The human gut microbiota, a complex community of microorganisms, plays a crucial role in maintaining gut health and overall physiological well-being. Disruptions to this delicate balance, such as those caused by antibiotic treatment, can lead to various health issues, most notably antibiotic-associated diarrhea (AAD). AAD arises from the non-specific killing action of antibiotics, which eliminates beneficial gut bacteria along with pathogens, resulting in dysbiosis. Probiotics, live microorganisms that confer health benefits when administered orally, are often used to mitigate this dysbiosis and restore gut microbial balance. However, the effectiveness of probiotics is significantly hampered by the very antibiotics intended to treat the underlying infection, as the probiotics are susceptible to the same antimicrobial agents. This study addresses this critical challenge by developing a protective nanocoating for probiotic bacteria that allows them to survive antibiotic exposure and effectively colonize the gut, thus ameliorating AAD. The core strategy is based on the use of a biocompatible 'nanoarmor' consisting of tannic acid (TA) and ferric ions (FeIII) that encapsulates individual probiotic cells, forming a barrier to antibiotics. This method contrasts with traditional encapsulation techniques that use polymeric particles, aiming for a simpler, safer, and more effective method with broad-spectrum protection against a range of antibiotics.

Extensive research highlights the importance of the gut microbiome in maintaining health and preventing various diseases. Studies have explored the use of probiotics as therapeutics for a wide range of gastrointestinal and other disorders, leveraging their ability to inhibit pathogen colonization and regulate gut bacterial composition. However, the concurrent use of probiotics and antibiotics presents a significant challenge, as antibiotics often kill the intended therapeutic bacteria. Previous research on encapsulation of probiotics has focused on various polymeric particles to enhance survival and colonization, but a simple, broadly protective, and biocompatible coating against diverse antibiotics was lacking. This gap in the literature prompted the investigation into the use of a novel polyphenol-based nanocoating as a protective layer for probiotic cells.

This study employed a multi-faceted approach, incorporating in vitro and in vivo experiments to evaluate the effectiveness of the 'probiotic nanoarmor'. Initially, *Escherichia coli* Nissle 1917 (EcN), a common probiotic strain, was selected as a model organism to test the nanoarmor's ability to protect against antibiotics. The nanoarmor was created using a biocompatible polyphenol-based assembly method, combining tannic acids (TA) and ferric ions (FeIII). Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were used to confirm the formation of a uniform nanoshell around the bacteria. Zeta potential measurements and X-ray photoelectron spectroscopy (XPS) further characterized the nanoarmor's properties. To assess antibiotic protection, armored and non-armored (naïve) EcN were exposed to six clinically relevant antibiotics at concentrations exceeding their minimum bactericidal concentrations (MBCs). Colony-forming unit (CFU) counting evaluated bacterial viability. The mechanism of protection was investigated using the Brunauer-Emmett-Teller (BET) method for nitrogen adsorption, cross-sectional TEM, and quartz crystal microbalance (QCM) to analyze interactions between the nanoarmor and antibiotic molecules. High-performance liquid chromatography (HPLC) was used to determine antibiotic concentrations in solutions treated with FeIII-TA aggregates. Furthermore, the study tested the feasibility of using lyophilized, armored bacteria encapsulated in enteric capsules, mimicking oral administration. In vivo experiments using levofloxacin-treated rats evaluated the efficacy of the armored probiotics in reducing AAD and improving gut health. Fecal samples were collected and analyzed for CFU counts, and enzyme-linked immunosorbent assays (ELISAs) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to measure inflammatory markers. Histological analysis of intestinal tissues provided further insights into the effects of the armored probiotics. Finally, adherence assays examined the interaction of armored probiotics with rat intestinal mucus. Additional biosafety studies involving the oral administration of armored probiotics to healthy rats were performed to assess any potential adverse effects.

The results demonstrate that the polyphenol-based nanoarmor effectively protects probiotic bacteria from a wide range of antibiotics. The nanoarmor, approximately 20 nm thick, uniformly coated the bacteria and was successfully applied to Gram-positive (*Lactobacillus casei*) and Gram-negative (EcN) bacteria, as well as a commercial blend of probiotic strains. In vitro experiments showed that armored bacteria exhibited significantly higher viability after exposure to six different antibiotics compared to naïve bacteria. This protective effect was observed for all three bacterial types tested. The mechanism of protection was found to be primarily due to the adsorption of antibiotic molecules by the nanoarmor, creating a low-concentration microenvironment around the bacteria. QCM analysis demonstrated significant binding of antibiotics to the FeIII-TA surface, suggesting multiple molecular interactions are involved in this process. The nanoarmor’s protection extended to lyophilized bacteria encapsulated in enteric capsules, simulating oral delivery. In vivo studies in rats treated with levofloxacin showed that the oral administration of armored probiotics significantly improved AAD symptoms compared to naïve probiotics. Armored probiotics resulted in higher bacterial colonization in the gut, a marked reduction in diarrhea severity (assessed by stool scores), and improved bodyweight gain. Furthermore, armored probiotics modulated the expression of pro- and anti-inflammatory cytokines, indicating an amelioration of intestinal inflammation associated with AAD. Histological analysis did not reveal any adverse effects of the nanoarmor on the rat's gut. Biosafety tests with healthy rats indicated that armored probiotics were not toxic.

This study successfully demonstrates a novel approach to protect probiotics from the deleterious effects of antibiotics, thereby significantly enhancing their effectiveness in mitigating AAD. The polyphenol-based nanoarmor offers a simple, safe, and broadly effective solution compared to existing techniques. The mechanism of action, based on antibiotic adsorption, is robust and generalizable across diverse antibiotic types and bacterial species. The successful translation of the nanoarmor strategy from in vitro studies to the in vivo setting highlights the potential of this technology for clinical applications. The observation that armored probiotics effectively colonize the gut even in the presence of levofloxacin underscores its potential for improving the treatment of AAD. The findings indicate that the nanoarmor does not significantly interfere with the normal functioning of the gut microbiota or impair probiotic adhesion to intestinal mucus. The study's strong evidence of reduced inflammation and improved clinical symptoms associated with AAD further validates the potential benefits of this technology.

This research presents a significant advancement in the field of probiotic therapy, offering a robust strategy to overcome the limitations of concurrent antibiotic and probiotic administration. The polyphenol-based nanoarmor provides a safe and effective means of protecting probiotics from antibiotics, improving their efficacy in preventing and treating AAD. Future research should focus on clinical trials to evaluate the nanoarmor's safety and efficacy in human patients, investigating various antibiotic combinations, and expanding its application to other therapeutic bacteria used in various medical procedures, such as fecal microbiota transplantation (FMT).

The study primarily focused on a limited set of antibiotics and bacterial strains. While the nanoarmor showed broad-spectrum protection, further research is needed to investigate its efficacy against a wider range of antibiotics and bacterial species commonly found in the gut. The in vivo study was conducted on rats, and the findings may not be directly generalizable to humans. Additional research is needed to address the long-term effects of the nanoarmor on the gut microbiome and to determine the optimal dosage and administration route for humans.