AureoBos’ Origin

In the biggest state of Mexico, Chihuahua, lies our tight-knit team, bound not only by our passion for science but also by our love for our community. One would say the way to someone’s heart is through their stomach, and we are certain about that. Our gastronomy is known worldwide and besides being delicious, it tells the story of the people living in our country. At least in Mexico, our kitchen wouldn’t be complete without a key ingredient: milk. Quesadillas, enchiladas, flan, jamoncillo and numerous delicacies come from this golden liquid.

During the project's early stages, we found ourselves inspired by the story of our teammate Alexa. She told us about the challenges her godmother, a dairy farmer, faced daily. This is when we delved into her issues, specifically into mastitis Udder inflammation, characterized by swelling, increased warmth, alterations in milk appearance, and potential systemic illness, results in physiological and/or anatomical changes to the udder. : a common and prevalent disease in dairy cattle. This caught our attention due to its significant multifaceted impact on dairy owners. Motivated by Chihuahua's prominence in the Mexican dairy industry¹, we recognized the global significance of addressing this issue. We were also inspired by the work of previous iGEM teams, particularly the 2017 TU Delft and the 2021 IISER Kolkata teams, whose project focuses on the diagnosis of mastitis. This led us to choose a different path, focusing on developing a non-antibiotic treatment for mastitis.

Mastitis reduces the quality and quantity of milk produced. Current treatments to eradicate this infection include antibiotics, which force producers to withdraw the milk, affecting their incomes and decreasing even more the supply of this food. Over 6 billion people worldwide trust their nutrition on milk which is rich in high-quality protein and vital nutrients that are easily absorbed by our bodies. These nutrients include fats, fatty acids, carbohydrates, sugars, vitamins, and minerals² as shown in Figure 1.

Figure 1. Nutrients of whole milk, excluding water. ³

Into the Udder: Impact of Bovine Mastitis

When we think about milk, we immediately associate it with cows, and that is not very far from reality, although it can also be obtained from buffaloes, camels or goats. 82% of total world milk is obtained from cattle.⁴ About 150 million households worldwide are engaged in dairy farming.⁵ In most developing countries, like Mexico, milk production is carried out by smallholders, exerting a significant impact on household livelihoods, food security and nutritional requirements. With an astonishing production of 550,498 thousand metric tons of milk worldwide as of July 2023,⁶ the dairy industry stands as a major employer worldwide, underscoring the critical importance of udder health.

Figure 2. Common ethiological agents of bovine mastitis.

Specific contagious pathogens stand out as significant contributors, mainly Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis as shown in Figure 3, microorganisms that causes damage on the milking tissue, causing irreversible damage to the cow’s milk peak production.

S. aureus, the predominant pathogen responsible for bovine mastitis, produces harmful toxins and enzymes. These include protein A, which hinders antibody recognition by neutrophils; β-lactamase inducing microabscess formation; and TSST-1 (toxic shock syndrome toxin-1), the major cause of toxic shock syndrome characterized by fever, rash and organ failure.^9,10

On the other hand, S. agalactiae is adept at forming biofilms and resides in the bovine gastrointestinal tract.¹¹ Lastly, S. uberis is associated with clinical and sub-clinical infections. It was reported that α-casein and β-casein in milk induce production of biofilms, which help S. uberis to persist under environmental stress and resist antibiotic treatment.¹²

Figure 3. S. agalactiae, S. aureus and S. uberis in the mastitis infection.

The impact of bovine mastitis extends beyond individual cows. When engaging with our stakeholders, we noticed that the absence of preventive measures such as proper hygiene in both the cowpens and the milking routine significantly raises the risk of infection.
Mastitis infections manifest in three forms: subclinical level is when the infection isn't visibly noticeable, and no symptoms can be detected; clinical level is the state, where mastitis symptoms become noticeable, and the cow's milk production decreases; chronic mastitis: This refers to a condition where the infection keeps coming back, and symptoms of clinical level reappears as shown in Figure 4. Subclinical mastitis, if not addressed, progresses to clinical stage. Untreated mastitis lowers milk production, increases financial losses, and can harm the cow's udder. Severe cases can even result in cow mortality.¹³

Figure 4. Classification of mastitis according to symptoms and signs.

Milk Is Money

Milk is a valuable commodity worldwide. The global dairy market was valued at 893 billion USD in 2022¹⁴, and milk production and consumption have been increasing in the last 5 years. As a result, at least 80% of the world's population consumes dairy products¹⁵.

Bovine mastitis results in substantial expenses encompassing treatment, animal care, and compromised milk yield. Milk composition, quantity, and quality are adversely affected, with enduring consequences such as impaired udders hindering future milk production for affected cows.⁸ For both the Mexican dairy sector and global economies, bovine mastitis stands as a leading cause of financial loss, estimated at $200 USD per cow each year.¹⁶ The National Mastitis Council of the United States estimated that losses in this country amount to two billion dollars per year, and an average of 200 dollars per cow per year.¹⁷ With rising dairy cattle populations, the disease's prevalence is poised to escalate, projecting the bovine mastitis market size to significantly impact the global economy by 2027. According to the research from the University of Glasgow, mastitis is estimated to cost around 19.7 billion dollars to 30 billion USD worldwide; these costs include diagnostics, milk loss, veterinary services, labor, therapeutics, among others.¹⁸

Conventional Treatments

Mastitis’ primary treatment consists of antibiotics, such as natural or synthetic penicillins. However, overuse of antibiotics in bovine mastitis can trigger resistance and non-response, raising costs and endangering public health.¹⁹

Figure 5. Commonly used antibiotics according to our stakeholders

In Mexico, a wide range of antibiotics are approved by the government for treating mastitis, as shown in Figure 5. However, there are several alternative treatments which offer various options to dairy farmers such as intramammary infusion, dry cow therapy, vaccination, bacteriophages, probiotics and herbal remedies.

Figure 6. Available treatments for bovine mastitis

Intramammary infusion of antibiotics is notable for effectively reducing the incidence of mastitis and improving milk quality. Antibiotics, although effective, have the disadvantage of requiring a withdrawal period after administration due to regulatory problems in many countries. This withdrawal period prohibits the collection of milk for human consumption, which leads to economic losses for farmers²⁰ The economic impact of mastitis can be considerable. To learn more about the existing solutions as shown above and the comparison of treatments against AureoBosyou can check our analysis and benchmarking in the entrepreneurship section.

The Risk Beyond

Due to their efficacy, antibiotics are still widely used by dairy cattle farmers. However, this widespread use leads to a significant release of antibiotics into milk, which poses a serious threat to human health.²⁰

Antibiotic resistance manifests itself innate or acquired, and plasmid-mediated resistance contributes significantly to its dissemination. In addition, bacterial biofilm growth offers protection against the effects of antibiotics, making inappropriate antibiotic stewardship counterproductive and increasing resistance.²¹ Despite the inevitability of antimicrobial resistance, the current situation is due to disproportionate and erroneous use of antibiotics.²² Alarmingly, about 4.95 million deaths in 2019 were related to bacterial resistance to antibiotics²³, underscoring the urgency of raising awareness of antibiotic use in our food chain. WHO has declared that antimicrobial resistance is one of the top 10 public health threats facing humanity.²⁴

The Landfill

Discussing mastitis inevitably brings attention to the substantial issue of food wastage. The persistence of food waste remains a significant challenge within the food supply chain, with a staggering one-third of produced nourishment being discarded and approximately 735 million people facing hunger.²⁵

Addressing the issue of hunger, tackling dairy industry losses becomes a significant stride in the ongoing battle to eradicate health and nutrition inequality. In the past decade, dairy consumption has seen an increase, however, in our country, milk production is largely dominated by bigger producers, leading to inflated product prices.¹ In response, AureoBos introduces an innovative approach to curbing milk losses and enhancing the availability of dairy derivatives.

The New Antimicrobial Era: Endolysins

As previously discussed, three main gram-positive bacteria are frequently linked with mastitis:S. aureus, S. uberis, and S. agalactiae .²⁶ The prevalence of multidrug-resistant strains, including methicillin-resistant Staphylococcus aureus (MRSA) among gram-positive bacteria are a significant healthcare concern. Consequently, there's a pressing need for new antimicrobial agents.²⁷

The most efficient antimicrobial agents come from submicroscopic entities: bacteriophages. Endolysins, which are enzymes present in bacteriophages' double-stranded DNA, capable of breaking down peptidoglycan in bacterial cell walls that lead to the bursting of bacterial cells, facilitating the release of new viral particles. These endolysins demonstrate notable potential as an alternative to antibiotics, displaying specific antimicrobial effects on gram-positive bacteria, even in the presence of antibiotic-resistant strains. AureoBos emerged from the realization that externally applying endolysins to bacterial cell walls leads to peptidoglycan degradation and swift bacterial cell lysis. ²⁸

Figure 7. Action mechanisms that our selected endolysins show.

