The endocannabinoid system (ECS), initially identified for its role in maintaining homeostasis, particularly in regulating brain function, has evolved into a complex orchestrator influencing various physiological processes beyond its original association with the nervous system. Notably, an expanding body of evidence emphasizes the ECS\'s crucial involvement in regulating immune responses. While the specific role of the ECS in bacterial infections remains under ongoing investigation, compelling indications suggest its active participation in host-pathogen interactions. Incorporating the ECS into the framework of bacterial pathogen infections introduces a layer of complexity to our understanding of its functions. While some studies propose the potential of cannabinoids to modulate bacterial function and immune responses, the outcomes inherently hinge on the specific infection and cannabinoid under consideration. Moreover, the bidirectional relationship between the ECS and the gut microbiota underscores the intricate interplay among diverse physiological processes. The ECS extends its influence far beyond its initial discovery, emerging as a promising therapeutic target across a spectrum of medical conditions, encompassing bacterial infections, dysbiosis, and sepsis. This review comprehensively explores the complex roles of the ECS in the modulation of bacteria, the host\'s response to bacterial infections, and the dynamics of the microbiome. Special emphasis is placed on the roles of cannabinoid receptor types 1 and 2, whose signaling intricately influences immune cell function in microbe-host interactions.

The bacillus Calmette-Guérin (BCG) is an attenuated bacterium derived from virulent Mycobacterium bovis. It is the only licensed vaccine used for preventing severe forms of tuberculosis in children. Besides its specific effects against tuberculosis, BCG administration is also associated with beneficial non-specific effects (NSEs) following heterologous stimuli in humans and mice. The NSEs from BCG could be related to both adaptive and innate immune responses. The latter is also known as trained immunity (TI), a recently described biological feature of innate cells that enables functional improvement based on metabolic and epigenetic reprogramming. Currently, the mechanisms related to BCG-mediated TI are the focus of intense research, but many gaps are still in need of elucidation. This review discusses the present understanding of TI induced by BCG, exploring signaling pathways that are crucial to a trained phenotype in hematopoietic stem cells and monocytes/macrophages lineage. It focuses on BCG-mediated TI mechanisms, including the metabolic-epigenetic axis and the inflammasome pathway in these cells against intracellular pathogens. Moreover, this study explores the TI in different immune cell types, its ability to protect against various intracellular infections, and the integration of trained innate memory with adaptive memory to shape next-generation vaccines.

Pathogens such as Staphylococcus aureus are able to survive in many types of host cells including phagocytes such as neutrophils and macrophages, thereby resulting in intracellular infections. Treatment of intracellular infections by conventional antimicrobials (e.g., antibiotics) is often ineffective due to low intracellular efficacy of the drugs. Thus, novel techniques which can enhance the activity of antimicrobials within cells are highly demanded. Our recent studies have shown that photochemical internalization (PCI) is a promising approach for improving the efficacy of antibiotics such as gentamicin against intracellular staphylococcal infection. In this chapter, we describe the protocols aiming to study the potential of PCI-antibiotic treatment for intracellular infections in vitro and in vivo using a RAW 264.7 cell infection model and a zebrafish embryo infection model. Proof of concept of this approach is demonstrated. The protocols are expected to prompt further development of PCI-antimicrobial based novel therapies for clinically challenging infectious diseases associated with intracellular survival of pathogens.

Recalcitrant respiratory tract infections caused by bacteria have emerged as one of the greatest health challenges worldwide. Aerosolized antimicrobial therapy is becoming increasingly attractive to combat such infections, as it allows targeted delivery of high drug concentrations to the infected organ while limiting systemic exposure. However, successful aerosolized antimicrobial therapy is still challenged by the diverse biological barriers in infected lungs. Nanoparticle-mediated pulmonary drug delivery is gaining increasing attention as a means to overcome the biological barriers and accomplish site-specific drug delivery by controlling release of the loaded drug(s) at the target site. With the aim to summarize emerging efforts in combating respiratory tract infections by using nanoparticle-mediated pulmonary delivery strategies, this review provides a brief introduction to the bacterial infection-related pulmonary diseases and the biological barriers for effective treatment of recalcitrant respiratory tract infections. This is followed by a summary of recent advances in design of inhalable nanoparticle-based drug delivery systems that overcome the biological barriers and increase drug bioavailability. Finally, challenges for the translation from exploratory laboratory research to clinical application are also discussed and potential solutions proposed.

