Secondary bacterial metabolites can regulate animal physiology, but the specific bacterial genes and molecules responsible for regulating, synthesizing, and transporting these molecules and the mechanisms by which they impact animal physiology often remain unclear. Here, we used Caenorhabditis elegans dauer formation as a gross morphological readout to identify bacterial mechanisms that promote development when animal TGF-β or insulin signaling is impaired. We found that the native C. elegans microbiome constituent, Pseudomonas lurida , suppresses dauer arrest and that this activity requires the two-component system, GacS/GacA, and the downstream massABC nonribosomal peptide synthetase cluster. Using sequential chromatography followed by mass spectrometry and NMR, we identified Massetolide F, a bacterial cyclic lipopeptide biosurfactant, as a massABC -dependent product. In addition, we found that purified Massetolide F was sufficient to restore dauer suppression to P. lurida ΔmassBC . We also found that surfactin C, a cyclic lipopeptide from B. subtilis , similarly suppressed dauer formation whereas the cyclic lipopeptide antibiotic daptomycin did not, indicating that dauer suppression is shared by a subset of cyclic lipopeptides rather than being a general property of the class. Lastly, we found that massABC -dependent Massetolide F production induces the expression of the acid sphingomyelinase asm-3 in C. elegans, that asm-3 is required for Massetolide F induced dauer suppression, and that loss of Massetolide F production also leads to reduced bacterial clearance in host animals. Together, these findings identify Massetolide F as a microbiome-derived molecule that promotes animal development and reveal an unexpected function for bacterial biosurfactants in regulating animal physiology.

The rise of bacterial resistance has prompted researchers to shift their focus toward alternative antimicrobial agents, including antimicrobial peptides (AMPs). Some naturally occurring AMPs are lipidated, showing similarity with clinically used antimicrobial agents such as daptomycin and polymyxins, which are lipidated peptides. In this study, we investigate the impact of various lipid modifications on nisin. Nisin is a well-studied 34-amino acid AMP, ribosomally synthesized and post-translational modified, which is effective against many Gram-positive bacteria and is widely used as a food preservative. Wild-type nisin and two truncated forms, nisin(1-31) and nisin(1-20) were conjugated at the C-terminus with either a C4, C6, C8, or C10 lipid tail. Antimicrobial activity was assessed against two Gram-positive strains: methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Our results show that the lipidation of wild-type nisin and nisin(1-31) reduced its antibacterial activity, with the longest chains exhibiting the lowest inhibitory effects. However, lipidation of the nisin fragment nisin(1-20), especially with C8 resulting in compound 12, enhanced its antibacterial activity and specificity against S. aureus species, making it four times more potent than wild-type nisin. Mode-of-action studies revealed that the lipidated construct 12 retained its ability to bind lipid II but does not induce pore formation in S. aureus. Notably, due to its short length, ring-protected structure, and lipid modification, the construct demonstrated improved proteolytic stability against chymotrypsin, trypsin and protease K. Our findings suggest that optimizing lipid-peptide combinations can lead to more effective, specific, and stable candidates for antimicrobial development.

Multidrug-resistant (MDR) Gram-negative bacteria pose significant therapeutic challenges due to their protective outer membranes and evolving resistance mechanisms. Exploiting bacterial iron uptake pathways via siderophore-antibiotic conjugates offers a promising strategy to enhance antibiotic efficacy. Here we synthesized siderophore-aztreonam, and siderophore-daptomycin conjugates and compared their activity to that of cefiderocol against select Gram-negative pathogens, including MDR Acinetobacter baumannii and Pseudomonas aeruginosa. Our in vitro studies demonstrated that siderophore conjugates exhibit comparable or superior antimicrobial activity to, with daptomycin conjugates notably retaining efficacy over extended periods. These findings suggest that conjugation with iron chelators that closely mimic natural siderophores can improve antibiotic delivery and activity, providing complementary options to existing therapies for multidrug-resistant Gram-negative infections.

Antimicrobial resistance (AMR) is a One Health challenge driven by microbial exchange among humans, animals and the environment. Zoological institutions offer useful settings for environmental AMR surveillance. This single-zoo cross-sectional study examined the occurrence, antimicrobial susceptibility and genomic characteristics of nontyphoidal Salmonella enterica (NTS) and clinically relevant Enterococcus spp. in faecal samples from 101 clinically healthy captive ungulates representing seven conservation-priority species at a major urban zoo in the United Arab Emirates. NTS was detected in 4/101 samples (3.9%), including serovars Schwarzengrund (n = 2), Kentucky (n = 1) and Chester (n = 1). Among the four recovered NTS isolates, all met the study MDR definition within the tested panel, including a Salmonella Kentucky ST198 isolate carrying multiple resistance genes and quinolone-associated mutations. Enterococcus spp. were detected in 77/101 samples (76.2%), dominated by Enterococcus faecium and Enterococcus casseliflavus (each 41.5%). Among 33 E. faecium/Enterococcus faecalis isolates tested phenotypically, resistance was generally low, with erythromycin and ciprofloxacin resistance each observed in 9.1%. One clinically important E. faecium isolate showed glycopeptide resistance and genetic markers associated with reduced daptomycin susceptibility. These single-institution cross-sectional data provide an initial regional baseline for AMR-relevant enteric bacteria in conservation-managed ungulates and identify priorities for broader longitudinal and interface-based surveillance.

