Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.

The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose.

Small molecules help intestinal pathogens navigate the complex human gastrointestinal tract to exploit favorable microhabitats. These small molecules provide spatial landmarks for pathogens to regulate synthesis of virulence caches and are derived from the host, ingested plant and animal material, and the microbiota. Their concentrations and fluxes vary along the length of the gut and provide molecular signatures that are beginning to be explored through metabolomics and genetics. However, while many small molecules have been identified and are reviewed here, there are undoubtedly others that may also profoundly affect how enteric pathogens infect their hosts.

New antibiotics are needed against antibiotic-resistant gram-negative bacteria. The repurposed antifungal drug, ciclopirox, equally blocks antibiotic-susceptible or multidrug-resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates, indicating that it is not affected by existing resistance mechanisms. Toward understanding how ciclopirox blocks growth, we screened E. coli mutant strains and found that disruption of genes encoding products involved in galactose salvage, enterobacterial common antigen synthesis, and transport of the iron binding siderophore, enterobactin, lowered the minimum inhibitory concentration of ciclopirox needed to block growth of the mutant compared to the isogenic parent strain. We found that ciclopirox induced enterobactin production and that this effect is strongly affected by the deletion of the galactose salvage genes encoding UDP-galactose 4-epimerase, galE, or galactose-1-phosphate uridylyltransferase, galT. As disruption of ECA synthesis activates the regulation of capsular synthesis (Rcs) phosphorelay, which inhibits bacterial swarming and promotes biofilm development, we test whether ciclopirox prevents activation of the Rcs pathway. Sub-inhibitory concentrations of ciclopirox increased swarming of the E. coli laboratory K12 strain BW25113 but had widely varying effects on swarming or surface motility of clinical isolate E. coli, A. baumannii, and K. pneumoniae. There was no effect of ciclopirox on biofilm production, suggesting it does not target Rcs. Altogether, our data suggest ciclopirox-mediated alteration of lipopolysaccharides stimulates enterobactin production and affects bacterial swarming.

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