The Association between Insertion Sequences and Antibiotic Resistance Genes

Insertion sequences (ISs) are abundant mobile genetic elements on bacterial genomes, responsible for mobilization of many genes, including antibiotic resistance genes (ARGs). As ARGs often occur in similar genetic contexts, understanding which ISs tend to be associated with known ARGs could be a first step toward discovering novel ARGs through predictive or experimental strategies. This could be valuable, as early identification of ARGs in pathogens could facilitate surveillance, confinement actions, molecular diagnostics, and drug development. Here, we present a comprehensive analysis of the association of specific ISs with known ARGs. A large collection of bacterial genomes was used to characterize the immediate context of 2,437 known ARGs and 3,768 ISs. While many ARGs were consistently found close to specific ISs, the contexts around all ISs were more variable. Nevertheless, a subset of individual ISs, as well as tentative composite transposons, showed significant associations with ARGs. These included, e.g., insertion sequences classified as IS6, Tn3, IS4, and IS1 that were not only strongly associated with diverse ARGs but also highly abundant in pathogens. Therefore, we conclude that the context of this subset of ISs and tentative composite transposons would be particularly valuable to discover novel ARGs through modeling or empirical approaches. A set of 1,891 metagenomes were analyzed to identify environments where those ISs commonly associated with ARGs were particularly abundant. The associations found in metagenomes were similar to those found in genomes.IMPORTANCE The emergence and spread of antibiotic resistance genes (ARGs) among pathogens threaten the prevention and treatment of bacterial infections as well as our food production chains. Early knowledge about mobile ARGs that are present in pathogens or that have the potential to become clinically relevant could help mitigate potential negative consequences. Recently, exploring integron gene cassettes was shown to be successful for identifying novel mobilized ARGs, some of which were already circulating in pathogens. Still, only a subset of ARGs is mobilized by integrons, and the contexts of other mobile genetic elements associated with ARGs remain unexplored. This includes insertion sequences (ISs) responsible for the mobilization of many ARGs. Our analyses identified ISs, species, and environments where ARG-IS relationships are particularly strong. This could be a first step to guide the discovery of novel ARGs, while also providing insights into mechanisms involved in the mobilization and transfer of ARGs

Approximate reasoning with fuzzy-syllogistic systems

The well known Aristotelian syllogistic system consists of 256 moods. We have found earlier that 136 moods are distinct in terms of equal truth ratios that range in τ=[0,1]. The truth ratio of a particular mood is calculated by relating the number of true and false syllogistic cases the mood matches. A mood with truth ratio is a fuzzy-syllogistic mood. The introduction of (n-1) fuzzy existential quantifiers extends the system to fuzzy-syllogistic systems nS, 1<n, of which every fuzzy-syllogistic mood can be interpreted as a vague inference with a generic truth ratio that is determined by its syllogistic structure. We experimentally introduce the logic of a fuzzy-syllogistic ontology reasoner that is based on the fuzzy-syllogistic systems nS. We further introduce a new concept, the relative truth ratio rτ=[0,1] that is calculated based on the cardinalities of the syllogistic cases

Predicting clinical resistance prevalence using sewage metagenomic data

Antibiotic resistance surveillance through regional and up-to-date testing of clinical isolates is a foundation for implementing effective empirical treatment. Surveillance data also provides an overview of geographical and temporal changes that are invaluable for guiding interventions. Still, due to limited infrastructure and resources, clinical surveillance data is lacking in many parts of the world. Given that sewage is largely made up of human fecal bacteria from many people, sewage epidemiology could provide a cost-efficient strategy to partly fill the current gap in clinical surveillance of antibiotic resistance. Here we explored the potential of sewage metagenomic data to assess clinical antibiotic resistance prevalence using environmental and clinical surveillance data from across the world. The sewage resistome correlated to clinical surveillance data of invasive Escherichia coli isolates, but none of several tested approaches provided a sufficient resolution for clear discrimination between resistance towards different classes of antibiotics. However, in combination with socioeconomic data, the overall clinical resistance situation could be predicted with good precision. We conclude that analyses of bacterial genes in sewage could contribute to informing management of antibiotic resistance. Karkman et al. explore how well available global sewage metagenomic data can predict clinical resistance prevalence using different models. A combination of sewage metagenomic data with socioeconomic factors predicts overall clinical resistance well, but still has limited ability to discriminate between resistance to different classes of antibiotics.Peer reviewe

