Longitudinal dynamics of farmer and livestock nasal and faecal microbiomes and resistomes.

Globally, half a billion people are employed in animal agriculture and are directly exposed to the associated microorganisms. However, the extent to which such exposures affect resident human microbiomes is unclear. Here we conducted a longitudinal profiling of the nasal and faecal microbiomes of 66 dairy farmers and 166 dairy cows over a year-long period. We compare farmer microbiomes to those of 60 age-, sex- and ZIP code-matched people with no occupational exposures to farm animals (non-farmers). We show that farming is associated with microbiomes containing livestock-associated microbes; this is most apparent in the nasal bacterial community, with farmers harbouring a richer and more diverse nasal community than non-farmers. Similarly, in the gut microbial communities, we identify more shared microbial lineages between cows and farmers from the same farms. Additionally, we find that shared microbes are associated with antibiotic resistance genes. Overall, our study demonstrates the interconnectedness of human and animal microbiomes.

Antibiotic perturbations to the gut microbiome.

Antibiotic-mediated perturbation of the gut microbiome is associated with numerous infectious and autoimmune diseases of the gastrointestinal tract. Yet, as the gut microbiome is a complex ecological network of microorganisms, the effects of antibiotics can be highly variable. With the advent of multi-omic approaches for systems-level profiling of microbial communities, we are beginning to identify microbiome-intrinsic and microbiome-extrinsic factors that affect microbiome dynamics during antibiotic exposure and subsequent recovery. In this Review, we discuss factors that influence restructuring of the gut microbiome on antibiotic exposure. We present an overview of the currently complex picture of treatment-induced changes to the microbial community and highlight essential considerations for future investigations of antibiotic-specific outcomes. Finally, we provide a synopsis of available strategies to minimize antibiotic-induced damage or to restore the pretreatment architectures of the gut microbial community.

Functional Metagenomics to Study Antibiotic Resistance.

The construction and screening of metagenomic expression libraries have a great potential to identify novel genes with desired functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.

Epidemiology of Plasmid Lineages Mediating the Spread of Extended-Spectrum Beta-Lactamases among Clinical Escherichia coli.

The prevalence of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Escherichia coli has been increasing, with this spread driven by ESBL-encoding plasmids. However, the epidemiology of ESBL-disseminating plasmids remains understudied, obscuring the roles of individual plasmid lineages in ESBL spread. To address this, we performed an in-depth genomic investigation of 149 clinical ESBL-like E. coli isolates from a tertiary care hospital. We obtained high-quality assemblies for 446 plasmids, revealing an extensive map of plasmid sharing that crosses time, space, and bacterial sequence type boundaries. Through a sequence-based network, we identified specific plasmid lineages that are responsible for the dissemination of major ESBLs. Notably, we demonstrate that IncF plasmids separate into 2 distinct lineages that are enriched for different ESBLs and occupy distinct host ranges. Our work provides a detailed picture of plasmid-mediated spread of ESBLs, demonstrating the extensive sequence diversity within identified lineages, while highlighting the genetic elements that underlie the persistence of these plasmids within the clinical E. coli population. IMPORTANCE The increasing incidence of nosocomial infections with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a significant threat to public health, given the limited treatment options available for such infections. The rapid ESBL spread is suggested to be driven by localization of the resistance genes on conjugative plasmids. Here, we identify the contributions of different plasmid lineages in the nosocomial spread of ESBLs. We provide further support for plasmid-mediated spread of ESBLs but demonstrate that some ESBL genes rely on dissemination through plasmids more than the others. We identify key plasmid lineages that are enriched in major ESBL genes and highlight the encoded genetic elements that facilitate the transmission and stable maintenance of these plasmid groups within the clinical E. coli population. Overall, our work provides valuable insight into the dissemination of ESBLs through plasmids, furthering our understating of factors underlying the increased prevalence of these genes in nosocomial settings.

A Tunable and Expandable Transactivation System in Probiotic Yeast Saccharomyces boulardii.

Precise transcriptional modulation is a key requirement for developing synthetic probiotics with predictably tunable functionalities. In this study, an expandable and tunable transactivation system was constructed and validated in probiotic yeast Saccharomyces boulardii. The use of nuclease-null Cas9 and scaffold RNA (scRNA) directed regulation enabled transactivation under the control of a synthetic promoter in S. boulardii. A synthetic promoter consisting of the scRNA target sequence and the core GAL7 promoter region restricted interference from the native galactose regulon. The system was readily expanded by introducing new target sequences to the promoter and scRNA. Complementarity between the promoter and scRNA, and binding specificity between scRNA and transcriptional activator, served as two layers of orthogonality of the transactivation. In addition, activator expression under the control of an inducible promoter enabled control of the transactivation via chemical inducer. The described system has the potential to enable engineering of probiotic yeast to more precisely perform therapeutic functions.

Structure of the 5' Untranslated Region of Enteroviral Genomic RNA.

Enteroviral RNA genomes share a long, highly structured 5' untranslated region (5' UTR) containing a type I internal ribosome entry site (IRES). The 5' UTR is composed of stably folded RNA domains connected by unstructured RNA regions. Proper folding and functioning of the 5' UTR underlies the efficiency of viral replication and also determines viral virulence. We have characterized the structure of 5' UTR genomic RNA from coxsackievirus B3 using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and base-specific chemical probes in solution. Our results revealed novel structural features, including realignment of major domains, newly identified long-range interactions, and an intrinsically disordered connecting region. Together, these newly identified features contribute to a model for enteroviral 5' UTRs with type I IRES elements that links structure to function during the hierarchical processes directed by genomic RNA during viral infection.IMPORTANCE Enterovirus infections are responsible for human diseases, including myocarditis, pancreatitis, acute flaccid paralysis, and poliomyelitis. The virulence of these viruses depends on efficient recognition of the RNA genome by a large family of host proteins and protein synthesis factors, which in turn relies on the three-dimensional folding of the first 750 nucleotides of the molecule. Structural information about this region of the genome, called the 5' untranslated region (5' UTR), is needed to assist in the process of vaccine and antiviral development. This work presents a model for the structure of the enteroviral 5' UTR. The model includes an RNA element called an intrinsically disordered RNA region (IDRR). Intrinsically disordered proteins (IDPs) are well known, but correlates in RNA have not been proposed. The proposed IDRR is a 20-nucleotide region, long known for its functional importance, where structural flexibility helps explain recognition by factors controlling multiple functional states.

Structural Identification and Kinetic Analysis of the in Vitro Products Formed by Reaction of Bisphenol A-3,4-quinone with N-Acetylcysteine and Glutathione.

Bisphenol A (BPA) has received considerable attention as an endocrine disrupting chemical and a possible substrate for genotoxic metabolites. BPA metabolism leads to formation of electrophilic o-quinones cable of binding to DNA and other endogenous nucleophiles. We have structurally identified the products resulting from the reaction of bisphenol A-3,4-quinone (BPAQ) with N-acetylcysteine (NAC) and glutathione (GSH). The major and minor isomers are both the result of 1,6-conjugate addition and are produced almost instantly in high yield. Reactions using 1.3 equiv of GSH showed the presence of a bis-glutathionyl adduct which was not observed using higher GSH concentration relative to BPAQ. NAC reactions with BPAQ showed no bis-N-acetylcysteinyl adducts. Stopped-flow kinetic analysis reveals the 1,6-conjugate additions to be reversible with a forward free energy of activation of 9.2 and 7.8 kcal/mol for the NAC and GSH reactions, respectively. The bimolecular forward rate constant at 19.4 °C was approximately three time faster for GSH compared to NAC, 1547 vs 496 M-1 s-1. The free energy of activation for the reverse reactions were similar, 11.7 and 11.2 kcal/mol for NAC and GSH, respectively. We plan to use this model system to further explore the mechanism of adduct formation between sulfur nucleophiles and o-quinones and the resulting chemical properties of both NAC and GSH adducts.