A few commonly used non-antibiotic drugs have recently been associated with changes in gut microbiome composition, but the extent of this phenomenon is unknown. We screened >1000 marketed drugs against 40 representative gut bacterial strains, and found that 24% of the drugs with human targets, including members of all therapeutic classes, inhibited the growth of at least one strain. Particular classes such as the chemically diverse antipsychotics were overrepresented. The effects of human-targeted drugs on gut bacteria are reflected on their antibiotic-like side effects in humans and are concordant with existing human cohort studies, providing in vivo relevance for our screen. Susceptibility to antibiotics and human-targeted drugs correlates across bacterial species, suggesting that non-antibiotics may promote antibiotic resistance. Our results provide a comprehensive resource for future research on drug-microbiome interactions, opening new paths for side effect control and drug repurposing, and broaden our view on antibiotic resistance.

No abstract

Bacterial metabolism plays a fundamental role in gut microbiota ecology and host-microbiome interactions. Yet the metabolic capabilities of most gut bacteria have remained unknown. Here we report growth characteristics of 96 phylogenetically diverse gut bacterial strains across 4 rich and 15 defined media. The vast majority of strains (76) grow in at least one defined medium, enabling accurate assessment of their biosynthetic capabilities. These do not necessarily match phylogenetic similarity, thus indicating a complex evolution of nutritional preferences. We identify mucin utilizers and species inhibited by amino acids and short-chain fatty acids. Our analysis also uncovers media for in vitro studies wherein growth capacity correlates well with in vivo abundance. Further value of the underlying resource is demonstrated by correcting pathway gaps in available genome-scale metabolic models of gut microorganisms. Together, the media resource and the extracted knowledge on growth abilities widen experimental and computational access to the gut microbiota.

RprA is a small regulatory RNA known to weakly affect the translation of σS (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase-induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5′-untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base-pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD-activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off – for instance, later in stationary phase or during biofilm formation – or by conditions that further activate RprA expression via the Rcs two-component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.

Antibiotics are used for fighting pathogens, but also target our commensal bacteria as a side effect, disturbing the gut microbiota composition and causing dysbiosis and disease [1][2][3] . Despite this well-known collateral damage, the activity spectrum of the different antibiotic classes on gut bacteria remains poorly characterized. Having monitored the activities of >1,000 marketed drugs on 38 representative species of the healthy human gut microbiome 4 , we here characterize further the 144 antibiotics therein, representing all major classes. We determined >800 Minimal Inhibitory Concentrations (MICs) and extended the antibiotic profiling to 10 additional species to validate these results and link to available data on antibiotic breakpoints for gut microbes. Antibiotic classes exhibited distinct inhibition spectra, including generation-dependent effects by quinolones and phylogeny-independence by βlactams. Macrolides and tetracyclines, two prototypic classes of bacteriostatic protein synthesis inhibitors, inhibited almost all commensals tested. We established that both kill different subsets of prevalent commensal bacteria, and cause cell lysis in specific cases. This species-specific activity challenges the long-standing divide of antibiotics into bactericidal and bacteriostatic, and provides a possible explanation for the strong impact of macrolides on the gut microbiota composition in animals 5-8 and humans [9][10][11] . To mitigate the collateral damage of macrolides and tetracyclines on gut commensals, we exploited the fact that drug combinations have species-specific outcomes in bacteria 12 and sought marketed drugs, which could antagonize the activity of these antibiotics in abundant gut commensal species. By screening >1,000 drugs, we identified several such antidotes capable of protecting gut species from these antibiotics without compromising their activity against relevant pathogens. Altogether, this study broadens our understanding of antibiotic action on gut commensals, uncovers a previously unappreciated and broad bactericidal effect of prototypical bacteriostatic antibiotics on gut bacteria, and opens avenues for preventing the collateral damage caused by antibiotics on human gut commensals..

Summary

Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment. Escherichia coli ZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of the zntA exporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation of zntA. Biochemical and physical analysis of ZntR R19A demonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands-zinc and DNAfunction together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.

The study shows that microbial-derived proteolytic activity has the capacity to contribute to mucosal homeostasis and may participate in the pathogenesis of inflammatory bowel disease.