Discovery of a novel integron-borne aminoglycoside resistance gene present in clinical pathogens by screening environmental bacterial communities

Background New antibiotic resistance determinants are generally discovered too late, long after they have irreversibly emerged in pathogens and spread widely. Early discovery of resistance genes, before or soon after their transfer to pathogens could allow more effective measures to monitor and reduce spread, and facilitate genetics-based diagnostics. Results We modified a functional metagenomics approach followed by in silico filtering of known resistance genes to discover novel, mobilised resistance genes in class 1 integrons in wastewater-impacted environments. We identified an integron-borne gene cassette encoding a protein that conveys high-level resistance against aminoglycosides with a garosamine moiety when expressed in E. coli. The gene is named gar (garosamine-specific aminoglycoside resistance) after its specificity. It contains none of the functional domains of known aminoglycoside modifying enzymes, but bears characteristics of a kinase. By searching public databases, we found that the gene occurs in three sequenced, multi-resistant clinical isolates (two Pseudomonas aeruginosa and one Luteimonas sp.) from Italy and China, respectively, as well as in two food-borne Salmonella enterica isolates from the USA. In all cases, gar has escaped discovery until now. Conclusion To the best of our knowledge, this is the first time a novel resistance gene, present in clinical isolates, has been discovered by exploring the environmental microbiome. The gar gene has spread horizontally to different species on at least three continents, further limiting treatment options for bacterial infections. Its specificity to garosamine-containing aminoglycosides may reduce the usefulness of the newest semisynthetic aminoglycoside plazomicin, which is designed to avoid common aminoglycoside resistance mechanisms. Since the gene appears to be not yet common in the clinics, the data presented here enables early surveillance and maybe even mitigation of its spread.publishedVersio

Minimal selective concentrations of tetracycline in complex aquatic bacterial biofilms

Selection pressure generated by antibiotics released into the environment could enrich for antibiotic resistance genes and antibiotic resistant bacteria, thereby increasing the risk for transmission to humans and animals. Tetracyclines comprise an antibiotic class of great importance to both human and animal health. Accordingly, residues of tetracycline are commonly detected in aquatic environments. To assess if tetracycline pollution in aquatic environments promotes development of resistance, we determined minimal selective concentrations (MSCs) in biofilms of complex aquatic bacterial communities using both phenotypic and genotypic assays. Tetracycline significantly increased the relative abundance of resistant bacteria at 10 μg/L, while specific tet genes (tetA and tetG) increased significantly at the lowest concentration tested (1 μg/L). Taxonomic composition of the biofilm communities was altered with increasing tetracycline concentrations. Metagenomic analysis revealed a concurrent increase of several tet genes and a range of other genes providing resistance to different classes of antibiotics (e.g. cmlA, floR, sul1, and mphA), indicating potential for co-selection. Consequently, MSCs for the tet genes of ≤ 1 μg/L suggests that current exposure levels in e.g. sewage treatment plants could be sufficient to promote resistance. The methodology used here to assess MSCs could be applied in risk assessment of other antibiotics as well

Five mucosal transcripts of interest in ulcerative colitis identified by quantitative real-time PCR: a prospective study

