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.

Despite the fact that fluorescence in situ hybridization (FISH) is a wellestablished 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.2

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.

30Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the 31 detection and characterization of microbial populations. FISH is affected by a wide 32 variety of abiotic and biotic variables and the way they interact with each other. This is

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.

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.

Bacterial resistance to antibiotics threatens the ability to treat life-threatening bloodstream infections. Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) able to inhibit essential genes can become an alternative to traditional antibiotics, as long as they are safely transported in human serum upon intravenous administration and they are carried across the multilayered bacterial envelopes, impermeable to ONs. In this study, fusogenic liposomes were considered to transport the ONs and promote their internalization in clinically relevant bacteria. Locked nucleic acids and 2’-OMethyl RNA were evaluated as model NAMs and formulated into DOTAP–DOPE liposomes followed by post-PEGylation. Our data showed a complexation stability between the post-PEGylated liposomes and the ONs of over 82%, during 24 h in native human serum, as determined by fluorescence correlation spectroscopy. Quantification by a lipid-mixing assay showed that liposomes, with and without post-PEGylation, fused with all bacteria tested. Such fusion promoted the delivery of a fraction of the ONs into the bacterial cytosol, as observed by fluorescence in situ hybridization and bacterial fractionation. In short, we demonstrated for the first time that liposomes can safely transport ONs in human serum and intracellularly deliver them in both Gram-negative and -positive bacteria, which holds promise towards the treatment of bloodstream infections.

IntroductionIn this study, we applied fluorescence in vivo hybridization (FIVH) using locked nucleic acid (LNA) probes targeting the bacterial rRNA gene for in vivo detection of H. pylori infecting the C57BL/6 mouse model. A previously designed Cy3_HP_LNA/2OMe_PS probe, complementary to a sequence of the H. pylori 16S rRNA gene, was used. First, the potential cytotoxicity and genotoxicity of the probe was assessed by commercial assays. Further, the performance of the probe for detecting H. pylori at different pH conditions was tested in vitro, using fluorescence in situ hybridization (FISH). Finally, the efficiency of FIVH to detect H. pylori SS1 strain in C57BL/6 infected mice was evaluated ex vivo in mucus samples, in cryosections and paraffin-embedded sections by epifluorescence and confocal microscopy.ResultsH. pylori SS1 strain infecting C57BL/6 mice was successfully detected by the Cy3_HP_LNA/2OMe_PS probe in the mucus, attached to gastric epithelial cells and colonizing the gastric pits. The specificity of the probe for H. pylori was confirmed by microscopy.ConclusionsIn the future this methodology can be used in combination with a confocal laser endomicroscope for in vivo diagnosis of H. pylori infection using fluorescent LNA probes, which would be helpful to obtain an immediate diagnosis. Our results proved for the first time that FIVH method is applicable inside the body of a higher-order animal.