Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.
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Today, we live in a world where every time we turn on our smartphones, we are inextricably tied by data, laws and flowing bytes to different countries. A world in which personal expressions are framed and mediated by digital platforms, and where new kinds of currencies, financial exchange and even labor bypass corporations and governments. Simultaneously, the same technologies increase governmental powers of surveillance, allow corporations to extract ever more complex working arrangements and do little to slow the construction of actual walls along actual borders. On the one hand, the agency of individuals and groups is starting to approach that of nation states; on the other, our mobility and hard-won rights are under threat. What tools do we need to understand this world, and how can art assist in envisioning and enacting other possible futures?This publication investigates the new relationships between states, citizens and the stateless made possible by emerging technologies. It is the result of a two-year EU-funded collaboration between Aksioma (SI), Drugo More (HR), Furtherfield (UK), Institute of Network Cultures (NL), NeMe (CY), and a diverse range of artists, curators, theorists and audiences. State Machines insists on the need for new forms of expression and new artistic practices to address the most urgent questions of our time, and seeks to educate and empower the digital subjects of today to become active, engaged, and effective digital citizens of tomorrow.Contributors: James Bridle, Max Dovey, Marc Garrett, Valeria Graziano, Max Haiven, Lynn Hershman Leeson, Francis Hunger, Helen Kaplinsky, Marcell Mars, Tomislav Medak, Rob Myers, Emily van der Nagel, Rachel O’Dwyer, Lídia Pereira, Rebecca L. Stein, Cassie Thornton, Paul Vanouse, Patricia de Vries, Krystian Woznicki.
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Matrix-assisted laser desorption/ionisation time of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.
New innovative methods to determine the DNA sequences of different bacterial species are rising. In the field of microbiology, these methods are very important since it is now possible to determine all the genetic characteristics of the bacterium in one step! This enables to define e.g. the species family, drug resistance or relatedness to other bacteria in outbreak evaluations which is necessary to efficiently treat the bacteria or target potential outbreaks. For many years, PCR-based methods have been the technique of choice to determine DNA sequences (including next-generation sequencing techniques). Recently, a new technique has been introduced to the market that is based on single molecule real-time sequencing (SMRT) with the possibility to determine the DNA sequence of a bacterium. This SMRT MinION sequencing technique is housed on an USB stick and is known for its user-friendliness and huge data output. However, before such a new technique can be implemented and presented in laboratories and used for educational purposes, methods should be harmonized and evaluated to proof its applicability. Harmonisation of the methodology regarding new laboratory techniques is very important to be able to compare results generated by different laboratories. A single consistent protocol, applied in each lab, is essential to obtain the best results in interlaboratory comparisons. During this KIEM-hbo project, we – i.e. Avans UAS, Maastricht University Medical Center and the company IS-diagnostics – will determine the DNA sequence of bacterial species and mixes thereof with a harmonized protocol for an interlaboratory comparison. We will compare this technique to the IS-PRO, an existing technology. Finally a workshop will be organized for medical technicians and other SMRT sequencing users to evaluate the protocols. This will, generate an up-to-date and harmonized sequencing protocol which can be expanded to future research and diagnostics in the different areas.
