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Biological safety is a critical requirement when working with or around infectious disease agents. To prevent exposures and keep staff and patients safe, laboratories and health care facilities rely on personal protective equipment, standard operating procedures (SOPs), and engineering controls. In an instant, however, a single inappropriate human behavior can negate any of these safeguards. This reference provides an important call to action for anyone who relies on safety plans to consider carefully the humans who must follow those plans. Written by an expert in behavioral biosafety training, Prepare and Protect offers a common-sense program for addressing and reducing the risk factors of human behavior. Learn how to Examine the safety culture of your organization and its approach to risk Motivate the compliance, adherence to rules, and community thinking that keep everyone safe Evaluate, validate, and verify SOPs and staff competence Create safety plans and safety training programs that connect outcomes to behaviors Provide leadership that translates the containment philosophy from words to actions The critical message of this book is illustrated and enriched by personal accounts from infectious disease pioneers, from lab safety directors and trainers to the researchers and health care workers directly affected by infectious hazards. If your work involves pathogenic microbes-whether treating patients in a hospital emergency department or conducting research in a biosafety level 2 or higher laboratory-or overseeing those who do these jobs, this resource will teach you how to develop a culture of biosafety through behavior.

Key Points CRISPR–Cas systems provide archaea and bacteria with adaptive immunity against viruses and plasmids. CRISPR–Cas genomic loci show extreme diversity in sequence and gene arrangement. We developed a computational approach for CRISPR–Cas classification, combining comparisons of Cas protein sequences and locus architectures. Two classes, five types and 16 subtypes of CRISPR–Cas systems were identified based on this approach. An automated classifier was developed for assigning CRISPR–Cas loci from sequenced genomes to specific subtypes. The evolution of CRISPR–Cas systems is marked by extensive horizontal transfer and recombination of functional modules. CRISPR–Cas systems provide bacteria and archaea with adaptive immunity to invading foreign DNA. In an Analysis article, Koonin and colleagues update a previous classification of these systems to incorporate the large volume of genomic data generated in recent years. The evolution of CRISPR– cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR– cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

Filamentous fungi are an emergent cause of severe infections in immunocompromised patients. Timely and accurate identification is crucial to initiate appropriate therapy. Traditional identification methods are time-consuming, labor-intensive, and operator-dependent. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry is a rapid and easy-to-perform identification method. The effectiveness of a commercial MALDI-TOF MS platform to identify filamentous fungi in a clinical laboratory was evaluated. The study included 67 fungal isolates from 35 species/species complexes, which were identified and confirmed in mycology reference laboratories; 32 derived from clinical samples, 34 from strain collections and one was an ATCC strain. The study used the VITEK.sup.® MS system (v3.2.0 database), after sample extraction by VITEK.sup.® MS Mould Kit. Results were classified as \"correct species\", \"correct species complex\", \"correct genus\"and \"no identification\". VITEK.sup.® MS correctly identified 91.0% of isolates (58.2% to species, 29.9% to species complex, and 1.5% to genus level only). In 82%, the result matched the species/species complex identified by reference methods. No misidentifications were observed. The kit was rapid and easy to use. In conclusion, the VITEK.sup.® MS system showed a high capability to accurately identify filamentous fungi in a clinical laboratory.

Faecal microbiota transplantation (FMT) is an important therapeutic option for Clostridium difficile infection. Promising findings suggest that FMT may play a role also in the management of other disorders associated with the alteration of gut microbiota. Although the health community is assessing FMT with renewed interest and patients are becoming more aware, there are technical and logistical issues in establishing such a non-standardised treatment into the clinical practice with safety and proper governance. In view of this, an evidence-based recommendation is needed to drive the practical implementation of FMT. In this European Consensus Conference, 28 experts from 10 countries collaborated, in separate working groups and through an evidence-based process, to provide statements on the following key issues: FMT indications; donor selection; preparation of faecal material; clinical management and faecal delivery and basic requirements for implementing an FMT centre. Statements developed by each working group were evaluated and voted by all members, first through an electronic Delphi process, and then in a plenary consensus conference. The recommendations were released according to best available evidence, in order to act as guidance for physicians who plan to implement FMT, aiming at supporting the broad availability of the procedure, discussing other issues relevant to FMT and promoting future clinical research in the area of gut microbiota manipulation. This consensus report strongly recommends the implementation of FMT centres for the treatment of C. difficile infection as well as traces the guidelines of technicality, regulatory, administrative and laboratory requirements.