Staphylococcus pettenkoferi has increasingly been reported as a human pathogen, with virulence potential recently confirmed in vivo. However, its genomic diversity and antimicrobial resistance remain insufficiently characterized. This study, conducted within the PETTENK-OS project of the ESCMID Study Group for Staphylococci and Staphylococcal Diseases (ESGS), aimed to characterize the genomic diversity and resistome of this underrecognized coagulase-negative staphylococcus (CoNS). Whole-genome sequencing was performed on 61 European clinical isolates (mainly bloodstream infections and osteomyelitis) from 17 hospitals in five countries, plus two reference strains. Antimicrobial susceptibility profiles were determined by disk diffusion and broth microdilution (2025 EUCAST breakpoints). Phylogenetic analyses clustered the 63 isolates into two major clades: Clade I (n = 51; subclades I-A, n = 47, and I-B, n = 4) and Clade II (subclades II-A, n = 8, and II-B, n = 3). Susceptibility rates were below 70% for cefoxitin, erythromycin, and levofloxacin, whereas high susceptibility (>90%) was observed for vancomycin, teicoplanin, dalbavancin, aminoglycosides, oxazolidinones, trimethoprim-sulfamethoxazole, fusidic acid, and tetracycline. Nearly all isolates (92.1%) were fosfomycin-resistant. Daptomycin MIC50/90 values (1/2 mg/L) were elevated, including eight resistant isolates carrying mutations in walKR, cls1, and/or cls2 genes. Twenty-two isolates (35.4%) were multidrug-resistant, and 18 (28.6%) were methicillin-resistant, all in subclade I-A and mecA-positive within SCCmec type IV. This study provides a comprehensive genomic and phenotypic assessment of antimicrobial resistance in S. pettenkoferi, revealing widespread multidrug-resistant lineages, including ceftobiprole- and daptomycin-resistant isolates across distinct clades. These findings underscore the need for continued surveillance and improved diagnostics for this increasingly encountered CoNS species.

To offset limitations of current methods of antibiofilm activity determination, including in vitro technical variations and the limited number of conditions assessable in animals, we describe a rabbit tissue-cage model allowing ex vivo screening of antimicrobials against in vivo-formed S. aureus biofilm. Cages containing six beads of polyethylene, titanium or steel served as support for S. aureus SH1000 biofilm formation. In vitro validation steps included: (i) quantification of bead-adherent bacteria after 24 h incubation in bacterial suspension, measured at 4.7 (95%CI, 4.5-4.9) log10 CFU/bead, with no difference between biomaterials, allowing the selection of polyethylene for animal experiments due to its lighter weight; and (ii) minimum concentration eradicating 90% of biofilm-embedded bacteria (MBEC90) determination by bead incubation in increasing concentrations of vancomycin, daptomycin and rifampicin, which were not significantly different compared to the reference method (MBECAssay[®]). In vivo validation included: (i) subcutaneous implantation of six cages per rabbit; (ii) infection with a range of different inocula (500/1000/2000 CFU/cage) before harvesting on day 7, 14 or 28 for quantification of bead-adherent bacteria. The 500 CFU/cage inoculum with a 14-day harvesting time provided the best balance between reproducibility and infection persistence and was therefore selected for ex vivo MBEC90 determination against in vivo-formed biofilms. Vancomycin, daptomycin, and rifampin MBEC90 values were 1.0, 1.5, and < 0.016 mg/L, respectively, and were significantly lower than those obtained against in vitro-formed biofilms. Compared with existing in vitro and in vivo studies, this innovative model of ex vivo determination of MBEC90 on in vivo-formed biofilm is likely to be more predictive of the in vivo anti-staphylococcal antibiofilm activity and requires a limited number of animals.

