1. Overview
As Staphylococcus aureus (S. aureus) is a common pathogen and a global challenge that is threatening people's health, our team focused on ways that can help protect public health from S. aureus contamination and infection. This idea is also inspired by norovirus outbreak that caused massive diarrhea in our school BNDS. Last year, BNDS_China 2022 designed a detective test kit that targeted the pathogenic bacteria Bacillus cereus. The test kit aimed to detect B. cereus contamination in food sources like homes, school canteens, and restaurants - common areas where outbreaks could occur. Based on the idea of detecting bacteria in food, we aimed to expand the existing bacteria-detection mode that could be applied to other dangerous bacteria to environmental detection and direct treatment of infections, which follow the possible actions of people suspecting the happening of a bacterial contamination. This is the origin of in vitro detection, in vivo detection, and in vivo elimination modules in our project design. Then, we started to gather feedback and suggestions in order to improve our project (feasibility) and make it more useful to a broader community (applicability).
To have a better understanding of the applicability and market demands of our products, we first released an online questionnaire to Beijing residents in June. The valuable result provided us advice and made us to keep thinking what could be done more to help people with their health. This also confirmed a need of convenient and safe tools to deal with community-associated MRSA (CA-MRSA).
Then, in July and August, we had meetings with two doctors to interview about their opinions on how our products could potentially benefit the hospital settings. These precious experiences greatly promoted our understanding on antibiotics usage in China hospitals and the need for a rapid pathogen detection. We learned that the superbug transmission in hospital may cause severe influence to patients in hospitals and is hard to be resolved. However, using alternative treatments other than conventional antibiotics may reduce the development of antibiotic resistance. The faster detection may promote more precise medicine prescriptions, decreasing the opportunity of broad-spectrum antibiotics usage and giving patients’ better treatments. Therefore, we expanded our potential target to hospital settings containing healthcare-associated MRSA (HA-MRSA).
To refine our technical approach and promote project's practicability, we regularly consulted our advisors about the capsule design. When we challenged ourselves to explore the new area in DNA aptamer, we decided to gain feedback from professional people to improve our product. Therefore, we outreached Prof. Luo and visited his lab in Hangzhou in August. This visiting was particularly informative - Prof. Luo and his students provided invaluable guidance that deepened our aptamer-based work.
2. Applicability
Questionnaire to Beijing Residents
Staphylococcus aureus (S. aureus) has gained worldwide attention due to both its high prevalence and its significant threat to public health. As one of the first and the most representative types of bacteria that developed antibiotic resistance through the overuse of antibiotics, S. aureus is responsible for a wide spectrum of infections that can lead to life-threatening conditions. It is estimated that approximately 140 million individuals, including 100 million children, are affected by S. aureus infections worldwide (Wu et al., 2016). Our team was also surprised to learn that about 30% of the human population asymptomatically carries S. aureus, putting them at risk of infection (Acton et al., 2008). It should be noted that S. aureus intestinal colonization has a high potential to facilitate staphylococcal infections and transmission in hospital settings (Piewngam & Otto, 2019). Thus, environmental contamination and intestinal colonization are two main areas that our team paid attention to. To promote food safety awareness and improve the situation regarding bacterial infections and antibiotic resistance, our team decided to design a test kit to detect and eliminate S. aureus both in vivo and in vitro. In order to assess the practical application of our expected product in real-life situations, we conducted an online questionnaire to investigate Beijing residents. Their valuable responses could help indicate real problems to solve and guide the improvement of our project's impact on the community around us in Beijing. Therefore, our team sincerely appreciates the participants of this questionnaire.
The questionnaire collected a total of 279 valid responses, with 86.74% of participants having experienced bacterial infection before. Among those with related experience, as high as 30.11% reported experiencing memorable infections more than 3 times, and about 11.47% experienced over 5 memorable infections. For the most severe consequence participants underwent after experiencing bacterial infections, 45.88% required a hospital visit and 16.49% were in severe conditions requiring hospital treatment for an extended period. The results regarding people's experiences with bacterial infection far exceeded our expectations. While contamination is commonly known to exist, quantifying its significant effects on human health through these data underscores the harmfulness of pathogens like S. aureus. This also strengthens the urgency and necessity of raising public awareness about food safety as well as making reliable, convenient detection and bactericidal products available.
