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Burkholderia thailandensis has emerged as a nonpathogenic surrogate for Burkholderia pseudomallei , the causative agent of melioidosis, and an important Gram-negative model bacterium for studying the biosynthesis and regulation of secondary metabolism. We recently reported that subinhibitory concentrations of trimethoprim induce vast changes in both the primary and secondary metabolome of B. thailandensis . In the current work, we show that the folate biosynthetic enzyme FolE2 is permissive under standard growth conditions but essential for B. thailandensis in the presence of subinhibitory doses of trimethoprim. Reasoning that FolE2 may serve as an attractive drug target, we screened for and identified ten inhibitors, including dehydrocostus lactone (DHL), parthenolide, and β-lapachone, all of which are innocuous individually but form a chemical-synthetic lethal combination with subinhibitory doses of trimethoprim. We show that DHL is a mechanism-based inhibitor of FolE2 and capture the structure of the covalently inhibited enzyme using X-ray crystallography. In vitro, the combination of subinhibitory trimethoprim and DHL is more potent than Bactrim, the current standard of care against melioidosis. Moreover, unlike Bactrim, this combination does not affect the growth of most commensal and beneficial gut bacteria tested, thereby providing a degree of specificity against B. pseudomallei . Our work provides a path for identifying antimicrobial drug targets and for utilizing binary combinations of molecules that form a toxic cocktail based on metabolic idiosyncrasies of specific pathogens., Competing Interests: Competing interests statement:M.R.S. is a consultant to Merck Co, which had no role in this study. M.R.S. is a co-founder of Cryptyx Bioscience, Inc, which had no role in this study. Some of the authors have filed a patent application related to the research reported in this article.

Reducing antibiotic levels in soil ecosystems is vital to curb the dissemination of antimicrobial resistance genes (ARGs) and mitigate global health threats. However, gaps persist in understanding how antibiotic resistome can be suppressed during antibiotic degradation. Herein, we investigate the efficacy of a biochar biofilm incorporating antibiotics-degrading bacterial strain (Arthrobacter sp. D2) to mitigate antibiotic resistome in non-manured and manure-amended soils with sulfadiazine (SDZ) and trimethoprim (TMP) contamination. Results show that biofilm enhanced SDZ degradation by 83.0% within three days and increased TMP attenuation by 55.4% over 60 days in non-manured soils. In the non-manured black soil, the relative abundance of ARGs increased initially after biofilm inoculation. However, by day 30, it decreased by 20.5% compared to the controls. Moreover, after 7 days, biofilm reduced TMP by 38.5% in manured soils and decreased the total ARG abundance by 19.0%. Thus, while SDZ degradation did not increase sulfonamide resistance genes, TMP dissipation led to a proliferation of insertion sequences and related TMP resistance genes. This study underscores the importance of antibiotic degradation in reducing related ARGs while cautioning against the potential proliferation and various ARGs transfer by resistant microorganisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen Streptococcus pneumoniae . We explored a collection of 662 S . pneumoniae genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the folA and sulA loci are responsible for TMP non-susceptibility in S. pneumoniae and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of in silico analysis-wet-lab validation-diagnostic tool building could be adapted to explore AMR in other combinations of bacteria-antibiotic., Importance: In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae . These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility., Competing Interests: The authors declare no conflict of interest.

Nucleocytoplasmic large DNA viruses (NCLDVs; also called giant viruses), constituting the phylum Nucleocytoviricota, can infect a wide range of eukaryotes and exchange genetic material with not only their hosts but also prokaryotes and phages. A few NCLDVs were reported to encode genes conferring resistance to beta‑lactam, trimethoprim, or pyrimethamine, suggesting that they are potential vehicles for the transmission of antibiotic resistance genes (ARGs) in the biome. However, the incidence of ARGs across the phylum Nucleocytoviricota, their evolutionary characteristics, their dissemination potential, and their association with virulence factors remain unexplored. Here, we systematically investigated ARGs of 1416 NCLDV genomes including those of almost all currently available cultured isolates and high-quality metagenome-assembled genomes from diverse habitats across the globe. We reveal that 39.5% of them carry ARGs, which is approximately 37 times higher than that for phage genomes. A total of 12 ARG types are encoded by NCLDVs. Phylogenies of the three most abundant NCLDV-encoded ARGs hint that NCLDVs acquire ARGs from not only eukaryotes but also prokaryotes and phages. Two NCLDV-encoded trimethoprim resistance genes are demonstrated to confer trimethoprim resistance in Escherichia coli. The presence of ARGs in NCLDV genomes is significantly correlated with mobile genetic elements and virulence factors., (© 2024. The Author(s).)

In this work, multicomponent trimethoprim-based pharmaceutical solid systems were developed by mechanochemistry, using coformers from the GRAS list and other active pharmaceutical ingredients. The choice of coformers took into account their potential to increase the aqueous solubility/dissolution rate of TMP or its antibacterial activity. All the binary systems were characterized by thermal analysis, powder X-ray diffraction and infrared spectroscopy, and 3 equimolar systems with FTIR pointing to salts, and 4 eutectic mixtures were identified. The intrinsic dissolution rate of TMP in combination with nicotinic acid (a salt) and with paracetamol (eutectic mixture) were 25% and 5% higher than for pure TMP, respectively. For both Gram-positive and -negative strains, the antibacterial activity of TMP with some of the coformers was improved, since the dosage used was lower than the TMP control. A significant increase in antibacterial activity against E. coli was found for the eutectic mixture with curcumin, with the best results being obtained for the eutectic and equimolar mixtures with ciprofloxacin. Combining trimethoprim with coformers offers an interesting alternative to using trimethoprim alone: multicomponent forms with enhanced TMP dissolution rates were identified, as well as combinations showing enhanced antibacterial activity relatively to the pure drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Antimicrobial resistance is a global health threat, compounded by the reduction in the discovery of new antibiotics. A repurposed drugs-based approach could provide a viable alternative for the treatment of multidrug-resistant (MDR) bacterial infections. In this study, we sought to evaluate the in vitro efficacy of a novel drug combination, polymyxin B/trimethoprim (PT) + rifampin on MDR isolates from patients with bacterial keratitis in India., Methods: Forty-three isolates, which included 20 Staphylococcus aureus , 19 Pseudomonas aeruginosa , 3 Pseudomonas stutzeri , and 1 Acinetobacter baumannii , were evaluated for their antibiotic resistance by minimum inhibitory concentration (MIC). Fractional Inhibitory Concentration Index (FICI) testing was performed to measure the antimicrobial impact of PT + rifampin in combination., Results: Among S. aureus isolates, 100% were resistant to at least 1 antibiotic class, 12 (60%) were MDR, and 14 (70%) were classified as methicillin-resistant. Among the gram-negative isolates, >90% were classified as MDR. Fractional Inhibitory Concentration (FIC) testing revealed that PT + rifampin was effective in completely inhibiting growth of all isolates while also displaying additive or synergistic activity in approximately 70% of the strains. Mean FICI values were 0.753 ± 0.311 and 0.791 ± 0.369 for S. aureus and gram-negative isolates, respectively, and a >2-fold reduction in MIC was measured for both PT and rifampin when tested in combination versus alone., Conclusions: Our data demonstrate the ability of PT + rifampin to eliminate all isolates tested, even those conferring MDR, highlighting the promise of this drug combination for the treatment of bacterial keratitis., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship., (© 2024. The Author(s).)

