Promising strategies employing nucleic acids as antimicrobial drugs.

Antimicrobial resistance (AMR) is a growing concern because it causes microorganisms to develop resistance to drugs commonly used to treat infections. This results in increased difficulty in treating infections, leading to higher mortality rates and significant economic effects. Investing in new antimicrobial agents is, therefore, necessary to prevent and control AMR. Antimicrobial nucleic acids have arisen as potential key players in novel therapies for AMR infections. They have been designed to serve as antimicrobials and to act as adjuvants to conventional antibiotics or to inhibit virulent mechanisms. This new category of antimicrobial drugs consists of antisense oligonucleotides and oligomers, DNAzymes, and transcription factor decoys, differing in terms of structure, target molecules, and mechanisms of action. They are synthesized using nucleic acid analogs to enhance their resistance to nucleases. Because bacterial envelopes are generally impermeable to oligonucleotides, delivery into the cytoplasm typically requires the assistance of nanocarriers, which can affect their therapeutic potency. Given that numerous factors contribute to the success of these antimicrobial drugs, this review aims to provide a summary of the key advancements in the use of oligonucleotides for treating bacterial infections. Their mechanisms of action and the impact of factors such as nucleic acid design, target sequence, and nanocarriers on the antimicrobial potency are discussed.

Fighting Methicillin-Resistant Staphylococcus aureus with Targeted Nanoparticles.

Antimicrobial resistance (AMR) is considered one of the greatest threats to global health. Methicillin-resistant Staphylococcus aureus (MRSA) remains at the core of this threat, accounting for about 90% of S. aureus infections widespread in the community and hospital settings. In recent years, the use of nanoparticles (NPs) has emerged as a promising strategy to treat MRSA infections. NPs can act directly as antibacterial agents via antibiotic-independent activity and/or serve as drug delivery systems (DDSs), releasing loaded antibiotics. Nonetheless, directing NPs to the infection site is fundamental for effective MRSA treatment so that highly concentrated therapeutic agents are delivered to the infection site while directly reducing the toxicity to healthy human cells. This leads to decreased AMR emergence and less disturbance of the individual's healthy microbiota. Hence, this review compiles and discusses the scientific evidence related to targeted NPs developed for MRSA treatment.

Engineered liposomes to deliver nucleic acid mimics in Escherichia coli.

Antisense oligonucleotides (ASOs) composed of nucleic acid mimics (NAMs) monomers are considered as potential novel therapeutic drugs against bacterial infections. However, bacterial envelopes are generally impermeable to naked oligonucleotides. Herein, liposomes loaded with NAMs-modified oligonucleotides (LipoNAMs) were evaluated to deliver ASOs in Escherichia coli. Specifically, we tested several surface modifications that included methoxyPEG conjugated to different lipid anchors or modification of the PEG distal ends with maleimide groups and antibodies. MethoxyPEG coated LipoNAMs showed low delivery efficiency for most bacteria, but maleimide-functionalized PEG LipoNAMs were able to deliver ASOs to nearly half of the bacterial population. Conjugation of antibodies to maleimide-functionalized PEG LipoNAMs increased 1.3-fold the delivery efficiency, enhancing the selectivity towards E. coli and biocompatibility. This work demonstrated for the first time that the coupling of antibodies to PEGylated liposomes can significantly improve the delivery of ASOs in E. coli, which might bring alternative routes for the treatment of bacterial infections in the future.

Liposome Delivery of Nucleic Acids in Bacteria: Toward In Vivo Labeling of Human Microbiota.

Development of specific probes to study the in vivo spatial distribution of microorganisms is essential to understand the ecology of human microbiota. Herein, we assess the possibility of using liposomes loaded with fluorescently labeled nucleic acid mimics (LipoNAMs) to image Gram-negative and Gram-positive bacteria. We proved that liposome fusion efficiencies were similar in both Gram-negative and Gram-positive bacteria but that the efficiency was highly dependent on the lipid concentration. Notably, LipoNAMs were significantly more effective for the internalization of oligonucleotides in bacteria than the fixation/permeabilization methods commonly used in vitro. Furthermore, a structural and morphological assessment of the changes on bacteria allowed us to observe that liposomes increased the permeability of the cell envelope especially in Gram-negative bacteria. Considering the delivery efficiency and permeabilization effect, lipid concentrations of approximately 5 mM should be selected to maximize the detection of bacteria without compromising the bacterial cellular structure.