Bacterial infections are an imminent global healthcare threat evolving from rapidly advancing bacterial defence mechanisms that antibiotics fail to overcome. Antibiotics have been designed for systemic administration to target planktonic bacteria, leading to difficulties in reaching the site of localized bacterial infection and an inability to overcome the biological, chemical and physical barriers of bacteria, including biofilms, intracellular infections and antimicrobial resistance. The amphiphilic, biomimetic and antimicrobial properties of lipids provide a promising toolbox to innovate and advance antimicrobial therapies, overcoming the barriers presented by bacteria in order to directly and effectively treat recalcitrant infections. Nanoparticulate lipid-based drug delivery systems can enhance antibiotic permeation through the chemical and physical barriers of bacterial infections, as well as fuse with bacterial cell membranes, release antibiotics in response to bacteria and act synergistically with loaded antibiotics to enhance the total antimicrobial efficacy. This review explores the barriers presented by bacterial infections that pose bio-pharmaceutical challenges to antibiotics and how different structural and functional mechanisms of lipids can enhance antimicrobial therapies. Different nanoparticulate lipid-based systems are presented as valuable drug delivery systems to advance the efficacy of antibiotics, including liposomes, liquid crystalline nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers and lipid nanocarriers. In summary, liquid crystalline nanoparticles are emerging with the greatest potential for clinical applications and commercial success as an \"all-rounder\" advanced lipid-based antimicrobial therapy that overcomes the multiple biological, chemical and physical barriers of bacteria.

Exploring the structure-activity relationship (SAR) at the cationic part of arylthiazole antibiotics revealed hydrazine as an active moiety. The main objective of the study is to overcome the inherited toxicity associated with the free hydrazine. A series of hydrocarbon bridges was inserted in between the groups, to separate the two amino groups. Hence, the aminomethylpiperidine-containing analog 16 was identified as a new promising antibacterial agent with efficient antibacterial and pharmacokinetic profiles. Briefly, compound 16 outperformed vancomycin in terms of the antibacterial spectrum against vancomycin-resistant staphylococcal and enterococcal strains with minimum inhibitory concentrations (MICs) ranging from 2 to 4 μg/mL, which is a faster bactericidal mode of action, completely eradicating the high staphylococcal burden within 6-8 h, and it has a unique ability to completely clear intracellular staphylococci. In addition, the initial pharmacokinetic assessment confirmed the high metabolic stability of compound 16 (biological half-life >4 h); it had a good extravascular distribution and maintained a plasma concentration higher than the average MIC value for over 12 h. Moreover, compound 16 significantly reduced MRSA burden in an in vivo MRSA skin infection mouse experiment. These attributes collectively suggest that compound 16 is a good therapeutic candidate for invasive staphylococcal and enterococcal infections. From a mechanistic point of view, compound 16 inhibited undecaprenyl diphosphate phosphatase (UppP) with an IC50 value of 29 μM.

Polymeric nanocarriers (PNs) have demonstrated to be a promising alternative to treat intracellular infections. They have outstanding performance in delivering antimicrobials intracellularly to reach an adequate dose level and improve their therapeutic efficacy. PNs offer opportunities for preventing unwanted drug interactions and degradation before reaching the target cell of tissue and thus decreasing the development of resistance in microorganisms. The use of PNs has the potential to reduce the dose and adverse side effects, providing better efficiency and effectiveness of therapeutic regimens, especially in drugs having high toxicity, low solubility in the physiological environment and low bioavailability. This review provides an overview of nanoparticles made of different polymeric precursors and the main methodologies to nanofabricate platforms of tuned physicochemical and morphological properties and surface chemistry for controlled release of antimicrobials in the target. It highlights the versatility of these nanosystems and their challenges and opportunities to deliver antimicrobial drugs to treat intracellular infections and mentions nanotoxicology aspects and future outlooks.

Compounds with high lipophilic properties are often associated with bad physicochemical properties, triggering many off-targets, and less likely to pass clinical trials. Two metabolically stable phenylthiazole antibiotic scaffolds having notable high lipophilic characters, one with alkoxy side chain and the other one with alkynyl moiety, were derivatized by inserting a cyclic amine at the lipophilic tail with the objective of improving physicochemical properties and the overall pharmacokinetic behavior. Only alkynyl derivatives with 4- or 5-membered rings showed remarkable antibacterial activity. The azetidine-containing compound 8 was the most effective and it revealed a potent antibacterial effect against 15 multi-drug resistant (MDR)-Gram positive pathogens including Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis and enterococci. Compound 8 was also highly effective in clearing 99.7% of the intracellular methicillin-resistant S. aureus (MRSA) harbored inside macrophages. In addition to the remarkable enhancement in aqueous solubility, the in vivo pharmacokinetic study in rats indicated that compound 8 can penetrate gut cells and reach plasma at a therapeutic concentration within 15 min and maintain effective plasma concentration for around 12 h. Interestingly, the main potential metabolite (compound 9) was also active as an antibacterial agent with potent antibiofilm activity.

Infectious diseases remain a major burden in today\'s world, causing high mortality rates and significant economic losses, with >9 million deaths per year predicted by 2030. Invasion of host cells by intracellular bacteria poses treatment challenges due to the poor permeation of antimicrobials into the infected cells. To overcome these limitations, mesoporous silica nanoparticles (MSNP) loaded with the antibiotic rifampicin were investigated as a nanocarrier system for the treatment of intracellular bacterial infection with specific interest in the influence of particle size on treatment efficiency. An intracellular infection model was established using small colony variants (SCV) of S. aureus in macrophages to systemically evaluate the efficacy of rifampicin-loaded MSNP against the pathogen as compared to a rifampicin solution. As hypothesized, the superior uptake of MSNP by macrophages resulted in an enhanced treatment efficacy of the encapsulated rifampicin as compared to free antibiotic. This study provides a potential platform to improve the performance of currently available antibiotics against intracellular infections.

Increasing antibiotic resistance in pathogenic microorganisms has led to renewed interest in bacteriophage therapy in both humans and animals. A \"Trojan Horse\" approach utilizing liposome encapsulated phages may facilitate access to phagocytic cells infected with intracellular pathogens residing therein, e.g., to treat infections caused by Mycobacterium tuberculosis, Listeria, Salmonella, and Staphylococcus sp. Additionally, liposome encapsulated phages may adhere to and diffuse within mucosa harboring resistant bacteria which are challenges in treating respiratory and gastrointestinal infections. Orally delivered phages tend to have short residence times in the gastrointestinal tract due to clinical symptoms such as diarrhea; this may be addressed through mucoadhesion of liposomes. In the present study we have evaluated the use of a microfluidic based technique for the encapsulation of bacteriophages in liposomes having mean sizes between 100 and 300 nm. Encapsulation of two model phages was undertaken, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). The yield of encapsulated T3 phages was 109 PFU/ml and for phage K was much lower at 105 PFU/ml. The encapsulation yield for E. coli T3 phages was affected by aggregation of T3 phages. S. aureus phage K was found to interact with the liposome lipid bilayer resulting in large numbers of phages bound to the outside of the formed liposomes instead of being trapped inside them. We were able to inactivate the liposome bound S. aureus K phages whilst retaining the activity of the encapsulated phages in order to estimate the yield of microfluidic encapsulation of large tailed phages. Previous published studies on phage encapsulation in liposomes may have overestimated the yield of encapsulated tailed phages. This overestimation may affect the efficacy of phage dose delivered at the site of infection. Externally bound phages would be inactivated in the stomach acid resulting in low doses of phages delivered at the site of infection further downstream in the gastrointestinal tract.