Figure 1. Have you ever experienced a bacterial infection (from consuming unsafe food or an exposed wound)?
Figure 2. Among the bacterial infections you or people around you have experienced, which had the most severe consequences?
When asked about their preferences for the next step after suspecting bacterial contamination in their homes or surroundings, only approximately half of the participants chose to first detect the extent of contamination. Surprisingly, 24% of them opted to clean the area with alcohol thoroughly, while 11% preferred to consume antibiotics only after symptoms appeared. While cleaning the home immediately may reduce the risk of exposure to harmful bacteria, it may not be efficient when the contamination source is food or water. The distribution of people's preferences highlights the need to raise awareness about food safety in familiar settings.
When asked about the possible primary reason for not choosing to detect the extent of contamination, a majority of the participants claimed that personal bacterial detection was considered inconvenient (67.74%) and expensive (33.33%) by people around them or themselves. Seeking help from detection services was also deemed less preferable (33.33%). Additionally, 58% of participants believed that as long as drugs could alleviate symptoms caused by bacterial infections, there was no need to detect potential contamination. Therefore, promoting reduced antibiotic usage while providing affordable and convenient detection methods becomes crucial for people's health. Besides, raising people's food safety awareness is also pivotal.
As potential consumers, participants identified convenience (78.85%) and accuracy (75.99%) as the two most important characteristics of a bacteria detection kit. They also valued high efficiency (62.37%) and affordability (56.27%) as additional advantages. Taking this feedback into consideration, our team made concerted efforts to incorporate the ideas of convenience, accuracy, efficiency, and cost-effectiveness in the design and implementation of the aptamer in vitro test kit.
Given that living biotherapeutics are not commonly used in clinical settings, we conducted a thorough inquiry into participants' acceptance of using engineered organisms inside the human body. The results showed that 59.5% of participants were open to the idea of using such organisms, provided that safety was assured. However, some concerns were raised regarding the balance of the intestinal microorganism community, potential transmission of engineered genes among bacteria, and the validity of biological testing in the intestinal environment. Taking these concerns into account, our team made adjustments to the design of the in vivo E. coli capsule to ensure both its functionality in releasing bactericidal substances and biosafety. The ultimate design of the capsule includes a kill-switch to prevent leakage, integration of engineered genes into the E. coli genome for genetic stability, and a self-lysis mechanism that allows the E. coli to release active bactericidal substances only when S. aureus is detected. By utilizing the self-lysis mechanism, the capsule can be effective both before and after intake, as the bactericidal substances remain protected inside the E. coli until they encounter S. aureus. In selecting the bactericidal substances, we considered criteria such as their safety for human use, avoidance of antibiotic resistance development, specificity in antimicrobial activity, and compatibility with the intestinal environment in terms of pH and temperature.
Remarkably, approximately 91.7% of participants expressed willingness to try an oral capsule containing S. aureus-killing E. coli that poses no biosafety concerns. This demonstrates a growing support for new technologies and a desire to improve quality of life. Participants mentioned specific occasions for using this capsule, such as when experiencing symptoms associated with bacterial contamination, like stomachaches, or when facing potential risks of bacterial contamination in their surroundings. However, participants were less likely to choose the capsule when consuming online or raw food in restaurants. This suggests that consumers place trust in unfamiliar food providers, emphasizing the need for strict supervision of food sources, cooking processes, and environmental sanitation by sellers.
Figure 3. If there is a possibility of harmful bacteria in your home or nearby environment (e.g., suspected spoiled food, possibly contaminated water), what would you choose to do?
Figure 4. Reasons for not opting for testing when oneself or someone nearby is at risk
Figure 5. If a bacterial detection reagent kit is available, what features would prompt you to purchase it?