Synergistic control of the risks posed by emerging antimicrobials and antibiotic resistance genes (ARGs) is crucial for ensuring ecological safety. Although electrogenic respiration can enhance the biodegradation of several antimicrobials and reduce ARGs accumulation, the association mechanisms of antimicrobial biodegradation (trimethoprim, TMP) with the fate of the antimicrobial resistome remain unclear. Here, the biotransformation pathway of TMP, microbial associations, and functional gene profiles (e.g., degradation, antimicrobial resistance, and electron transfer) were analyzed. The results showed that the microbial electrogenic respiration significantly enhanced the biodegradation of TMP, especially with a cosubstrate sodium acetate supply. Electroactive bacteria enriched in the electrode biofilm positively correlated with potential TMP degraders dominated in the planktonic communities. These cross-niche microbial associations may contribute to the accelerated catabolism of TMP and extracellular electron transfer. Importantly, the evolution and dissemination of overall ARGs and mobile genetic elements (MGEs) were significantly weakened due to the enhanced cometabolic biodegradation of TMP. This study provides a promising strategy for the synergistic control of the water ecological risks of antimicrobials and their resistome, while also highlighting new insights into the association of antimicrobial biodegradation with the evolution of the resistome in an electrically integrated biological process.

Resistance mechanisms are a shelter for Acinetobacter baumannii to adapt to our environment which causes difficulty for the infections to be treated and WHO declares this organism on the top of pathogens priority for new drug development. The most common mechanism that develops drug resistance is the overexpression of the efflux pump, especially Resistance-nodulation-cell division (RND) family, to almost most antibiotics. The study is designed to detect RND efflux pump genes in A. baumannii, and its correlation to multidrug resistance, in particular, the carbapenems resistance Acinetobacter baumannii (CRAB), and using different inhibitors that restore the antibiotic susceptibility of imipenem. Clinical A. baumannii isolates were recovered from different Egyptian hospitals in Intensive care unit (ICU). The expression of genes in two strains was analyzed using RT-PCR before and after inhibitor treatment. About 100 clinical A. baumannii isolates were recovered and identified and recorded as MDR strains with 75% strains resistant to imipenem. adeB, adeC, adeK, and adeJ were detected in thirty- seven the carbapenems resistance Acinetobacter baumannii (CRAB) strains. Cinnamomum verum oil, Trimethoprim, and Omeprazole was promising inhibitor against 90% of the carbapenems resistance Acinetobacter baumannii (CRAB) strains with a 2-6-fold decrease in imipenem MIC. Downregulation of four genes was associated with the addition of those inhibitors to imipenem for two the carbapenems resistance Acinetobacter baumannii (CRAB) (ACN15 and ACN99) strains, and the effect was confirmed in 24 h killing kinetics. Our investigation points to the carbapenems resistance Acinetobacter baumannii (CRAB) strain's prevalence in Egyptian hospitals with the idea to revive the imipenem activity using natural and chemical drugs as inhibitors that possessed high synergistic activity., (© 2024. The Author(s).)

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus . Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis , exerts whole cell activity against M. abscessus . Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus . As observed in M. tuberculosis , PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus . PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus ., Competing Interests: C.J.B., A.E.M., E.B.N., D.B.O., and R.W. are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

There is growing consensus that under physiological conditions, collecting duct H + secretion is independent of epithelial Na + channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H + excretion by blocking renal H + -K + -ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H + excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K + retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K + depletion was used to increase renal H + -K + -ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H + -K + -ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K + depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H + -K + -ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H + excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H + excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K + diet highlights the importance of renal H + -K + -ATPase-mediated H + secretion in states of K + depletion. NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K + retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na + channel-dependent K + retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H + secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H + excretion.

Importance: Bacteria employ a number of mechanisms to adapt to antibiotics. Mutations in transcriptional regulators alter the expression levels of genes that can change the susceptibility of bacteria to antibiotics. Two-component signaling proteins are a major class of signaling molecule used by bacteria to regulate transcription. In previous work, we found that mutations in MgrB, a feedback regulator of the PhoQP two-component system, conferred trimethoprim tolerance to Escherichia coli . Here, we elucidate how mutations in MgrB have a domino-like effect on the gene regulatory network of E. coli . As a result, pervasive perturbation of gene regulation ensues. Depending on the environmental context, this pervasive deregulation is either adaptive or maladaptive. Our study sheds light on how deregulation of gene expression can be beneficial for bacteria when challenged with antibiotics, and why regulators like MgrB may have evolved in the first place., Competing Interests: The authors declare no conflict of interest.

The opportunistic apicomplexan parasite Toxoplasma gondii is the etiologic agent for toxoplasmosis, which can infect a widespread range of hosts, particularly humans and warm-blooded animals. The present chemotherapy to treat or prevent toxoplasmosis is deficient and is based on diverse drugs such as atovaquone, trimethoprim, spiramycine, which are effective in acute toxoplasmosis. Therefore, a safe chemotherapy is required for toxoplasmosis considering that its responsible agent, T. gondii, provokes severe illness and death in pregnant women and immunodeficient patients. A certain disadvantage of the available treatments is the lack of effectiveness against the tissue cyst of the parasite. A safe chemotherapy to combat toxoplasmosis should be based on the metabolic differences between the parasite and the mammalian host. This article covers different relevant molecular targets to combat this disease including the isoprenoid pathway (farnesyl diphosphate synthase, squalene synthase), dihydrofolate reductase, calcium-dependent protein kinases, histone deacetylase, mitochondrial electron transport chain, etc., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo., (© 2023. The Author(s).)

This study investigates the effects, conversions, and resistance induction, following the addition of 150 μg·L -1 of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), in a laboratory-scale micro-aerated anaerobic membrane bioreactor (MA-AnMBR). TMP and SMX were removed at 97 and 86%, indicating that micro-aeration did not hamper their removal. These antibiotics only affected the pH and biogas composition of the process, with a significant change in pH from 7.8 to 7.5, and a decrease in biogas methane content from 84 to 78%. TMP was rapidly adsorbed onto the sludge and subsequently degraded during the long solids retention time of 27 days. SMX adsorption was minimal, but the applied hydraulic retention time of 2.6 days was sufficiently long to biodegrade SMX. The levels of three antibiotic-resistant genes (ARGs) (sul1, sul2, and dfrA1) and one mobile genetic element biomarker (intI1) were analyzed by qPCR. Additions of the antibiotics increased the relative abundances of all ARGs and intI1 in the MA-AnMBR sludge, with the sul2 gene folding 15 times after 310 days of operation. The MA-AnMBR was able to reduce the concentration of antibiotic-resistant bacteria (ARB) in the permeate by 3 log.

Trimethoprim (TMP) is a low-cost, widely prescribed antibiotic. Its effectiveness is increasingly challenged by the spread of genes coding for TMP-resistant dihydrofolate reductases: dfrA, and the lesser-known, evolutionarily unrelated dfrB. Despite recent reports of novel variants conferring high level TMP resistance (dfrB10 to dfrB21), the prevalence of dfrB is still unknown due to underreporting, heterogeneity of the analyzed genetic material in terms of isolation sources, and limited bioinformatic processing. In this study, we explored a coherent set of shotgun metagenomic sequences to quantitatively estimate the abundance of dfrB gene variants in aquatic environments. Specifically, we scanned sequences originating from influents and effluents of municipal sewage treatment plants as well as river-borne microbiomes. Our analyses reveal an increased prevalence of dfrB1, dfrB2, dfrB3, dfrB4, dfrB5, and dfrB7 in wastewater microbiomes as compared to freshwater. These gene variants were frequently found in genomic neighborship with other resistance genes, transposable elements, and integrons, indicating their mobility. By contrast, the relative abundances of the more recently discovered variants dfrB9, dfrB10, and dfrB13 were significantly higher in freshwater than in wastewater microbiomes. Moreover, their direct neighborship with other resistance genes or markers of mobile genetic elements was significantly less likely. Our findings suggest that natural freshwater communities form a major reservoir of the recently discovered dfrB gene variants. Their proliferation and mobilization in response to the exposure of freshwater communities to selective TMP concentrations may promote the prevalence of high-level TMP resistance and thus limit the future effectiveness of antimicrobial therapies., (© 2023. The Author(s).)

