Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii.

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

The Citizen Phage Library: Rapid Isolation of Phages for the Treatment of Antibiotic Resistant Infections in the UK.

Antimicrobial resistance poses one of the greatest threats to global health and there is an urgent need for new therapeutic options. Phages are viruses that infect and kill bacteria and phage therapy could provide a valuable tool for the treatment of multidrug-resistant infections. In this study, water samples collected by citizen scientists as part of the Citizen Phage Library (CPL) project, and wastewater samples from the Environment Agency yielded phages with activity against clinical strains Klebsiella pneumoniae BPRG1484 and Enterobacter cloacae BPRG1482. A total of 169 and 163 phages were found for K. pneumoniae and E. cloacae, respectively, within four days of receiving the strains. A third strain (Escherichia coli BPRG1486) demonstrated cross-reactivity with 42 E. coli phages already held in the CPL collection. Seed lots were prepared for four K. pneumoniae phages and a cocktail combining these phages was found to reduce melanisation in a Galleria mellonella infection model. The resources and protocols utilised by the Citizen Phage Library enabled the rapid isolation and characterisation of phages targeted against multiple strains. In the future, within a clearly defined regulatory framework, phage therapy could be made available on a named-patient basis within the UK.

Gamifying simulated nursing education: a digital technology approach to enhancing pedagogy and student experience.

Higher educational institutions have responded to a shortage of clinical placements for students by adopting innovative approaches, such as the use of simulated learning environments. The integration of gamification in simulated placements presents a promising opportunity to enrich and diversify the learning experience. A series of game-based resources to support simulated practice learning was developed by the academic team at the University of Bolton. This study involved evaluating the experiences of students who engaged in these interactive scenarios to assess the potential impact of these digital interventions on learning. The findings indicate that the approach had a significant impact on student learning, improving both their knowledge and their confidence in applying procedures in practice. These findings are of particular significance since it is commonly considered that students in fields such as nursing, which are known for their emphasis on human-centred care, place less value on digital learning technologies.

Academic and Attendance Outcomes after Participation in a School Breakfast Program.

BACKGROUND: Little is known about how school breakfast programs are associated with school attendance and academic performance. This study evaluated Dallas Independent School District's (DISD) breakfast after the bell (BATB) program that provides breakfast for both habitually tardy and non-tardy students on (1) academic performance and (2) student attendance over 2 school years. METHODS: A pre-post study design evaluated the impact of the BATB program in elementary/middle/high schools on student attendance and academic outcomes. Paired t-tests evaluated changes in outcomes between 2017 to 2018 and 2018 to 2019 school years. RESULTS: The analytical sample consisted of 30,493 students (70.32% BATB participants, 50.47% male, 68.78% Hispanic). BATB participants were over 2.5 times more likely to attend school versus non-BATB participants (aOR = 2.55; 95% confidence interval [CI]: 2.23-2.92; p < .001). Compared to pre-participation (2017-2018), unadjusted models showed 2018 to 2019 BATB participants' mean reading scores increased from 1502.72 to 1545.76 during the 2018 to 2019 academic year (p < .001). There were no significant changes in reading and math scores over the 2-year implementation after adjustment. CONCLUSIONS: Results here showed that a school breakfast program that is housed in a large public school system that serves predominantly low resource, ethnically diverse students is associated with increased student attendance.

In vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure) against polymicrobial communities and individual bacterial strains derived from diabetic foot infections.

