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Introducing novel antibiotics is essential to counter the spread of drug-resistant pathogens. Here, we report the discovery of 4′-thiothymidine (4′sT), a nucleoside antibiotic from the nematode symbiont Photorhabdus asymbiotica , identified through activation of a silent biosynthetic gene cluster. 4′sT features an unusual 4′-thiosugar moiety. We identified its biosynthetic gene cluster, including a radical SAM enzyme presumably involved in sulfur incorporation. 4′sT exhibits strain-selective activity that is governed by differences in thymidine metabolism rather than variations in the molecular target. These findings expand our knowledge of antibiotics with unusual selective activity.

Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra , a betaproteobacterium; odilorhabdins, from Xenorhabdus , are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli , which we identified as a novel natural product antibiotic, 3′-amino 3′-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk , kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. IMPORTANCE Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express in vitro . To gain access to silent operons, we first fractionated supernatant from Photorhabdus and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3′-amino 3′-deoxyguanosine (ADG), active against E. coli . ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.

Enterococcus faecalis is a resilient Gram-positive bacterium commonly associated with persistent infections in endodontics, including root canal treatment failures. This dissertation explores the genetic determinants of antibiotic resistance and persister formation in E. faecalis, as well as its survival strategies under antibiotic stress. Understanding these mechanisms is essential for developing more effective therapeutic approaches in endodontic and clinical settings.Chapter 1 provides an overview of E. faecalis as a significant pathogen in endodontics, detailing its ability to survive harsh environments and form biofilms, which contribute to its persistence in root canals. The chapter further discusses the bacterium’s intrinsic resistance to common antibiotics and its ability to acquire resistance genes through horizontal gene transfer, with a focus on vancomycin-resistant strains. Additionally, the phenomenon of persister cell formation, which allows E. faecalis to survive in a dormant state under antibiotic pressure, is introduced as a key factor in treatment failure.Chapter 2 investigates the genetic basis of persister formation in E. faecalis using the Dunny transposon mutant library. The library, containing mutants covering approximately 70% of the E. faecalis genome, was screened for genes associated with persister formation under antibiotic stress. Genes such as PhoU, uvrD/REP, and Fis were identified as potential candidates for regulating persister formation. However, experimental results showed no significant difference in persister formation between knockout mutants and the wild-type strain, suggesting that other genetic factors may play a critical role in this process.Chapter 3 focuses on the identification of antibiotic resistance genes in E. faecalis using the same transposon mutant library. A screening for resistance to ampicillin led to the identification of several mutants with resistance-associated genes, including sensor histidine kinases and phosphate-binding proteins. These findings highlight the complexity of resistance mechanisms in E. faecalis, with metabolic regulation, stress responses, and biofilm formation playing integral roles in survival under antibiotic pressure.Chapter 4 presents experiments assessing the microbial contamination and disinfection efficacy of gutta-percha (GP) cones in endodontics. While GP cones are generally clean, they can harbor bacteria, which may lead to infections if not properly disinfected. The effectiveness of sodium hypochlorite (NaOCl) as a disinfecting agent was evaluated in clinical settings. 16S rRNA sequencing revealed that NaOCl treatment significantly reduced bacterial contamination on GP cones, underscoring the importance of disinfection protocols in preventing infection during root canal therapy.This dissertation provides insights into the genetic determinants of antibiotic resistance and persister formation in E. faecalis, contributing to the understanding of its role in endodontic infections. It also emphasizes the need for improved disinfection strategies to ensure the safety and success of root canal treatments. Further exploration of the genetic factors involved in E. faecalis persistence and resistance is necessary to develop novel therapeutic strategies to combat this challenging pathogen.

The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity.Evybactin is an antimicrobial natural product that targets DNA gyrase, where it binds to a site overlapping with synthetic thiophene poisons and exerts selectivity for Mycobacterium tuberculosis via its transport mechanism into the cell.

With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra, a β-proteobacterium; odilorhabdins, from Xenorhabdus are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone on of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only a small portion of their biosynthetic gene clusters (BGC) is expressed in vitro. In order to access their silent operons, a new approach of “fractionate first, screen later” was used. Extracts from a small library of isolates were separated into fractions, resulting in 200-fold concentrated material, and then screened for antimicrobial activity using differential screening specifically to discover compounds that act selectively against Gram negative species. This approach resulted in the discovery of 3’-amino-3’-deoxyguanosine (ADG) and compounds ADC70-72. ADG, which shows selective activity against Escherichia coli is produced by a silent operon that would have been missed by conventional screening approaches. The biosynthetic gene cluster (BGC) of ADG that shows homology to the BGC of puromycin is identified and upon heterologous expression, induction of this BGC results in the production of ADG in E. coli. Mutants resistant to ADG map to gsk and gmk, which encode kinases of guanosine. In vitro and in vivo biochemical analysis shows that ADG is a prodrug that is sequentially converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG inhibits RNA synthesis. This inhibition occurs through incorporation of ADG-TP into a growing RNA chain, interrupting transcription. Microscopy assays show that ADG treatment results in the inhibition of the cell division as well, apparently through interfering with the GTPase activity of FtsZ by ADG-TP. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG is restricted to E. coli and closely related species, explaining the selectivity of the compound. Due to a high structural similarity between ADG and guanosine and the fact that ADG is converted to ADG-TP inside the E. coli cells, there are probably numerous targets for ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs which are rare among natural compounds. While ADG is explored in-depth in this dissertation, future studies are required to elucidate the structures, minimal inhibitory concentrations (MIC), and mode of actions for compounds ADC70-72.

