The resistome and microbiome of wastewater treatment plant workers - The AWARE study.

Urban wastewater treatment plants harbor a large collection of antibiotic resistant enteric bacteria. It is therefore reasonable to hypothesize that workers at such plants would possess a more diverse set of resistant enteric bacteria, compared to the general population. To address this hypothesis, we have compared the fecal microbiome and resistome of 87 workers at wastewater treatment plants (WWTPs) from Romania and the Netherlands to those of 87 control individuals, using shotgun metagenomics. Controlling for potential confounders, neither the total antibiotic resistance gene (ARG) abundance, nor the overall bacterial composition were significantly different between the two groups. If anything, the ARG richness was slightly lower in WWTP workers, and in a stratified analysis the total ARG abundance was significantly lower in Dutch workers compared to Dutch control participants. We identified country of residence, together with recent antibiotic intake in the Dutch population, as the largest contributing factors to the total abundance of ARGs. A striking side-finding was that sex was associated with carriage of disinfectant resistance genes, with women in both Romania and the Netherlands having significantly higher abundance compared to men. A follow up investigation including an additional 313 publicly available samples from healthy individuals from three additional countries showed that the difference was significant for three genes conferring resistance to chemicals commonly used in cosmetics and cleaning products. We therefore hypothesize that the use of cosmetics and, possibly, cleaning products leads to higher abundance of disinfectant resistance genes in the microbiome of the users. Altogether, this study shows that working at a WWTP does not lead to a higher abundance or diversity of ARGs and no large shifts in the overall gut microbial composition in comparison to participants not working at a WWTP. Instead, other factors such as country of residence, recent antibiotic intake and sex seem to play a larger role.

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.

Causal and Associational Language in Observational Health Research: A Systematic Evaluation.

We estimated the degree to which language used in the high-profile medical/public health/epidemiology literature implied causality using language linking exposures to outcomes and action recommendations; examined disconnects between language and recommendations; identified the most common linking phrases; and estimated how strongly linking phrases imply causality. We searched for and screened 1,170 articles from 18 high-profile journals (65 per journal) published from 2010-2019. Based on written framing and systematic guidance, 3 reviewers rated the degree of causality implied in abstracts and full text for exposure/outcome linking language and action recommendations. Reviewers rated the causal implication of exposure/outcome linking language as none (no causal implication) in 13.8%, weak in 34.2%, moderate in 33.2%, and strong in 18.7% of abstracts. The implied causality of action recommendations was higher than the implied causality of linking sentences for 44.5% or commensurate for 40.3% of articles. The most common linking word in abstracts was "associate" (45.7%). Reviewers' ratings of linking word roots were highly heterogeneous; over half of reviewers rated "association" as having at least some causal implication. This research undercuts the assumption that avoiding "causal" words leads to clarity of interpretation in medical research.

Planning a Collection of Virtual Patients to Train Clinical Reasoning: A Blueprint Representative of the European Population.

BACKGROUND: Virtual patients (VPs) are a suitable method for students to train their clinical reasoning abilities. We describe a process of developing a blueprint for a diverse and realistic VP collection (prior to VP creation) that facilitates deliberate practice of clinical reasoning and meets educational requirements of medical schools. METHODS: An international and interdisciplinary partnership of five European countries developed a blueprint for a collection of 200 VPs in four steps: (1) Defining the criteria (e.g., key symptoms, age, sex) and categorizing them into disease-, patient-, encounter- and learner-related, (2) Identifying data sources for assessing the representativeness of the collection, (3) Populating the blueprint, and (4) Refining and reaching consensus. RESULTS: The blueprint is publicly available and covers 29 key symptoms and 176 final diagnoses including the most prevalent medical conditions in Europe. Moreover, our analyses showed that the blueprint appears to be representative of the European population. CONCLUSIONS: The development of the blueprint required a stepwise approach, which can be replicated for the creation of other VP or case collections. We consider the blueprint an appropriate starting point for the actual creation of the VPs, but constant updating and refining is needed.

International Travel as a Risk Factor for Carriage of Extended-Spectrum ß-Lactamase-Producing Escherichia coli in a Large Sample of European Individuals-The AWARE Study.