Introduction. Diabetic foot infection (DFI) is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. DFIs are often complicated by ischaemia in the affected limb, the presence of polymicrobial biofilms and increasingly the occurrence of antibiotic resistant bacteria.Hypothesis/Gap statement. Antibiotic loaded beads could inhibit the growth of polymicrobial DFI communities with differing compositions in vitro.Aim. This study investigates the in vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure, Biocomposites Ltd., UK) against polymicrobial DFI communities and individual bacterial strains derived from DFIs.Methodology. Debrided tissue obtained from the base of infected diabetic foot ulcers was homogenised and spread over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates. Calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar and following incubation, zones of inhibition (ZOI) were measured. For individual bacterial strains isolated from the infected tissue, calcium sulfate beads containing vancomycin, gentamicin, flucloxacillin or rifampicin and beads containing a combination of vancomycin and gentamicin or flucloxacillin and rifampicin were tested for their ability to inhibit growth.Results. Calcium sulfate beads loaded with a combination of vancomycin and gentamicin were able to inhibit bacterial growth from all polymicrobial tissue homogenates tested, with ZOI diameters ranging from 15 to 40 mm. In the case of individual bacterial strains, beads containing combinations of vancomycin and gentamicin or flucloxacillin and rifampicin were able to produce ZOI with Gram-positive facultatitive anaerobic strains such as Staphylococcus aureus and Enterococcus faecalis, Gram-negative facultative anaerobic strains such as Pseudomonas aeruginosa and obligate anaerobic strains such as Finegoldia magna even where acquired resistance to one of the antibiotics in the combination was evidenced.Conclusion. The local use of calcium sulfate beads containing a combination of two antibiotics demonstrated high efficacy against polymicrobial DFI communities and individual DFI bacterial strains in in vitro zone of inhibition tests. These results show promise for clinical application, but further research and clinical studies are required.

A guide to the NMC emergency standards for nurse education during the current deployment of student nurses.

The Nursing and Midwifery Council (NMC) recognises the important contribution that nursing students are making to the national response to the COVID-19 pandemic. This article reports on the Greater Manchester Supervision and Delegation Framework, providing practical guidance for students and practice staff (practice supervisor/practice assessor and registered nurse) on how to support student nurses who have opted into a paid (deployed) healthcare role. The framework operationalises NMC emergency standards for Nursing and Midwifery education, enabling students to complete their pre-registration undergraduate or postgraduate nursing programme while also supporting the healthcare workforce (NMC, 2020).

Training Student Pharmacists in Medication Dispensing and Checking Within a High-Fidelity Clinical Environment.

Objective. To describe the implementation of and student pharmacist experience with high-fidelity training in medication dispensing and clinical checking processes and procedures. Methods. An actual dispensary in a large teaching hospital was used as the setting in which to provide undergraduate student pharmacists the opportunity to develop skills and knowledge regarding the process and procedures of medication dispensing and clinical checking. The sessions were facilitated by a member of the hospital pharmacy team and overseen by a faculty member. Students were required to conduct legal and clinical checks and process anonymized hospital prescriptions. Students recorded their experiences in their professional portfolio as blog entries, which were then discussed with academic staff members on a biweekly basis. Content analysis of the blogs was conducted to investigate the student experience. Results. Forty-eight students attended 30 hours of dispensing sessions over a 20-week period from September to April. Content analysis of 102 blog entries demonstrated students reporting observations and experiences which mapped to the three components of communities of practice: the domain of pharmacy practice, the working dynamics of a community of professionals within the workplace, and the commonality of practice and shared repertoire of resources. Conclusion. The high-fidelity teaching of students about the dispensing and checking process in an actual dispensary environment supports student pharmacists' knowledge and skills development. The work-based nature of this approach also provides experiential learning opportunities for students to observe and participate in the professional community of practice.

Recurrent vertebral fractures in a young adult: a closer look at bone health in type 1 diabetes mellitus.