As Beijing residents are more likely to use E. coli capsule or aptamer detection kit to tackle community contamination of CA-MRSA in daily lives, we hoped to gather more feedback from potential stakeholders' opinions on their expectations for our products. We contacted some participants who left their contact information and were willing to answer questions. Many shared experiences with bacterial infection. One person described frequent diarrhea, making trips to the hospital inconvenient. Not wishing to use antibiotics regularly, he expressed support for our probiotic capsule. Another had frequent vomiting due to infection, recovering after drinking fluids and a probiotic drug. A young participant contracted Helicobacter pylori from family but was not prescribed antibiotics due to unstable gut flora. These stories reinforced that when facing infection symptoms, people desire convenient and harmless treatments. Living biotherapeutics are already used to stabilize gut flora and treat diarrhea by ingesting probiotics. Our research on the probiotic drug Zheng Chang Sheng led us to propose Bacillus subtilis as a Gram-positive chassis, as it inhibits S. aureus colonization through colonization resistance. When asked about expectations, participants frequently mentioned "safety", "quality", "convenience", and "specificity". Some suggested the capsule would benefit older adults. Many inquired about specifically targeting S. aureus given the microbiome diversity in the intestines. Participants also offered distributing information on bacterial contamination prevention and applying this detection and treatment approach to other common infections like erysipelas.
In conclusion, our questionnaire revealed the urgent need for a more convenient and affordable S. aureus detection kit and a safe E. coli capsule capable of suppressing S. aureus colonization in the intestine. By providing consumers with choices to protect their food safety and contribute to the post-antibiotic era by reducing unnecessary antibiotic consumption, we aim to address these needs and enhance overall well-being.
Figure 6. Would you be willing to use live biological testing/sterilization products, and if not, why?
Figure 7. If there were commercially available edible sterilization capsules that have bactericidal effects with high specificity (S. aureus) and do not pose any biosecurity issues, would you buy and keep them at home?
Figure 8. In what scenarios would you choose to consume sterilization capsules?
3. Applicability
Visit Hospital and Interview Healthcare Workers (Gastroenterologist and Dermatologist)
Our team also spoke to a number of doctors in hospitals to enquire about the feasibility and applicability of our project in hospital settings. We first interviewed Dr. Li Yunchao, a gastroenterologist at Emergency General Hospital. Then, we had an online meeting with dermatologist Dr. Ma Haili from Yantaishan Hospital. We started the interviews by learning about the general situation in hospitals regarding to bacterial infections. Then we discussed with doctors and asked for their opinions on our project's potential impact on protecting people's health from daily actions, facing the challenge of antibiotic resistance, and providing assistance in hospital situations.
Dr. Li:
Figure 9. The entrance of the hospital's colorectal outpatient department.
Figure 10. BNDS-China team member Ziyue Lei interviewing Dr. Li.
Figure 11. Dr. Li (left 2) with BNDS-China team members Huyang Zhang (left 1), Ziyue Lei (left 3), and Xinyi Gao (left 4).
Dr. Ma:
To summarize, we learned from interviews that:
1. S. aureus (MRSA) poses severe threats in healthcare settings due its prevalence and potential for transmission, especially among vulnerable patient populations like those with weak immune systems, ICU patients, and infants. The broad use of antibiotics in hospitals and daily life has contributed to strong antibiotic resistance development in MRSA. There is a need for alternative approaches to antibiotics to both treat infections and reduce further resistance issues.
2. Using endolysins and AMP LL-37 delivered via an oral capsule represents a good alternative to conventional antibiotics. While different antibiotic levels are used to slow resistance, MRSA demonstrates how resistance can emerge. Introducing an endolysin-containing capsule could raise awareness around prudent antibiotic use at both the patient and societal level.
3. Rapid detection of bacterial infections is also crucial in healthcare settings for prescribing precise treatment and protecting patient health. Our designed aptamer test kit offers high detection efficiency (detection shows result in 5-30 min) and could fulfill this need in hospitals (detection may take 1-3 days). Given this demand, the application of our aptamer kit could be expanded to healthcare settings. Future work will involve incorporating additional aptamers to detect other common and dangerous bacterial pathogens.
4. Using our aptamer kit and E. coli capsule in family and daily settings before eating also provides preventive benefits by potentially reducing infection rates and associated medical costs. Storing these products at home may increase health awareness and promote habits that protect against community bacterial contamination. Instruction materials coupled with the products could emphasize behaviors supporting such protection.
