Gamification as an educational tool to address antimicrobial resistance: a systematic review.

Background: Antimicrobial resistance (AMR) poses a serious threat to global healthcare, and inadequate education has been identified as a major challenge by the WHO. The human , animal and agricultural sectors contribute to the emergence of AMR. Gamification has emerged as an innovative tool to improve knowledge and change behaviours. Our study provides an overview of the literature on existing games in prescribers' education across the One Health sectors, with a particular focus on the impact of gamification on learning. Methods: Using the PRISMA guidelines, we searched Cochrane, PubMed, Scopus and Google Scholar for articles related to gamification for future prescribers of antimicrobials from inception until 28 March 2023. Retrieval and screening of articles was done using a structured search protocol with strict inclusion/exclusion criteria. Results: A total of 120 articles were retrieved, of which 6 articles met the inclusion criteria for final analysis. High-income countries had the most studies, with one global study incorporating low- to middle-income countries. All games were evaluated in the human sector. Board and card games, featuring scoring and point systems, were the most prevalent game types. Most games focused on improving knowledge and prescribing behaviours of medical students, with bacteria or antibiotics as the only content. All studies highlighted the significant potential of gamification in mitigating AMR, promoting antimicrobial stewardship, and improving retention of information compared with conventional lectures. Conclusions: Our review found an absence of studies in the animal and environmental sectors, disproportionately focused on medical students with questionable sample size, inadequate assessment of game content and effectiveness, and opportunities for game developers.

Off-Target Integron Activity Leads to Rapid Plasmid Compensatory Evolution in Response to Antibiotic Selection Pressure.

Integrons are mobile genetic elements that have played an important role in the dissemination of antibiotic resistance. Under stress, the integron can generate combinatorial variation in resistance cassette expression by cassette reshuffling, accelerating the evolution of resistance. However, the flexibility of the integron integrase site recognition motif hints at potential off-target effects of the integrase on the rest of the genome that may have important evolutionary consequences. Here, we test this hypothesis by selecting for increased-piperacillin-resistance populations of Pseudomonas aeruginosa with a mobile integron containing a difficult-to-mobilize ß-lactamase cassette to minimize the potential for adaptive cassette reshuffling. We found that integron activity can decrease the overall survival rate but also improve the fitness of the surviving populations. Off-target inversions mediated by the integron accelerated plasmid adaptation by disrupting costly conjugative genes otherwise mutated in control populations lacking a functional integrase. Plasmids containing integron-mediated inversions were associated with lower plasmid costs and higher stability than plasmids carrying mutations albeit at the cost of a reduced conjugative ability. These findings highlight the potential for integrons to create structural variation that can drive bacterial evolution, and they provide an interesting example showing how antibiotic pressure can drive the loss of conjugative genes. IMPORTANCE Tackling the public health challenge created by antibiotic resistance requires understanding the mechanisms driving its evolution. Mobile integrons are widespread genetic platforms heavily involved in the spread of antibiotic resistance. Through the action of the integrase enzyme, integrons allow bacteria to capture, excise, and shuffle antibiotic resistance gene cassettes. This integrase enzyme is characterized by its ability to recognize a wide range of recombination sites, which allows it to easily capture diverse resistance cassettes but which may also lead to off-target reactions with the rest of the genome. Using experimental evolution, we tested the off-target impact of integron activity. We found that integrons increased the fitness of the surviving bacteria through extensive genomic rearrangements of the plasmids carrying the integrons, reducing their ability to spread horizontally. These results show that integrons not only accelerate resistance evolution but also can generate extensive structural variation, driving bacterial evolution beyond antibiotic resistance.

An analysis of 45 large-scale wastewater sites in England to estimate SARS-CoV-2 community prevalence.

