AbstractAntimicrobial Resistance Research Articles

Recent developments in antibiotic contamination of animal products, soil, and water worldwide

Abstract Antimicrobial resistance (AMR), facilitated by antibiotic consumption, remains one of the biggest threats to global health and food security. The burgeoning AMR has an estimated forecast of 10 million deaths and 100 trillion USD economic losses annually worldwide by 2050 if no urgent actions are taken. The indiscriminate use of antibiotics in food animal production plays an expressive role in the AMR crisis. This paper compiles information regarding antibiotics and AMR in animals, animal-derived products, and agriculture-impacted environment. A holistic approach is needed to mitigate the burden of AMR within the context of human-animal-environment. Currently there are few approaches to this problem such as nanotechnology, anaerobic digestion, biochar composting, and alternatives to antibiotic treatments (like herbal plant extracts, probiotics, vaccines, enzymes, and antimicrobial peptides) have been developed. However, there are gaps in knowledge about AMR and areas for improvement are obvious. There is no a clear path to put an end to the persistent trends of AMR. Despite the trends for stricter regulation on the use of antibiotics worldwide, they find their way into food animal production, water, and soil as a result of misuses in many countries. We need to acknowledge the antibiotic contamination and/or AMR as a silent pandemic, and we are challenged to adopt a global approach to reducing and improving their use.

Antimicrobial Resistance, One Health Interventions and the Least Restrictive Alternative Principle

Abstract Antimicrobial resistance (AMR) is increasingly recognised as a threat to human, animal and environmental health. In an effort to counter this threat, several intervention plans have been proposed and implemented by states and organisations such as the WHO. A One Health policy approach, which targets multiple domains (healthcare, animal husbandry and the environment), has been identified as useful for curbing AMR. Johnson and Matlock have recently argued that One Health policies in the AMR context require special ethical justification because of the so-called least restrictive alternative principle. This article analyses and rejects two assumptions that this argument relies on. The first assumption is that One Health policies are generally more restrictive than their alternatives because they target more domains and impact more people. The second assumption is that the least restrictive alternative principle has a special normative importance in that it establishes a systematic presumption in favour of the least restrictive policy options. Once these assumptions are rejected, the use of One Health policies on AMR can be justified more easily than Johnson and Matlock argue.

Developing an ethical evaluation framework for coercive antimicrobial stewardship policies

Abstract Antimicrobial resistance (AMR) has been declared one of the top ten global public health threats facing humanity. To address AMR, coercive antimicrobial stewardship policies are being enacted in some settings. These policies, like all in public health, require ethical justification. Here, I introduce a framework for ethically evaluating coercive antimicrobial stewardship policies on the basis of ethical justifications (and their limitations). I consider arguments from effectiveness; duty of easy rescue; tragedy of the commons; responsibility-tracking; the harm principle; paternalism; justice and development; a precautionary approach; and professional duties. I consider how these justifications might form the basis for developing a comprehensive ethical framework, and the need for this to be context-specific and aligned with the priorities, evidence and needs of the particular jurisdictions in which a policy is to be enacted. I demonstrate how the ethical justifications might be used by reference to an example policy of the EU ban on the use of certain human-critical antibiotics for livestock, before concluding with challenges for further development of the framework.

A novel method for evaluating the depletion of veterinary pharmaceuticals using radioisotopes

