Integrated Human practices

Choosing the stakeholders

In light of the pervasive issue of antibiotic resistance, we decided to begin our investigation by focusing on healthcare facilities, such as hospitals, clinics, dental practices, and medical research institutes within our territory. Our interest was about learning the very practical nature of the problem, thereby shedding light on its day-to-day implications. To this end, we opted to engage with healthcare professionals to explore their perspectives and experiences.

Our primary objective was to address the significant gap that often exists between medical research and clinical practice. As a multidisciplinary team, we were well-positioned to identify this divide, with our passion for research serving as a driving force to seek remedies. The presence of medical expertise within our team provided us with an initial understanding of clinical practices and enabled us to accurately comprehend the practical aspects of clinical problems. Simultaneously, the biotechnological and engineering components were fundamental in crafting an effective solution within a limited time frame.

Notably, our team functioned as a cohesive unit, without any one individual assuming a dominant role. Every facet of the project evolved through close collaboration among diverse team members. These are the foundational principles that guided our selection of the information that warranted further exploration, ultimately shaping our project into its optimal form.

Managing stakeholders

To accomplish our goal, we devised a short questionnaire designed to gather the following information:

• The prevalence of clinically significant infections encountered in their professional practice.
• The extent of awareness regarding antibiotic resistance within the healthcare community.
• The willingness to consider our proposed system as a therapeutic intervention.

The questionnaire consisted of the following questions:

1. What is your profession?
2. What is your specialization? (completed or in progress)
3. How long have you been working in the healthcare sector? (number of years of work + number of years of specialization, if applicable)
4. Which institute do you work at? (please provide the name and location)
5. How often do you encounter antibiotic-resistant infections?
6. What is the most common causative agent of bacterial infections you have treated?
7. How many of the bacterial infections you have treated occurred post-surgery or hospitalization (approximate percentage)?
8. How many of the infections you have treated required a change of antibiotics due to the ineffectiveness of the initial treatment?
9. Which antibiotic do you most frequently use?
10. How aware do you consider yourself of the issue of antibiotic resistance and the existence of multi-drug resistant bacteria?
11. Have you ever heard of bacteriophages, viruses that infect only bacteria?
12. Have you ever heard of phage therapy?
13. If it were possible, how willing would you be to use the therapy described above in conjunction with a common antibiotic if the antibiotics you have used are not effective or minimally effective?
14. What is the main reason for your choice?
15. Among the following information, which do you consider the most important to communicate to the patient before administering the described viral therapy?
16. Would you be willing to participate in a brief interview within the next 2 months to further discuss your thoughts on this matter? (a 15-minute conversation via Zoom or in person, whichever you prefer).

The questionnaire was answered by a total of 75 medical professionals. From their answers we elaborated the informations reported in the graphs below:

This graph shows the percentage of infections that need a change of the antibiotic, split by the bacteria that the specialist treats in most cases. The majority of the specialists admit to treating mostly Escherichia coli and we can appreciate only his variability, this bacteria needs a change of antibiotic in about 0-50% of the cases.

We also made some graphs to understand how the different specializations of the person could change the answer to some of the questions.

Here in the x-axis is reported how many infections needed a change of the antibiotic due to the ineffectiveness of the first one that was administered. The x-axis is split by the several specializations that answered our questionnaire. Structured medical is the most present among our stakeholders, almost half of them need to change the antibiotic administrate in 25-50% of their cases.

In the last graph, the x-axis represents the number of infections due to post-surgery or hospitalization, split by the several specialists who answered the questionnaire. Considering the two specializations which are mostly present, structured medical and nursing, we can see a similar distribution between the three ranges <25%, 25-50%, and 50-75%. This graph evidences the huge amount of infections that affect several patients after they had surgery or have been hospitalized.

Interviews

Individual interviews were conducted with professionals who volunteered to gain a more profound understanding of our concept and to provide enhanced contributions to the project. These interviews were conducted without any form of compensation and strictly adhered to the privacy policies and conditions outlined by our University.

For the purpose of more accurate transcription, all interviews were audio-recorded; subsequently, the audio recordings were deleted after transcription. A standardized set of questions (outlined below) was posed to all interviewees. Nonetheless, the direction of each interview diverged based on the specific field of expertise of the participant and their individual curiosities. Consequently, not all questions were consistently addressed, leading to a more in-depth exploration of specific topics.

The track we generally followed was composed of the following questions:

1. Which other bacterial species do you frequently encounter in the infections you treat, in addition to the most common one identified in the survey?
2. Which organ or system do the infections you typically treat affect?
3. If you recall, which antibiotic has been most frequently ineffective in your experience?
4. If you've had to change antibiotic therapy, which alternative antibiotic did you administer?
5. What would you like to know about our therapy before deciding whether to use it or not?
6. Would you find it more beneficial for this system to be applied as a therapeutic treatment or as a prophylactic treatment (e.g., a gel to be applied on venous catheters before use, on surgical instruments before an operation, etc.)?
7. In what manner do you believe it would be most effective to administer this therapy to a patient (oral, intramuscular, intravenous), and why?
8. What aspects of this therapy are least convincing to you, and why?

All data were collected and analyzed by team members only, under the supervision and responsibility of Marco Costanzo e Niccolò Venturini degli Esposti.

