Suppression PCR-Based Selective Enrichment Sequencing for Pathogen and Antimicrobial Resistance Detection on Cell-Free DNA in Sepsis-A Targeted, Blood Culture-Independent Approach for Rapid Pathogen and Resistance Diagnostics in Septic Patients.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. The rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcomes in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we targeted eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested in an experimental research study on the first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be additionally detected. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time-to-results to 11-13 h and suggesting diagnostic potential in sepsis.

Molecular detection of blaCTX-M gene to predict phenotypic cephalosporin resistance and clinical outcome of Escherichia coli bloodstream infections in Vietnam.

BACKGROUND: Blood stream infections (BSI) caused by Extended Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae is a clinical challenge leading to high mortality, especially in developing countries. In this study, we sought to describe the epidemiology of ESBL-producing Escherichia coli strains isolated from Vietnamese individuals with BSI, to investigate the concordance of genotypic-phenotypic resistance, and clinical outcome of ESBL E. coli BSI. METHODS: A total of 459 hospitalized patients with BSI were screened between October 2014 and May 2016. 115 E. coli strains from 115 BSI patients were isolated and tested for antibiotic resistance using the VITEK®2 system. The ESBL phenotype was determined by double disk diffusion method following the guideline of Clinical and Laboratory Standards Institute. Screening for beta-lactamase (ESBL and carbapenemase) genes was performed using a multiplex-PCR assay. RESULTS: 58% (67/115) of the E. coli strains were ESBL-producers and all were susceptible to both imipenem and meropenem. Resistance to third-generation cephalosporin was common, 70% (81/115) were cefotaxime-resistant and 45% (52/115) were ceftazidime-resistant. blaCTX-M was the most common ESBL gene detected (70%; 80/115) The sensitivity and specificity of blaCTX-M-detection to predict the ESBL phenotype was 87% (76-93% 95% CI) and 54% (39-48% 95% CI), respectively. 28%% (22/80) of blaCTX-M were classified as non-ESBL producers by phenotypic testing for ESBL production. The detection of blaCTX-M in ESBL-negative E. coli BSI was associated with fatal clinical outcome (27%; 6/22 versus 8%; 2/26, p = 0.07). CONCLUSION: A high prevalence of ESBL-producing E. coli isolates harbouring blaCTX-M was observed in BSI patients in Vietnam. The genotypic detection of blaCTX-M may have added benefit in optimizing and guiding empirical antibiotic therapy of E. coli BSI to improve clinical outcome.

One Health in Somalia: Present status, opportunities, and challenges.

One Health (OH) is an integrated approach aiming at improving the health of people, animals, and ecosystems. It recognizes the interconnectedness of human health with the health of animals, plants, and the environment. Since Somali people's livelihoods are mainly based on livestock, agriculture, marine resources, and their shared environment, OH-oriented initiatives could significantly impact the country toward reducing complex problems affecting the health of humans, animals, and the environment. The term "One Health" was first introduced into the global scientific community in September 2004 and in 2013 in Somalia. After ten years, there is still a long road ahead for implementing the OH approach in the country. Herein, we present the status, opportunities, and challenges of OH in Somalia and recommend ways to promote and institutionalize it. The country has been involved in various OH initiatives solely driven by external funding, focusing on research, capacity development, and community interventions, apart from university-led initiatives such as Somali One Health Centre. In Somalia, OH initiatives face numerous challenges, ranging from limited infrastructure and resources to weak governance and institutional capacity. We urge the Somali government to address these challenges and prioritize OH as the main approach to tackling critical health issues. We suggest the Somali government institutionalize and implement OH actions at all administrative levels, including Federal, State, District, and community, through a mechanism to improve multisectoral coordination and collaboration to predict, prevent, detect, control, and respond to communicable and non-communicable diseases at the human-animal-ecosystem interface for improving health outcomes for all.

An elephant in the room? Explaining agenda-setting in antimicrobial resistance policies in 30 European countries.