Bacteria that reside in the gastrointestinal tract must adapt to the antibacterial properties of bile salts (BSs) and other compounds. We investigated five putative transporters of the enteric-colonizing Enterococcus faecalis for their roles in adaptation to BS and antibiotic resistance. Transcript levels of ef2593 and ef0575 increased over fivefold, and ef0420 increased 12-fold when exposed to 0.02% and 0.1% BS concentrations. No change in the expression of ef2592 or ef1814 was seen. E. faecalis wild-type MMH594 overexpressing efflux pumps by plasmid constructs (EF2592, EF2593, EF0575, EF0420) or by a pre-exposure to 0.02% BS (EF2593 or EF0420) showed a higher CFU/mL than wild type alone when exposed to 0.3% BS, indicating adaptation of E. faecalis to resist a higher BS level in the medium, and that E. faecalis BS response involved multiple transporters. In MIC assays, the following overexpressors also showed a twofold increase in antibiotic resistance: EF2592 and EF2593 (daptomycin, vancomycin); EF0420 (daptomycin, oritavancin); and EF1814 (daptomycin, levofloxacin, oritavancin, and vancomycin). While no change in EF0575-overexpressor, mutant 0575 showed a significant increase in susceptibility to daptomycin, linezolid, levofloxacin, and oritavancin. In addition, nisin-induced overexpression of several transporters from a plasmid in Lactococcus lactis also increased the MICs of multiple antibiotics, most notably for EF1814, with a 4- to 16-fold increase in the MICs of daptomycin, levofloxacin, linezolid, and vancomycin. These data indicate that expression of multiple E. faecalis transporters responds to bile salts and may contribute to survival and colonization in the gut, as well as causing low-level multidrug resistance.Bile salts are key antimicrobial components of the gastrointestinal tract, and bacterial adaptation to bile salt stress is critical for host colonization and persistence. In this study, we demonstrate that multiple E. faecalis transporters are induced by bile salts and contribute to adaptation at physiologically relevant bile salt concentrations. Importantly, several of these transporters also confer low-level resistance to clinically important antibiotics, including daptomycin, vancomycin, levofloxacin, linezolid, and oritavancin. These findings suggest that transport systems selected for survival in the intestinal environment may have the unintended consequence of increasing antibiotic tolerance and multidrug resistance. By linking host-associated environmental stress with antimicrobial resistance phenotypes, this work advances our understanding of how gut colonization factors can influence bacterial persistence and therapeutic outcomes. The identified transporters represent potential targets for strategies to limit both intestinal colonization and antibiotic resistance in E. faecalis.

Maintaining the efficacy of β-lactam antibiotics against Staphylococcus aureus is a clinical priority given the prevalence of methicillin-resistant S. aureus (MRSA). We previously showed that the pyrimidine analogs 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd) synergize with β-lactams. Here, we extended this by evaluating additional nucleotide metabolism-targeting agents. Gemcitabine (Gem) and mitomycin C (Mito), such as 5-FU and 5-FUrd, exhibited intrinsic anti-MRSA activity and potentiated β-lactams, whereas the purine analog 6-thioguanine (6-TG) showed distinct, often antagonistic effects. Transcriptomic analysis revealed that pyrimidine-targeting agents repress lysine and glutamate biosynthesis, while 6-TG induced these pathways, implicating amino acid metabolism in β-lactam potentiation. Consistent with this, pyrimidine analogs also suppressed GlmS expression, potentially limiting UDP-GlcNAc production required for cell wall synthesis, and synergized with fosfomycin. Fluorescence microscopy confirmed that the potentiation of oxacillin activity by pyrimidine-targeting agents, but not 6-TG, was accompanied by impaired peptidoglycan synthesis. Additionally, glutathione-mediated attenuation of killing implicated reactive oxygen species in the bactericidal activity of cloxacillin combinations. Finally, these agents displayed strong antibiofilm activity, further enhanced in combination with daptomycin and rifampicin. Together, these findings highlight the potential of pyrimidine analogs to potentiate cell wall-targeting antibiotics and identify an important role for modulation of cell wall precursor pathways in this anti-MRSA activity.

Daptomycin (DAP) is a cyclic lipopeptide drug for intravenous application against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). In spite of numerous studies, its mode of action remains controversial. This is partially due to the fact that no attention has been paid to the strong dependency of DAP activity on the cell count-referred to as the inoculum effect when minimal inhibitory concentrations (MICs) are concerned. The first objective is to quantify the inoculum effect of DAP acting against MRSA and, on this basis, review comparisons of data recorded at different cell counts. The data imply that the aqueous DAP concentration in equilibrium with minimally growth-inhibited MRSA is 0.24 μg/mL and the corresponding inhibitory drug load bound per bacterium is Re[bac∗] = 1.0E+7. To directly compare phenomena observed at different cell counts, the samples must agree in Re[bac∗], not in DAP concentration. The second objective has been to render data on liposomal model systems quantitatively comparable to in vivo data. This is achieved by the liposome-MIC-competition assay combining bacteria and liposomes in a single MIC experiment. It suggests an equivalent phosphatidylglycerol concentration of 3.5E-8 μM/(colony-forming units [CFU]/mL), giving rise to a re-assignment of model studies to comparable in vivo systems. Finally, the data obtained here are combined with common estimates regarding the lipid numbers per bacterium, suggesting that the majority of the bacteria-bound DAP is not localized in the membrane but most likely in the cell wall. Off-target binding is a crucial issue for membrane-active antibiotics.