A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.

We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.

Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant.

Fecal microbiota transplantation (FMT) is an emerging therapy for recurrent or refractory Clostridioides difficile infection and is being actively investigated for other conditions. We describe two patients in whom extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bacteremia occurred after they had undergone FMT in two independent clinical trials; both cases were linked to the same stool donor by means of genomic sequencing. One of the patients died. Enhanced donor screening to limit the transmission of microorganisms that could lead to adverse infectious events and continued vigilance to define the benefits and risks of FMT across different patient populations are warranted.

Shiga Toxin-Producing Escherichia coli Transmission via Fecal Microbiota Transplant.

Fecal microbiota transplantation (FMT) is recommended therapy for multiply recurrent Clostridioides difficile infection. We report adverse events in 7 patients who received FMT from a stool donor who was colonized with Shiga toxin-producing Escherichia coli (STEC). No patients died of FMT-transmitted STEC. Improved screening can likely avoid future transmission.

Multiple Copies of qnrA1 on an IncA/C2 Plasmid Explain Enhanced Quinolone Resistance in an Escherichia coli Mutant.

In a previous study, mutants with enhanced ciprofloxacin resistance (Cipr) were selected from Escherichia coli J53/pMG252 carrying qnrA1 Strain J53 Cipr 8-2 showed an increase in the copy number and transcription level of qnrA1 We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cipr 8-2 were almost identical, except for the region containing qnrA1 that in pMG252A contained 4 additional copies of the qnrA1-qacEΔ1-sul1-ISCR1 region.

Information capacity of specific interactions.

Specific interactions are a hallmark feature of self-assembly and signal-processing systems in both synthetic and biological settings. Specificity between components may arise from a wide variety of physical and chemical mechanisms in diverse contexts, from DNA hybridization to shape-sensitive depletion interactions. Despite this diversity, all systems that rely on interaction specificity operate under the constraint that increasing the number of distinct components inevitably increases off-target binding. Here we introduce "capacity," the maximal information encodable using specific interactions, to compare specificity across diverse experimental systems and to compute how specificity changes with physical parameters. Using this framework, we find that "shape" coding of interactions has higher capacity than chemical ("color") coding because the strength of off-target binding is strongly sublinear in binding-site size for shapes while being linear for colors. We also find that different specificity mechanisms, such as shape and color, can be combined in a synergistic manner, giving a capacity greater than the sum of the parts.

Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture.

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the "Barr body." Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called "superdomains," such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called "superloops." DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4 We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes.

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

Plasmids and genes contributing to high-level quinolone resistance in Escherichia coli.

INTRODUCTION: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations. METHODS: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6')1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids. RESULTS: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6')1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26. CONCLUSION: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences.

Community-acquired in name only: A cluster of carbapenem-resistant Acinetobacter baumannii in a burn intensive care unit and beyond.

OBJECTIVE: To describe an investigation into 5 clinical cases of carbapenem-resistant Acinetobacter baumannii (CRAB). DESIGN: Epidemiological investigation supplemented by whole-genome sequencing (WGS) of clinical and environmental isolates. SETTING: A tertiary-care academic health center in Boston, Massachusetts. PATIENTS OR PARTICIPANTS: Individuals identified with CRAB clinical infections. METHODS: A detailed review of patient demographic and clinical data was conducted. Clinical isolates underwent phenotypic antimicrobial susceptibility testing and WGS. Infection control practices were evaluated, and CRAB isolates obtained through environmental sampling were assessed by WGS. Genomic relatedness was measured by single-nucleotide polymorphism (SNP) analysis. RESULTS: Four clinical cases spanning 4 months were linked to a single index case; isolates differed by 1-7 SNPs and belonged to a single cluster. The index patient and 3 case patients were admitted to the same room prior to their development of CRAB infection, and 2 case patients were admitted to the same room within 48 hours of admission. A fourth case patient was admitted to a different unit. Environmental sampling identified highly contaminated areas, and WGS of 5 environmental isolates revealed that they were highly related to the clinical cluster. CONCLUSIONS: We report a cluster of highly resistant Acinetobacter baumannii that occurred in a burn ICU over 5 months and then spread to a separate ICU. Two case patients developed infections classified as community acquired under standard epidemiological definitions, but WGS revealed clonality, highlighting the risk of burn patients for early-onset nosocomial infections. An extensive investigation identified the role of environmental reservoirs.

Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments.

Hi-C experiments explore the 3D structure of the genome, generating terabases of data to create high-resolution contact maps. Here, we introduce Juicer, an open-source tool for analyzing terabase-scale Hi-C datasets. Juicer allows users without a computational background to transform raw sequence data into normalized contact maps with one click. Juicer produces a hic file containing compressed contact matrices at many resolutions, facilitating visualization and analysis at multiple scales. Structural features, such as loops and domains, are automatically annotated. Juicer is available as open source software at http://aidenlab.org/juicer/.