The association between type 1 diabetes mellitus (T1DM) and bone health has garnered interest over the years. Fracture risk is known to be increased in individuals with T1DM, although bone health assessment is not often performed in the clinical setting. We describe the case of a 21-year-old male with longstanding T1DM with multilevel vertebral fractures on imaging, after presenting with acute back pain without apparent trauma. Dual-energy X-ray absorptiometry (DXA) revealed significantly reduced bone mineral density at the lumbar spine and femoral neck. Extensive investigations for other secondary or genetic causes of osteoporosis were unremarkable, apart from moderate vitamin D deficiency. High-resolution peripheral quantitative computed tomography and bone biospy revealed significant alterations of trabecular bone microarchitecture. It later transpired that the patient had sustained vertebral fractures secondary to unrecognised nocturnal hypoglycaemic seizures. Intravenous zoledronic acid was administered for secondary fracture prevention. Despite anti-resorptive therapy, the patient sustained a new vertebral fracture after experiencing another hypoglycaemic seizure in his sleep. Bone health in T1DM is complex and not well understood. There are significant challenges in the assessment and management of osteoporosis in T1DM, particularly in young adults, where fracture prediction tools have not been validated. Clinicians should be aware of hypoglycaemia as a significant risk factor for fracture in patients with T1DM. LEARNING POINTS: Type 1 diabetes mellitus (T1DM) is a secondary cause of osteoporosis, characterised by reduced bone mass and disturbed bone microarchitecture.Hypoglycaemic seizures generate sufficient compression forces along the thoracic column and can cause fractures in individuals with compromised bone quality.Unrecognised hypoglycaemic seizures should be considered in patients with T1DM presenting with fractures without a history of trauma.Patients with T1DM have increased fracture risk and risk factors should be addressed. Evaluation of bone microarchitecture may provide further insights into mechanisms of fracture in T1DM.Further research is needed to guide the optimal screening and management of bone health in patients with T1DM.

Formalising multidisciplinary peer review: developing a haematological malignancy-specific electronic proforma and standard operating procedure to facilitate procedural efficiency and evidence-based clinical practice.

BACKGROUND: Multidisciplinary team (MDT) meetings aimed at facilitating peer review have become standard practice in oncology. However, there is scant literature on the optimal structure and conduct of such meetings. AIMS: To develop a process for formal peer review of patients with haematological malignancies and to audit any resulting changes made to the management recommendations of the treating physician. METHODS: A standard operating procedure (SOP) for MDT meetings was developed essentially to integrate clinical peer review with weekly pathology and radiology meetings. The centrepiece is the electronic submission of a patient-specific proforma (Microsoft InfoPath) prior to the meeting. It serves as the template for presentation, discussion and recording of recommendations and conclusions. The final verified document is stored in the electronic patient record, and a copy is sent to the general practitioner. The proposed management plans were compared to the consensus recommendations of the meeting for the first 4 years since inception. RESULTS: Both SOP and proforma underwent continual improvements. These provided the framework for the conduct of a robust weekly MDT meeting for peer review of the management of patients with haematological malignancies. On 20% of occasions, patient management plans were altered to optimise patient care as a direct consequence on peer review at the MDT. CONCLUSION: Our streamlined process, in its ultimate format, has provided a mature and efficient forum for formal peer review in a genuine multidisciplinary environment. Both initial data and informal feedback support its ongoing activity as an integral component of delivering quality patient care.

Epithelial and stromal cell urokinase plasminogen activator receptor expression differentially correlates with survival in rectal cancer stages B and C patients.

Urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for colorectal cancer (CRC) patient survival. However, CRC uPAR expression remains controversial, especially regarding cell types where uPAR is overexpressed (e.g., epithelium (uPARE) or stroma-associated cells (uPARS)) and associated prognostic relevance. In this study, two epitope-specific anti-uPAR monoclonal antibodies (MAbs) could discriminate expression of uPARE from uPARS and were used to examine this association with survival of stages B and C rectal cancer (RC) patients. Using immunohistochemistry, MAbs #3937 and R4 were used to discriminate uPARE from uPARS respectively in the central and invasive frontal regions of 170 stage B and 179 stage C RC specimens. Kaplan-Meier and Cox regression analyses were used to determine association with survival. uPAR expression occurred in both epithelial and stromal compartments with differential expression observed in many cases, indicating uPARE and uPARS have different cellular roles. In the central and invasive frontal regions, uPARE was adversely associated with overall stage B survival (HR = 1.9; p = 0.014 and HR = 1.5; p = 0.031, respectively) reproducing results from previous studies. uPARS at the invasive front was associated with longer stage C survival (HR = 0.6; p = 0.007), reflecting studies demonstrating that macrophage peritumoural accumulation is associated with longer survival. This study demonstrates that different uPAR epitopes should be considered as being expressed on different cell types during tumour progression and at different stages in RC. Understanding how uPARE and uPARS expression affects survival is anticipated to be a useful clinical prognostic marker of stages B and C RC.

