The initial inspiration for our project came from the book The Perfect Predator, which team members read while studying Acinetobacter baumannii. We creatively incorporated this inspiration into the design of our homepage. The Perfect Predator mainly recounts the story of epidemiologist Stephanie Strathdee and her husband, Tom Patterson. During their trip to Egypt, Tom unfortunately contracted the superbug, Acinetobacter baumannii, turning their trip into a race against time and a battle with death.
Through our school's microbiology testing course, we learned that the evolution of pathogens is not limited to antibiotic resistance alone. In fact, there are multiple co-evolutionary pathways, including the enhancement of virulence and the continuous evolution of resistance. The emergence of highly resistant and highly virulent pathogens has reduced the number of available antibiotics, increasing the threat to vulnerable groups such as the elderly, pregnant women, and infants. Moreover, in everyday life, the seemingly safe environment around us is filled with hidden dangers, such as opportunistic pathogens, reminding us to remain vigilant and not underestimate these threats.
Several key superbug pathogens, as warned by the World Health Organization (WHO), include carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Klebsiella pneumoniae, and rifampicin-resistant Mycobacterium tuberculosis, which are primarily found in hospital environments. In recent years, increasing reports indicate that many highly resistant and highly virulent strains are gradually spreading from hospitals into the community.
Given China's current trend of population aging, the elderly and pregnant women are at higher risk of carrying or being infected by these pathogens due to their frequent medical visits compared to the general population. It is also worth noting that many pathogens exhibit geographical specificity, such as KL49-type carbapenem-resistant Acinetobacter baumannii, which is mainly concentrated in Guangzhou, and the Beijing strain of Mycobacterium tuberculosis, which is primarily distributed in Southeast Asia.
Due to the impact of the COVID-19 pandemic, mask-wearing frequency has significantly increased in the post-pandemic era, especially in hospitals, where patients and healthcare workers have developed a habit of wearing masks. This has effectively reduced the risk of airborne transmission of pathogens. However, pathogens may still exist on surfaces and in human excretions, entering the body through excretory pathways and causing infection.
China's population density is relatively high, and public restrooms, including those in hospitals, are predominantly pit latrines. Whether it's public restrooms, trash bins, garbage stations, or sewage areas, these facilities offer limited protection against pathogens and are insufficient in providing efficient defense.
For instance, if a pregnant woman visiting a hospital for a prenatal check-up is a carrier of carbapenem-resistant Acinetobacter baumannii (CRAB), the pathogens she leaves behind after using the restroom could potentially infect the next user. This alternation not only spreads the pathogens but also poses a risk to the newborn or other family members, including the elderly, throughout the pregnancy.
Therefore, designing an efficient, safe, eco-friendly, and low-cost preventive measure in areas with high risks of cross-transmission through fecal excretions is an issue worth addressing.
Current preventive measures mainly rely on physical or chemical methods, such as ultraviolet (UV) light, hydrogen peroxide, alcohol, and bleach disinfectants, with most cleaning being performed manually. Although some places have implemented periodic robot cleaning, and many toilets now have self-cleaning functions, it is still difficult to achieve effective and long-lasting antibacterial measures in many public places across China, such as public restrooms and trash bins. An environmentally friendly, safe, inexpensive, and effective solution is still being sought. Chemical disinfection methods rely on manual intervention, making it difficult to achieve real-time and efficient sterilization. Furthermore, chemical disinfectants can have potential effects on the environment, as well as on human skin and respiratory systems. Physical disinfection methods, such as high-temperature or UV sterilization toilets, have yet to be widely adopted.
To address the aforementioned challenges, we launched the "Protector Plan," focusing on bacteria-based treatments. We began by screening engineered strains and designing multiple modification plans. These plans are divided into three main directions: enhancing bacterial defense mechanisms, increasing offensive capabilities, and designing complementary and synergistic bacterial populations with a focus on environmental protection and human safety.
Different types of bacteria have various capsular polysaccharide structures on their surfaces. From a synthetic biology perspective, it is now possible to engineer a strain to produce different types of capsular polysaccharides. For example, Escherichia coli Nissle 1917 can naturally produce K5-type heparin-like capsules, and by introducing different genes, it can produce K4-type chondroitin sulfate capsules, among others.
More importantly, genetic modifications can increase capsule production (e.g., by overexpressing pgmA and galU) and speed up the transport of capsular polysaccharides from the inside of the cell to the outside (e.g., by overexpressing KpsT and KpsE), thereby increasing the amount of polysaccharides attached to the bacterial surface (FliC). As the capsule thickens, our engineered strain can better resist adverse environments, improve adhesion, and enhance resistance to desiccation. Additionally, when external nutrients are scarce, capsular polysaccharides can serve as carbon or nitrogen sources for the bacteria. The thicker capsule also effectively protects the bacteria from phage adsorption and reduces the likelihood of attacks by pathogenic bacteria, especially those mediated by the T6SS system.
Bacterial adhesion is often a prerequisite for colonization in the environment. Type 1 pili are the most common and well-characterized adhesive surface organelles of enterobacteria. These pili are thin, rod-shaped surface structures about 7 nm wide and 1 μm long, composed of heteropolymers of four different subunits. Research shows that the receptor recognition element of type 1 pili is the 30-kDa FimH protein, which enhances bacterial adhesion, and the overexpression of FimH leads to increased biofilm formation. The ΔfimE mutant can overexpress type 1 pili, promoting microcolony formation. Within these microcolonies, cells are protected from contact-dependent killing mechanisms like those mediated by the T6SS system, improving the survival rate of engineered bacteria.
Escherichia coli Nissle 1917 (EcN) has been used as a probiotic to combat intestinal diseases for decades. Among various commonly used engineered E. coli strains, we selected EcN for modification, both for its proven safety and its relatively high offensive capabilities. Research shows that EcN can effectively inhibit a variety of pathogens, including Pseudomonas aeruginosa, Yersinia, Salmonella, Shigella, and enterohemorrhagic E. coli (EHEC). This inhibitory ability is primarily attributed to EcN's production of microcins, siderophores, and adhesins.
To enhance the long-range offensive capabilities of our engineered strain, we overexpressed EcN's own microcin genes (mcmA and mchB) and their corresponding immunity protein genes (mcmI and mchI), testing whether these modifications could improve EcN's long-range antibacterial activity.
Through whole-genome sequencing analysis, we found that EcN has a relatively complete T6SS system and a Contact-Dependent Growth Inhibition (CDI) system. However, compared to the T6SS-active Escherichia coli 042, EcN lacks the tagh gene, which has been proven to play an important regulatory role in the T6SS system of Vibrio cholerae. Therefore, we introduced the tagh gene from E. coli 042 into EcN via plasmid to observe its effects on EcN's offensive capabilities. Additionally, we overexpressed the cdiB gene in EcN's CDI system to further evaluate whether it could enhance its attack potency.
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[3] Margot Marie Dessartine, Artemis Kosta, Thierry Doan, Éric Cascales, Jean-Philippe Côté. Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks. mBio. 2024 Apr 10;15(4):e0255323.
[4] Yi Ma, Wei Fu, Bin Hong, Xinfeng Wang, Shoujin Jiang, Jufang Wang. Antibacterial MccM as the Major Microcin in Escherichia coli Nissle 1917 against Pathogenic Enterobacteria. Int J Mol Sci. 2023 Jul 20;24(14):11688.
[5] Ahmad M Aljohani, Cecile El-Chami, Muna Alhubail, Ruth G Ledder, Catherine A O'Neill, Andrew J McBain. Escherichia coli Nissle 1917 inhibits biofilm formation and mitigates virulence in Pseudomonas aeruginosa. Front Microbiol. 2023 Mar 8:14:1108273.
