevention (ERP) is a psychological intervention delivered by mental health professionals to help individuals with bipolar disorder (BD) recognise and manage early warning signs for mania and depression. ERP has an emerging evidence base and is recommended as good practice for mental health professionals. However, without highly perceived value to both those receiving (services users) or delivering it (health professionals), implementation will not occur. The aim of this study is to determine what values of ERP are perceived by service users (SUs) and mental health professionals (care coordinators, CCs) providing community case management.
A nested qualitative study design was employed as part of a randomised controlled trial of ERP. Semi-structured interviews were conducted with a purposive sub-sample of 21 CCs and 21 SUs, and an iterative approach used to develop a framework of conceptual categories that was applied systematically to the data.
The process of implementing and receiving ERP was valued by both SUs and CCs for three similar sets of reasons: improved understanding of BD (where a knowledge deficit of BD was perceived), enhanced working relationships, and improved ways of managing the condition. There were some differences in the implications these had for both CCs and SUs who also held some reservations.
CCs and SUs perceive similar value in early warning signs interventions to prevent relapse, and these have particular benefits to them. If this perceived value is maintained, CCs and SUs in routine practice may use ERP long-term.
NICE guidelines suggest that patients with Chronic Fatigue Syndrome/Myalgic Encephalitis (CFS/ME) should be managed in Primary Care. Practice Nurses are increasingly being involved in the management of long-term conditions, so are likely to also have a growing role in managing CFS/ME. However their attitudes to, and experiences of patients with CFS/ME and its management must be explored to understand what barriers may exist in developing their role for this group of patients. The aim of this study was to explore Practice Nurses' understanding and beliefs about CFS/ME and its management.
Semi-structured interviews with 29 Practice Nurses. Interviews were transcribed verbatim and an iterative approach used to develop themes from the dataset.
Practice nurses had limited understanding about CFS/ME which had been largely gained through contact with patients, friends, personal experiences and the media rather than formal training. They had difficulty seeing CFS/ME as a long term condition. They did identify a potential role they could have in management of CFS/ME but devalued their own skills in psychological intervention, and suggested counselling would be an appropriate therapeutic option. They recognised a need for further training and on going supervision from both medical and psychological colleagues. Some viewed the condition as contentious and held pejorative views about CFS/ME. Such scepticism and negative attitudes will be a significant barrier to the management of patients with CFS/ME in primary care.
The current role of Practice Nurses in the ongoing management of patients with CFS/ME is limited. Practice Nurses have little understanding of the evidence-base for treatment of CFS/ME, particularly psychological therapies, describing management options in terms of advice giving, self-help or counselling. Practice Nurses largely welcomed the potential development of their role in this area, but identified barriers and training needs which must be addressed to enable them to feel confident managing of patients with this condition. Training must begin by addressing negative attitudes to patients with CFS/ME.
Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNA's), delivered intranasally, could prove a valuable alternative for infection control. siRNA's against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model.
siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection.
siRNA treatment has potential for both prevention and early treatment of EHV-1 infections.
The successful introduction of new methods for managing medically unexplained symptoms in primary care is dependent to a large degree on the attitudes, experiences and expectations of practitioners. As part of an exploratory randomised controlled trial of reattribution training, we sought the views of participating practitioners on patients with medically unexplained symptoms, and on the value of and barriers to the implementation of reattribution in practice.
A nested attitudinal survey and qualitative study in sixteen primary care teams in north-west England. All practitioners participating in the trial (n = 74) were invited to complete a structured survey. Semi-structured interviews were undertaken with a purposive sub-sample of survey respondents, using a structured topic guide. Interview transcripts were used to identify key issues, concepts and themes, which were grouped to construct a conceptual framework: this framework was applied systematically to the data.
Seventy (95%) of study participants responded to the survey. Survey respondents often found it stressful to work with patients with medically unexplained symptoms, though those who had received reattribution training were more optimistic about their ability to help them. Interview participants trained in reattribution (n = 12) reported that reattribution increased their confidence to practice in a difficult area, with heightened awareness, altered perceptions of these patients, improved opportunities for team-building and transferable skills. However general practitioners also reported potential barriers to the implementation of reattribution in routine clinical practice, at the level of the patient, the doctor, the consultation, diagnosis and the healthcare context.
Reattribution training increases practitioners' sense of competence in managing patients with medically unexplained symptoms. However, barriers to its implementation are considerable, and frequently lie outside the control of a group of practitioners generally sympathetic to patients with medically unexplained symptoms and the purpose of reattribution. These findings add further to the evidence of the difficulty of implementing reattribution in routine general practice.
General practitioners’ (GPs) communication with patients presenting medically unexplained symptoms (MUS) has the potential to somatize patients’ problems and intensify dependence on medical care. Several reports indicate that GPs have negative attitudes about patients with MUS. If these attitudes deter participation in training or other methods to improve communication, practitioners who most need help will not receive it.
To identify how GPs’ attitudes to patients with MUS might inhibit their participation with training to improve management.
GPs (N = 33) who had declined or accepted training in reattribution techniques in the context of a research trial.
GPs were interviewed and their accounts analysed qualitatively.
Although attitudes that devalued patients with MUS were common in practitioners who had declined training, these coexisted, in the same practitioners, with evidence of intuitive and elaborate psychological work with these patients. However, these practitioners devalued their psychological skills. GPs who had accepted training also described working psychologically with MUS but devalued neither patients with MUS nor their own psychological skills.
GPs’ attitudes that suggested disengagement from patients with MUS belied their pursuit of psychological objectives. We therefore suggest that, whereas negative attitudes to patients have previously been regarded as the main barrier to involvement in measures to improve patient management, GPs devaluing of their own psychological skills with these patients may be more important.
medically unexplained symptoms; general practitioners; management; psychological skills; attitudes