Correlations between integrin ανß6 expression and clinico-pathological features in stage B and stage C rectal cancer.

Integrin ανß6 is highly expressed in a range of human cancers and frequently correlates with patient survival. This study examines correlations between ανß6 expression and patient clinico-pathological features in Stage B and Stage C rectal cancer, including overall survival. Expression of ανß6 was measured in 362 Stage B or C rectal cancer tissue samples at the tumour central region, invasive tumour front and adjacent non-neoplastic mucosa using immunohistochemistry. Distribution of ανß6 was found to be significantly higher at the invasive front compared to central regions of the tumour (p<0.001) or adjacent non-neoplastic mucosa (p<0.001) suggesting ανß6 plays a role in tumour cell invasion. However, integrin ανß6 expression was not associated with clinico-pathological features or overall survival indicating it is not an independent prognostic marker differentiating Stage B or C rectal cancer. Previous ανß6 studies have suggested the expression of ανß6 is involved in the earlier stages (i.e. Stages A/B) of tumour progression rather than the later stages (i.e. Stages C/D). However, our study has revealed that in rectal cancer ανß6 expression does not increase between Stages B and C, but may occur earlier, namely before or during Stage B cancer.

Role for MyD88, TLR2 and TLR9 but not TLR1, TLR4 or TLR6 in experimental autoimmune encephalomyelitis.

The potential roles of TLRs in the cause and pathogenesis of autoimmune CNS inflammation remain contentious. In this study, we examined the effects of targeted deletions of TLR1, TLR2, TLR4, TLR6, TLR9, and MyD88 on the induction of myelin oligodendrocyte glycoprotein 35-55 (MOG(35-55)) peptide/CFA/pertussis toxin-induced autoimmune encephalomyelitis. Although C57BL/6.Tlr1(-/-), C57BL/6.Tlr4(-/-) and C57BL/6.Tlr6(-/-) mice showed normal susceptibility to disease, signs were alleviated in female C57BL/6.Tlr2(-/-) and C57BL/6.Tlr9(-/-) mice and C57BL/6.Tlr2/9(-/-) mice of both sexes. C57BL/6.Myd88(-/-) mice were completely protected. Lower clinical scores were associated with reduced leukocyte infiltrates. These results were confirmed by passive adoptive transfer of disease into female C57BL/6.Tlr2(-/-) and C57BL/6.Tlr9(-/-) mice, where protection in the absence of TLR2 was associated with fewer infiltrating CD4(+) cells in the CNS, reduced prevalence of detectable circulating IL-6, and increased proportions of central (CD62L(+)) CD4(+)CD25(+)Foxp3(+) regulatory T cells. These results provide a potential molecular mechanism for the observed effects of TLR signaling on the severity of autoimmune CNS inflammation.

Role of SLAM in NKT cell development revealed by transgenic complementation in NOD mice.

Allelic variation of SLAM expression on CD4(+)CD8(+) thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4(+)CD8(+) thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4(+)CD8(+) thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4(+)CD8(+) thymocytes and produce larger numbers of NKT cells. Transgenic enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradoxically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells.

Allelic variation of Ets1 does not contribute to NK and NKT cell deficiencies in type 1 diabetes susceptible NOD mice.