Antibiotic resistance (AR) is currently a major threat to global health, calling for a One Health approach to be properly understood, monitored, tackled, and managed. Potential risk factors for AR are often studied in specific high-risk populations, but are still poorly understood in the general population. Our aim was to explore, describe, and characterize potential risk factors for carriage of Extended-Spectrum Beta-Lactamase-resistant Escherichia coli (ESBL-EC) in a large sample of European individuals aged between 16 and 67 years recruited from the general population in Southern Germany, the Netherlands, and Romania. Questionnaire and stool sample collection for this cross-sectional study took place from September 2018 to March 2020. Selected cultures of participants' stool samples were analyzed for detection of ESBL-EC. A total of 1183 participants were included in the analyses: 333 from Germany, 689 from the Netherlands, and 161 from Romania. Travels to Northern Africa (adjusted Odds Ratio, aOR 4.03, 95% Confidence Interval, CI 1.67-9.68), Sub-Saharan Africa (aOR 4.60, 95% CI 1.60-13.26), and Asia (aOR 4.08, 95% CI 1.97-8.43) were identified as independent risk factors for carriage of ESBL-EC. Therefore, travel to these regions should continue to be routinely asked about by clinical practitioners as possible risk factors when considering antibiotic therapy.

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.

Antibiotic Resistance in Wastewater Treatment Plants and Transmission Risks for Employees and Residents: The Concept of the AWARE Study.

Antibiotic resistance has become a serious global health threat. Wastewater treatment plants may become unintentional collection points for bacteria resistant to antimicrobials. Little is known about the transmission of antibiotic resistance from wastewater treatment plants to humans, most importantly to wastewater treatment plant workers and residents living in the vicinity. We aim to deliver precise information about the methods used in the AWARE (Antibiotic Resistance in Wastewater: Transmission Risks for Employees and Residents around Wastewater Treatment Plants) study. Within the AWARE study, we gathered data on the prevalence of two antibiotic resistance phenotypes, ESBL-producing E. coli and carbapenemase-producing Enterobacteriaceae, as well as on their corresponding antibiotic resistance genes isolated from air, water, and sewage samples taken from inside and outside of different wastewater treatment plants in Germany, the Netherlands, and Romania. Additionally, we analysed stool samples of wastewater treatment plant workers, nearby residents, and members of a comparison group living ≥1000 m away from the closest WWTP. To our knowledge, this is the first study investigating the potential spread of ESBL-producing E. coli, carbapenemase-producing Enterobacteriaceae, and antibiotic resistance genes from WWTPs to workers, the environment, and nearby residents. Quantifying the contribution of different wastewater treatment processes to the removal efficiency of ESBL-producing E. coli, carbapenemase-producing Enterobacteriaceae, and antibiotic resistance genes will provide us with evidence-based support for possible mitigation strategies.

Do wastewater treatment plants increase antibiotic resistant bacteria or genes in the environment? Protocol for a systematic review.

BACKGROUND: Antibiotic resistance is a global public health threat. Water from human activities is collected at wastewater treatment plants where processes often do not sufficiently neutralize antibiotic resistant bacteria and genes, which are further shed into the local environment. This protocol outlines the steps to conduct a systematic review based on the Population, Exposure, Comparator and Outcome (PECO) framework, aiming at answering the question "Are antimicrobial-resistant enterobacteriaceae and antimicrobial resistance genes present (O) in air and water samples (P) taken either near or downstream or downwind or down-gradient from wastewater treatment plants (E), as compared to air and water samples taken either further away or upstream or upwind or up-gradient from such wastewater treatment plant (C)?" Presence of antimicrobial-resistant bacteria and genes will be quantitatively measured by extracting their prevalence or concentration, depending on the reviewed study. METHODS: We will search PubMed, EMBASE, the Cochrane database and Web of Science for original articles published from 1 Jan 2000 to 3 Sep 2018 with language restriction. Articles will undergo a relevance and a design screening process. Data from eligible articles will be extracted by two independent reviewers. Further, we will perform a risk of bias assessment using a decision matrix. We will synthesize and present results in narrative and tabular form and will perform a meta-analysis if heterogeneity of results allows it. DISCUSSION: Antibiotic resistance in environmental samples around wastewater treatment plants may pose a risk of exposure to workers and nearby residents. Results from the systematic review outlined in this protocol will allow to estimate the extend of exposure, to inform policy making and help to design future studies.

An educational intervention to improve knowledge about prevention against occupational asthma and allergies using targeted maximum likelihood estimation.