Abstract Antimicrobial resistance (AMR) as one of the world’s most pressing public health problems needs immediate attention, because it has the ability to affect the human healthcare, agriculture, and veterinary industries. Despite warnings about overuse and their implications, antimicrobials are overprescribed worldwide for humans and animals, which leads to the promotion of resistant microorganisms such as bacteria. Food is a medium for exposure to or transfer of residues of the drugs and can contribute to the burden of the pharmaceuticals associated with development of AMR. Studying on residues of veterinary drugs in foods is essential in the fight against AMR. Herein, we introduce a new method for visualizing the residues of a veterinary drug in animal matrices using radionuclides, called “Depletion Imaging”. Amoxicillin was chosen to be the first antimicrobial for this study. The drug was labeled with [62Zn/65Zn] ZnCl2. Radiolabelled amoxicillin and non-labeled amoxicillin were administrated to rainbow trout fish simultaneously. To enable visualization of the remaining residues of amoxicillin in fish, In-vivo positron emission tomography (PET) imaging was done at different intervals from 30 min to 21 days after administration. Evaluation of the amount of radiolabelled amoxicillin in fish was done using a high purity germanium (HPGe) nuclear detector, and enzyme linked immunosorbent assay (ELISA) technique was used for the non-labeled drug. In this study, a comprehensive method for in-house production of zinc radioisotopes was also included. The results showed that depletion imaging and biodistribution study based on gamma spectroscopy of radionuclides in tissues, is a precise method for accurate understanding of the drug’s distribution, metabolic and excretory profile.

Inter-plasmid transfer of antibiotic resistance genes accelerates antibiotic resistance in bacterial pathogens.

Antimicrobial resistance is a major threat for public health. Plasmids play a critical role in the spread of antimicrobial resistance via horizontal gene transfer between bacterial species. However, it remains unclear how plasmids originally recruit and assemble various antibiotic resistance genes (ARGs). Here, we track ARG recruitment and assembly in clinically relevant plasmids by combining a systematic analysis of 2420 complete plasmid genomes and experimental validation. Results showed that ARG transfer across plasmids is prevalent, and 87% ARGs were observed to potentially transfer among various plasmids among 8229 plasmid-borne ARGs. Interestingly, recruitment and assembly of ARGs occur mostly among compatible plasmids within the same bacterial cell, with over 88% of ARG transfers occurring between compatible plasmids. Integron and insertion sequences drive the ongoing ARG acquisition by plasmids, especially in which IS26 facilitates 63.1% of ARG transfer events among plasmids. In vitro experiment validated the important role of IS26 involved in transferring gentamicin resistance gene aacC1 between compatible plasmids. Network analysis showed four beta-lactam genes (blaTEM-1, blaNDM-4, blaKPC-2, and blaSHV-1) shuffling among 1029 plasmids and 45 clinical pathogens, suggesting that clinically alarming ARGs transferred accelerate the propagation of antibiotic resistance in clinical pathogens. ARGs in plasmids are also able to transmit across clinical and environmental boundaries, in terms of the high-sequence similarities of plasmid-borne ARGs between clinical and environmental plasmids. This study demonstrated that inter-plasmid ARG transfer is a universal mechanism for plasmid to recruit various ARGs, thus advancing our understanding of the emergence of multidrug-resistant plasmids.

One Health governance principles for AMR surveillance: a scoping review and conceptual framework

Abstract Antimicrobial resistance (AMR) is a pressing global health issue with serious implications for health, food security, and livelihoods. Collective action, from local to global, that draws on the One Health (OH) approach to facilitate collaboration between the human, animal, and environmental sectors is required to inform initiatives to mitigate AMR. For AMR surveillance, this involves applying an intersectoral, multistakeholder perspective to guide the co-creation of knowledge and policy around the collection, analysis, and application of surveillance data to detect, monitor, and prevent AMR health threats. Currently, there is little available evidence on how to operationalize a OH approach to support integrated AMR surveillance systems, or on how the governance of such systems facilitates intersectoral action on AMR. We conducted a scoping review of the literature to identify the governance domains most relevant to applying the OH approach to the design and evaluation of AMR surveillance systems. We found that governance is a crucial component of the development of surveillance systems equipped to tackle complex, structural issues such as AMR. The governance domains identified include participation, coordination and collaboration, management, sustainability, accountability and transparency, and equity. These domains are relevant throughout all stages of policy design, implementation, and evaluation of AMR surveillance systems. Equity is both a domain and an essential component of the other domains. All the domains are interdependent and co-constitutive, so that progress in one domain can accelerate progress in another. The conceptual framework presented in this article can inform the design and evaluation of OH AMR governance systems and other complex health challenges that have similar barriers and facilitators to OH governance. The qualitative evaluation questions developed for each domain facilitate assessment of the breadth (the range of actors involved in governance) and depth (how meaningful their engagement is) for each domain relevant to OH governance. Finally, the prioritization of formal, sustainable, and democratic governance of AMR can help to facilitate achievement of the sustainable development goals (SDGs) and promote conservation of the use of antimicrobials for future generations.

