Engineered CRISPR biofilms to enhance gut microbiome and combat antibiotic resistance in space environments

Abstract:

Space environments lead to difficult challenges in human physiology, such as gastrointestinal problems (Akinsuyi, et al. 2024). For astronauts who spend time in such conditions, their gut health becomes a critical factor. While there are some studies on the side effects of space flights conducted such as bone loss and muscle atrophy, there are limited studies available on the effects of spaceflight on astronaut microbiome (Tesei, et al. 2022). However, it is important to understand the effects of space on microbiome health, because in long duration space missions susceptibility to illness and infection are increased (Cowen, et al. 2024).

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Thus, this research proposal aims to achieve engineering biofilms from microorganisms derived from the gut in order to enhance gut microbiome and designing CRISPR regulated kill switches to combat antibiotic resistance, all occurring collectively in space environments.

 

Introduction:

The nature of spaceflight missions such as radiation, microgravity, and psychological stress all contribute to decreased microbiome health (Akinsuyi, et al. 2024). The human microbiome consists of bacteria, eukaryotes, viruses, and archaea and alterations caused to the microbiome results in further harming human health as well as disease pathogenesis (Ogunrinola, et al. 2020).

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On the other hand, antibiotic resistance is a rising global concern, which can amplify in space where immune systems become more compromised (Genome Enumeration of Antibiotic Resistance in Space [NASA], 2024). In the confined environment of a spacecraft, antibiotics are less effective and infections become more threatening. Thus, this research proposes the use of CRISPR-Cas9 technology to genetically engineer probiotic bacteria such as Lactobacillus rhamnosus GG and E. coli Nissle 1917 to create biofilms that act beneficial to the gut. These biofilms not only strengthen intestinal lining and promote microbial stability, but also suppress antibiotic resistant bacteria such as ampicillin-resistant E. coli and methicillin-resistant Staphylococcus epidermidis. By integrating CRISPR, the probiotics will include features such as:

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● The ability to secrete antimicrobial peptides targeting resistant strains

● CRISPR interference (CRISPRi) systems to silence resistance genes in surrounding bacteria

● Built in kill switches that ensure safety and prevent uncontrolled colonization

 

Literature Review:

Emerging probiotic, Lactobacillus rhamnosus GG has been studied for its effects on non-pathogenic E. Coli biofilm and treated by gluten, xylitol, and lactose, and from the biofilm interaction ratio index (BIRI), it can be concluded that there were changes in the multispecies biofilm’s formation as a result of the bacteria’s relations with each other ( Kwiecińska-Piróg, et al. 2024). Moreover, L. rhamnosus combined with another probiotic strain (L. paracassei) is able to enhance the adhesion effect of intestinal epithelium cells (Verdenelli, et al. 2009). Based on the research by Kwiecińska-Piróg, Joanna., et al. (2024), the results indicate that L. rhamnosus and E. Coli could achieve a similar effect on intestinal epithelial cells. Additionally, another study conducted by Choudhury, Ankan,. Et al. (2021), notes that by using bioengineering on probiotics to release antimicrobial peptides (AMP) against pathogens, it is possible to the eliminate harmful species whilst not disrupting microbiome of the stomach, offering a novel way of achieving not only probiotics that can help in strengthening stomach lining but also in fighting against viral and bacterial infections.

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While there have been many studies conducted on areas such as CRISPR engineered probiotics and antibiotic resistant modified probiotics, there is a lack in research of the combination of these topics as well as the usage of biofilms created from modified probiotics to increase gut health in space exploration and combat antibiotic resistance which this research aims to solve.

 

Methodology:​ 

Probiotic strains L. rhamnosus GG and E. coli Nissle 1917, will be cultured under aerobic conditions using MRS medium and Luria-Bertani broth, respectively. The strains will be tested for compatibility in dual species biofilm formation by using static crystal violet assays in 96-well plates

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Each strain will be engineered using CRISPR-Cas9 plasmid systems with the following modifications:

● Insertion of genes encoding antimicrobial peptides (e.g., nisin, LL-37) to target antibiotic resistant pathogens.
 

● CRISPR interference (CRISPRi) constructs to silence resistance genes such as bla (β-lactamase) and mecA in dual cultured resistant strains like ampicillin-resistant E. coli and Methicillin-resistant Staphylococcus (MRSE).
 

● Kill switch circuits responsive to environmental cues (e.g., oxygen level, nutrient depletion, etc) to prevent uncontrolled growth or mutation in case of unexpected behavior.

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To stimulate microgravity environments, biofilms will be grown on gut epithelial surfaces in Rotating Wall Vessel (RWV) bioreactors or clinostats. The formation, stability, and morphology of the biofilms will be analyzed using scanning electron microscopy and confocal laser scanning microscopy.

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Engineered biofilms will be dual cultured with antibiotic resistant E. coli and Staphylococcus epidermidis strains. The effectiveness of AMP secretion and CRISPRi gene silencing will be assessed by Colony-forming unit (CFU) counts, qPCR for resistance gene expression, and AMP concentration via ELISA or mass spectrometry.

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The impact of engineered strains on healthy gut bacteria will be tested using fecal samples in an in vitro gut model (e.g., SHIME). Microbial diversity will be analyzed by 16S rRNA sequencing to ensure that the engineered biofilms do not disrupt the native microbiome. Kill switch function will be tested under stress-inducing conditions (e.g., temperature shock, nutrient deprivation). Growth inhibition and self destruction will be quantified via OD600 and live/dead cell staining.

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References :

Akinsuyi, O. S., Xhumari, J., Ojeda, A., & Roesch, L. F.W (2024). Gut permeability among Astronauts during Space missions. Life Sciences in Space Research, 41, 171-180. ScienceDirect. https://www.sciencedirect.com/science/article/pii/S2214552424000300

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Cowen, D., Zhang, R., & Komorowski, M. (2025, April). Infections in long-duration space missions. The Lancet Microbe, 6(4). https://www.thelancet.com/journals/lanmic/home

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Kwiecińska-Piróg, J., Chomont, K., Fydrych, D., Stawarz, J., Bogiel, T., Przekwas, J., & Komkowska, E. G. (2024, May 3). How xylitol, gluten, and lactose change human gut microbiota Escherichia coli and Lactobacillus rhamnosus GG biofilm. Nutrition, 124. ScienceDirect. https://www-sciencedirect-com.sierracollege.idm.oclc.org/science/article/pii/S089990072 4000960

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National Aeronautics and Space Administration. (2024, February 22). Genomic Enumeration of Antibiotic Resistance in Space (GEARS) (C. Kaiser, Ed.). https://science.nasa.gov/biological-physical/investigations/gears/

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Ogunrinola, G. A., Oyewale, J. O., Oshamika, O. O., & Olasehinde, G. I. (2020). The Human Microbiome and Its Impacts on Health. International journal of microbiology, 2020, 8045646. https://doi.org/10.1155/2020/8045646

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Tesei, D., Jewszynko, A., Lynch, A. M., & urbaniak, C. (2022, March 28). Understanding the Complexities and Changes of the Astronaut Microbiome for Successful Long-Duration Space Missions (C. H. House, Ed.). Life (Basel). https://pmc.ncbi.nlm.nih.gov/articles/PMC9031868/