The oxidative modification of low-density lipoprotein (LDL) in the arterial wall plays a pivotal role in the initiation and progression of atherosclerosis which is a complex and progressive disorder. Paraoxonase1 (PON1), which is required for lipid metabolism, is believed to protect LDL from oxidation. The relationship between PON1 gene Leusin55Methionin (L55M) and Glutamine192Arginine (Q192R) polymorphisms in western Iranians with atherosclerosis and its association with enzyme activity and oxidized low-density lipoprotein (oxLDL) were examined in the present study. In this study, blood specimens were collected from 145 healthy individuals and 154 patients with atherosclerosis proven by angiography referred to Shahid Madani Hospital, Khorramabad, Iran. Genomic deoxy ribonucleic acid (DNA) was extracted from whole blood. For all the subjects, restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was carried out for the detection of L55M and Q192R polymorphisms. PON1 enzyme activity and the level of oxLDL were also evaluated. There was a 3.114-fold increase in the risk of developing atherosclerosis in the subjects presenting the PON1L55M, MM genotype compared to those with the LL genotype (OR 3.114; 95% CI 1.412–6.870). PON1Q192R polymorphism in the PON1 gene was not associated with atherosclerosis. Patients with atherosclerosis had significantly higher oxLDL and reduced PON1 enzyme activity (P < 0.05) compared to the controls. There was no association between the type of genotype, enzyme activity, and oxLDL level. It has been concluded that PON1L55M polymorphism and MM genotype are associated with an increased risk of coronary artery disease (CAD) in Iranian patients with atherosclerosis. We did not find any relationship between PON1Q192R polymorphism and atherosclerosis.

Introduction Hospitalization of a patient in the intensive care unit is considered a stressful event for family members, which can lead to psychological reactions and changes in care needs. Managing this experience and difficult conditions requires the use of appropriate coping strategies. Therefore, the present study was conducted to investigate the relationship between coping strategies, care needs, and life expectancy in the families of patients hospitalized in the intensive care unit. Methods The present descriptive-analytical study was conducted among the family members of patients hospitalized in the intensive care units and medical education centers of Qazvin city, Iran, in 2023–2024. 198 family members were included in the study using the Convenience sampling method. Schneider, Billings-Moss, and Lasky questionnaires were used, respectively, to collect data on patients’ life expectancy, coping strategies, and family care needs. Statistical analysis was performed using mean, standard deviation, frequency, percentage, and SPSS software version 22. To investigate the relationship between variables, univariate regression was first used; then, multivariate regression was used to adjust for the effect of confounding variables. The significance level of all tests was considered less than or equal to 0.05. Results Based on the study’s results, the total score of participants’ coping strategies was 50.45 ± 10.18. The results of the present study showed that the participants’ care needs, with a score of 134.08 ± 12.77, are higher than the average. Life expectancy was above average with a total score of 29.01 ± 5.02. The results of multiple regression showed that, with a unit increase in coping strategies, the participants’ life expectancy increased by 0.56 (β = 0.56 (0.29, 0.83), p < 0.001). However, no significant relationship was observed between coping strategies and care needs (β = 0.08 (-0.02, 0.19), P  = 0.146). Conclusion The findings showed that the care needs and life expectancy of these people are above average, and coping strategies have a positive and significant relationship with life expectancy. Given the limitations of cross-sectional studies in inferring causal relationships, further studies are needed to identify the potential role of coping strategies in improving life expectancy. It is suggested that future studies examine the impact of targeted interventions that enhance coping strategies on the care needs and life expectancy of family members of patients hospitalized in intensive care units.

Background It has been indicated that Angiotensin-Converting Enzyme Insertion/Deletion (ACE I/D) polymorphism (rs4646994) could be regarded as a genetic factor that raises the risk of CAD through its impact on the activity of Angiotensin-Converting Enzyme (ACE) and angiotensin II level. The present study seeks to examine the relationship between ACE I/D polymorphism with the risk of atherosclerosis. Moreover, its potential effects on ACE activity and oxLDL level are investigated. Methods In this study, 145 healthy individuals and 154 patients (143 males and 156 females) were selected among the subjects referred to Shahid Madani Hospital. Atherosclerosis was determined in all subjects with gold standard angiography. Blood samples were collected, used to isolate white blood cells (WBC) and serum separation. The DNA was extracted and the polymorphism was determined by polymerase chain reaction (PCR). The enzyme activity was measured using high-performance liquid chromatography (HPLC). Results This study indicated that patients with atherosclerosis had higher levels of oxidized Low-Density Lipoprotein (oxLDL) and ACE activity ( P  < 0.05) as compared to controls. Although we found a significant association between ACE I/D polymorphism genotype and the allele with atherosclerosis in the male group, there were no association when the entire patient group was compared to the entire control group. Conclusion Our study revealed the ACE I/D polymorphism of the ACE gene may not be an independent risk factor in the development of atherosclerosis and evaluation of ACE activity level is more important in evaluating the risk of disease. The researchers found no relation between ACE I/D polymorphism and atherosclerosis and also between types of genotype, ACE activity, and OxLDL level.