<p>Abstract</p> <p>Background</p> <p>The cause and pathophysiology of ulcerative colitis are both mainly unknown. We have previously used whole-genome microarray technique on biopsies obtained from patients with ulcerative colitis to identifiy 5 changed mucosal transcripts. The aim of this study was to compare mucosal expressions of these five transcripts in ulcerative colitis patients vs. controls, along with the transcript expression in relation to the clinical ulcerative colitis status.</p> <p>Methods</p> <p>Colonic mucosal specimens from rectum and caecum were taken at ambulatory colonoscopy from ulcerative colitis patients (<it>n </it>= 49) with defined inflammatory activity and disease extension, and from controls (<it>n </it>= 67) without inflammatory bowel disease. The five mucosal transcripts aldolase B, elafin, MST-1, simNIPhom and SLC6A14 were analyzed using quantitative real-time PCR.</p> <p>Results</p> <p>Significant transcript differences in the rectal mucosa for all five transcripts were demonstrated in ulcerative colitis patients compared to controls. The grade of transcript expression was related to the clinical disease activity.</p> <p>Conclusion</p> <p>The five gene transcripts were changed in patients with ulcerative colitis, and were related to the disease activity. The known biological function of some of the transcripts may contribute to the inflammatory features and indicate a possible role of microbes in ulcerative colitis. The findings may also contribute to our pathophysiological understanding of ulcerative colitis.</p

Carriage of ESBL-producing Enterobacterales in wastewater treatment plant workers and surrounding residents - the AWARE Study

To investigate whether wastewater treatment plant (WWTP) workers and residents living in close proximity to a WWTP have elevated carriage rates of ESBL-producing Enterobacterales, as compared to the general population. From 2018 to 2020, we carried out a cross-sectional study in Germany, the Netherlands, and Romania among WWTP workers (N = 344), nearby residents (living ≤ 300~m away from WWTPs; N = 431) and distant residents (living ≥ 1000~m away = reference group; N = 1165). We collected information on potential confounders via questionnaire. Culture of participants' stool samples was performed with ChromID®-ESBL agar plates and species identification with MALDI-TOF-MS. We used logistic regression to estimate the odds ratio (OR) for carrying ESBL-producing E. coli (ESBL-EC). Sensitivity analyses included stratification by country and interaction models using country as secondary exposure. Prevalence of ESBL-EC was 11% (workers), 29% (nearby residents), and 7% (distant residents), and higher in Romania (28%) than in Germany (7%) and the Netherlands (6%). Models stratified by country showed that within the Romanian population, WWTP workers are about twice as likely (aOR = 2.34, 95% CI: 1.22-4.50) and nearby residents about three times as likely (aOR = 3.17, 95% CI: 1.80-5.59) to be ESBL-EC carriers, when compared with distant residents. In stratified analyses by country, we found an increased risk for carriage of ESBL-EC in Romanian workers and nearby residents. This effect was higher for nearby residents than for workers, which suggests that, for nearby residents, factors other than the local WWTP could contribute to the increased carriage

Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance

There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.(1)Peer reviewe

Wastewater treatment plants, an “escape gate” for ESCAPE pathogens

Antibiotics are an essential tool of modern medicine, contributing to significantly decreasing mortality and morbidity rates from infectious diseases. However, persistent misuse of these drugs has accelerated the evolution of antibiotic resistance, negatively impacting clinical practice. The environment contributes to both the evolution and transmission of resistance. From all anthropically polluted aquatic environments, wastewater treatment plants (WWTPs) are probably the main reservoirs of resistant pathogens. They should be regarded as critical control points for preventing or reducing the release of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic-resistance genes (ARGs) into the natural environment. This review focuses on the fate of the pathogens Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae spp. (ESCAPE) in WWTPs. All ESCAPE pathogen species, including high-risk clones and resistance determinants to last-resort antibiotics such as carbapenems, colistin, and multi-drug resistance platforms, were detected in wastewater. The whole genome sequencing studies demonstrate the clonal relationships and dissemination of Gram-negative ESCAPE species into the wastewater via hospital effluents and the enrichment of virulence and resistance determinants of S. aureus and enterococci in WWTPs. Therefore, the efficiency of different wastewater treatment processes regarding the removal of clinically relevant ARB species and ARGs, as well as the influence of water quality factors on their performance, should be explored and monitored, along with the development of more effective treatments and appropriate indicators (ESCAPE bacteria and/or ARGs). This knowledge will allow the development of quality standards for point sources and effluents to consolidate the WWTP barrier role against the environmental and public health AR threats