Factors and challenges contributing to antimicrobial resistance in East African pastoral settings and importance of One Health approach

Abstract Antimicrobial resistance (AMR) is acknowledged as a One Health dilemma because resistant microbial and genes are swiftly appearing and spreading worldwide across humans, animals and the environment. Livestock owners in East African pastoral regions face significant financial and socio-economic challenges owing to animal diseases that can be particularly severe in remote areas, where communities rely solely on their livestock for subsistence. Unfortunately, providing veterinary services in these regions is challenging because of the inadequate infrastructure and limited resources. Consequently, pastoralists may resort to self-medicating their animals, often using antimicrobials in ways that can contribute to the emergence of AMR. Prolonged use, overuse, or misuse of antimicrobial drugs in both human and animal populations in low-income East African pastoral settings is a significant factor that contributes to the emergence of AMR in these regions. Similarly, a significant factor contributing to the spread of AMR in pastoral regions is the disregard for withdrawal periods by pastoralists, owing to a lack of awareness about AMR. Inadequate sanitation protocols and limited access to uncontaminated water sources are additional factors contributing to the dissemination of AMR pathogens in both animals and humans. This leads to critical public health concerns that hinder effective antimicrobial treatment, reduce animal productivity, and have economic consequences for pastoralists. Addressing the challenges associated with AMR requires a comprehensive, multi-sectoral approach that can facilitate the coordinated surveillance and monitoring of AMR, promote the rational use of antimicrobials, advocate for alternative disease prevention and management strategies, and raise awareness among communities and healthcare professionals in pastoral areas about the emergence of AMR. The challenges and factors contributing to AMR in pastoral settings are complex; however, the One Health approach offers a promising strategy for reducing the risk of AMR. This review aims to present a comprehensive overview of the factors and challenges associated with AMR in pastoral settings, and proposes the adoption of a One Health approach as a solution to this pressing issue. One Health impact statement The review highlights that antimicrobial resistance (AMR) in East African pastoral settings is a significant issue that contributes to the emergence and spread of AMR worldwide. Prolonged use, overuse, or misuse of antimicrobials in human and animal populations is a significant factor in the emergence of AMR in these settings. The challenges and factors contributing to AMR in pastoral settings are intricate and call for a multi-faceted approach that encourages rational use of antimicrobials, promotes alternative prevention and management methods, and enhances awareness among Eastern African pastoral communities. The article highlights the significance of adopting the One Health approach in mitigating the hazards of AMR in these settings.

Efficacy of Sitafloxacin for Mycoplasma genitalium in an Era of Increasing Antimicrobial Resistance

Abstract Antimicrobial resistance in Mycoplasma genitalium is rising globally and antimicrobial options are limited. We evaluated the efficacy of sitafloxacin regimens for macrolide-resistant M genitalium at Melbourne Sexual Health Centre, Australia, between January 2017 and February 2022. Before June 2017, patients received doxycycline followed by sitafloxacin; subsequently, patients received doxycycline followed by combined doxycycline + sitafloxacin. Of 229 patients treated with a sitafloxacin regimen, 80.6% experienced microbial cure. Sitafloxacin cured 94.2% of infections that had not previously failed moxifloxacin and 69.5% of infections that had; prior failure of moxifloxacin was associated with an 8-fold odds of sitafloxacin failure. There was no difference in cure between sequential monotherapy and combination therapy when patients were stratified by past failure of moxifloxacin (P > .05); however, small numbers limited comparisons. Sitafloxacin was well tolerated and still achieved 70% cure in patients in whom moxifloxacin had failed. These data highlight the benefit of incorporating relevant fluoroquinolone resistance markers into assays to assist clinical decision making.

