Assessment of undergraduate medical students' understanding and application of ethics: must do better.

Current methods used to assess newly qualified doctors have limited ability to assess reasoning about complex issues. This editorial looks at the points this raises in relation to the new medical licensing assessment.

The medical licensing assessment will fall short of determining whether a UK medical graduate behaves ethically.

UK medical graduates will soon need to pass the medical licensing assessment, which assesses skills and knowledge in ethics using multiple choice questions (eg single best answer questions) and objective structured clinical examination. However, educational leaders have recognised that these methods lack the sophistication needed to accurately assess medical ethics. The reasons are two-fold. First, there may be a knowledge and practice gap in medical schools when it comes to preparing students for the assessment. To this end, this article shares peer advice about how best to use objective structured clinical examinations and single best answer questions for assessing medical ethics to help prepare students for the medical licensing assessment. Second, the design of the assessment is unlikely to adequately measure graduates' ethical values and behaviour in real world scenarios. Further work is needed to design assessments that are sophisticated enough to examine candidates' ethical reasoning and their actual behaviour.

Antibiofilm and immunomodulatory resorbable nanofibrous filing for dental pulp regenerative procedures.

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

Microtiter plate assays to assess antibiofilm activity against bacteria.

Bacterial biofilms demonstrate high broad-spectrum adaptive antibiotic resistance and cause two thirds of all infections, but there is a lack of approved antibiofilm agents. Unlike the standard minimal inhibitory concentration assay to assess antibacterial activity against planktonic cells, there is no standardized method to evaluate biofilm inhibition and/or eradication capacity of novel antibiofilm compounds. The protocol described here outlines simple and reproducible methods for assessing the biofilm inhibition and eradication capacities of novel antibiofilm agents against adherent bacterial biofilms grown in 96-well microtiter plates. It employs two inexpensive dyes: crystal violet to stain adhered biofilm biomass and 2,3,5-triphenyl tetrazolium chloride to quantify metabolism of the biofilm cells. The procedure is accessible to any laboratory with a plate reader, requires minimal technical expertise or training and takes 4 or 5 d to complete. Recommendations for how biofilm inhibition and eradication results should be interpreted and presented are also described.

Murine Model of Sinusitis Infection for Screening Antimicrobial and Immunomodulatory Therapies.

The very common condition of sinusitis is characterized by persistent inflammation of the nasal cavity, which contributes to chronic rhinosinusitis and morbidity of cystic fibrosis patients. Colonization by opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa triggers inflammation that is exacerbated by defects in the innate immune response. Pathophysiological mechanisms underlying initial colonization of the sinuses are not well established. Despite their extensive use, current murine models of acute bacterial rhinosinusitis have not improved the understanding of early disease stages due to analytical limitations. In this study, a model is described that is technically simple, allows non-invasive tracking of bacterial infection, and screening of host-responses to infection and therapies. The model was modified to investigate longer-term infection and disease progression by using a less virulent, epidemic P. aeruginosa cystic fibrosis clinical isolate LESB65. Tracking of luminescent bacteria was possible after intranasal infections, which were sustained for up to 120 h post-infection, without compromising the overall welfare of the host. Production of reactive oxidative species was associated with neutrophil localization to the site of infection in this model. Further, host-defense peptides administered by Respimat® inhaler or intranasal instillation reduced bacterial burden and impacted disease progression as well as cytokine responses associated with rhinosinusitis. Thus, future studies using this model will improve our understanding of rhinosinusitis etiology and early stage pathogenesis, and can be used to screen for the efficacy of emerging therapies pre-clinically.

Human organoid biofilm model for assessing antibiofilm activity of novel agents.

Bacterial biofilms cause 65% of all human infections and are highly resistant to antibiotic therapy but lack specific treatments. To provide a human organoid model for studying host-microbe interplay and enabling screening for novel antibiofilm agents, a human epidermis organoid model with robust methicillin-resistant Staphylococcus aureus (MRSA) USA300 and Pseudomonas aeruginosa PAO1 biofilm was developed. Treatment of 1-day and 3-day MRSA and PAO1 biofilms with antibiofilm peptide DJK-5 significantly and substantially reduced the bacterial burden. This model enabled the screening of synthetic host defense peptides, revealing their superior antibiofilm activity against MRSA compared to the antibiotic mupirocin. The model was extended to evaluate thermally wounded skin infected with MRSA biofilms resulting in increased bacterial load, cytotoxicity, and pro-inflammatory cytokine levels that were all reduced upon treatment with DJK-5. Combination treatment of DJK-5 with an anti-inflammatory peptide, 1002, further reduced cytotoxicity and skin inflammation.

Antibiofilm peptides: overcoming biofilm-related treatment failure.

Health leaders and scientists worldwide consider antibiotic resistance among the world's most dangerous pathogens as one of the biggest threats to global health. Antibiotic resistance has largely been attributed to genetic changes, but the role and recalcitrance of biofilms, largely due to growth state dependent adaptive resistance, is becoming increasingly appreciated. Biofilms are mono- and multi-species microbial communities embedded in an extracellular, protective matrix. In this growth state, bacteria are transcriptionally primed to survive extracellular stresses. Adaptations, affecting metabolism, regulation, surface charge, immune recognition and clearance, allow bacteria to thrive in the human body and withstand antibiotics and the host immune system. Biofilms resist clearance by multiple antibiotics and have a major role in chronic infections, causing more than 65% of all infections. No specific antibiofilm agents have been developed. Thus, there is a pressing need for alternatives to traditional antibiotics that directly inhibit and/or eradicate biofilms. Host defence peptides (HDPs) are small cationic peptides that are part of the innate immune system to both directly kill microbes but also function to modulate the immune response. Specific HDPs and their derivatives demonstrate broad-spectrum activity against biofilms. In vivo biofilm assays show efficacy in abscess, respiratory, in-dwelling device, contact lens and skin infection models. Further progress has been made through the study of ex vivo organoid and air-liquid interface models to better understand human infections and treatment while relieving the burden and complex nature of animal models. These avenues pave the way for a better understanding and treatment of the underlying cause of chronic infections that challenge the healthcare system.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacteriumavium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® - a commercial MAP bacterin - we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer's patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Surfing motility is a complex adaptation dependent on the stringent stress response in Pseudomonas aeruginosa LESB58.

Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick, viscous mucus, containing the glycoprotein mucin, that can lead to progressive airway obstruction. Recently, we demonstrated that the presence of mucin induced a rapid surface adaptation in motile bacteria termed surfing motility, which data presented here indicates is very different from swarming motility. Pseudomonas aeruginosa, the main colonizing pathogen in CF, employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. We used motility-based assays and RNA-Seq to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain). Motility experiments revealed that an LESB58 stringent response mutant (ΔrelAΔspoT) was unable to surf. Transcriptional profiling of ΔrelAΔspoT mutant cells from surfing agar plates, when compared to wild-type cells from the surfing edge, revealed 2,584 dysregulated genes. Gene Ontology and KEGG enrichment analysis revealed effects of the stringent response on amino acid, nucleic acid and fatty acid metabolism, TCA cycle and glycolysis, type VI secretion, as well as chemotaxis, cell communication, iron transport, nitrogen metabolic processes and cyclic-di-GMP signalling. Screening of the ordered PA14 transposon library revealed 224 mutants unable to surf and very limited overlap with genes required for swarming. Mutants affecting surfing included two downstream effector genes of the stringent stress response, the copper regulator cueR and the quinolone synthase pqsH. Both the cueR and pqsH cloned genes complemented the surfing deficiency of ΔrelAΔspoT. Our study revealed insights into stringent stress dependency in LESB58 and showed that surfing motility is stringently-controlled via the expression of cueR and pqsH. Downstream factors of the stringent stress response are important to investigate in order to fully understand its ability to colonize and persist in the CF lung.