In late 2015, the first example of a transferrable polymyxin resistance mechanism in Gram-negative pathogens, MCR-1, was reported. Since that report, MCR-1 has been described to occur in many Gram-negative pathogens, and the mechanism of MCR-1-mediated resistance was rapidly determined: an ethanolamine is attached to lipid A phosphate groups, rendering the membrane more electropositive and repelling positively charged polymyxins. Acquisition of MCR-1 is clinically significant because polymyxins are frequently last-line antibiotics used to treat extensively resistant organisms, so acquisition of this mechanism might lead to pan-resistant strains. Therefore, the ability to inhibit MCR-1 and restore polymyxin sensitivity would be an important scientific advancement. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense molecules that were designed to target mRNA, preventing translation. Peptide conjugation enhances cellular entry, but they are positively charged, so we tested our lead antibacterial PPMOs by targeting an essential Escherichia coli gene, acpP, and demonstrated that they were still effective in mcr-1-positive E. coli strains. We then designed and synthesized two PPMOs targeted to mcr-1 mRNA. Five clinical mcr-1-positive E. coli strains were resensitized to polymyxins by MCR-1 inhibition, reducing MICs 2- to 16-fold. Finally, therapeutic dosing of BALB/c mice with MCR-1 PPMO combined with colistin in a sepsis model reduced morbidity and bacterial burden in the spleen at 24 h and offered a survival advantage out to 5 days. This is the first example of a way to modulate colistin resistance with an antisense approach and may be a viable strategy to combat this globally emerging antibiotic resistance threat.

Background Specific antimicrobial breast pocket irrigations have been proven over the past 20 years to reduce the incidence of capsular contracture by a factor of 10, and the emergence of breast implant–associated anaplastic large cell lymphoma (BIA-ALCL) and its link to bacteria/technique has created renewed interest in different antimicrobial breast pocket preparation agents. Our previous studies have identified that both Betadine-containing and non-Betadine-containing antimicrobial irrigations provide excellent broad-spectrum bacterial coverage. The current science of BIA-ALCL has implicated the Gram-negative microbiome as a key in pathogenesis. Objectives The aim of this study was to revisit the antimicrobial effectiveness of clinically utilized Betadine and non-Betadine solutions, along with other antimicrobial agents that have not yet been tested, against multiple organisms, including additional common Gram-negative bacteria associated with chronic breast implant infections/inflammation. Methods Current and new antimicrobial breast irrigations were tested via standard techniques for bactericidal activity against multiple Gram-positive and Gram-negative strains. Test results are detailed and clinical recommendations for current antimicrobial irrigations are provided. Results Betadine-containing irrigations were found to be superior according to the testing performed. Conclusions There are quite few misconceptions with regard to antimicrobial breast pocket irrigation. These are discussed and final evidence-based recommendations for practice are given.

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens in immunocompromised individuals, such as those with cystic fibrosis (CF) or chronic granulomatous disease (CGD). Burkholderia are intrinsically resistant to many antibiotics and the lack of antibiotic development necessitates novel therapeutics. Peptide-conjugated phosphorodiamidate morpholino oligomers are antisense molecules that inhibit bacterial mRNA translation. Targeting of PPMOs to the gene acpP, which is essential for membrane synthesis, lead to defects in the membrane and ultimately bactericidal activity. Exploration of additional PPMO sequences identified the ATG and Shine-Dalgarno sites as the most efficacious for targeting acpP. The CF lung is a complex microenvironment, but PPMO inhibition was still efficacious in an artificial model of CF sputum. PPMOs had low toxicity in human CF cells at doses that were antibacterial. PPMOs also reduced the bacterial burden in the lungs of immunocompromised CyBB mice, a model of CGD. Finally, the use of multiple PPMOs was efficacious in inhibiting the growth of both Burkholderia and Pseudomonas in an in vitro model of coinfection. Due to the intrinsic resistance of Burkholderia to traditional antibiotics, PPMOs represent a novel and viable approach to the treatment of Burkholderia infections.

Cefiderocol is a siderophore cephalosporin with potent antibacterial activity against a broad range of Gram-negative pathogens, including multi-drug resistant strains. Siderophore antibiotics bind ferric iron and utilize iron transporters to cross the cell membrane. In the biofilm setting, where antibiotic resistance is high but iron scavenging is important, cefiderocol may have advantageous antimicrobial properties. In this study, we compared the antimicrobial activity of cefiderocol to seven commonly used antibiotics in well-characterized multi-drug resistant pathogens, then determined their efficacy in the biofilm setting. MIC90 values were consistently lower for cefiderocol in all strains tested compared to other antibiotics (ceftolozane-tazobactam, ceftazidime-avibactam, ceftazidime, piperacillin-tazobactam, imipenem, tobramycin, clarithromycin). Cefiderocol treatment displayed a superior reduction in Pseudomonas aeruginosa biofilm (93%, p<0.0001) compared to other antibiotics (49-82%). Cefiderocol was generally as effective or superior in reducing biofilm in other pathogens depending on the pathogen-antibiotic combination. There was a trend towards greater biofilm reduction when the antibiotic dose was increased or with increased frequency of antibiotic treatment. We conclude that cefiderocol effectively reduces biofilm and is a potent inhibitor of planktonic growth across a range of Gram-negative medically important pathogens.

Overexpression of bacterial efflux pumps is a driver of increasing antibiotic resistance in Gram-negative pathogens. The AcrAB–TolC efflux pump has been implicated in resistance to a number of important antibiotic classes including fluoroquinolones, macrolides, and β-lactams. Antisense technology, such as peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), can be utilized to inhibit expression of efflux pumps and restore susceptibility to antibiotics. Targeting of the AcrAB–TolC components with PPMOs revealed a sequence for acrA, which was the most effective at reducing antibiotic efflux. This acrA–PPMO enhances the antimicrobial effects of the levofloxacin and azithromycin in a panel of clinical Enterobacteriaceae strains. Additionally, acrA–PPMO enhanced azithromycin in vivo in a K. pneumoniae septicemia model. PPMOs targeting the homologous resistance-nodulation-division (RND)-efflux system in P. aeruginosa, MexAB–OprM, also enhanced potency to several classes of antibiotics in a panel of strains and in a cell culture infection model. These data suggest that PPMOs can be used as an adjuvant in antibiotic therapy to increase the efficacy or extend the spectrum of useful antibiotics against a variety of Gram-negative infections.