Antimicrobial resistance threatening animal-human health in Papua New Guinea

ABSTRACT Antimicrobial resistance (AMR) has the potential to reverse gains made in modern medicine and seriously jeopardize the lives of people and animals from infections that were once readily treatable (FAO/OIE 2015). Adopting a one health approach is critical to better understand and mitigate factors driving the emerging concern of AMR. Although the development and transmission of AMR in wildlife is not well understood, there is growing evidence that it is associated with proximity to humans or domestic animals. Papua New Guinea (PNG) faces some unique challenges for zoonotic spillover disease events and transmission of AMR between animals and humans. These challenges arise from the erosion of wildlife habitats due to deforestation and close contact of wildlife with villagers and their village-based livestock raised under free-range systems. Papua New Guinea’s human health system and the animal health field and laboratory services are resource-limited and are facing many human and animal disease challenges. The Fleming Fund Country Grant, implemented by the Burnet Institute Australia, is working with Papua New Guinea government and industry stakeholders to tackle AMR by encouraging information sharing, raising awareness, and supporting laboratory capacity in human and animal health for effective AMR surveillance. In addition, national legislation is being strengthened to support implementation of the Papua New Guinea Antimicrobial Resistance National Action Plan (Government of Papua New Guinea 2019) to encourage the judicious use and effective monitoring of antimicrobial medicines in humans and animals.

209 Prevalence of Antimicrobial Resistance Genes and Mobile Genetic Elements in the Swine Gut Microbiome

Abstract Antimicrobial resistance (AMR) is one of the most pressing threats to public health. It contributes to over a million deaths a year currently and by 2050 it is expected to cause more annual death than cancer. The swine industry has taken important steps to address the AMR crisis by phasing out the use of antimicrobial growth promoters. Nonetheless, the use of antimicrobials for controlling infectious diseases on swine farms remains high, particularly during the nursery stage of pig production. This has raised concerns about the potential role of the swine industry in the emergence and spread of antimicrobial resistance genes (ARGs) capable of conferring resistance to medically important antimicrobials. While traditionally ARGs have been studied in the context of pathogens, there is growing evidence that the natural microbiome of humans and animals can also serve as a significant reservoir of ARGs. Of particular importance is the association of ARGs with different classes of mobile genetic elements (MGEs) which can facilitate the horizontal transfer of genes among commensal bacteria and pathogens. In this study, we evaluated the diversity and distribution of ARGs and their associated MGEs in the swine gut microbiome. To achieve this, we isolated and sequenced the whole genomes of 130 unique bacterial isolates from stool, digesta, and mucosa of healthy adult sows and their piglets. Screening of genomes for the presence of ARGs was performed using the Resistance Gene Identifier (RGI) and Comprehensive Antibiotic Resistance Database (CARD), resulting in the identification of 323 ARGs across 117 genomes. Among these, genes predicted to confer resistance against tetracycline, lincosamide, beta-lactams, and fluoroquinolone were the most prevalent across all genomes. Importantly, we observed that several strictly anaerobic species belonging to gut-specific lineages including Lachnospiraceae, Megasphaeraceae, and Desulfovibrionaceae contained large ARGs pools spanning more than five different classes of antimicrobials. Further, we screened the flanking regions of ARGs for the presence of signature genes of MGEs including recombinases, phage integrases and capsids, and type IV secretion system. A total of 60 ARGs were identified to be associated with different classes of MGEs, including integrative and conjugative elements (ICEs; n = 30), bacteriophages (n = 8), integrative mobile elements (IMEs; n = 6), and plasmids (n = 3). Overall, our study highlights that ARGs, including those conferring resistance to medically important antimicrobials, are prevalent across different lineages of the swine gut microbiota. The association of the identified ARGs with MGEs warrants further in vitro experiments to evaluate the risk of transmission of resistance to zoonotic pathogens capable of infecting humans and animals.

