Metrics & MoreArticle Recommendations CONSPECTUS: It has been over half a century since the last class of antibiotics active against the most problematic Gram-negative bacteria was approved by the Food and Drug Administration (FDA). The major challenge with developing antibiotics to treat these infections is not drugtarget engagement but rather the inability of most small molecules to traverse the Gram-negative membranes, be retained, and accumulate within the cell. Despite an abundance of lead compounds, limited understanding of the physicochemical properties needed for compound accumulation (or avoidance of efflux) in Gram-negative bacteria has precluded a generalizable approach for developing Gram-negative antibiotics. Indeed, in many instances, despite years of intensive derivatization efforts and the synthesis of hundreds of compounds aimed at building in Gram-negative activity, little or no progress has been made in expanding the spectrum of activity for many Gram-positive-only antibiotics. In this Account, we describe the discovery and successful applications of a promising strategy for enhancing the accumulation of Gram-positive-only antibiotics as a means of imbuing compounds with broad-spectrum activity. Utilizing a prospective approach examining the accumulation in Escherichia coli for more than 180 diverse compounds, we found that small molecules have an increased likelihood to accumulate in E. coli when they contain an ionizable Nitrogen, have low Threedimensionality, and are Rigid. Implementing these guidelines, codified as the "eNTRy rules" and assisted by web application www. entry-way.org, we have facilitated compound entry and systematically built Gram-negative activity into Gram-positive-only antibiotics. Though each antibiotic will have case-specific considerations, we describe a set of important criteria to consider when selecting candidate Gram-positive-only antibiotics for conversion to Gram-negative-active versions via the eNTRy rules. As detailed herein, using this blueprint the spectrum of activity was expanded for three antibiotic classes that engage three different biological targets: DNA gyrase inhibitor 6DNM, FabI inhibitor Debio-1452, and FMN riboswitch inhibitor Ribocil C. In each scenario, the eNTRy rules guided the synthesis of key analogues predisposed to accumulate in Gram-negative bacteria leading to compounds that display antibiotic activity (minimum inhibitory concentrations (MIC) ≤8 μg mL −1 ) against E. coli and other Gram-negative ESKAPE pathogens. While the eNTRy rules will continue to be refined and enhanced as more accumulation data is gathered, on the basis of these collective results and on other examples not covered herein it is clear that the eNTRy rules are actionable for the development of novel broad-spectrum antibiotics from Gram-positive-only compounds. By enabling the prediction of compound accumulation, the eNTRy rules should facilitate the process of discovering and developing novel antibiotics active against Gramcontinued...

Genomic studies and experiments with permeability-deficient strains have revealed a variety of biological targets that can be engaged to kill Gram-negative bacteria. However, the formidable outer membrane and promiscuous efflux pumps of these pathogens prevent many candidate antibiotics from reaching these targets. One such promising target is the enzyme FabI, which catalyzes the rate-determining step in bacterial fatty acid biosynthesis. Notably, FabI inhibitors have advanced to clinical trials for Staphylococcus aureus infections but not for infections caused by Gram-negative bacteria. Here, we synthesize a suite of FabI inhibitors whose structures fit permeation rules for Gram-negative bacteria and leverage activity against a challenging panel of Gram-negative clinical isolates as a filter for advancement. The compound to emerge, called fabimycin, has impressive activity against >200 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, and does not kill commensal bacteria. X-ray structures of fabimycin in complex with FabI provide molecular insights into the inhibition. Fabimycin demonstrates activity in multiple mouse models of infection caused by Gram-negative bacteria, including a challenging urinary tract infection model. Fabimycin has translational promise, and its discovery provides additional evidence that antibiotics can be systematically modified to accumulate in Gram-negative bacteria and kill these problematic pathogens.

This study was designed to determine the prevalence and microbial load of Listeria spp., Escherichia coli O157 and Salmonella spp. in ready-to-eat products in supermarkets across Trinidad. The microbial load was assessed using the total aerobic plate count (TAPC) per g/ml of foods and prevalence of Escherichia coli O157 and Salmonella spp. determined using conventional methods. For Listeria monocytogenes, immunomagnetic separation (IMS), TECRA (enzymelinked immunosorbent assay, ELISA) and conventional methods were used. The log 10 mean ± sd TAPC per g or ml was highest for vegetables (11.0±11.6), and lowest for seafood (5.2±5.7) (p < 0.05). The prevalence of L. monocytogenes was 1.7 %. Sixteen (4.5%) of 153 samples yielded E. coli but all samples were negative for Salmonella spp. and E. coli O157. The high microbial load and isolation of L. monocytogenes and E. coli from popular RTE foods could pose a health risk to consumers in the country.

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Single-experiment analysis of phospholipid compositional gradients reveals diffusion coefficients, phase separation parameters, and binding densities as a function of localized lipid mixture. Compositional gradients are formed by directed self assembly where rapid-prototyping techniques (i.e., additive manufacturing or laser-cutting) prescribe lipid geometries that self-spread, heal and mix by diffusion.