Antimicrobial resistances (AMR) present a particularly challenging cross-sectoral policy problem, affecting human and animal health as well as the environment. Compared to the actual problem pressure, the public awareness for AMR is comparatively low and the issue has not been high on the political agenda in most. Given the rising problem pressure, we aim to find out as to what degree and under which conditions political parties bring AMR on the political agenda. By means of multilevel logit regressions based on 173 electoral manifestos in 30 European countries from 2015 to 2020, we explore the conditions that explain whether AMR are taken up in manifestos. The empirical findings indicate firstly that AMR are only addressed by political parties in Northern and Western Europe, in no case in Eastern, and only in one case in Southern Europe, though resistant bacteria are more widely spread in the latter. Secondly, Green parties are those who are most likely to address the AMR challenge. Thirdly, vote share is positively associated with AMR agenda-setting, while EU membership is insignificant and the national average on antibiotics consumption is negatively related to AMR agenda-setting. Finally, AMR are surprisingly mainly perceived as a problem of the agricultural policy subsystem despite its cross-sectoral policy character. The study makes theoretical and empirical contributions: regarding theory, the article shows that typical variables that are used for agenda-setting are less explanatory for complex intersectoral policies. This is also accompanied by the empirical contribution: since problem awareness and complexity of policy problems are correlated, AMR are reduced to an agricultural issue and as such, it is taken over by political parties that have expertise on agricultural-environmental topics.

Does diagnostic testing always decrease antibiotics prescriptions?

BACKGROUND: Empiric prescription to treat infectious diseases in community care settings has caused antibiotics to be overprescribed, increasing antimicrobial resistance (AMR). To reduce antibiotics prescription, the use of point-of-care diagnostic testing (POCT) has been suggested. METHODS: We present a stylized static theoretical economic model to analyse whether the use of POCT always decreases antibiotics prescriptions. We consider the interaction of a group of doctors who differ in their level of concern about AMR when prescribing with a firm selling a POCT, and we characterize the price set by the manufacturer and doctors' decision to employ POCT. RESULTS: We found that the number of antibiotics prescriptions is not always lower. This result depends on the distribution of the doctors' concern about AMR as there is a proportion of doctors who use POCT and then prescribe antibiotics while other doctors change their prescribing behaviour after using POCT and stop giving antibiotics to patients who do not benefit from them. When the proportion of patients who need antibiotic treatment is higher than the proportion of doctors who use POCT and stop prescribing unnecessary antibiotics, the number of antibiotics prescriptions is larger. Our analysis also shows that the use of POCT improves health outcomes. CONCLUSIONS: We should be very careful when we assert that POCT reduces antibiotics prescriptions as there are situations in which the opposite effect occurs.

The value of biofilm testing to guide antimicrobial stewardship in chronic respiratory diseases.

Introduction: Biofilm production is an important yet currently overlooked aspect of diagnostic microbiology that has implications for antimicrobial stewardship. In this study, we aimed to validate and identify additional applications of the BioFilm Ring Test® (BRT) for Pseudomonas aeruginosa (PA) isolates from patients with bronchiectasis (BE). Materials and methods: Sputa were collected from BE patients who had at least one PA positive culture in the previous year. We processed the sputa to isolate both mucoid and non-mucoid PA, and determined their susceptibility pattern, mucA gene status, and presence of ciprofloxacin mutations in QRDR genes. The Biofilm production index (BPI) was obtained at 5 and 24 hours. Biofilms were imaged using Gram staining. Results: We collected 69 PA isolates, including 33 mucoid and 36 non-mucoid. A BPI value below 14.75 at 5 hours predicted the mucoid PA phenotype with 64% sensitivity and 72% specificity. Conclusion: Overall, our findings suggest that the fitness-cost associated with the mucoid phenotype or ciprofloxacin resistance is shown through a time-dependent BPI profile. The BRT has the potential to reveal biofilm features with clinical implications.

The Bacterial Urban Resistome: Recent Advances.

Cities that are densely populated are reservoirs of antibiotic resistant genes (ARGs). The overall presence of all resistance genes in a specific environment is defined as a resistome. Spatial proximity of surfaces and different hygienic conditions leads to the transfer of antibiotic resistant bacteria (ARB) within urban environments. Built environments, public transportation, green spaces, and citizens' behaviors all support persistence and transfer of antimicrobial resistances (AMR). Various unique aspects of urban settings that promote spread and resilience of ARGs/ARB are discussed: (i) the role of hospitals and recreational parks as reservoirs; (ii) private and public transportation as carriers of ARGs/ARB; (iii) the role of built environments as a hub for horizontal gene transfer even though they support lower microbial biodiversity than outdoor environments; (iv) the need to employ ecological and evolutionary concepts, such as modeling the fate of a specific ARG/ARB, to gain enhanced health risk assessments. Our understanding and our ability to control the rise of AMR in an urban setting is linked to our knowledge of the network connecting urban reservoirs and the environment.

