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The drug-impermeable bacterial membrane in Gram-negative pathogens limits antibiotic access to intracellular drug targets. To expand our rapidly waning antibiotic arsenal, one approach is to improve the intracellular delivery of drugs with historically poor accumulation in Gram-negative bacteria. To do so, we engineered macromolecular potentiators to permeabilize the Gram-negative membrane to facilitate drug influx. Potentiators, known as WD40, were synthesized by grafting multiple copies of a cationic α-helical antimicrobial peptide, WLBU2, onto a dextran polymer scaffold. WD40 enabled drug uptake in the model pathogen P. aeruginosa, a capability that was not observed with unmodified WLBU2 peptide. WD40 was able to reduce minimum inhibitory concentrations of a drug panel by up to 3 orders of magnitude. Hydrophobic and highly three-dimensional antibiotics exhibited the greatest potentiation. Antibiotic activity was potentiated in several clinical strains and resulted in sensitization of drug-resistant strains to rifampin, a drug not previously used for Gram-negative infections.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Among the most urgent public health threats is the worldwide emergence of carbapenem-resistant Enterobacteriaceae.sup.1-4, which are resistant to the antibiotic class of 'last resort'. In the United States and Europe, carbapenem-resistant strains of the Klebsiella pneumoniae ST258 (ref. .sup.5) sequence type are dominant, endemic.sup.6-8 and associated with high mortality.sup.6,9,10. We report the global evolution of pathogenicity in carbapenem-resistant K. pneumoniae, resulting in the repeated convergence of virulence and carbapenem resistance in the United States and Europe, dating back to as early as 2009. We demonstrate that K. pneumoniae can enhance its pathogenicity by adopting two opposing infection programs through easily acquired gain- and loss-of-function mutations. Single-nucleotide polymorphisms in the capsule biosynthesis gene wzc lead to hypercapsule production, which confers phagocytosis resistance, enhanced dissemination and increased mortality in animal models. In contrast, mutations disrupting capsule biosynthesis genes impair capsule production, which enhances epithelial cell invasion, in vitro biofilm formation and persistence in urinary tract infections. These two types of capsule mutants have emerged repeatedly and independently in Europe and the United States, with hypercapsule mutants associated with bloodstream infections and capsule-deficient mutants associated with urinary tract infections. In the latter case, drug-tolerant K. pneumoniae can persist to yield potentially untreatable, persistent infection. Mutations in Klebsiella pneumoniae that lead to gain or loss of capsule production affect pathogenicity and associate with bloodstream or urinary tract infections, respectively., Author(s): Christoph M. Ernst [sup.1] [sup.2] [sup.3] , Julian R. Braxton [sup.1] [sup.2] [sup.3] , Carlos A. Rodriguez-Osorio [sup.1] [sup.2] [sup.3] , Anna P. Zagieboylo [sup.1] [sup.2] [sup.3] , Li [...]

Dysregulation of the immune response to bacterial infection can lead to sepsis, a condition with high mortality. Multiple whole-blood gene-expression studies have defined sepsis-associated molecular signatures, but have not resolved changes in transcriptional states of specific cell types. Here, we used single-cell RNA-sequencing to profile the blood of people with sepsis (n = 29) across three clinical cohorts with corresponding controls (n = 36). We profiled total peripheral blood mononuclear cells (PBMCs, 106,545 cells) and dendritic cells (19,806 cells) across all subjects and, on the basis of clustering of their gene-expression profiles, defined 16 immune-cell states. We identified a unique CD14.sup.+ monocyte state that is expanded in people with sepsis and validated its power in distinguishing these individuals from controls using public transcriptomic data from subjects with different disease etiologies and from multiple geographic locations (18 cohorts, n = 1,467 subjects). We identified a panel of surface markers for isolation and quantification of the monocyte state and characterized its epigenomic and functional phenotypes, and propose a model for its induction from human bone marrow. This study demonstrates the utility of single-cell genomics in discovering disease-associated cytologic signatures and provides insight into the cellular basis of immune dysregulation in bacterial sepsis. Single-cell transcriptomic analysis identifies a distinct gene signature associated with peripheral monocyte populations that distinguishes people with sepsis from those with sterile inflammation and uninfected controls., Author(s): Miguel Reyes [sup.1] [sup.2] , Michael R. Filbin [sup.1] [sup.3] , Roby P. Bhattacharyya [sup.1] [sup.4] , Kianna Billman [sup.1] , Thomas Eisenhaure [sup.1] , Deborah T. Hung [sup.1] [...]

Formation of the inflammasome, a scaffolding complex that activates caspase-1, is important in numerous diseases. Pyroptotic cell death induced by anthrax lethal toxin (LT) is a model for inflammasome-mediated caspase-1 activation. We discovered 7-desacetoxy-6,7-dehydrogedunin (7DG) in a phenotypic screen as a small molecule that protects macrophages from LT-induced death. Using chemical proteomics, we identified protein kinase R (PKR) as the target of 7DG and show that RNAi knockdown of PKR phenocopies treatment with 7DG. Further, we show that PKR's role in ASC assembly and caspase-1 activation induced by several different inflammasome stimuli is independent of PKR's kinase activity, demonstrating that PKR has a previously uncharacterized role in caspase-1 activation and pyroptosis that is distinct from its reported kinase-dependent roles in apoptosis and inflammasome formation in lipopolysaccharide-primed cells. Remarkably, PKR has different roles in two distinct cell death pathways and has a broad role in inflammasome function relevant in other diseases.