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Antibiotic resistance in bacteria remains a major problem and environments that help to maintain such resistance, represent a significant problem to infection control in the community. Restrooms have always been regarded as potential sources of infectious diseases and we suggest they have the potential to sustain bacterial \"resistomes\". Recent studies have demonstrated the wide range of different bacterial phyla that can be found in non-healthcare restrooms. In our study we focused on the Staphylococci. These species are often skin contaminants on man and have been reported as common restroom isolates in recent molecular studies. We collected samples from 18 toilets sited in 4 different public buildings. Using MALDI-TOF-MS and other techniques, we identified a wide range of antibiotic resistant Staphylococci and other bacteria from our samples. We identified 19 different Staphylococcal species within our isolates and 37.8% of the isolates were drug resistant. We also identified different Staphylococcal species with the same antibiograms inhabiting the same restrooms. Bacterial \"resistomes\" are communities of bacteria often localised in specific areas and within these environments drug resistance determinants may be freely transferred. Our study shows that non-healthcare restrooms are a source of antibiotic resistant bacteria where a collection of antibiotic resistance genes in pathogenic and non-pathogenic bacteria could form a resistome containing a \"nexus of genetic diversity\"

Single-cell proteomics confidently quantifies cellular heterogeneity, however quantification of post-translational modifications, such as those deposited on histone proteins, remains elusive. Here, we develop a robust mass spectrometry-based method for the unbiased analysis of single-cell histone post-translational modifications (sc-hPTM). sc-hPTM identifies both single- and combinatorial histone post-translational modifications (67 peptidoforms in total), which includes nearly all frequently studied histone post-translational modifications with comparable reproducibility to traditional bulk experiments. As a proof of concept, we treat cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrate that our method can i) distinguish between treated and untreated cells, ii) identify sub-populations of cells with heterogeneous response to the treatment, and iii) reveal differential co-regulation of histone post-translational modifications in the context of drug treatment. The sc-hPTM method enables comprehensive investigation of chromatin heterogeneity at single-cell resolution and provides a further understanding of the histone code. Chromatin structure is regulated by chemical modifications of histone proteins, but measuring these at single-cell resolution has been challenging. Here, the authors develop a mass spectrometry-based method to profile histone modifications in individual cells, revealing chromatin heterogeneity and differential co-regulation.

Background Three-dimensional (3D) cell culture has emerged as an alternative approach to 2D flat culture to model more accurately the phenotype of solid tissue in laboratories. Culturing cells in 3D more precisely recapitulates physiological conditions of tissues, as these cells reduce activities related to proliferation, focusing their energy consumption toward metabolism and homeostasis. Results Here, we demonstrate that 3D liver spheroids are a suitable system to model chromatin dynamics and response to epigenetics inhibitors. To delay necrotic tissue formation despite proliferation arrest, we utilize rotating bioreactors that apply active media diffusion and low shearing forces. We demonstrate that the proteome and the metabolome of our model resemble typical liver functions. We prove that spheroids respond to sodium butyrate (NaBut) treatment, an inhibitor of histone deacetylases (HDACi), by upregulating histone acetylation and transcriptional activation. As expected, NaBut treatment impaired specific cellular functions, including the energy metabolism. More importantly, we demonstrate that spheroids reestablish their original proteome and transcriptome, including pre-treatment levels of histone acetylation, metabolism, and protein expression once the standard culture condition is restored after treatment. Given the slow replication rate (> 40 days) of cells in 3D spheroids, our model enables to monitor the recovery of approximately the same cells that underwent treatment, demonstrating that NaBut does not have long-lasting effects on histone acetylation and gene expression. These results suggest that our model system can be used to quantify molecular memory on chromatin. Conclusion Together, we established an innovative cell culture system that can be used to model anomalously decondensing chromatin in physiological cell growth and rule out epigenetics inheritance if cells recover the original phenotype after treatment. The transient epigenetics effects demonstrated here highlight the relevance of using a 3D culture model system that could be very useful in studies requiring long-term drug treatment conditions that would not be possible using a 2D cell monolayer system.

Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer–promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions. CRISPRi-based screen identifies enhancers that nonlinearly regulate lineage-specifying transcription factors during definitive endoderm differentiation. A new CTCF-loop-constrained Interaction Activity (CIA) model is better at predicting functional enhancers than previous Hi-C-based enhancer–promoter contact frequency models.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii . We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T . gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite’s genome, including more accurate mapping of known genes and the identification of new genes and cis -regulatory elements. Motif analysis reveals not only the known T . gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T . gondii’s perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T . gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

Background Coagulase negative staphylococci (CoNS) are important reservoirs of antibiotic resistance genes and associated mobile genetic elements and are believed to contribute to the emergence of successful methicillin resistant Staphylococcus aureus (MRSA) clones. Although, these bacteria have been linked to various ecological niches, little is known about the dissemination and genetic diversity of antibiotic resistant CoNS in general public settings. Methods Four hundred seventy-nine samples were collected from different non-healthcare/general public settings in various locations ( n  = 355) and from the hands of volunteers ( n  = 124) in London UK between April 2013 and Nov 2014. Results Six hundred forty-three staphylococcal isolates belonging to 19 staphylococcal species were identified. Five hundred seventy-two (94%) isolates were resistant to at least one antibiotic, and only 34 isolates were fully susceptible. Sixty-eight (11%) mecA positive staphylococcal isolates were determined in this study. SCC mec types were fully determined for forty-six isolates. Thirteen staphylococci (19%) carried SCC mec V, followed by 8 isolates carrying SCC mec type I (2%), 5 SCC mec type IV (7%), 4 SCC mec type II (6%), 1 SCC mec type III (2%), 1 SCC mec type VI (2%), and 1 SCC mec type VIII (2%). In addition, three isolates harboured a new SCC mec type 1A, which carried combination of class A mec complex and ccr type 1. MLST typing revealed that all S. epidermidis strains possess new MLST types and were assigned the following new sequence types: ST599, ST600, ST600, ST600, ST601, ST602, ST602, ST603, ST604, ST605, ST606, ST607 and ST608. Conclusions The prevalence of antibiotic resistant staphylococci in general public settings demonstrates that antibiotics in the natural environments contribute to the selection of antibiotic resistant microorganisms. The finding of various SCC mec types in non-healthcare associated environments indicates the complexity of SCC mec . We also report on new MLST types that were assigned for all S. epidermidis isolates, which demonstrates the genetic variability of these isolates.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps—(1) immobilization of a single cell on solid substrate, (2) hypotonic lysis of immobilized single cell, and (3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

Staphylococcus epidermidis is a major opportunistic pathogen primarily recovered from device-associated healthcare associated infections (DA-HAIs). Although S. epidermidis and other coagulase-negative staphylococci (CoNS) are less virulent than Staphylococcus aureus, these bacteria are an important reservoir of antimicrobial resistance genes and resistance-associated mobile genetic elements that can be transferred between staphylococcal species. We report a whole genome sequence of a multidrug resistant S. epidermidis (strain G6_2) representing multilocus sequence type (ST) 59 and isolated from an environmental sampling of a hotel room in London, UK. The genome of S. epidermidis G6_2 comprises of a 2408357 bp chromosome and six plasmids, with an average G+C content of 32%. The strain displayed a multi-drug resistance phenotype which was associated with carriage of 7 antibiotic resistance genes (blaZ, mecA, msrA, mphC, fosB, aacA-aphD, tetK) as well as resistance-conferring mutations in fusA and ileS. Antibiotic resistance genes were located on plasmids and chromosome. Comparative genomic analysis revealed that antibiotic resistance gene composition found in G6_2 was partly preserved across the ST59 lineage.

Cutler and Vijg remind us of the publication that laid the foundation for genetic mutation theories of ageing.