The NOD mouse is a well characterized model of type 1 diabetes that shares several of the characteristics of Ets1-deficient targeted mutant mice, viz: defects in TCR allelic exclusion, susceptibility to a lupus like disease characterized by IgM and IgG autoantibodies and immune complex-mediated glomerulonephritis, and deficiencies of NK and NKT cells. Here, we sought evidence for allelic variation of Ets1 in mice contributing to the NK and NKT cell phenotypes of the NOD strain. ETS1 expression in NK and NKT cells was reduced in NOD mice, compared to C57BL/6 mice. Although NKT cells numbers were significantly correlated with ETS1 expression in both strains, NKT cell numbers were not linked to the Ets1 gene in a first backcross from NOD to C57BL/6 mice. These results indicate that allelic variation of Ets1 did not contribute to variation in NKT cell numbers in these mice. It remains possible that a third factor not linked to the Ets1 locus controls both ETS1 expression and subsequently NK and NKT cell phenotypes.

Congenic analysis of the NKT cell control gene Nkt2 implicates the peroxisomal protein Pxmp4.

Type 1 NKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. Herein, we report the production and characterization of a NOD.Nkrp1(b).Nkt2b(b) congenic mouse strain, which has increased thymic and peripheral NKT cells, a decreased incidence of type 1 diabetes, and enhanced cytokine responses in vivo and increased proliferative responses in vitro following challenge with alpha-galactosylceramide. The 19 highly differentially expressed candidate genes within the congenic region identified by microarray expression analyses included Pxmp4. This gene encodes a peroxisome-associated integral membrane protein whose only known binding partner is Pex19, an intracellular chaperone and component of the peroxisomal membrane insertion machinery encoded by a candidate for the NKT cell control gene Nkt1. These findings raise the possibility that peroxisomes play a role in modulating glycolipid availability for CD1d presentation, thereby influencing NKT cell function.

Slamf1, the NKT cell control gene Nkt1.

Invariant NKT cells play a critical role in controlling the strength and character of adaptive immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. In this study, we report the production and characterization of a NOD.Nkrp1(b).Nkt1(b) congenic mouse strain, apply microarray expression analyses to limit candidate genes within the 95% confidence region, identify Slamf1 (encoding signaling lymphocyte activation molecule) and Slamf6 (encoding Ly108) as potential candidates, and demonstrate retarded signaling lymphocyte activation molecule expression during T cell development of NOD mice, resulting in reduced expression at the CD4(+)CD8(+) stage, which is consistent with decreased NKT cell production and deranged tolerance induction in NOD mice.

Genetic control of NKT cell numbers.

NKT cells play a critical role in shaping the character and strength of a wide range of immune responses, including those against pathogens, tumours, allografts and autologous tissues. Because numbers of NKT cells affect clinical outcomes in a wide range of disease models, and this characteristic demonstrates allelic variation, the mapping of the locations and identification of the coding sequences of these genes has become a matter of significant importance. Here, we review the results to date that examine the effects of targeted deletion of a number of candidate genes, as well as the congenic and genetic linkage analyses that have attempted to localize allelic loci that affect NKT cell numbers. Although a number of candidate genes have been examined, there is no evidence that any of these contribute to variation in NKT cell numbers in natural populations. Two of the most important genetic regions controlling NKT cell numbers are Nkt1 on chromosome 1, which may contribute to lupus susceptibility, and Nkt2 on chromosome 2, which appears to contribute to diabetes susceptibility. Of great interest is a third locus on chromosome 18, identified in a novel congenic line, which can confer an absolute deficiency in this important immunoregulatory lymphocyte population.

Type 1 diabetes and NKT cells: a report on the 3rd International Workshop on NKT cells and CD1-mediated antigen presentation, September 2004, Heron Island, QLD, Australia.

NKT cells play a major role in regulating the vigor and character of a broad range of immune responses. Defects in NKT cell numbers and function have been associated with type 1 diabetes, especially in the NOD mouse model. The 3rd International Workshop on NKT Cells and CD1-Mediated Antigen Presentation provided an opportunity for researchers in the field of NKT cell biology to discuss their latest results, many of which have direct relevance to understanding the etiology and pathogenesis of diabetes.