Analysis of Extended Spectrum Beta Lactamase (ESBL) Genes of Non-Invasive ESBL Enterobacterales in Southeast Austria in 2017.

Extended spectrum beta lactamases producing Enterobacteriaceae are a major player in the antibiotic resistance challenge. In general, the situation regarding antibiotic resistance in Austria is very good compared to many other countries. Perhaps this is why there is a lack of data on the distribution of ESBL genes in the clinical setting. The aim of this study was to collect data on ESBL genes from a larger sample of human non-invasive clinical isolates from one region in Austria. In total, 468 isolates from different sample materials isolated at the Medical University of Graz from 2017 were examined. The most frequent organisms were Escherichia coli and Klebsiella pneumoniae. Among the enzymes produced, CTX-M-15 was clearly dominant, exotic ESBLs were only represented by three Proteus mirabilis isolates harboring genes for VEB-6 and one P. mirabilis for CTX-M-2, respectively. Compared to other countries, the results are in line with the expectations. The data help to better classify the many studies from the non-clinical field in Austria and to shift the focus slightly away from the exotic results and sample sites.

Educational Activities for Students and Citizens Supporting the One-Health Approach on Antimicrobial Resistance.

Antibiotic resistance is one of the biggest threats to global health, food security and development. Urgent action is needed at all levels of society to reduce the impact and spread of antibiotic resistance. For a more sustaining approach, education in children, college students, citizens and caregivers are essential. The One-Heath approach is a collaborative, multisectoral and transdisciplinary strategy in which, no single organizations or sector can address the issue of antimicrobial resistance at the human-environment interface alone. Within this strategy, education plays a central role. In this scoping review, we highlighted a range of learning activities on antibiotic resistance as part of the One-Health approach. In particular, those applications that can be introduced to a wide audience to help arrest the current crisis for the next generation. The review identifies a high number of teaching opportunities: board and role-play games, round tables, musicals, e-learning and environmental experiments to couple with more curricula and formal education to inform a diverse group of audiences.

Prevalence, Virulence Gene Distribution and Alarming the Multidrug Resistance of Aeromonas hydrophila Associated with Disease Outbreaks in Freshwater Aquaculture.

The study aims to evaluate the infection prevalence, virulence gene distribution and antimicrobial resistance of Aeromonas hydrophila associated in diseased outbreaks of cultured freshwater fish in Northern Vietnam. The confirmed A. hydrophila were screened for the presence of the five pitutative-virulence genes including aerolysin (aerA), hemolysin (hlyA), cytotonic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), and heat-stable enterotoxin (ast), and examined the susceptibility to 16 antibiotics. A total of 236 A. hydrophila isolates were recovered and confirmed from 506 diseased fish by phenotypic tests, PCR assays, and gyrB, rpoB sequenced analyses, corresponding to the infection prevalence at 46.4%. A total of 88.9% of A. hydrophila isolates harbored at least one of the tested virulence genes. The genes aerA and act were most frequently found (80.5% and 80.1%, respectively) while the ast gene was absent in all isolates. The resistance to oxacillin, amoxicillin and vancomycin exhibited the highest frequencies (>70%), followed by erythromycin, oxytetracycline, florfenicol, and sulfamethoxazole/trimethoprim (9.3-47.2%). The multiple antibiotic resistance (MAR) index ranged between 0.13-0.88 with 74.7% of the isolates having MAR values higher than 0.2. The results present a warning for aquaculture farmers and managers in preventing the spread of A. hydrophila and minimizing antibiotic resistance of this pathogen in fish farming systems.

Microbiome dynamics during the HI-SEAS IV mission, and implications for future crewed missions beyond Earth.

BACKGROUND: Human health is closely interconnected with its microbiome. Resilient microbiomes in, on, and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein, we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a 1 year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. RESULTS: Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance, and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crew members were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crew members within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. CONCLUSIONS: This study highlights main routes of microbial transfer, interaction of the crew, and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors. Video abstract.

One Health Approach to Address Zoonotic Diseases.

