More Like this

1. Bacterial efflux pump modulators prevent bacterial growth in macrophages and under broth conditions that mimic the host environment

   https://doi.org/10.1128/mbio.02492-23  

   Allgood, Samual C; Su, Chih-Chia; Crooks, Amy L; Meyer, Christian T; Zhou, Bojun; Betterton, Meredith D; Barbachyn, Michael R; Yu, Edward W; Detweiler, Corrella S (December 2023, mBio)

   Hughes, Kelly T (Ed.)

   ABSTRACT New approaches for combating microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen,Salmonella entericaserotype Typhimurium (S. Typhimurium), in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy. Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome,acrABis required for growth, the EPM analogs are bacteriostatic, and the EPM analogs increase the potency of antibiotics. These data suggest that under macrophage-like conditions, the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics. IMPORTANCEBacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics. 

   more » « less
2. A nutrient bottleneck controls antibiotic efficacy in structured bacterial populations

   https://doi.org/10.1038/s41467-026-69625-4  

   Hancock, Anna M; Dill-Macky, Arabella S; Moore, Jenna A; Day, Catherine; Donia, Mohamed S; Datta, Sujit S (December 2026, Nature Communications)

   Abstract Antibiotic resistance is a growing global health threat. Although antibiotic activity is well studied in homogeneous liquid cultures, many infections are caused by spatially structured multicellular populations where consumption of scarce nutrients establishes strong spatial variations in their abundance. These nutrient variations have long been hypothesized to help bacterial populations tolerate antibiotics, since liquid culture studies link antibiotic tolerance to metabolic activity, and thus, local nutrient availability. Here, we test this hypothesis by visualizing cell death in structuredEscherichia colipopulations exposed to select nutrients and antibiotics. We find that nutrient availability acts as a bottleneck to antibiotic killing, causing death to propagate through the population as a traveling front. By integrating our measurements with biophysical theory and simulations, we establish quantitative principles that explain how collective nutrient consumption can limit the progression of this “death front,” protecting a population from a nominally deadly antibiotic dose. While increasing nutrient supply can overcome this bottleneck, in some cases, excess nutrient unexpectedlypromotesthe regrowth of resistant cells. Altogether, this work provides a key step toward predicting and controlling antibiotic treatment of spatially structured bacterial populations, yielding biophysical insights into collective behavior and guiding strategies for effective antibiotic stewardship. 

   more » « less
3. Single-cell heterogeneity underpins antagonistic antibiotic interactions

   https://doi.org/10.1038/s44320-025-00163-9  

   Teuber_Carvalho, João Pedro; Schultz, Daniel (January 2026, Molecular Systems Biology)

   Using combinations of existing antibiotics is a promising strategy to treat bacterial infections. Although some drugs act synergistically, other drug combinations inhibit microbial growth less than what is expected from their individual effects. Ciprofloxacin and tetracycline display such antagonistic interaction. In a new study, Broughton and colleagues (Broughton et al, 2025) used single-cell microfluidics to show that exposing E. coli cells to a combination of ciprofloxacin and tetracycline results in highly heterogeneous outcomes. The survival of single cells is linked to the expression of moderate levels of the SOS response, which fixes the double-strand DNA breaks caused by ciprofloxacin. High expression of the SOS response was found only among dying cells. Tetracycline then counteracts ciprofloxacin by increasing the proportion of cells that survive treatment within the low-SOS subpopulation. These findings highlight the importance of single-cell studies in understanding the phenotypic heterogeneity that emerges during antibiotic responses, which decide the success of treatments. 

   more » « less
4. Antibiotic Susceptibility of Escherichia coli Cells during Early-Stage Biofilm Formation

   https://doi.org/10.1128/JB.00034-19  

   Gu, Huan; Lee, Sang Won; Carnicelli, Joseph; Jiang, Zhaowei; Ren, Dacheng; O'Toole, George (September 2019, Journal of Bacteriology)

   ABSTRACT Bacteria form complex multicellular structures on solid surfaces known as biofilms, which allow them to survive in harsh environments. A hallmark characteristic of mature biofilms is the high-level antibiotic tolerance (up to 1,000 times) compared with that of planktonic cells. Here, we report our new findings that biofilm cells are not always more tolerant to antibiotics than planktonic cells in the same culture. Specifically, Escherichia coli RP437 exhibited a dynamic change in antibiotic susceptibility during its early-stage biofilm formation. This phenomenon was not strain specific. Upon initial attachment, surface-associated cells became more sensitive to antibiotics than planktonic cells. By controlling the cell adhesion and cluster size using patterned E. coli biofilms, cells involved in the interaction between cell clusters during microcolony formation were found to be more susceptible to ampicillin than cells within clusters, suggesting a role of cell-cell interactions in biofilm-associated antibiotic tolerance. After this stage, biofilm cells became less susceptible to ampicillin and ofloxacin than planktonic cells. However, when the cells were detached by sonication, both antibiotics were more effective in killing the detached biofilm cells than the planktonic cells. Collectively, these results indicate that biofilm formation involves active cellular activities in adaption to the attached life form and interactions between cell clusters to build the complex structure of a biofilm, which can render these cells more susceptible to antibiotics. These findings shed new light on bacterial antibiotic susceptibility during biofilm formation and can guide the design of better antifouling surfaces, e.g., those with micron-scale topographic structures to interrupt cell-cell interactions. IMPORTANCE Mature biofilms are known for their high-level tolerance to antibiotics; however, antibiotic susceptibility of sessile cells during early-stage biofilm formation is not well understood. In this study, we aim to fill this knowledge gap by following bacterial antibiotic susceptibility during early-stage biofilm formation. We found that the attached cells have a dynamic change in antibiotic susceptibility, and during certain phases, they can be more sensitive to antibiotics than planktonic counterparts in the same culture. Using surface chemistry-controlled patterned biofilm formation, cell-surface and cell-cell interactions were found to affect the antibiotic susceptibility of attached cells. Collectively, these findings provide new insights into biofilm physiology and reveal how adaptation to the attached life form may influence antibiotic susceptibility of bacterial cells. 

   more » « less
5. 18β-Glycyrrhetinic Acid Induces Metabolic Changes and Reduces Staphylococcus aureus Bacterial Cell-to-Cell Interactions

   https://doi.org/10.3390/antibiotics11060781  

   Weaver, Alan J.; Borgogna, Timothy R.; O’Shea-Stone, Galen; Peters, Tami R.; Copié, Valérie; Voyich, Jovanka; Teintze, Martin (June 2022, Antibiotics)

   The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18β-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D 1H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell–cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics. 

   more » « less