Delivery of Oligonucleotides into Bacteria by Fusogenic Liposomes.

Oligonucleotides able to hybridize bacterial RNA via in situ hybridization may potentially act as new antimicrobials, replacing antibiotics, and as fast in vivo diagnostic probes, outperforming current clinical methodologies. Nonetheless, oligonucleotides are not able to efficiently permeate the multi-layered bacterial envelope to reach their target RNA in the cytosol. Cationic fusogenic liposomes are here suggested as vehicles to enable the internalization of oligonucleotides in bacteria. Here, we describe the formulation of DOTAP-DOPE liposomes, their complexation with small negatively charged oligonucleotides, and the evaluation of the intracellular delivery of the oligonucleotides in bacteria. This strategy uncovers the potential of performing FISH in vivo for real-time detection and treatment of infections.

Computational Resources and Strategies to Construct Single-Molecule Models of FISH.

Currently, the interactions occurring between oligonucleotides and the cellular envelope of bacteria are not fully resolved at the molecular level. Understanding these interactions is essential to gain insights on how to improve the internalization of the tagged oligonucleotides during fluorescence in situ hybridization (FISH). Agent-based modeling (ABM) is a promising in silico tool to dynamically simulate FISH and bring forward new knowledge on this process. Notably, it is important to simulate the whole bacterial cell, including the different layers of the cell envelope, given that the oligonucleotide must cross the envelope to reach its target in the cytosol. In addition, it is also important to characterize other molecules in the cell to best emulate the cell and represent molecular crowding. Here, we review the main information that should be compiled to construct an ABM on FISH and provide a practical example of an oligonucleotide targeting the 23S rRNA of Escherichia coli .

Nanomaterials and molecular transporters to overcome the bacterial envelope barrier: Towards advanced delivery of antibiotics.

With the dramatic consequences of bacterial resistance to antibiotics, nanomaterials and molecular transporters have started to be investigated as alternative antibacterials or anti-infective carrier systems to improve the internalization of bactericidal drugs. However, the capability of nanomaterials/molecular transporters to overcome the bacterial cell envelope is poorly understood. It is critical to consider the sophisticated architecture of bacterial envelopes and reflect how nanomaterials/molecular transporters can interact with these envelopes, being the major aim of this review. The first part of this manuscript overviews the permeability of bacterial envelopes and how it limits the internalization of common antibiotic and novel oligonucleotide drugs. Subsequently we critically discuss the mechanisms that allow nanomaterials/molecular transporters to overcome the bacterial envelopes, focusing on the most promising ones to this end - siderophores, cyclodextrins, metal nanoparticles, antimicrobial/cell-penetrating peptides and fusogenic liposomes. This review may stimulate drug delivery and microbiology scientists in designing effective nanomaterials/molecular transporters against bacterial infections.

Intracellular delivery of oligonucleotides in Helicobacter pylori by fusogenic liposomes in the presence of gastric mucus.

The rising antimicrobial resistance contributes to 25000 annual deaths in Europe. This threat to the public health can only be tackled if novel antimicrobials are developed, combined with a more precise use of the currently available antibiotics through the implementation of fast, specific, diagnostic methods. Nucleic acid mimics (NAMs) that are able to hybridize intracellular bacterial RNA have the potential to become such a new class of antimicrobials and additionally could serve as specific detection probes. However, an essential requirement is that these NAMs should be delivered into the bacterial cytoplasm, which is a particular challenge given the fact that they are charged macromolecules. We consider these delivery challenges in relation to the gastric pathogen Helicobacter pylori, the most frequent chronic infection worldwide. In particular, we evaluate if cationic fusogenic liposomes are suitable carriers to deliver NAMs across the gastric mucus barrier and the bacterial envelope. Our study shows that DOTAP-DOPE liposomes post-PEGylated with DSPE-PEG (DSPE Lpx) can indeed successfully deliver NAMs into Helicobacter pylori, while offering protection to the NAMs from binding and inactivation in gastric mucus isolated from pigs. DSPE Lpx thus offer exciting new possibilities for in vivo diagnosis and treatment of Helicobacter pylori infections.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: The effect of pH, dextran sulfate and probe concentration.

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Effect of Native Gastric Mucus on in vivo Hybridization Therapies Directed at Helicobacter pylori.

Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2'-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers-the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria.

Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect.

Despite the fact that fluorescence in situ hybridization (FISH) is a well-established technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization - temperature, time and formamide concentration - in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations.