An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl- xylR -inducible promoter, the pSpac- lacI system, and the arabinose-inducible P _BAD promoter, but each of these systems has limitations in its application to Staphylococcus aureus . In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter:: gfp _uvr fusion carried on a shuttle plasmid, we demonstrated that dose-dependant tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

Pseudomonas aeruginosa must often overcome a high concentration of oxidants to successfully infect the human host. We report here the results of a transcriptome profiling comparing cells treated with H ₂ O ₂ and untreated controls. The data indicate that the early response of P. aeruginosa to H ₂ O ₂ consists of an upregulation of protective mechanisms and a downregulation of primary metabolism.

SoxR is a transcriptional regulator that controls an oxidative stress response in Escherichia coli . The regulator is primarily activated by superoxide anion-dependent oxidation. Activated SoxR turns on transcription of a single gene, soxS , which encodes a transcriptional regulator that activates a regulon that includes dozens of oxidative stress response genes. SoxR homologues have been identified in many bacterial species, including the opportunistic pathogen Pseudomonas aeruginosa . However, the expected SoxR partner, SoxS, has not been found in P. aeruginosa . Thus, the primary gene target(s) of P. aeruginosa SoxR is unknown and the involvement of this regulator in the oxidative stress response of the bacterium remains unclear. We utilized transcriptome profiling to identify the P. aeruginosa SoxR regulon and constructed and characterized an unmarked P. aeruginosa Δ soxR mutant. We provide evidence indicating that P. aeruginosa SoxR activates a six-gene regulon in response to O ₂ ^·− -induced stress. The regulon includes three transcriptional units: (i) the recently identified mexGHI - ompD four-gene operon, which encodes a multidrug efflux pump system involved in quorum-sensing signal homeostasis; (ii) gene PA3718, encoding a probable efflux pump; and (iii) gene PA2274, encoding a probable monooxygenase. We also demonstrate that P. aeruginosa SoxR is not a key regulatory player in the oxidative stress response. Finally, we show that P. aeruginosa SoxR is required for virulence in a mouse model of intrapulmonary infection. These results demonstrate that the E. coli -based SoxRS paradigm does not hold in P. aeruginosa and foster new hypotheses for the possible physiological role of P. aeruginosa SoxR.

Background: Phage therapy, a treatment utilizing bacteriophages to combat bacterial infections, is gaining attention as a promising alternative to antibiotics, particularly for managing antibiotic-resistant bacteria. This study aims to provide a comprehensive review of phage therapy by examining its safety, efficacy, influencing factors, future prospects, and regulatory considerations. The study also seeks to identify strategies for optimizing its application and to propose a systematic framework for its clinical implementation. Methods: A comprehensive analysis of preclinical studies, clinical trials, and regulatory frameworks was undertaken to evaluate the therapeutic potential of phage therapy. This included an in-depth assessment of key factors influencing clinical outcomes, such as infection site, phage–host specificity, bacterial burden, and immune response. Additionally, innovative strategies—such as combination therapies, bioengineered phages, and phage cocktails—were explored to enhance efficacy. Critical considerations related to dosing, including inoculum size, multiplicity of infection, therapeutic windows, and personalized medicine approaches, were also examined to optimize treatment outcomes. Results: Phage therapy has demonstrated a favorable safety profile in both preclinical and clinical settings, with minimal adverse effects. Its ability to specifically target harmful bacteria while preserving beneficial microbiota underpins its efficacy in treating a range of infections. However, variable outcomes in some studies highlight the importance of addressing critical factors that influence therapeutic success. Innovative approaches, including combination therapies, bioengineered phages, expanded access to diverse phage banks, phage cocktails, and personalized medicine, hold significant promise for improving efficacy. Optimizing dosing strategies remains a key area for enhancement, with critical considerations including inoculum size, multiplicity of infection, phage kinetics, resistance potential, therapeutic windows, dosing frequency, and patient-specific factors. To support the clinical application of phage therapy, a streamlined four-step guideline has been developed, providing a systematic framework for effective treatment planning and implementation. Conclusion: Phage therapy offers a highly adaptable, targeted, and cost-effective approach to addressing antibiotic-resistant infections. While several critical factors must be thoroughly evaluated to optimize treatment efficacy, there remains significant potential for improvement through innovative strategies and refined methodologies. Although phage therapy has yet to achieve widespread approval in the U.S. and Europe, its accessibility through Expanded Access programs and FDA authorizations for food pathogen control underscores its promise. Established practices in countries such as Poland and Georgia further demonstrate its clinical feasibility. To enable broader adoption, regulatory harmonization and advancements in production, delivery, and quality control will be essential. Notably, the affordability and scalability of phage therapy position it as an especially valuable solution for developing regions grappling with escalating rates of antibiotic resistance.

Two genes of the sigB operon, rsbU and rsbV , were deleted in an rsbU ⁺ strain (FDA486) to evaluate the contribution of these two genes to ς ^B activity in Staphylococcus aureus. The ς ^B protein level and the transcription of two ς ^B -dependent promoters ( sigB and sarA P3 transcripts) were analyzed in the constructed mutants. A deletion of the first gene ( rsbU ) within the sigB operon led only to a partial reduction in ς ^β activity. A deletion of the second gene ( rsbV ) resulted in a more dramatic reduction in the ς ^B protein level and its activity than did the deletion of rsbU , thus indicating that RsbV can be activated independent of RsbU. In the parental strain, the ς ^B -dependent transcript initiated upstream of rsbV was 28-fold higher than the ς ^A -dependent transcript originating from the rsbU promoter. The level of the ς ^B -dependent transcript decreased up to 50% in the rsbU mutant and up to 90% in the rsbV mutant compared with the transcript in the wild type. The yellow pigment of S . aureus colonies, a ς ^B -dependent phenotype, was partially reduced in the rsbU and rsbV mutants, whereas alpha-hemolysin was increased. Additionally, the sarA P3 promoter activity of the parental strain was induced to a higher level in response to pH 5.5 than was that of the rsbU or rsbV mutant, indicating that RsbU is the major activator of the ς ^B response to acid stress. Using a tetracycline-inducible system to modulate the expression of RsbW, we progressively repressed pigment production, presumably by reducing the free ς ^B level. Collectively, our data indicated that RsbU and RsbV in S . aureus contributed to different levels of ς ^B protein expression and varying ς ^B activities. Although RsbV can activate ς ^B independent of RsbU, RsbU remains the major activator of ς ^B during acid stress.