Accurate surveillance of the COVID-19 pandemic can be weakened by under-reporting of cases, particularly due to asymptomatic or pre-symptomatic infections, resulting in bias. Quantification of SARS-CoV-2 RNA in wastewater can be used to infer infection prevalence, but uncertainty in sensitivity and considerable variability has meant that accurate measurement remains elusive. Here, we use data from 45 sewage sites in England, covering 31% of the population, and estimate SARS-CoV-2 prevalence to within 1.1% of estimates from representative prevalence surveys (with 95% confidence). Using machine learning and phenomenological models, we show that differences between sampled sites, particularly the wastewater flow rate, influence prevalence estimation and require careful interpretation. We find that SARS-CoV-2 signals in wastewater appear 4-5 days earlier in comparison to clinical testing data but are coincident with prevalence surveys suggesting that wastewater surveillance can be a leading indicator for symptomatic viral infections. Surveillance for viruses in wastewater complements and strengthens clinical surveillance, with significant implications for public health.

Understanding and managing uncertainty and variability for wastewater monitoring beyond the pandemic: Lessons learned from the United Kingdom national COVID-19 surveillance programmes.

The COVID-19 pandemic has put unprecedented pressure on public health resources around the world. From adversity, opportunities have arisen to measure the state and dynamics of human disease at a scale not seen before. In the United Kingdom, the evidence that wastewater could be used to monitor the SARS-CoV-2 virus prompted the development of National wastewater surveillance programmes. The scale and pace of this work has proven to be unique in monitoring of virus dynamics at a national level, demonstrating the importance of wastewater-based epidemiology (WBE) for public health protection. Beyond COVID-19, it can provide additional value for monitoring and informing on a range of biological and chemical markers of human health. A discussion of measurement uncertainty associated with surveillance of wastewater, focusing on lessons-learned from the UK programmes monitoring COVID-19 is presented, showing that sources of uncertainty impacting measurement quality and interpretation of data for public health decision-making, are varied and complex. While some factors remain poorly understood, we present approaches taken by the UK programmes to manage and mitigate the more tractable sources of uncertainty. This work provides a platform to integrate uncertainty management into WBE activities as part of global One Health initiatives beyond the pandemic.

Integron activity accelerates the evolution of antibiotic resistance.

Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to 'evolve on demand' in response to antibiotic pressure. To test this hypothesis, we inserted a custom three-cassette integron into Pseudomonas aeruginosa and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.

From urinary tract infections to bacterial pneumonia, many diseases can now be treated through a course of antibiotics. Yet bacteria have evolved to respond to this threat, gaining new antibiotic resistance genes that allow them to evade the drugs. Addressing this growing issue requires to either discover new antibiotics, or to stop resistance before it emerges ­ a strategy that can only work if scientists know exactly how this mechanism takes place. For bacteria, it is a waste of resources to produce the proteins that confer resistance if antibiotics are absent. In fact, doing so can decrease their chance to survive and reproduce. A genetic element known as an integron can help to manage that burden. This piece of genetic information is formed of a succession of 'cassettes' containing antibiotic resistance genes. More proteins are made from the genes present at the start of the integron, compared to the ones towards the end. When bacteria encounter antibiotics, an enzyme called integrase is activated, allowing the organisms to shuffle the order of their cassettes in the integron. It is thought ­ but not yet proven ­ that this mechanism helps bacteria to activate their resistance 'on demand'. To find out, Souque et al. engineered the bacteria Pseudomonas aeruginosa to carry a custom integron with three cassettes, each helping the organism to resist to a different antibiotic. In addition, only half of the bacteria had a working integrase and could therefore shuffle their gene cassettes. The organisms were then exposed to an increasing amount of the antibiotics for which the cassette in the last position provided resistance. The bacteria with a working integrase survived longer than those without, as they were able to shuffle their cassettes and move the useful antibiotic resistance gene into top position. In addition, the cassettes carrying the genes to resist to other types of antibiotics were excised from the genetic information and lost. Understanding integrons could guide future antibiotic treatment strategies, for instance by combining antibiotics with chemicals that block integrase activity. It might also be possible to force bacteria to delete resistance cassettes by cycling through different antibiotics.

Macropinosomes are Key Players in Early Shigella Invasion and Vacuolar Escape in Epithelial Cells.

Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) to study the highly transient events of S. flexneri's early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial pathogens.