Machine learning to predict ceftriaxone resistance using single nucleotide polymorphisms within a global database of Neisseria gonorrhoeae genomes.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is an urgent global health issue. Machine learning (ML) is a powerful tool that can aid in identifying mutations and predicting their impact on AMR. The study aimed to use ML models to predict ceftriaxone susceptibility and decreased susceptibility (S/DS). A public database of N. gonorrhoeae genomes with minimum inhibitory concentration (MIC) data was used to evaluate seven ML models using 97 single nucleotide polymorphisms (SNPs) known to be associated with ceftriaxone resistance. Ceftriaxone MICs ≤ 0.064 mg/L were classified as susceptible, and ceftriaxoneMICs > 0.064 mg/L were classified as DS. The contributions of individual SNPs to predict S/DS were calculated using SHapley Additive exPlanation (SHAP) values. An ML model was retrained using different combinations of SNPs with the highest SHAP values. The performance of ML models was assessed using different metrics including area under the curve (AUC) and balanced accuracy (bAcc). The ML analyses included 9,540 N. gonorrhoeae genomes; 368 (0.04%) were classified as DS. Of the models evaluated, the model trained with a random forest classifier had the highest performance (AUC 0.965; bAcc 0.926). A model retrained the top five SNPs, according to SHAP values, demonstrated a similar performance (AUC 0.916; bAcc 0.879) as the model with 97 SNPs. An ML approach using mutations in N. gonorrhoeae can be used to predict S/DS to ceftriaxone. The results highlight a practical application of ML to identify mutations most associated with S/DS to ceftriaxone, which can aid in the development of assays to predict AMR. IMPORTANCE Antimicrobial resistance in Neisseria gonorrhoeae is an urgent global health issue. The objectives of the study were to use a global collection of 12,936 N. gonorrhoeae genomes from the PathogenWatch database to evaluate different machine learning models to predict ceftriaxone susceptibility/decreased susceptibility using 97 mutations known to be associated with ceftriaxone resistance. We found the random forest classifier model had the highest performance. The analysis also reported the relative contributions of different mutations within the ML model predictions, allowing for the identification of the mutations with the highest importance for ceftriaxone resistance. A machine learning model retrained with the top five mutations performed similarly to the model using all 97 mutations. These results could aid in the development of molecular tests to detect resistance to ceftriaxone in N. gonorrhoeae. Moreover, this approach could be applied to building and evaluating machine learning models for predicting antimicrobial resistance in other pathogens.

One Health strategies for addressing antimicrobial resistance: an exclusive emphasis on Bulgaria

Abstract Antimicrobial resistance (AMR) is a public health challenge, primarily driven by the excessive use of antibiotics (ABs). While the European Union (EU) has successfully reduced ABs consumption since 2009, Bulgaria (BG) reports an increase in ABs use in both community and hospital settings. According to the 2021 Eurobarometer, the consumption rate of ABs among Bulgarians is twice as high as that in Germany, with a significant number taking ABs without a prescription. Moreover, only 41% of Bulgarians are aware that ABs do not kill viruses, and 30% took ABs for COVID-19 (as compared to 1% in Finland). This highlights an urgent need for interventions to tackle AMR in BG, using One Health (OH) approaches that consider the complex interplay between human, animal, and environmental health. The data presented aims to alert a wide range of stakeholders that AMR is not just a clinical or national issue but it has cross-border implications. It emphasizes the role that everyone must play in preserving the efficacy of ABs. Strategies for sustainable OH solutions: Researchers and educators should incorporate AMR and OH in their core responsibilities, providing relevant training and raising community awareness regarding prudent ABs use. Encouraging integrative medicine approaches, including traditional practices such as phytotherapy, can help delay the ABs prescriptions. Research is also necessary to support OH legislative changes that restrict ABs sales. Particularly, Bulgaria is among the few EU countries that have not approved an OH Action Plan against AMR or an inter-sectoral OH mechanism between veterinary, food, and health authorities. OH remains relatively unfamiliar in BG. Despite this, there is a political will to develop national OH policies, as demonstrated in the recently adopted National Health Strategy 2030. Urgent actions are needed to address AMR in BG. Using EU's AMR activities as a model, this analysis is a step towards a regional OH project. Key messages • AMR is a pressing public health problem in Bulgaria with cross-border implications. One Health strategies are needed urgently to address it. EU's AMR activities can be a model. • Bulgarian researchers, educators, and policymakers must make AMR their central responsibility. OH Action Plan against AMR and an inter-sectoral collaboration are the keys to success.