4. Feasibility
Visit Nucleic Acid Aptamer Selection Center in Hangzhou and Interview Prof. Luo
Purpose
Our team aims to detect and eliminate S. aureus both in vitro and in vivo. For in vitro detection, a DNA aptamer that targets S. aureus is required as the core component of the test kit. Initially, we wanted to screen out the DNA aptamers of S. aureus through the systematic evolution of ligands by exponential enrichment (SELEX). However, by doing literature reviews, we found that it took about 10-15 rounds[1] to complete aptamer SELEX while the success rate of merely 30%. The longer-than-affordable experiment period and unpredictable return motivated us to change the aptamer selection method. As an alternative solution, we found the in-silico selection method which only required computer simulation and subsequent verifications such as DNA nano-switch and ELONA. However, we know little about the technical detail of the selection process, so we reached out to Prof. Luo, a professor at the Institute of Cancer and Basic Medicine, Chinese Academy of Sciences, and the Vice Director of Nucleic acid aptamer Selection Center, whom we initially familiarized via BiliBili. We first conducted several short interviews through E-mail and WeChat. Then, we visited the Nucleic acid aptamer Selection Center Prof.Luo worked at on 16th April and did a further in-person interview with Mr. Jiang, one of the students of Prof. Luo.
Figure 12. Contact Prof. Luo through E-mail for the first time
Major gains from the interview and the contributions to our project
1. From in-silico selection method to aptamer verification
Our team initially wanted to use the in-silico selection method to select the DNA aptamers of S. aureus. Indeed, in the long run, the silico selection method should be more important. However, Prof. Luo and Mr. Jiang said the in-silico selection method was not recommended for high school student for the following reasons.
·The in-silico selection method needs computing resources that laptops and home desktops are not able to run.
·The simulation of the interaction between nucleic acid and proteins requires a large amount of previous data. However, there are fewer experiments done so far on the interaction between nucleic acid and protein, so the requirement of data size is not satisfied. Thus, it is still very difficult to mimic the interaction between the three-dimensional structure of DNA aptamer and the cell wall protein of S. aureus only through the in-silico method. The accuracy cannot be ensured.
Therefore, our team choose to use a previously identified aptamer to design the test kit and do verification experiments only.
Figure 13. The visit to the Nucleic acid aptamer Selection Center
2. From selecting the whole bacteria as the target to one surface protein as a target
Our team originally wanted to choose the whole S. aureus bacteria as the target and select the corresponding aptamers. However, Prof. Luo and Mr. Jiang suggested that we could choose one of the bacteria’s surface proteins as a target. If bacteria are directly targeted for screening, the screening failure probability is high due to the complex surface of bacteria with many molecules. If proteins are used to experiment, it will be easier.
Therefore, our team did further research and decided to use Protein A which has specificity to S. aureus as the target.
3. From ELONA to EMSA for aptamers verification
Originally, our team would like to use DNA nano-switch and ELONA to verify the in-silico-selected DNA aptamer. However, Prof. Luo and Mr. Jiang said that the ELONA method was not very recommended since it was one of the very frontiers. The accuracy of verification cannot be ensured. To address this issue, Mr. Jiang mentioned a traditional verification method-EMSA-for the detection of the interaction between nucleic acid and proteins. He explained the principles of EMSA and offered us some advice on how to design appropriate control groups. Furthermore, he provided additional verification methods such as flow cytometry and plasma resonance. More specifically, he not only showed the basic principles of these two methods but also listed out the cost and time spent on doing one round of verification which was really helpful to our final decision on the chosen experiment method.
Finally, our team decided to use EMSA as the verification method to test the DNA aptamer of Protein A. We designed the experimental groups and control groups in accordance with the suggestion from Mr. Jiang. (Figure 3.b)
Figure 14. (a) Mr. Jiang was explaining the principles of Flow Cytometry
References
Piewngam, P., & Otto, M. (2019, March 26). Probiotics to preventStaphylococcus aureusdisease? Gut Microbes, 11(1), 94-101. https://doi.org/10.1080/19490976.2019.1591137
Wu, S., Duan, N., Gu, H., Hao, L., Ye, H., Gong, W., & Wang, Z. (2016, June 24). A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins. Toxins, 8(7), 176. https://doi.org/10.3390/toxins8070176
Acton, D. S., Tempelmans Plat-Sinnige, M. J., van Wamel, W., de Groot, N., & van Belkum, A. (2008, August 8). Intestinal carriage of Staphylococcus aureus: how does its frequency compare with that of nasal carriage and what is its clinical impact? European Journal of Clinical Microbiology & Infectious Diseases, 28(2). https://doi.org/10.1007/s10096-008-0602-7