Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime–avibactam, meropenem–vaborbactam, and imipenem–relebactam; blaOXA-48 like–positive isolates were 100% susceptible to ceftazidime–avibactam; and metallo β-lactamase–positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime–avibactam, meropenem–vaborbactam, imipenem–relebactam, and ceftolozane–tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer β-lactamase inhibitors

Multifaceted Genome Control by Set1 Dependent and Independent of H3K4 Methylation and the Set1C/COMPASS Complex

<div><p>Histone modifiers are critical regulators of chromatin-based processes in eukaryotes. The histone methyltransferase Set1, a component of the Set1C/COMPASS complex, catalyzes the methylation at lysine 4 of histone H3 (H3K4me), a hallmark of euchromatin. Here, we show that the fission yeast <i>Schizosaccharomyces pombe</i> Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways. Set1 employs its RNA-binding RRM2 and catalytic SET domains to repress <i>Tf2</i> retrotransposons and pericentromeric repeats while relying on its H3K4me function to maintain transcriptional repression at the silent mating type (<i>mat</i>) locus and subtelomeric regions. These repressive functions of Set1 correlate with the requirement of Set1C components to maintain repression at the <i>mat</i> locus and subtelomeres while dispensing Set1C in repressing <i>Tf2s</i> and pericentromeric repeats. We show that the contributions of several Set1C subunits to the states of H3K4me diverge considerably from those of <i>Saccharomyces cerevisiae</i> orthologs. Moreover, unlike <i>S. cerevisiae</i>, the regulation of Set1 protein level is not coupled to the status of H3K4me or histone H2B ubiquitination by the HULC complex. Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of <i>Tf2s</i> into <i>Tf</i> bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation. Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.</p></div

Set1 represses different classes of repetitive elements via distinct functional domains.

<p>(A) Schematic of <i>S. pombe</i> Set1 protein domain architecture. (B) H3K4 methylation (H3K4me) in <i>set1</i> domain mutants. Mono (H3K4me1), di (H3K4me2), and tri (H3K4me3) methylation of H3K4 was analyzed from histone extracts of indicated <i>set1</i> mutant strains by western blotting. Full-length FLAG-set1 and the indicated deleted domain mutants of <i>set1</i> contain an N-terminal FLAG (3×) epitope. <i>set1F^H3K4me-</i> denotes an H3K4me null mutant due to the presence of a FLAG (3×) epitope at the C-terminus of <i>set1</i>. (C) <i>Tf2</i> repression requires intact RRM2 and SET domains. (D–F) Set1 represses heterochromatic loci dependent and independent of H3K4me. Expression of <i>Tf2</i> ORF and heterochromatic loci was analyzed by qRT-PCR. Fold changes relative to wildtype were normalized by <i>act1</i> expression. (s.d., error bars; <i>n</i> = 3). Pericentromeric repeat <i>dg</i> (<i>cen</i>), silent mating type <i>cenH</i> (<i>mat</i>), subtelomeric <i>prl70</i> (<i>subtel</i>).</p

Model for the roles of Set1C in genome control in <i>S. pombe</i>.

<p>Set1 exerts its multifaceted genome control at euchromatin and heterochromatin dependent and independent of H3K4 methylation and the Set1C complex. At euchromatin, Set1 operates as part of the Set1C/COMPASS complex that is responsible for H3K4me distribution at active RNA polymerase II genes. At interspersed <i>Tf2s</i> and heterochromatic loci, Set1 has a repressive role mediated through two distinct pathways: H3K4me/Set1C-dependent repression at the silent <i>mat</i> locus and subtelomeres, and H3K4me/Set1C-independent repression at <i>Tf2s</i> and pericentromeric heterochromatin. Our findings also anticipate the presence of novel non-histone H3K4 substrates involved in the repression of <i>Tf2s</i> and pericentromeric repeats that could be partly mediated via Set1 association with Pol II nascent transcripts. In addition, Set1C and H3K4me have a distinct genome organization role at <i>Tf2s</i> by antagonizing the activity of the histone H3K4 acetyltransferase Mst1 to maintain the integrity of <i>Tf</i> bodies.</p

Set1 localization at euchromatic and heterochromatic targets is largely unaffected by certain domain deletions or H3K4me status.