Phages differ substantially in the bacterial hosts that they infect. Their host range is determined by the specific structures that they use to target bacterial cells. Tailed phages use a broad range of receptor-binding proteins, such as tail fibres, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors. Recent technical advances and new structure–function insights have begun to unravel the molecular mechanisms and temporal dynamics that govern these interactions. Here, we review the current understanding of the targeting machinery and mechanisms of tailed phages. These new insights and approaches pave the way for the application of phages in medicine and biotechnology and enable deeper understanding of their ecology and evolution.

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species. Lactobacillus is a lactic acid bacteria and has a wide range of application from use in probiotic food production to biotherapeutics. Here, the authors sequence and compare the genomes of 213 different Lactobacillus strains and related genera, and provide new insight into phylogenomic organization and adaptive immunity elements in this bacteria family.

Testing 21 different fecal DNA extraction protocols in multiple laboratories results in a standardized protocol with the potential to improve comparability across human gut microbiome studies. Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

Multiplexed genome editing is simplified by harnessing the ability of Cpf1 to process its own pre-crRNA. Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

Metabolic syndrome is characterized by a constellation of comorbidities that predispose individuals to an increased risk of developing cardiovascular pathologies as well as type 2 diabetes mellitus1. The gut microbiota is a new key contributor involved in the onset of obesity-related disorders2. In humans, studies have provided evidence for a negative correlation between Akkermansia muciniphila abundance and overweight, obesity, untreated type 2 diabetes mellitus or hypertension3–8. Since the administration of A. muciniphila has never been investigated in humans, we conducted a randomized, double-blind, placebo-controlled pilot study in overweight/obese insulin-resistant volunteers; 40 were enrolled and 32 completed the trial. The primary end points were safety, tolerability and metabolic parameters (that is, insulin resistance, circulating lipids, visceral adiposity and body mass). Secondary outcomes were gut barrier function (that is, plasma lipopolysaccharides) and gut microbiota composition. In this single-center study, we demonstrated that daily oral supplementation of 1010A. muciniphila bacteria either live or pasteurized for three months was safe and well tolerated. Compared to placebo, pasteurized A. muciniphila improved insulin sensitivity (+28.62 ± 7.02%, P = 0.002), and reduced insulinemia (−34.08 ± 7.12%, P = 0.006) and plasma total cholesterol (−8.68 ± 2.38%, P = 0.02). Pasteurized A. muciniphila supplementation slightly decreased body weight (−2.27 ± 0.92 kg, P = 0.091) compared to the placebo group, and fat mass (−1.37 ± 0.82 kg, P = 0.092) and hip circumference (−2.63 ± 1.14 cm, P = 0.091) compared to baseline. After three months of supplementation, A. muciniphila reduced the levels of the relevant blood markers for liver dysfunction and inflammation while the overall gut microbiome structure was unaffected. In conclusion, this proof-of-concept study (clinical trial no. NCT02637115) shows that the intervention was safe and well tolerated and that supplementation with A. muciniphila improves several metabolic parameters.

Fecal microbiota transplantation (FMT) has shown efficacy in treating recurrent Clostridium difficile infection and is increasingly being applied to other gastrointestinal disorders, yet the fate of native and introduced microbial strains remains largely unknown. To quantify the extent of donor microbiota colonization, we monitored strain populations in fecal samples from a recent FMT study on metabolic syndrome patients using single-nucleotide variants in metagenomes. We found extensive coexistence of donor and recipient strains, persisting 3 months after treatment. Colonization success was greater for conspecific strains than for new species, the latter falling within fluctuation levels observed in healthy individuals over a similar time frame. Furthermore, same-donor recipients displayed varying degrees of microbiota transfer, indicating individual patterns of microbiome resistance and donor-recipient compatibilities.