10.B. Workshop: Combatting AMR: European collaboration to ensure sustained access to effective antibiotics

Abstract Antimicrobial resistance (AMR) is one of the greatest public health threats facing the world today. The health and economic impacts of AMR are a major challenge for all healthcare systems and societies worldwide. It is estimated that antibiotic-resistant infections are responsible for a substantial number of deaths in Europe each year, with a health burden comparable to that of influenza, tuberculosis and HIV/AIDS combined and a cost of about €1.1 billion to the healthcare systems of EU/EEA countries. Holistic and multicomponent strategies in accordance with the ‘One Health’ perspective are key to curbing the development and spread of AMR. One crucial component of such strategies is ensuring broad and timely availability of effective antibiotics; this captures both investing in R&D for new antibiotics and safeguarding continued access to new and existing products. Scientific, economic, structural and regulatory barriers continue to limit the development of novel antibiotics with innovative characteristics despite intensified international efforts. Indeed, only a handful of new antibiotics have been developed and launched over the last few decades, and almost none have any innovative characteristics. This means that they are vulnerable to cross-resistance with existing antibiotics. At the same time, while ensuring timely access to new and existing antibiotics is critical, new antibiotics are often only launched in larger markets while supply-chain problems or withdrawals due to low profitability impact the availability of older antibiotics as well. This panel session aims to discuss the challenges facing health systems regarding how to foster research and development for new antibiotics and ensuring sustainable access to new and pre-existing antibiotics in the context of addressing AMR. In particular, it will aim to highlight avenues for collaboration and the potential role of the EU for addressing these challenges. Following a short presentation of the key findings of a policy brief published by the European Observatory on Health Systems and Policies at the request of the Swedish Presidency of the Council of the European Union, participants will hear reflections from the national and international levels and discuss potential implications and ways forward. Panelists will reflect on the relevant priorities of the upcoming Belgian Presidency of the Council of the European Union in the first half of 2024, as well as the WHO Regional Office for Europe's Regional Roadmap on AMR. Key messages • Fostering innovation and ensuring sustained access to effective antibiotics are paramount for addressing AMR. • Collaboration across countries, supported by the EU and the WHO will be key to achieving these goals.

Drivers of inappropriate antibiotic use in low- and middle-income countries.

Antimicrobial resistance (AMR) is a global security threat that accounts for about 700 000 deaths annually. Studies have shown that antimicrobial resistance could result in a 2% to 3.5% reduction in global Gross Domestic Product by 2050 and a loss of between 60 and 100 trillion US dollars, worth of economic output resulting in significant and widespread human suffering. Low- and middle-income countries (LMICs) will be worse hit by an unchecked rise of AMR. For example, it is predicted that AMR could kill about 4.1 million people in Africa by 2050 if it is not curbed. Similarly rising rates of AMR will lead to increased treatment costs and an inability to attain universal health coverage, in LMICs with fragile health systems. Sadly, AMR is driven by the inappropriate use of antimicrobials, especially antibiotics. Inappropriate antibiotic use is a pertinent problem in LMICs where regulatory frame works are weak. Inappropriate antibiotic use in LMICs is a multifaceted problem that cuts across clinical and veterinary medicine and agriculture. Therefore, efforts geared at curbing inappropriate antibiotic use in LMICs must identify the factors that drive this problem (i.e. inappropriate antibiotic use) in these countries. A clear knowledge of these factors will guide effective policy and decision making to curb inappropriate antibiotic use and ultimately AMR. The focus of this review is to discuss the factors that drive inappropriate antibiotic use in LMICs.

Challenges and opportunities in the implementation of an antimicrobial stewardship program in Nepal

Abstract Antimicrobial resistance (AMR) continues to be a serious global public issue. Unnecessary and inappropriate use of antimicrobials has been identified as a major contributing factor for AMR. Implementation of antimicrobial stewardship programs (ASPs) is valued as a key strategy to combat AMR. Although ASP is a key intervention to improve appropriate use of antibiotics, there is limited experience and research to describe its implementation in low-income countries such as Nepal. Grande International Hospital (GIH) is the first health organization in Nepal to implement and sustain a multidisciplinary ASP and infection control program. Challenges faced in implementing ASP include lack of acceptance from physicians, lack of knowledge regarding antibiotic prescribing, lack of staff for ASP activities, limitations in diagnostic testing to inform ASP, and limitations in antibiotic choice due to antibiotic unavailability. Our ASP includes the following components: an ASP committee, an antibiotic prescribing reference guide with dosage recommendations, inpatient formulary restriction system, educational outreach and programming for physicians and other stakeholders, and periodic review and revision of the program and reference guide. The ASP provided opportunities to address several knowledge gaps across our healthcare institution including improved knowledge and competency regarding rational use of antibiotics, access to quality medicines and better care to patients. It is our hope that, by describing the challenges and opportunities we experienced while implementing our ASP, we can support and encourage other institutions to adapt and implement ASPs in Nepal and other resource-limited settings.

A call to action-stopping antimicrobial resistance.

Antimicrobial resistance is a public health emergency and represents an impending pandemic. Implementing the lessons learned from responding to the COVID-19 pandemic is essential in accelerating the development of new antimicrobials and therapeutic strategies, rapid diagnostics and improved vaccines. A rededicated, coordinated and collaborative global effort of all stakeholders in academia, healthcare, government agencies, industry, finance and philanthropy is essential to halt the continued spread of antimicrobial resistance and to prevent further morbidity and mortality from drug-resistant pathogens.

MEGARes and AMR++, v3.0: an updated comprehensive database of antimicrobial resistance determinants and an improved software pipeline for classification using high-throughput sequencing.

Antimicrobial resistance (AMR) is considered a critical threat to public health, and genomic/metagenomic investigations featuring high-throughput analysis of sequence data are increasingly common and important. We previously introduced MEGARes, a comprehensive AMR database with an acyclic hierarchical annotation structure that facilitates high-throughput computational analysis, as well as AMR++, a customized bioinformatic pipeline specifically designed to use MEGARes in high-throughput analysis for characterizing AMR genes (ARGs) in metagenomic sequence data. Here, we present MEGARes v3.0, a comprehensive database of published ARG sequences for antimicrobial drugs, biocides, and metals, and AMR++ v3.0, an update to our customized bioinformatic pipeline for high-throughput analysis of metagenomic data (available at MEGLab.org). Database annotations have been expanded to include information regarding specific genomic locations for single-nucleotide polymorphisms (SNPs) and insertions and/or deletions (indels) when required by specific ARGs for resistance expression, and the updated AMR++ pipeline uses this information to check for presence of resistance-conferring genetic variants in metagenomic sequenced reads. This new information encompasses 337 ARGs, whose resistance-conferring variants could not previously be confirmed in such a manner. In MEGARes 3.0, the nodes of the acyclic hierarchical ontology include 4 antimicrobial compound types, 59 resistance classes, 233 mechanismsand 1448 gene groups that classify the 8733 accessions.

'To be, or not to be'-The dilemma of 'silent' antimicrobial resistance genes in bacteria.