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Two series of quinazolinone derivatives were designed and synthesized as dihydrofolate reductase (DHFR) inhibitors. All compounds were evaluated for their antibacterial and antitumor activities. Antibacterial activity was evaluated against three strains of Gram-positive and Gram-negative bacteria. Compound 3d exhibited the highest inhibitory activity against Staphylococcus aureus DHFR (SaDHFR) with IC 50 of 0.769 ± 0.04 μM compared to 0.255 ± 0.014 μM for trimethoprim. Compound 3e was also more potent than trimethoprim against Escherichia coli DHFR (EcDHFR) with IC 50 of 0.158 ± 0.01 μM and 0.226 ± 0.014 μM, respectively. Compound 3e exhibited a promising antiproliferative effect against most of the tested cancer cells. It also showed potent activity against leukemia (CCRF-CEM, and RPMI-8226); lung NCI-H522, and CNS U251 with GI% of 65.2, 63.22, 73.28, and 97.22, respectively. The cytotoxic activity of compound 3e was almost half the activity of doxorubicin against CCRF-CEM cell line with IC 50 of 1.569 ± 0.06 μM and 0.822 ± 0.03 µM, respectively. In addition, compound 3e inhibited human DHFR with IC 50 value of 0.527 ± 0.028 µM in comparison to methotrexate (IC 50  = 0.118 ± 0.006 µM). Compound 3e caused an arrest of the cell cycle mainly at the S phase and caused a rise in the overall apoptotic percentage from 2.03% to 48.51%. (23.89-fold). Treatment of CCRF-CEM cells with compound 3e produced a significant increase in the active caspase-3 level by 6.25-fold compared to untreated cells. Molecular modeling studies were performed to evaluate the binding pattern of the most active compounds in the bacterial and human DHFR., (© 2023 Wiley Periodicals LLC.)

Trimethoprim is predicted to inhibit several thiamine transporters, including the primary thiamine intestinal absorptive transporter, ThTR-2, and the hepatic and renal organic cation transporters, OCT1, OCT2, and MATEs. To investigate the effect of trimethoprim on thiamine absorption, studies were conducted in cells, mice, and healthy volunteers and supported by use of real-world data. In a randomized, crossover clinical study, seven healthy volunteers were given a single oral dose of thiamine or thiamine plus trimethoprim, followed by blood sampling. The thiamine area under the curve (AUC) increased with trimethoprim co-administration (P value = 0.031). Similar results were seen in mice. Trimethoprim appeared to act on thiamine absorption through inhibition of hepatic OCT1 as evidenced from its ability to modulate levels of isobutyrylcarnitine and propionylcarnitine, OCT1 biomarkers identified from metabolomic analyses. Real-world data further supported this finding, showing an association between trimethoprim use and higher levels of triglycerides, LDL cholesterol, and total cholesterol, consistent with OCT1 inhibition (P values: 2.2 × 10 -16 , 5.75 × 10 -7 , and 5.82 × 10 -7 , respectively). These findings suggest that trimethoprim increases plasma levels of thiamine by inhibiting hepatic OCT1. Trimethoprim reduced urinary excretion and clearance of biomarkers for OCT2 and MATEs, consistent with inhibition of renal organic cation transporters. This inhibition did not appear to play a role in the observed increases in thiamine levels. This study highlights the potential for drug-nutrient interactions involving transporters, in addition to transporters' established role in drug-drug interactions., (© 2023 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host's environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replaced Escherichia coli's chromosomal folA gene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons of folA genes from trimethoprim-sensitive Listeria grayi and trimethoprim-resistant Neisseria sicca. The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5'-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5'-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host's environment in shaping the codon bias compatibility of horizontally transferred genes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

With the subsisting restrictions on the use of antibiotics in poultry production, the use of plant extracts has shown some promising antimicrobial capacity similar to antibiotics; however, such capacity is largely dependent on their total polyphenol concentration and profile. Given the emerging antimicrobial potential of red osier dogwood (ROD) extract, the study aimed to investigate the pharmacodynamic effect of ROD extract on the ileal and cecal microbiota of broiler chickens challenged orally with Salmonella Enteritidis (SE). A 21 d 4 × 2 factorial experiment was conducted based on 2 main factors, including diets and SE challenge. A total of 384 one-day-old mixed-sex Cobb-500 broiler chicks were randomly allotted to 4 dietary treatments; Negative control (NC), NC + 0.075 mg trimethoprim-sulfadiazine (TMP/SDZ)/kg of diet, and NC containing either 0.3 or 0.5% ROD extract. On d 1, half of the birds were orally challenged with 0.5 mL of phosphate-buffered saline (Noninfected group) and the remaining half with 0.5 mL of 3.1 × 10 5 CFU/mL SE (Infected group). Dietary treatments were randomly assigned to 8 replicate cages at 6 birds/cage. On d 21, 10 birds/treatment were euthanized and eviscerated to collect ileal and cecal digesta for gut microbiota analysis. The ileal and cecal microbiota was dominated by phyla Firmicutes, Proteobacteria, and Actinobacteriota. The SE infection decreased (P < 0.05) the relative abundance of Proteobacteria and Actinobacteriota in the ileum and ceca, respectively, however, it increased (P < 0.05) Proteobacteria in the ceca. Both 0.3 and 0.5% ROD extracts (P < 0.05) depressed the relative abundance of Actinobacteriota in the ileum but marginally improved (P < 0.05) it in the ceca compared to the TMP/SDZ treatment. Dietary TMP/SDZ increased (P < 0.05) genus Bifidobacterium at the ileal and cecal segments compared to other treatments. Dietary 0.3 and 0.5% marginally improved (P < 0.05) Bifidobacterium in the ceca and depressed (P < 0.05) Weissella and was comparably similar to TMP/SDZ in the ileum. Regardless of the dietary treatments and SE infection, alpha diversity differed (P < 0.05) between ileal and cecal microbiota. Beta diversity was distinct (P < 0.05) in both ileal and cecal digesta along the SE infection model. Conclusively, both ROD extract levels yielded a pharmacodynamic effect similar to antibiotics on ileal and cecal microbiota., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Concerns about antibiotic-resistant Gram-negative pathogens are escalating, and accordingly siderophore-based intracellular antibiotic delivery is attracting more attention as an effective means to overcome these infections. Despite the successful clinical translation of this strategy, the delivery potential of siderophores has been limited to periplasm targeting, and this has appreciably restricted the repertoire of applicable antibiotics. To overcome this shortcoming of the current technology, this study focused on investigating the capability of simple bidentate catechol analogs to function as vehicles for cytoplasmic antibiotic delivery. Specifically, by employing trimethoprim, an inhibitor of dihydrofolate reductase located in the cytoplasm, as a model antibiotic, a chemical library of chelator-antibiotic conjugates featuring four different catechol analogs was prepared. Then, their various pharmacological properties and antimicrobial activities were evaluated. Analysis of these characterization data led to the identification of the active conjugates exhibiting notable iron- and trimethoprim-dependent potency against Escherichia coli . Further characterization of these hit molecules using E. coli mutant strains revealed that 2,3-dihydroxybenzoate could effectively deliver several corresponding conjugates to the cytoplasm by exploiting the siderophore uptake machineries present across the outer and inner membranes, originally designated for the native siderophore of E. coli , enterobactin. Considering the synthetic simplicity, such a catechol analog could have appreciable usage in potentiating cytoplasm-active antibiotics against recalcitrant Gram-negative pathogens.