<p>Enrichment of Set1 was determined by chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) using primers targeting (A) the highly expressed actin gene (<i>act1</i>) promoter, (B) the 5′ end of <i>Tf2</i> open reading frames, (C) the pericentromeric <i>dg</i> repeat of chromosome II (<i>cen</i>), (D) the silent mating type <i>cenH</i> (<i>mat</i>), and (E) the chromosome I subtelomeric <i>prl70</i> (<i>subtel</i>). Mock denotes untagged wildtype Set1 strains. Percent enrichment of target amplification compared to input (whole cell extract) was calculated using the 2^−ΔΔCt method following normalization by primers targeting mitochondrial DNA (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004740#s4" target="_blank">Materials and Methods</a>). (s.d., error bars; n = 2 biological×2 qPCR replicates).</p

Stability of Set1 proteins is uncoupled from the status of H3K4me and H2Bub.

<p>Set1 proteins containing an N-terminal FLAG epitope were analyzed by immunoblotting (IB) in cells deficient in (A) individual components of Set1C, (B) histone H3K4 mutants, or (C) HULC/H2Bub mutants. Alpha tubulin (loading control) was detected by anti-tubulin antibody (tat-1).</p

Set1C and H3K4me are required for the integrity of <i>Tf</i> bodies.

<p>(A, B) Declustering of <i>Tf2s</i> in (A) <i>set1</i> mutant with defects in H3K4me and (B) Set1C mutants. Fluorescence <i>in situ</i> hybridization (FISH) analysis was performed using a FISH probe corresponding to the ∼3.6 kb coding region of <i>Tf2</i> elements. Representative FISH images from indicated strains (top panels). Quantitative FISH analysis of observed <i>Tf2</i> foci/cell in indicated strains (bar graph; bottom panels). Number of cells analyzed per strain (<i>n</i>). Except for <i>sdc1</i>Δ, <i>Tf2</i> declustering in all mutant strains compared to WT was significant (<i>p</i><0.005, chi-square test).</p

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer

Epidemiologic Investigation of a Cluster of Neuroinvasive Bacillus cereus Infections in 5 Patients With Acute Myelogenous Leukemia

Background. Five neuroinvasive Bacillus cereus infections (4 fatal) occurred in hospitalized patients with acute myelogenous leukemia (AML) during a 9-month period, prompting an investigation by infection control and public health officials. Methods. Medical records of case-patients were reviewed and a matched case-control study was performed. Infection control practices were observed. Multiple environmental, food, and medication samples common to AML patients were cultured. Multilocus sequence typing was performed for case and environmental B cereus isolates. Results. All 5 case-patients received chemotherapy and had early-onset neutropenic fevers that resolved with empiric antibiotics. Fever recurred at a median of 17 days (range, 9–20) with headaches and abrupt neurological deterioration. Case-patients had B cereus identified in central nervous system (CNS) samples by (1) polymerase chain reaction or culture or (2) bacilli seen on CNS pathology stains with high-grade B cereus bacteremia. Two case-patients also had colonic ulcers with abundant bacilli on autopsy. No infection control breaches were observed. On case-control analysis, bananas were the only significant exposure shared by all 5 case-patients (odds ratio, 9.3; P = .04). Five environmental or food isolates tested positive for B cereus, including a homogenized banana peel isolate and the shelf of a kitchen cart where bananas were stored. Multilocus sequence typing confirmed that all case and environmental strains were genetically distinct. Multilocus sequence typing-based phylogenetic analysis revealed that the organisms clustered in 2 separate clades. Conclusions. The investigation of this neuroinvasive B cereus cluster did not identify a single point source but was suggestive of a possible dietary exposure. Our experience underscores the potential virulence of B cereus in immunocompromised hosts