Antimicrobial resistance is a serious threat to public health that dramatically undermines our ability to treat bacterial infections. Microorganisms exhibit resistance to different drug classes by acquiring resistance determinants through multiple mechanisms including horizontal gene transfer. The presence of drug resistance genotypes is mostly associated with corresponding phenotypic resistance against the particular antibiotic. However, bacterial communities harbouring silent antimicrobial resistance genes-genes whose presence is not associated with a corresponding resistant phenotype do exist. Under suitable conditions, the expression pattern of such genes often revert and regain resistance and could potentially lead to therapeutic failure. We often miss the presence of silent genes, since the current experimental paradigms are focused on resistant strains. Therefore, the knowledge on the prevalence, importance and mechanism of silent antibiotic resistance genes in bacterial pathogens are very limited. Silent genes, therefore, provide an additional level of complexity in the war against drug-resistant bacteria, reminding us that not only phenotypically resistant strains but also susceptible strains should be carefully investigated. In this review, we discuss the presence of silent antimicrobial resistance genes in bacteria, their relevance and their importance in public health.

Multi-drug and β-lactam resistance in Escherichia coli and food-borne pathogens

Abstract Antimicrobial resistance (AMR) is one of the top ten global public health threats facing humanity by menacing the effective use of available antimicrobials for treatment of an ever-increasing range of infections caused by bacteria. Its potential recognize the transmission to humans by contact with commensal and zoonotic resistant bacteria from animal and food sources. The aim of this study is to describe multi-drug resistance (MDR) profile in Campylobacter (n = 541), Salmonella (n = 919) and commensal Escherichia coli (n = 2777) isolated from animal and food samples between 2014 and 2019 in Portugal. Antimicrobial susceptibility testing results to fluoroquinolones/quinolones, macrolides, 3rd generation cephalosporins, polymyxins, carbapenems, penicillins, aminoglycosides, tetracyclines, sulphonamides, trimethoprim and chloramphenicol were clustered using k-modes. Clusters were described by population (broilers, broilers meat, turkeys, pigs, pig meat), AMR classification (mono/dual-resistance, MDR to 3-4, 5-6 and ≥7), β-lactamases production, sample stage (farm, slaughterhouse, processing plant, retail), season, and year. Overall, the results suggest an escalating MDR behavior from farm to post-farm stages in all bacteria, including E. coli MDR to critically important antimicrobials, and that Salmonella (fluoro)quinolones resistance may be associated with broilers. Most ESBL/AmpC producing E. coli were resistant to both C3G and C4G in isolates from animal and food samples (respectively, 77%; n = 846; and 74%; n = 188). The statistical method, K-modes, offers an overall visualization of the data with the identification of AMR profiles that have been further described using surveillance variables. Our results provide relevant information to support policy and decision making to tackle MDR in farm and post-farm stages. Key messages • An escalating multi drug resistance behavior from farm to post-farm stages. • Escherichia coli multi drug resistance to critically important antimicrobials.

Nonessential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress.

Antimicrobial resistance develops as a major problem in infectious diseases treatment. While antibiotic resistance mechanisms are usually studied using lethal antibiotic doses, lower doses allowing bacterial growth are now considered as factors influencing the development and selection of resistance. Starting with a high-density Tn insertion library in Vibrio cholerae and following its evolution by TN-seq in the presence of subinhibitory concentrations of antibiotics, we discovered that RNA modification genes can have opposite fates, being selected or counter-selected. We, thus have undertaken the phenotypic characterization of 23 transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications deletion mutants, for which growth is globally not affected in the absence of stress. We uncover a specific involvement of different RNA modification genes in the response to aminoglycosides (tobramycin and gentamicin), fluoroquinolones (ciprofloxacin), β-lactams (carbenicillin), chloramphenicol, and trimethoprim. Our results identify t/rRNA modification genes, not previously associated to any antibiotic resistance phenotype, as important factors affecting the bacterial response to low doses of antibiotics from different families. This suggests differential translation and codon decoding as critical factors involved in the bacterial response to stress.