The world of animals, humans, and environment is interlinked, giving rise to a number of benefits as well as a spread in zoonosis and multifactorial chronic diseases. With the emergence of antimicrobial resistances and environmental pollution, addressing these diseases needs an interdisciplinary and intersectoral expertise. "One Health (OH)" refers to such collaboration between local, national, and global experts from public health, health care, forestry, veterinary, environmental, and other related disciplines to bring about optimal health for humans, animals, and environment. The concept of OH is still in embryonic stage in India and increasingly gaining importance. The Government of India has taken some initiatives to tackle burgeoning problems such as antimicrobial resistance, zoonotic diseases, and food safety using the OH approach, but there are several challenges at the level of implementation. The major bottlenecks in implementing OH include absence of a legal framework to implement OH, poor coordination among different governmental and private agencies, lack of proper surveillance of animal diseases, poor data-sharing mechanism across sectors, and limited budget. Implementing systematic zoonotic surveillance; regulated antibiotic use among humans and animals; development of a zoonotic registry in the country; constitution of a wide network of academic, research, pharmaceutical, and various implementation stakeholders from different sectors is the need of the hour to effectively use OH in order to combat increasing zoonotic diseases.

Anticipating Xenogenic Pollution at the Source: Impact of Sterilizations on DNA Release From Microbial Cultures.

The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121°C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.

Association of farm-related factors with characteristics profiles of extended-spectrum ß-lactamase- / plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolates from German livestock farms.

Resistance to ß-lactam antibiotics, including third-generation cephalosporins, is of major concern for animal and human health. In this study, extended-spectrum ß-lactamase (ESBL) / plasmid-mediated AmpC (pAmpC) ß-lactamase -producing Escherichia coli isolates from German livestock farms were characterised and associations of these isolate characteristics with farm-related factors were investigated across different types of livestock. A total of 469 isolates originating from 150 farms (34 broiler farms, 38 fattening pig farms, 43 dairy cattle farms, 35 beef cattle farms) was included in the analyses. ESBL-gene family, phylogroup and phenotypic antimicrobial susceptibility for several antimicrobial agents were determined. This data was used to define different profiles characterising the isolates. Multivariate analyses using a distance-based non-parametric approach were performed to investigate associations between the profiles of the isolates and farm-related factors (e.g. management, husbandry, and environment of the farms). Co-occurrence of ESBL-gene families were not found in any of the isolates analysed. Sixty-eight percent of the isolates carried blaCTX-M variant genes. The frequency of phylogroups was as follows: A (55%), B1 (35%), D (17%) and B2 (3%). The most frequent phenotypic non-wildtype profile was non-wildtype status of solely cefepime (27%). Profiles of isolates from broilers differed substantially from those of other isolates. Associations between farm-related factors and characteristics profiles differed, depending on the isolate characteristics included in the analyses. Some factors describing the farm environment, like waterfowl in the surrounding of the farm, were associated with all tested profiles. The epidemiological method applied defines distances between isolates on basis of isolate characteristics data and is capable of analysing associations between isolate characteristics and epidemiological factors. As additional data, such as plasmid characteristics, gene type, or sequence information could be included in future studies, the method is suitable to identify points of action to reduce the occurrence of antimicrobial resistant bacteria.

Antimicrobial Resistance Genes in Pigeons from Public Parks in Costa Rica.

Antimicrobial resistance is known to be an emerging problem, but the extent of the issue remains incomplete. The aim of this study was to determine the presence or absence of nine resistance genes (blaTEM , catI, mecA, qnrS, sulI, sulII, tet(A), tet(Q), vanA) in the faeces of 141 pigeons from four urban parks in Alajuela, Guadalupe, Tres Ríos and San José in Costa Rica. The genes were identified by real-time PCR directly from enema samples. About 30% of the samples were positive for genes catI and sulI; between 13% and 17% were positive for qnrS, sulII, tet(A) and tet(Q); and 4% were positive for blaTEM . The mecA and vanA genes were not detected. The average of antimicrobial resistance genes detected per pigeon was 2. Eight different patterns of resistance were identified, without differences in the sampling areas, being the most common pattern 2 (sulII positive samples). During rainy season, the genes more frequently found were sulI and tet(A). In conclusion, the urban inhabiting pigeons tested are currently carrying antimicrobial resistance genes, potentially acting as reservoirs of resistant bacteria and vectors to humans. To the authors' knowledge, this is the first study carried out on direct detection of resistance genes in the digestive metagenomes of pigeons.