Design

Grounded in the principles of synthetic biology, our project is fundamentally centered on the bioengineering of recombinant fusion proteins within prokaryotic cells.

Our approach involves the use of endolysins Bacteriophage-encoded lytic enzymes responsible for breaking down the peptidoglycan within the bacterial cell wall ²⁹ and albumin binding domains, resulting as a product of novel fusion proteins with great antimicrobial potential. Fusion proteins are a novel technology that involve the genetic combination of two or more domains from proteins. This changes its properties according to each component added. One of its main applications is biopharmaceuticals, since the protein can acquire the ability to target specific molecules or cells.³⁰

For gram-positive bacteria, we utilize two endolysins: LysCSA13 and LysK. These endolysins possess distinct components, including the N-terminal catalytic domain and the C-terminal cell wall binding domain, which confers bacterial specificity.³¹

Nevertheless, gram-negative bacteria represent a challenge for endolysins due to the non-permeable lipopolysaccharide layer in their cell wall. Artilysins, endolysins fused with lipopolysaccharide destabilizer peptides, are able to disrupt and penetrate this layer, lysing cells in the process.³² By fusing endolysin LysSS, polycationic nonapeptide and CeCA, an antimicrobial peptide, artilysins start forming part of our project.

However, a significant limitation is the remarkably short in vivo half-life of these endolysins, lasting only 20 minutes.³³ This brief duration significantly impedes their potential to serve as effective active pharmaceutical agents: it is unfeasible to encompass the entire process of production, purification, distribution, storage, and other essential stages for successful application and market availability. Thus, we aim to extend its half-life by incorporating an albumin-binding domain, thereby enhancing their viability and potential impact.³⁴

All these enzymes will have a sole purpose: find mastitis causing pathogens in the udder. AureoBos’s mechanism is simple: an alginate chitosan gel solution encapsulating our three endolysins will be injected directly into the udder via intramammary infusion.³⁵

Methodology

The World in Our Project

Despite the challenge in quantifying milk loss due to mastitis accurately, our stakeholders emphasize its significant impact. We are dedicated to a noble mission: to pioneer a synthetic biology-based treatment of bovine mastitis using endolysins to counteract food waste and improve food safety. Aiming to minimize milk loss while also addressing the limitations and risks associated with antibiotic use, AureoBos primary goal is to offer an effective, safe alternative benefiting dairy farmers of all sizes. Our vision is simple yet profound: to showcase the antimicrobial potential of endolysins, remarkable proteins that combat antibiotic-resistant strains.³⁸

We embarked on this journey by immersing ourselves in the world of mastitis, actively engaging with stakeholders who shape and are impacted by our project. This field research has become the bedrock of our laboratory work, providing us with invaluable insights that by mathematical modeling the lifecyle of our solution guides our path to success. Each step we took was enhanced by the concerns, needs, and knowledge shared by our stakeholders. We envision our treatment transcending as a startup. We offer a solution that addresses the multifaceted challenges faced by local, national, and international dairy farmers, making a meaningful contribution to their well-being and sustainability.

AureoBos’ mission aligns with an anti-waste focus, acknowledging the importance of Sustainable Development Goal 2: Zero Hunger³⁹, helping through the collection of safe and nutritious milk. Our responsibility to preserve Chihuahua's culture fuels our commitment to effect positive and sustainable changes both locally and beyond. Our project exemplifies how synthetic biology, an unfamiliar field to many, can significantly contribute to community well-being. Through genetic engineering and biotechnological tools, we aim to combat bovine mastitis, benefiting local industries and advancing scientific knowledge. AureoBos showcases how synthetic biology can propel wellness progress. The team draws inspiration from our stakeholders’ contributions and strives to create a brighter, healthier future for the dairy industry and the global community.