The most suitable method to treat hydrocephalus disease is to insert a shunt catheter that drains the cerebral spinal fluid (CSF); however, shunt implantation is often associated with various bacterial infections. In this study, antibiotic-loaded nanospheres were prepared using the solvent evaporation technique and coated on an antibiotic-impregnated shunt surface to promote shunt antibacterial properties. Clindamycin (CDM) and rifampicin (RIF) were in combination loaded in a single nanosphere, whereas trimethoprim (TMP) was loaded individually in triblock copolymers [(d,l-lactide-random-ε-caprolactone)-block-poly(ethylene glycol)-block-(d,l-lactide-random-ε-caprolactone)] (PLEC). The drug-loading content, encapsulation efficiency, yield, size, and zeta potential of the antibiotic-loaded nanospheres were measured. The results showed that the drug-loading content of clindamycin- and rifampicin-loaded nanospheres (CDM/RIF-NPs) was approximately 3% and 8%, respectively, at a drug to polymer ratio of 1:2. In addition, trimethoprim-loaded nanospheres (TMP-NPs) showed nearly 7% drug loading at equal drug and polymer ratios. The amount of drug release was determined before and after the coating of nanospheres on the shunt surface. In addition, in silico molecular docking studies indicated the good chemical interaction of these antibiotics with PLEC, and the results were consistent with those of impregnation studies. Antibacterial tests of coated external ventricular drainage showed antibacterial activity for up to 21 days.

Background: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids., Methods: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0-D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14-D21), during 5 days of oral TMS treatment (D22-D26) and after discharge from the hospital up to 6 months later (D27-D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples., Results: Hospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3")-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased 6 months later., Conclusions: Oral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimise long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment. Video Abstract., (© 2023. The Author(s).)

The looming threat of a "post-antibiotic era" has been caused by a rapid rise in antibacterial resistance and subsequent depletion of effective antibiotic agents in the clinic. An efficient strategy to address this shortfall lies in the reengineering of pre-existing and commercially available antibiotic drugs. This is exemplified by dimerization, a design concept in which two pharmacophores are covalently linked to form a new chemical entity. The cage hydrocarbons cubane (1), bicyclo[2.2.2]octane (BCO) (2), adamantane (3), and bicyclo[1.1.1]pentane (BCP) (4) present themselves as an attractive family of linkers in this regard. In this report, all four hydrocarbon cages were employed as linkers in a series of dimers based on the commercially available antibiotics trimethoprim and tedizolid. A detailed synthetic roadmap for the protection and deprotection of each pharmacophore is outlined. Several members of the trimethoprim series showed activity on par with that of their trimethoprim progenitor, although this was not the case for the tedizolid series. The design strategy outlined herein highlights the utility of the group as a platform for the rapid and modular construction of future novel antibiotics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The valorization of poultry byproducts, like feathers (processed to feather meal), in animal feed could contribute to the presence of veterinary drugs, including antibiotics. An animal study was carried out to study the fate of sulfadiazine, trimethoprim, and oxytetracycline in feathers, plasma, and droppings of broiler chickens. Cage and floor housing, different from current farm practices, were studied. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A longer presence of antibiotics was observed in feathers compared to plasma, with sulfadiazine being present the most. The internal presence (via blood) and the external presence (via droppings) of antibiotics in/on feathers were shown. Analysis of Escherichia coli populations, from droppings and feathers, highlighted that resistant bacteria could be transferred from droppings to feathers in floor-housed animals. The overall results suggest that feathers are a potential reservoir of antimicrobial residues and could contribute to the selection of antibiotic-resistant bacteria in the environment, animals, and humans.

Objective: To determine the minimum inhibitory concentration (MIC) distribution of antibacterial drugs and the susceptibility of non-tuberculous mycobacterial (NTM) isolates to provide a reference basis for the clinical selection of an effective starting regimen. Methods: The common clinical isolates of NTM in the respiratory tract, which met the standards of the American Thoracic Society for NTM lung disease, were collected. The MICs of 81 isolates were determined using the microbroth dilution method (Thermo Fisher Scientific, USA), as recommended by the Clinical and Laboratory Standards Institute, USA. Results: Included were 43 Mycobacterium avium complex (MAC) strains, 24  M. abscessus complex (MAB) strains, and 14  M. kansasii strains. The sensitivity rates of MAC to clarithromycin and amikacin were 81.4% and 79.1%, respectively, while the sensitivity rates to linezolid and moxifloxacin were only 20.9% and 9.3%; the MIC of rifabutin was the lowest (MIC50% was just 2 μg/mL). After incubation for 3-5 days, the sensitivity rate of MAB to clarithromycin was 87.5%; this decreased to 50% after 14 days' incubation. Most of them were susceptible to amikacin (91.6%), and most were resistant to moxifloxacin (95.8%), ciprofloxacin (95.8%), imipenem (95.8%), amoxicillin/clavulanate (95.8%), tobramycin (79.1%), doxycycline (95.8%) and trimethoprim/sulfamethoxazole (95.8%). intermediate (83.3%) and resistant (16.7%) to cefoxitin. The susceptibility to linezolid was only 33.3%. The sensitivity and resistance breakpoints of tigecycline were set to ≤0.5 and ≥8 μg/mL, respectively, and the sensitivity and resistance rates were 50% and 0%, respectively. M. kansasii was susceptible to clarithromycin, amikacin, linezolid, moxifloxacin, rifampicin and rifabutin (100%). Discussion: In Wenzhou, clarithromycin, amikacin and rifabutin have good antibacterial activity against MAC, while linezolid and moxifloxacin have high resistance. Amikacin and tigecycline have strong antibacterial activity against MAB, while most other antibacterial drugs are resistant to varying degrees. Most antibacterial drugs are susceptible to M . kansasii and have good antibacterial activity. Conclusion: The identification of NTM species and the detection of their MICs have certain guiding values for the treatment of NTM lung disease.Key MessageThe three most common respiratory non-tuberculous mycobacterial (NTM) isolates with clinical significance in the Wenzhou area were tested for drug susceptibility. The broth microdilution method was used to determine the minimum inhibitory concentration distribution of antibacterial drugs and the susceptibility of NTM isolates to provide a reference basis for the clinical selection of an effective starting regimen.

Antimicrobial-resistant Salmonella enterica poses a significant public health concern worldwide. However, the dissemination of Salmonella enterica among food animals in eastern China has not been fully addressed. Here, we demonstrated the antimicrobial resistance (AMR) patterns and the whole-genome characterization of 105 S. enterica isolates from 1,480 fecal samples and anal swabs collected from 22 different farms (chickens, ducks, and pigs) and two live animal markets located in Zhejiang and Fujian Provinces in eastern China in 2019. The prevalence of isolates in duck farms (19.17%, 23/120) was statistically significantly higher ( P <  0.001) than that in chicken farms (6.61%, 37/523) and pig farms (3.50%, 7/200). Among these isolates, 75.26% (79/105) were multidrug resistant, with the highest rates of resistance to tetracycline (76.20%) and ampicillin (67.62%) and the lowest resistance rate to meropenem (0.00%). The serotypes were consistent with sequence types and were closely related to the sampling animal species and sites. S. enterica serotype Kentucky (20.95%, 22/105) was the most frequent serotype and harbored more AMR patterns and genes than others. Furthermore, IncFII(S) and IncHI2 were the most prevalent replicons. A total of 44 acquired AMR genes were found. Among those genes, aac(6')-Iaa , bla TEM-1B , floR , dfrA14 , fosA7 , mph (A), qnrS1 , sul1 , tet (A), and ARR-3 were the dominant AMR genes mediating the AMR toward aminoglycosides, β-lactams, phenicol, trimethoprim, fosfomycin, macrolide, quinolone, sulfonamides, tetracycline, and rifampin, respectively. The consistency of acquired AMR genes with AMR phenotypes for ampicillin, ceftiofur, ceftazidime, meropenem, sulfamethoxazole-trimethoprim, and tetracycline was >90%. Together, our study highlights the application of whole-genome sequencing to assess veterinary public health threats. IMPORTANCE Public health is a significant concern in China, and the foodborne pathogen Salmonella, which is spread via the animal-borne food chain, plays an important role in the overall disease burden in China annually. The development of advanced sequencing technologies has introduced a new way of understanding emerging pathogens. However, the routine surveillance application of this method in China remains in its infancy. Here, we applied a pool of all isolates from the prevalence data in Zhejiang and Fujian for whole-genome sequencing and combined these data with the cutting-edge bioinformatic analysis pipeline for one-step determination of the complete genetic makeup for all 105 genomes. The illustrated method could provide a cost-effective approach, without labor-intensive laboratory characterization, for predicting serotypes, genotypes, plasmid types, antimicrobial resistance genes, and virulence genes, and thus would provide essential knowledge for emerging pathogens. Our findings and perspectives are essential for delivering updated knowledge on foodborne pathogens in an understudied region in China.

Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion protein's abundance. However, in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ∼1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of fluorescence-activated cell sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as "C12", contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD ( i.e. , R12Y/G67S/Y100I). This domain was similarly responsive to TMP with respect to dose response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors toward its basal destabilization. However, the combination of all the C12 mutations was required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to IκBα and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD and improving the use of DDs overall, not only as a chemical biology tool but for gene therapy avenues as well.

The co-exposure of antibiotics has important effects on antibiotic resistance genes (ARGs) and microbial community aggregation in wastewater treatment plants (WWTPs). However, it is unclear whether differences in historical antibiotic exposure stress can determine responses of microbes and ARGs to combined antibiotics. By selecting a high concentration (30 mg·L -1 ) of sulfamethoxazole (SMX) and trimethoprim (TMP) as historical exposure stress conditions, the effects of SMX and TMP-combined pollution on ARGs, bacterial communities, and their interactions were explored in short-term experiments. Based on high-throughput quantitative PCR, a total of 13 ARGs were detected, and the absolute abundance was 2.21-5.42 copies·μL -1 (logarithm, DNA, the same below). Among them, sul2, ermB, mefA , and tetM-01 were the main subtypes in the samples, and the absolute abundance was between 2.95 and 5.40 copies·μL -1 . The combined contamination of SMX and TMP could cause the enrichment of ARGs and mobile genetic elements (MGEs); however, their effects on each subtype were different, and the historical legacy effect of SMX was higher than that of TMP. Under the different exposure histories, the co-occurrence and co-exclusion patterns existed between ARGs. Moreover, MGEs (especially intI-1 ) were significantly correlated with sulfonamides ( sul1 and sul2 ), tetracyclines[ tet(32) ], and macrolide-lincosamide-streptogramin (MLSB) resistance genes ( ermB ). Based on the full-scale classification of microorganisms, it was found that the microbial community structure of various groups responded differently to combined pollution, and the conditionally abundant taxa (CAT) were obviously enriched. Thauera, Pseudoxanthomonas, and Paracoccus were the dominant resistant bacterial genera. Furthermore, a total of 31 potential hosts of ARGs were identified with network analysis, which were dominated with conditionally rare taxa (CRT). Particularly, Candidatus&#95;Alysiosphaera and Fusibacter were positively correlated with most of the ARGs, being the common protentional hosts. Importantly, some rare genera (RT, Variibacter, Aeromonas, Cloacibacterium , etc.) were potential hosts of transposon IS613 , which played an important role in the proliferation and spread of ARGs. In conclusion, this study revealed the legacy effects of historical antibiotic stress on ARGs and their hosts, which could provide new ideas and theoretical basis for reducing ARGs pollution in WWTPs.

Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double-strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could resensitize antibiotic-resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic-resistance mutations., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (m D cTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for m D cTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and m D cTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations., Competing Interests: Declarations of interest S. Suzuki, A.N., T.Y., and S.T. are co-inventors of a patent application related to this work. The other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

BACKGROUNDSeveral molecular imaging strategies can identify bacterial infections in humans. PET affords the potential for sensitive infection detection deep within the body. Among PET-based approaches, antibiotic-based radiotracers, which often target key bacterial-specific enzymes, have considerable promise. One question for antibiotic radiotracers is whether antimicrobial resistance (AMR) reduces specific accumulation within bacteria, diminishing the predictive value of the diagnostic test.METHODSUsing a PET radiotracer based on the antibiotic trimethoprim (TMP), [11C]-TMP, we performed in vitro uptake studies in susceptible and drug-resistant bacterial strains and whole-genome sequencing (WGS) in selected strains to identify TMP resistance mechanisms. Next, we queried the NCBI database of annotated bacterial genomes for WT and resistant dihydrofolate reductase (DHFR) genes. Finally, we initiated a first-in-human protocol of [11C]-TMP in patients infected with both TMP-sensitive and TMP-resistant organisms to demonstrate the clinical feasibility of the tool.RESULTSWe observed robust [11C]-TMP uptake in our panel of TMP-sensitive and -resistant bacteria, noting relatively variable and decreased uptake in a few strains of P. aeruginosa and E. coli. WGS showed that the vast majority of clinically relevant bacteria harbor a WT copy of DHFR, targetable by [11C]-TMP, and that despite the AMR, these strains should be "imageable." Clinical imaging of patients with [11C]-TMP demonstrated focal radiotracer uptake in areas of infectious lesions.CONCLUSIONThis work highlights an approach to imaging bacterial infection in patients, which could affect our understanding of bacterial pathogenesis as well as our ability to better diagnose infections and monitor response to therapy.TRIAL REGISTRATIONClinicalTrials.gov NCT03424525.FUNDINGInstitute for Translational Medicine and Therapeutics, Burroughs Wellcome Fund, NIH Office of the Director Early Independence Award (DP5-OD26386), and University of Pennsylvania NIH T32 Radiology Research Training Grant (5T32EB004311-12).

Endogenous biomarkers for transporter-mediated drug-drug interaction (DDI) predictions represent a promising approach to facilitate and improve conventional DDI investigations in clinical studies. This approach requires high sensitivity and specificity of biomarkers for the targets of interest (e.g., transport proteins), as well as rigorous characterization of their kinetics, which can be accomplished utilizing physiologically-based pharmacokinetic (PBPK) modeling. Therefore, the objective of this study was to develop PBPK models of the endogenous organic cation transporter (OCT)2 and multidrug and toxin extrusion protein (MATE)1 substrates creatinine and N 1 -methylnicotinamide (NMN). Additionally, this study aimed to predict kinetic changes of the biomarkers during administration of the OCT2 and MATE1 perpetrator drugs trimethoprim, pyrimethamine, and cimetidine. Whole-body PBPK models of creatinine and NMN were developed utilizing studies investigating creatinine or NMN exogenous administration and endogenous synthesis. The newly developed models accurately describe and predict observed plasma concentration-time profiles and urinary excretion of both biomarkers. Subsequently, models were coupled to the previously built and evaluated perpetrator models of trimethoprim, pyrimethamine, and cimetidine for interaction predictions. Increased creatinine plasma concentrations and decreased urinary excretion during the drug-biomarker interactions with trimethoprim, pyrimethamine, and cimetidine were well-described. An additional inhibition of NMN synthesis by trimethoprim and pyrimethamine was hypothesized, improving NMN plasma and urine interaction predictions. To summarize, whole-body PBPK models of creatinine and NMN were built and evaluated to better assess creatinine and NMN kinetics while uncovering knowledge gaps for future research. The models can support investigations of renal transporter-mediated DDIs during drug development., (© 2022 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Dose-response relationships are a general concept for quantitatively describing biological systems across multiple scales, from the molecular to the whole-cell level. A clinically relevant example is the bacterial growth response to antibiotics, which is routinely characterized by dose-response curves. The shape of the dose-response curve varies drastically between antibiotics and plays a key role in treatment, drug interactions, and resistance evolution. However, the mechanisms shaping the dose-response curve remain largely unclear. Here, we show in Escherichia coli that the distinctively shallow dose-response curve of the antibiotic trimethoprim is caused by a negative growth-mediated feedback loop: Trimethoprim slows growth, which in turn weakens the effect of this antibiotic. At the molecular level, this feedback is caused by the upregulation of the drug target dihydrofolate reductase (FolA/DHFR). We show that this upregulation is not a specific response to trimethoprim but follows a universal trend line that depends primarily on the growth rate, irrespective of its cause. Rewiring the feedback loop alters the dose-response curve in a predictable manner, which we corroborate using a mathematical model of cellular resource allocation and growth. Our results indicate that growth-mediated feedback loops may shape drug responses more generally and could be exploited to design evolutionary traps that enable selection against drug resistance., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background and Purpose: The zinc finger transcription factor Snail is aberrantly activated in many human cancers and strongly associated with poor prognosis. As a transcription factor, Snail has been traditionally considered an 'undruggable' target. Here, we identified a potent small-molecule inhibitor of Snail, namely trimethoprim, and investigated its potential antitumour effects and the underlying mechanisms., Experimental Approach: The inhibitory action of trimethoprim on Snail protein and the related molecular mechanisms were revealed by molecular docking, biolayer interferometry, immunoblotting, immunoprecipitation, qRT-PCR, pull-down and cycloheximide pulse-chase assays. The anti-proliferative and anti-metastatic effects of trimethoprim via targeting Snail were tested in multiple cell-based assays and animal models., Key Results: This study identified trimethoprim, an antimicrobial drug, as a potent antitumour agent via targeting Snail. Molecular modelling analysis predicted that trimethoprim directly binds to the arginine-174 pocket of Snail protein. We further discovered that trimethoprim strongly interrupts the interaction of Snail with CREB-binding protein (CBP)/p300, which consequently suppresses Snail acetylation and promotes Snail degradation through the ubiquitin-proteasome pathway. Furthermore, trimethoprim sufficiently inhibited the proliferation, epithelial-mesenchymal transition (EMT) and migration of cancer cells in vitro via specifically targeting Snail. More importantly, trimethoprim effectively reduced Snail-driven tumour growth and metastasis to vital organs such as lung, bone and liver., Conclusions and Implications: These findings indicate, for the first time, that trimethoprim suppresses tumour growth and metastasis via targeting Snail. This study provides insights for a better understanding of the anticancer effects of trimethoprim and offers a potential anticancer therapeutic agent for clinical treatment., (© 2021 The British Pharmacological Society.)

Background: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture., Methods: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94)., Results: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC 90  > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%)., Conclusions: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing., (© 2022. The Author(s).)

Purpose: Pseudomonas aeruginosa is a leading cause of corneal infections. Recently, we discovered an antimicrobial drug combination, polymyxin B/trimethoprim (PT) + rifampin, that displayed impressive efficacy toward P. aeruginosa in both in vitro and in vivo studies. As such, this combination was further evaluated as a potential keratitis therapeutic through testing the combination's efficacy against a diverse set of P. aeruginosa clinical isolates., Methods: Minimum inhibitory concentrations (MICs) of moxifloxacin, levofloxacin, erythromycin, tobramycin, PT, polymyxin B (alone), trimethoprim (alone), and rifampin were determined for 154 ocular clinical P. aeruginosa isolates, 90% of which were derived from corneal scrapings. Additionally, the efficacy of PT + rifampin was evaluated utilizing fractional inhibitory concentration (FIC) testing., Results: While 100% of isolates were resistant to erythromycin (average MIC 224 ± 110 µg·mL-1) and trimethoprim (alone) (206 ± 67.3 µg·mL-1), antibiotic resistance was generally found to be low: moxifloxacin (2% of isolates resistant; average MIC 1.08 ± 1.61 µg·mL-1), levofloxacin (3.9%; 1.02 ± 2.96 µg·mL-1), tobramycin (1%; 0.319 ± 1.31 µg·mL-1), polymyxin B (0%; 0.539 ± 0.206 µg·mL-1), PT (0%; 0.416 ± 0.135 µg·mL-1), and rifampin (0%; 23.4 ± 6.86 µg·mL-1). Additionally, FIC testing revealed that PT + rifampin eradicated 100% of isolates demonstrating additive or synergistic activity in 95% of isolates (average FIC index 0.701 ± 0.132)., Conclusions: The drug combination of PT + rifampin was effective against a large panel of clinically relevant P. aeruginosa strains and, as such, may represent a promising therapeutic for P. aeruginosa keratitis., Translational Relevance: This work furthers the preclinical development of a novel antibiotic combination for the treatment of corneal infections (bacterial keratitis).

Burkholderia cepacia complex (Bcc) in airways of patients with cystic fibrosis (CF) is associated with an increased morbidity and mortality. A huge range of intrinsic antimicrobial resistances challenges the treatment of Bcc infections. The aim was to assess the susceptibility of Bcc to ceftazidime/avibactam and standard drugs for the treatment for CF patients and to determine the respective genomic determinants of resistance. Bcc isolates ( n  = 64) from a prospective multicenter study of CF airway pathogens (2004-2020, Germany) were subjected to broth microdilution and minimal inhibitory concentrations were interpreted with European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute breakpoints. A synergism between aztreonam and avibactam was tested using ceftazidime/avibactam disks with or without aztreonam. Plasmids and chromosomes of all isolates were screened for antimicrobial resistance genes. The highest susceptibility rate was detected for trimethoprim/sulfamethoxazole (83%), followed by ceftazidime/avibactam (78%), ceftazidime (53%), levofloxacin (39%) and meropenem (27%). The median inhibition zone diameters of ceftazidime-avibactam and ceftazidime/avibactam plus aztreonam were equal. This was in line with the absence of known class B metallo-β-lactamases in any of the isolates. The majority of isolates carried bla penA (98%) and bla ampC (86%). Trimethoprim/sulfamethoxazole and ceftazidime/avibactam showed high susceptibility rates. Aztreonam in combination with ceftazidime/avibactam had no synergistic effect in our Bcc isolates.

Antibiotics are some of the most widely used drugs. Their release in the environment is of great concern since their consumption is a major factor for antibiotic resistance, one of the most important threats to human health. Their occurrence and fate in agricultural systems have been extensively investigated in recent years. Yet whilst their biotic and abiotic degradation pathways have been thoroughly researched, their biotransformation pathways in plants are less understood, such as in case of trimethoprim. Although trimethoprim has been reported in the environment, its fate in higher plants still remains unknown. A bench-scale experiment was performed and 30 trimethoprim metabolites were identified in lettuce (Lactuca sativa L.), of which 5 belong to phase I and 25 to phase II. Data mining yielded a list of 1018 ions as possible metabolite candidates, which was filtered to a final list of 87 candidates. Molecular structures were assigned for 19 compounds, including 14 TMP metabolites reported for the first time. Alongside well-known biotransformation pathways in plants, additional novel pathways were suggested, namely, conjugation with sesquiterpene lactones, and abscisic acid as a part of phase II of plant metabolism. The results obtained offer insight into the variety of phase II conjugates and may serve as a guideline for studying the metabolization of other chemicals that share a similar molecular structure or functional groups with trimethoprim. Finally, the toxicity and potential contribution of the identified metabolites to the selective pressure on antibiotic resistance genes and bacterial communities via residual antimicrobial activity were evaluated., (© 2022. The Author(s).)

Escherichia coli is the most studied among those bacteria causing urinary tract infections. This study was aimed to find out antibacterial activity and minimum inhibitory concentration of selected antibacterial agents against E. coli isolates of hospitalized UTI patients. The specimens were inoculated on Eosin Methylene Blue medium. E. coli isolates were identified via colonial morphology, biochemical testing and API-20 kit. The susceptibility pattern of antibacterial agents was determined applying disc diffusion method (Kirby-Bauer) and dilution tube method. Among all, 38.82% (n=158/407) specimens were positive for E. coli, while the rest showed either no growth or exhibited colonies other than E. coli. while observing the susceptibility pattern, Imipenem was found the most effective (73.42%) antibacterial agent, followed by nitrofurantoin (52.53%), cefpirome (44.94%) and tazobactam/ piperacillin (44.94%), whereas the E. coli isolates were highly resistant to sulfamethoxazole/trimethoprim (71.52%), followed by Amoxicillin-clavulanic acid (67.72%), nalidixic acid (66.46%) and Tobramycin (62.03%), when tested by disc diffusion method. The isolates were susceptible to cefpirome (39.87%) and tobramycin (39.87%) and resistant to sulfamethoxazole/trimethoprim (75.32%), followed by levofloxacin (61.39%), when tested by tube dilution method. The study concluded high degree of resistance against Sulfamethoxazole/trimethoprim, in contrast, cephalosporin and Imipenem exhibited good potency which can be recommended for UTI.

MDR UPEC has become a global health challenge. Our study investigates the pairwise interactions among FOS, COL, NIT and TRI against 29 UPEC strains using the checkerboard method. The synergistic combinations are further evaluated for their bactericidal effects against the most resistant strain (MRS) using the time-kill method. The results showed that 100% of these strains were resistant to TRI and NIT, whereas 75·86% of them were susceptible to FOS and COL. Among all tested strains, only seven strains were highly resistant to all used antibiotics. Remarkably, FOS/COL, COL/NIT and COL/TRI combinations represent the most effective synergistic (fractional inhibitory concentration index <1) combinations against the seven strains at MICs lower than the susceptible breakpoint ranges, followed by FOS/NIT and FOS/TRI, which achieved synergistic interactions against 1/7 and 2/7 of these strains. Importantly, the bactericidal effects (reduction ≥3·0 log 10  CFU per ml) were only observed with FOS/COL, COL/NIT and COL/TRI combinations against MRS after 24 h of post-treatment. Our data suggested that FOS/COL, COL/NIT and COL/TRI combinations could be a promising option against MDR UPEC infections. Additionally, FOS/NIT and FOS/TRI probably represent a good option for MDR UPEC with lower MICs., (© 2021 The Society for Applied Microbiology.)

Antimicrobial resistance presents a significant health care crisis. The mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) confers resistance to the clinically important antifolate trimethoprim (TMP). Propargyl-linked antifolates (PLAs), next generation DHFR inhibitors, are much more resilient than TMP against this F98Y variant, yet this F98Y substitution still reduces efficacy of these agents. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of the NADPH cofactor. To understand the molecular basis of F98Y-mediated resistance and how PLAs' inhibition drives NADPH isomeric states, we used protein design algorithms in the osprey protein design software suite to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. Here, we present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs' inhibition, while other PLAs remain unaffected by this resistance mechanism., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: B.R.D. and M.S.F. are founders of Ten63 Therapeutics. V.G.F. reports personal fees (consultancy) from Novartis, Novadigm, Durata, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., Cerexa, Tetraphase, Trius, MedImmune, Bayer, Theravance, Basilea, Affinergy, Janssen, xBiotech, Contrafect, Regeneron, Basilea, Destiny, Amphliphi Biosciences, Integrated Biotherapeutics, C3J. V.G.F. has grants received or pending to his institution from following entities: NIH, MedImmune, Cerexa, Forest, Actavis, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Cubist, Merck, Medical Biosurfaces, Locus, Affinergy, Contrafect, Karius, Genentech, Regeneron, Basilea, Janssen. V.G.F. received educational fees from Green Cross, Cubist, Cerexa, Durata, Theravance, Debiopharm. V.G.F. received royalties from UpToDate. V.G.F. has a pending patent on host gene expression signature diagnostic for sepsis. B.R.D. was previously a guest editor for PLoS Comp. Biol.

Background: Urinary tract infection (UTI) is one of the common infections in childhood. Prompt diagnosis and treatment reduces the risk of complications. The choice of antibiotic to treat UTI varies from region to region. Rational use and appropriately chosen antibiotic reduces the emergence of resistant uropathogens., Objective: We investigated the resistance pattern of uropathogens for commonly used antibiotics to treat UTI locally., Methods: Data was collected between 2009 and 2019 on all infants and children under 16 years of age with culture proven UTI. Results were compared with previously published figures between 2002 and 2008., Results: A total of 1002 samples were analysed (91/year). Male to female ratio was 1:4.6. About 94% of the samples grew E. coli. As before, high resistance rates were recorded to Amoxicillin and Trimethoprim (Z = -0.325: P = 0.7452; not significant). Overall, average resistance has decreased for Nitrofurantoin from 10% between 2002 and 2008 to 5.84% between 2009 and 2019 (Z = 3.002: P = 0.0027). On the other hand, Cefalexin resistance has increased from 7.4 to 14.56% between the two study periods (Z = -4.2: P = < 0.0002)., Conclusion: Despite rising resistance rates, we recommend that Cefalexin should cautiously remain the antibiotic of choice for empirically treating uncomplicated urinary tract infections in secondary care pending urine culture. Nitrofurantoin should be reserved for treating non-coliform/atypical UTIs or multi-drug resistant UTIs. There is an ongoing need for clinicians in all geographic regions to continue to monitor antibiotic resistance rates every few years., (© 2021. Royal Academy of Medicine in Ireland.)

