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Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria ( Staphylococcus aureus and Streptococcus pneumoniae ) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC 90 ≤ 0.06 μg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae , as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

BACKGROUND: Polymyxin B and E (colistin) have been pivotal in the treatment of XDR Gram-negative bacterial infections; however, resistance has emerged. A structurally related lipopeptide, octapeptin C4, has shown significant potency against XDR bacteria, including polymyxin-resistant strains, but its mode of action remains undefined. OBJECTIVES: We sought to compare and contrast the acquisition of resistance in an XDR Klebsiella pneumoniae (ST258) clinical isolate in vitro with all three lipopeptides to potentially unveil variations in their mode of action. METHODS: The isolate was exposed to increasing concentrations of polymyxins and octapeptin C4 over 20 days. Day 20 strains underwent WGS, complementation assays, antimicrobial susceptibility testing and lipid A analysis. RESULTS: Twenty days of exposure to the polymyxins resulted in a 1000-fold increase in the MIC, whereas for octapeptin C4 a 4-fold increase was observed. There was no cross-resistance observed between the polymyxin- and octapeptin-resistant strains. Sequencing of polymyxin-resistant isolates revealed mutations in previously known resistance-associated genes, including crrB, mgrB, pmrB, phoPQ and yciM, along with novel mutations in qseC. Octapeptin C4-resistant isolates had mutations in mlaDF and pqiB, genes related to phospholipid transport. These genetic variations were reflected in distinct phenotypic changes to lipid A. Polymyxin-resistant isolates increased 4-amino-4-deoxyarabinose fortification of lipid A phosphate groups, whereas the lipid A of octapeptin C4-resistant strains harboured a higher abundance of hydroxymyristate and palmitoylate. CONCLUSIONS: Octapeptin C4 has a distinct mode of action compared with the polymyxins, highlighting its potential as a future therapeutic agent to combat the increasing threat of XDR bacteria.

INTRODUCTION: The octapeptins are a family of cyclic lipopeptides first reported in the 1970s then largely ignored. At the time, their reported antibiotic activity against polymyxin-resistant bacteria was a curiosity. Today the advent of widespread drug resistance in Gram-negative bacteria has prompted their ‘rediscovery’. The paucity of new antibiotics in the clinical pipeline is coupled with a global spread of increasing antibiotic resistance, particularly to meropenem and polymyxins B and E (colistin). AREAS COVERED: We review the original discovery of octapeptins, their recent first chemical syntheses, and their mode of action, then discuss their potential as a new class of antibiotics to treat extensively drug resistant (XDR) Gram-negative infections, with direct comparisons to the closely related polymyxins. EXPERT COMMENTARY: Cyclic lipopeptides in clinical use (polymyxin antibiotics) have significant dose-limiting nephrotoxicity inherent to their chemotype. This toxicity has prevented improved polymyxin analogs from progressing to the clinic, and tainted the perception of lipopeptide antibiotics in general. We argue that the octapeptins are fundamentally different from the polymyxins, with a disparate mode of action, spectra of action against MDR and XDR bacteria and a superior pre-clinical safety profile. They represent early stage candidates that can help prime the antibiotic discovery pipeline.

Background Rapid diagnosis and appropriate treatment is imperative in bacterial sepsis due increasing risk of mortality with every hour without appropriate antibiotic therapy. Atypical infections with fastidious organisms may take more than 4 days to diagnose leading to calls for improved methods for rapidly diagnosing sepsis. Capnocytophaga canimorsus is a slow-growing, fastidious gram-negative bacillus which is a common commensal within the mouths of dogs, but rarely cause infections in humans. C. canimorsus sepsis risk factors include immunosuppression, alcoholism and elderly age. Here we report on the application of emerging nanopore sequencing methods to rapidly diagnose an atypical case of C. canimorsus septic shock. Case presentation A 62 year-old female patient was admitted to an intensive care unit with septic shock and multi-organ failure six days after a reported dog bite. Blood cultures were unable to detect a pathogen after 3 days despite observed intracellular bacilli on blood smears. Real-time nanopore sequencing was subsequently employed on whole blood to detect Capnocytophaga canimorsus in 19 h. The patient was not immunocompromised and did not have any other known risk factors. Whole-genome sequencing of clinical sample and of the offending dog’s oral swabs showed near-identical C. canimorsus genomes. The patient responded to antibiotic treatment and was discharged from hospital 31 days after admission. Conclusions Use of real-time nanopore sequencing reduced the time-to-diagnosis of Capnocytophaga canimorsus in this case from 6.25 days to 19 h. Capnocytophaga canimorsus should be considered in cases of suspected sepsis involving cat or dog contact, irrespective of the patient’s known risk factors. Electronic supplementary material The online version of this article (10.1186/s12879-019-4173-2) contains supplementary material, which is available to authorized users.

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including nineteen polymyxin-resistant and five polymyxin-susceptible strains, underwent whole genome sequencing. Approximately 90% of polymyxin resistance was enabled by alterations upstream or within mgrB. The most common mutation identified was an insertion at nucleotide position 75 in mgrB via an ISKpn26-like element in the ST258 lineage and ISKpn13 in one ST11 isolate. Three strains acquired an IS1 element upstream of mgrB and another strain had an ISKpn25 insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D31E) affecting mgrB. Complementation assays revealed all mgrB perturbations contributed to resistance. Missense mutations in phoQ (T281M, G385C) were also found to facilitate resistance. Several variants in phoPQ co-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance conferring mutations, including the ISKpn26-like insertion colonising with a novel mutation in pmrB (P158R), both confirmed via complementation assays. We also characterized a new multi-drug resistant Klebsiella quasipneumoniae strain ST2401 which was susceptible to polymyxins. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins in K. pneumoniae.DATA SUMMARYWhole genome sequencing of the 24 clinical isolates has been deposited under BioProject PRJNA307517 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA307517).IMPACT STATEMENTKlebsiella pneumoniae contributes to a high abundance of nosocomial infections and the rapid emergence of antimicrobial resistance hinders treatment. Polymyxins are predominantly utilized to treat multidrug-resistant infections, however, resistance to the polymyxins is arising. This increasing prevalence in polymyxin resistance is evident especially in Greece and Brazil. Identifying the genomic variations conferring resistance in clinical isolates from these regions assists with potentially detecting novel alterations and tracing the spread of particular strains. This study commonly found mutations in the gene mgrB, the negative regulator of PhoPQ, known to cause resistance in KP. In the remaining isolates, missense mutations in phoQ were accountable for resistance. Multiple novel mutations were detected to be segregating with mgrB perturbations. This was either due to a mixed heterogeneous sample of two polymyxin-resistant strains, or because of multiple mutations within the same strain. Of interest was the validation of novel mutations inphoPQ segregating with a previously known ISKpn26-like element in disrupted mgrB isolates. Complementation of these phoPQ mutations revealed a reduction in minimum inhibitory concentrations and suggests the first evidence of partial suppressor mutations in KP. This research builds upon our current understanding of heteroresistance, lineage specific mutations and regulatory variations relating to polymyxin resistance.

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment.