Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

CRTH2 Mediates Profibrotic Macrophage Differentiation and Promotes Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a particularly deadly form of pulmonary fibrosis of unknown cause. In patients with IPF, high serum and lung concentrations of CHI3L1 (chitinase 3 like 1) can be detected and are associated with poor survival. However, the roles of CHI3L1 in these diseases have not been fully elucidated. We hypothesize that CHI3L1 interacts with CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells) to stimulate profibrotic macrophage differentiation and the development of pulmonary fibrosis and that circulating blood monocytes from patients with IPF are hyperresponsive to CHI3L1-CRTH2 signaling. We used murine pulmonary fibrosis models to investigate the role of CRTH2 in profibrotic macrophage differentiation and fibrosis development and primary human peripheral blood mononuclear cell culture to detect the difference of monocytes in the responses to CHI3L1 stimulation and CRTH2 inhibition between patients with IPF and normal control subjects. Our results showed that null mutation or small-molecule inhibition of CRTH2 prevents the development of pulmonary fibrosis in murine models. Furthermore, CHI3L1 stimulation induces a greater increase in CD206 expression in IPF monocytes than control monocytes. These results demonstrated that monocytes from patients with IPF appear to be hyperresponsive to CHI3L1 stimulation. These studies support targeting the CHI3L1-CRTH2 pathway as a promising therapeutic approach for IPF and that the sensitivity of blood monocytes to CHI3L1-induced profibrotic differentiation may serve as a biomarker that predicts responsiveness to CHI3L1- or CRTH2-based interventions.

Transcriptomics Reveals How Minocycline-Colistin Synergy Overcomes Antibiotic Resistance in Multidrug-Resistant Klebsiella pneumoniae.

Multidrug-resistant Gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using transcriptome sequencing (RNA-Seq), we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.

Large-scale, in-house production of viral transport media to support SARS-CoV-2 PCR testing in a multi-hospital healthcare network during the COVID-19 pandemic.

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for SARS-CoV-2 RT-PCR testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on Ct values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 EUA assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least four months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting including use of staged, assembly line for VTM transport tube production.

Large-Scale, In-House Production of Viral Transport Media To Support SARS-CoV-2 PCR Testing in a Multihospital Health Care Network during the COVID-19 Pandemic.

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-PCR (RT-PCR) testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on cycle threshold (CT ) values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization (EUA) assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least 4 months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting, including use of a staged assembly line for VTM transport tube production.