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of unclear mechanisms. In Escherichia coli, thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild-type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection in both thyA mutants denied thymidine, as well as in trimethoprim-treated wild-type (WT) cells, under the following three conditions: (i) intracellular iron chelation, (ii) mutational inactivation of hydrogen peroxide (HP) scavenging, and (iii) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division, which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. IMPORTANCE Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyperthymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS-positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS-positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 μg/mL and 32 μg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of XbaI-digested genomic DNA were observed in the qnrS-positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical XbaI-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13-positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1-positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of XbaI-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Although glutathione (GSH) has been shown to influence the antimicrobial effects of many kinds of antibiotics, little is known about its role in relation to trimethoprim (TMP), a widely used antifolate. In this study, several genes related to glutathione metabolism were deleted in different Escherichia coli strains (i.e., O157:H7 and ATCC 25922), and their effects on susceptibility to TMP were tested. The results showed that deleting gshA , gshB , grxA , and cydD caused TMP resistance, and deleting cydD also caused resistance to other drugs. Meanwhile, deleting gshA , grxA , and cydD resulted in a significant decrease of the periplasmic glutathione content. Supplementing exogenous GSH or further deleting glutathione importer genes ( gsiB and ggt ) restored TMP sensitivity to Δ cydD . Subsequently, the results of quantitative-reverse transcription PCR experiments showed that expression levels of acrA , acrB , and tolC were significantly upregulated in both Δ grxA and Δ cydD . Correspondingly, deleting cydD led to a decreased accumulation of TMP within bacterial cells, and further deleting acrA , acrB , or tolC restored TMP sensitivity to Δ cydD . Inactivation of CpxR and SoxS, two transcriptional factors that modulate the transcription of acrAB - tolC , restored TMP sensitivity to Δ cydD . Furthermore, mutations of gshA , gshB , grxA , cydC , and cydD are highly prevalent in E. coli clinical strains. Collectively, these data suggest that reducing the periplasmic glutathione content of E. coli leads to increased expression of acrAB - tolC with the involvement of CpxR and SoxS, ultimately causing drug resistance. To the best of our knowledge, this is the first report showing a linkage between periplasmic GSH and drug resistance in bacteria. IMPORTANCE After being used extensively for decades, trimethoprim still remains one of the key accessible antimicrobials recommended by the World Health Organization. A better understanding of the mechanisms of resistance would be beneficial for the future utilization of this drug. It has been shown that the AcrAB-TolC efflux pump is associated with trimethoprim resistance in E. coli clinical strains. In this study, we show that E. coli can sense the periplasmic glutathione content with the involvement of the CpxAR two-component system. As a result, reducing the periplasmic glutathione content leads to increased expression of acrA , acrB , and tolC via CpxR and SoxS, causing resistance to antimicrobials, including trimethoprim. Meanwhile, mutations in the genes responsible for periplasmic glutathione content maintenance are highly prevalent in E. coli clinical isolates, indicating a potential correlation of the periplasmic glutathione content and clinical antimicrobial resistance, which merits further investigation.

Antimicrobial resistance (AMR) has become a serious public and economic threat. The rate of bacteria acquiring AMR surpasses the rate of new antibiotics discovery, projecting more deadly AMR infections in the future. The Pathogen Box is an open-source library of drug-like compounds that can be screened for antibiotic activity. We have screened molecules of the Pathogen Box against Vibrio cholerae, the cholera-causing pathogen, and successfully identified two compounds, MMV687807 and MMV675968, that inhibit growth. RNA-seq analyses of V. cholerae after incubation with each compound revealed that both compounds affect cellular functions on multiple levels including carbon metabolism, iron homeostasis, and biofilm formation. In addition, whole-genome sequencing analysis of spontaneous resistance mutants identified an efflux system that confers resistance to MMV687807. We also identified that the dihydrofolate reductase is the likely target of MMV675968 suggesting it acts as an analog of trimethoprim but with a MIC 14-fold lower than trimethoprim in molar concentration. In summary, these two compounds that effectively inhibit V. cholerae and other bacteria may lead to the development of new antibiotics for better treatment of the cholera disease. IMPORTANCE Cholera is a serious infectious disease in tropical regions causing millions of infections annually. Vibrio cholerae, the causative agent of cholera, has gained multi-antibiotic resistance over the years, posing greater threat to public health and current treatment strategies. Here we report two compounds that effectively target the growth of V. cholerae and have the potential to control cholera infection.

Trimethoprim and sulfamethoxazole are prescribed for a broad spectrum of bacteria. However, the use of these medicines is restricted due to the risk of microbial resistance in the body. Nanotechnology is a strategy for overcoming this problem by helping develop novel drug delivery systems. This study aims to assess the ability of Fe 3 O 4 /Ag and Fe 3 O 4 @SiO 2 /Ag nanoparticles to improve efficiency of the traditional formulation of trimethoprim and sulfamethoxazole. Fe 3 O 4 /Ag and Fe 3 O 4 @SiO 2 /Ag were found to have sphere-like morphologies with average sizes of 33.2 and 35.1 nm, respectively. The values of the zeta potential for the pure sulfamethoxazole and trimethoprim were -30.6 and -10.0 mV, respectively, which increased to zero or even larger positive values after being conjugated with the NPs. The study of the release kinetics showed that 64.7% of the medicines were released from the carriers within 40 days. The values of MIC for sulfamethoxazole, trimethoprim, Fe 3 O 4 /Ag/sulfamethoxazole, Fe 3 O 4 /Ag/trimethoprim, Fe 3 O 4 @SiO 2 /Ag/sulfamethoxazole, and Fe 3 O 4 @SiO 2 /Ag/trimethoprim against Escherichia coli were calculated to be 12, 9, 4, 4, 4, and 4 μg/mL, respectively. Besides, the relevant values against Staphylococcus aureus were measured to be 12, 9, 4, 4, 3, and 2 μg/mL, respectively. The use of synthesized nanomaterials for the delivery of these antibiotics leads to smaller doses compared to their traditional forms., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Trimethoprim and sulfamethoxazole are used commonly together as cotrimoxazole for the treatment of urinary tract and other infections. The evolution of resistance to these and other antibacterials threatens therapeutic options for clinicians. We generated and analysed a chemical-biology-whole-genome data set to predict new targets for antibacterial combinations with trimethoprim and sulfamethoxazole. For this we used a large transposon mutant library in Escherichia coli BW25113 where an outward-transcribing inducible promoter was engineered into one end of the transposon. This approach allows regulated expression of adjacent genes in addition to gene inactivation at transposon insertion sites, a methodology that has been called TraDIS- Xpress . These chemical genomic data sets identified mechanisms for both reduced and increased susceptibility to trimethoprim and sulfamethoxazole. The data identified that over-expression of FolA reduced trimethoprim susceptibility, a known mechanism for reduced susceptibility. In addition, transposon insertions into the genes tdk , deoR , ybbC , hha , ldcA , wbbK and waaS increased susceptibility to trimethoprim and likewise for rsmH , fadR , ddlB , nlpI and prc with sulfamethoxazole, while insertions in ispD , uspC , minC , minD , yebK , truD and umpG increased susceptibility to both these antibiotics. Two of these genes' products, Tdk and IspD, are inhibited by AZT and fosmidomycin respectively, antibiotics that are known to synergise with trimethoprim. Thus, the data identified two known targets and several new target candidates for the development of co-drugs that synergise with trimethoprim, sulfamethoxazole or cotrimoxazole. We demonstrate that the TraDIS- Xpress technology can be used to generate information-rich chemical-genomic data sets that can be used for antibacterial development.