PURPOSE: Occupational asthma and allergies are potentially preventable diseases affecting 5-15% of the working population. However, the use of preventive measures is often insufficient. The aim of this study was to estimate the average treatment effect of an educational intervention designed to improve the knowledge of preventive measures against asthma and allergies in farm apprentices from Bavaria (Southern Germany). METHODS: Farm apprentices at Bavarian farm schools were asked to complete a questionnaire evaluating their knowledge about preventive measures against occupational asthma and allergies (use of personal protective equipment, personal and workplace hygiene measures). Eligible apprentices were randomized by school site to either a control or an intervention group. The intervention consisted of a short educational video about use of preventive measures. Six months after the intervention, subjects were asked to complete a post-intervention questionnaire. Of the 116 apprentices (70 intervention group, 46 control group) who answered the baseline questionnaire, only 47 subjects (41%; 17 intervention group, 30 control group) also completed the follow-up questionnaire. We, therefore, estimated the causal effect of the intervention using targeted maximum likelihood estimation. Models were controlled for potential confounders. RESULTS: Based on the targeted maximum likelihood estimation, the intervention would have increased the proportion of correct answers on all six preventive measures by 18.4% (95% confidence interval 7.3-29.6%) had all participants received the intervention vs. had they all been in the control group. CONCLUSIONS: These findings indicate the improvement of knowledge by the educational intervention.

The role of adiponectin in endothelial dysfunction and hypertension.

It has been two decades since the discovery of adiponectin, and today its role in insulin resistance, inflammation, and atherosclerosis are areas of major interest. Production of adiponectin is reduced in all inflammatory processes and states of insulin resistance such as obesity, type 2 diabetes mellitus, and coronary artery disease. Adiponectin regulates carbohydrate metabolism, and may also regulate vascular homeostasis by affecting important signaling pathways in endothelial cells and modulating inflammatory responses in the subendothelial space. Clinical studies have demonstrated a relationship between serum adiponectin concentrations and the activity of the renin-angiotensin-aldosterone system (RAAS), causing changes in blood pressure. Antihypertensive therapy with angiotensin II receptor blockers (ARBs) has been demonstrated to increase adiponectin levels in 3-6 months. Adiponectin has also been shown to play a role in cardiac injury in modulation of pro-survival reactions, cardiac energy metabolism, and inhibition of hypertrophic remodeling. The effects of adiponectin on the cardiovascular system are believed to be partially mediated by the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and cyclooxygenase-2 (COX-2) pathways, reducing endothelial cell apoptosis, promoting nitric oxide production, decreasing tumor necrosis factor-alpha (TNF-α) activity, and preventing atherosclerotic proliferation and smooth muscle cell migration. Further evaluation of biologically active forms of adiponectin and its receptor should help to clarify how obesity affects the cardiovascular system.

Single-nucleotide polymorphisms in adiponectin, AdipoR1, and AdipoR2 genes: insulin resistance and type 2 diabetes mellitus candidate genes.

It has already been a decade and a half since the discovery of adiponectin and its role as an insulin sensitizer and only 7 years since its receptors, AdipoR1 and AdipoR2, were described. A single-nucleotide polymorphism (SNP) is a DNA sequence variation that affects only one nucleotide; it may vary from one population to another with different predisposing factors to diseases and other ailments. Once some of the effects of adiponectin and its receptors were known, it was not long until an effort was made to find the associations between specific SNPs of the genes of this hormone and its receptors as genetic risk factors for insulin resistance, type 2 diabetes mellitus, and metabolic syndrome, although these genes were investigated as possible candidates related to the development of these metabolic disorders. All of these possible associations were studied in different populations from France, Finland, the United Kingdom, North America, and Japan, showing hardly concluding results, and because of that it is highly controversial to directly associate one of the genes mentioned above to insulin resistance, type 2 diabetes mellitus, and metabolic syndrome. All of these inconsistencies lead to a review that summarizes the SNPs of the genes of adiponectin, AdipoR1, and AdipoR2 that are mostly related to insulin resistance syndrome, type 2 diabetes mellitus, and metabolic syndrome, although presenting the possible factors that should be taken into account to homogenize the results obtained until now.

Incretin-based therapy for type 2 diabetes mellitus: pancreatic and extrapancreatic effects.

The prevalence of type 2 diabetes mellitus has increased at an alarming rate in recent years. Recent estimations project that 366 million people could have diabetes by 2030. The incretin system emerges as a new target for type 2 diabetes therapy, and new molecules are being approved for its use in humans since the year 2005. These agents could be divided into 2 main groups, glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 inhibitors. Endogenous GLP-1 is an incretin hormone composed by a 30-amino acid peptide and is secreted from L-cells in distal small intestine in response to calorie intake, causing a glucose-dependent ß-cell response resulting in a restoration of the first-phase insulin response. Additionally, GLP-1 regulates glucagon production, which leads to inhibition of glucogenolysis and gluconeogenesis in the liver. Synthetic molecules such as exenatide and liraglutide have been developed to bind GLP-1 receptor and mimic GLP-1 effects in pancreatic cells and other target organs.