Tosoni Alvise, MD

• Interview date: 10/07/2023
• Profession: Medical Doctor specialized in Pediatric Intensive Care
• Working at: Azienda Ospedaliera Università di Padova

Following our conversation with Dr. Tosoni, we recognised that the possible applications of our system to treat human bacterial infections were a lot more than we thought. While our initial objective for the interview was to gain insights into how to better align our project with the needs of healthcare professionals, it ultimately evolved into a forum for the exploration of novel therapeutic approaches suggested by Dr. Tosoni.

During the interview, Dr. Tosoni delineated the practical and clinical challenges inherent in his specific professional context, namely, the intensive pediatric care setting. In order to effectively perform his duties in this demanding environment, he emphasized the imperative need for pharmaceutical agents that are not only fast-acting but also dependable and easy to administer. Given the critical conditions frequently encountered by patients and the limited time available for intervention, any margin for error is effectively non-existent.

The most salient aspect of Dr. Tosoni's interview resided not in the questions we posed to him, but rather in the ones he directed towards us. He sought clarification regarding the duration required for our therapy to reach target cells and restore antibiotic susceptibility. The temporal aspect, concerning the "time" factor, had not been previously contemplated within our research. Upon consulting the existing literature on the in vitro and in vivo applications of the CRISPR/Cas system, we came to the realization that, in the best-case scenario, the process would necessitate several hours, contingent upon the injection site and the location of the infection.

Dr. Tosoni subsequently introduced a compelling real-world scenario. He proposed that our system could prove highly beneficial for critical patients in need of surgery. Post-surgery, patients typically receive a substantial dose of antibiotics to forestall nosocomial infections. Dr. Tosoni suggested that doctors could administer the engineered phage a few hours or even a day prior to the surgical procedure. This pre-surgical treatment could render potential infections more susceptible to antibiotics, particularly in patients with compromised immune systems. Another pivotal question posed by Dr. Tosoni pertained to the scenario in which the phage fails to reach all bacterial cells.

To sum up, Dr. Tososi made us reevaluate our approach to prophylaxis: whereas we had initially focused on physical surfaces, Dr. Tosoni emphasized the importance of pre-treatment before surgery, particularly when followed by immunosuppressive therapy.

Prof. Telmo Pievani

• Interview date: 17/07/2023
• Profession: Full Professor of Philosophy of Biological Sciences at the Department of Biology and Past President of the Italian Society of Evolutionary Biology.
• Working at: University of Padua

After allowing us to share our experience with our peers during one of his Bioethical lessons, Prof. Telmo Pievani has been willing to help us with the evolutionary and ethical aspects of our research by answering some questions. His suggestions have been precious and helped us refine the following interviews.

The part of the interview on the evolutionary aspects of our system took the form of a debate during which the Professor gave us positive feedback as we were expressing our hypothesis on the advantages given by our approach based on the use of a non-lytic phage, CRISPRi and variation of phage tropism (this parts can be discovered in their proper wiki sections).

Is it right to ask professionals whether they are inclined to use our tool or not?

Asking whether a person is not very inclined or very inclined is a little too direct. In this case the bioethical profile is so nuanced that it would be more correct to ask: "In your opinion, does this line of research present bioethical problems? If so, which ones? Otherwise, on what basis would professionals be inclined or not?"

We are conducting interviews with healthcare professionals asking their opinions about antibiotic resistance and our tool. What about interviewing patients, too?

It’s difficult to ask patients. They are in a delicate phase and questionnaires to people who are in need are controversial. Another thing would be dealing with rare diseases: in those cases, communities of patients are formed and it is possible to conduct interviews, while bacterial infections could happen to anyone, it’s not a chronic condition. Moreover, what could be the added value given by these interviews? This is the question you must ask yourself before.

For an eventual project scale-up, we are thinking about adapting our tool to other sectors, such as the agricultural and zootechnical ones. How could we frame the issue in a questionnaire? Which would be the main aspects to be underlined?

Talking about the environmental impact is fundamental. It would be useful to ask if your tool can contribute to decreasing the use of antibiotics. Nowadays, antibiotics can be isolated everywhere from the environment, even from exotic animals.

Bertoldo Marzia Katj, Nurse

• Interview date: 25/07/2023
• Profession: Nurse in Dialysis Department
• Working at: Azienda Ospedaliera Università di Padova

Nurse Marzia conveyed to us that her professional responsibilities involve caring for patients who frequently experience blood infections, notably those undergoing dialysis, a chronic treatment often necessitating the use of a Central Venous Catheter (CVC). The presence of a CVC poses a heightened risk of blood infections. In our discussion with Nurse Marzia, we discerned a compelling imperative to tailor interventions according to the specific needs of patients.

Marzia emphasized that a therapy such as the one under consideration could prove more advantageous if administered orally for preventive purposes, while intravenous administration would be preferred for treatment. In the former scenario, she expounded, non-invasive treatments are essential to address a broad patient population. Conversely, in the latter scenario, a swift and efficient mode of administration is required to combat the infection expeditiously.

Sarah Di Sopra & Iannis Bekas

• Interview date: 07/08/2023
• Profession: respectively, Biologist and Medical Doctor specializing in Microbiology
• Working at: Azienda Ospedaliera Università di Padova

We conducted an interview with two healthcare professionals, both of whom were specializing in microbiology at our university. This duo comprises a medical doctor, Dr Iannis Bekas, and a biologist, Sarah di Sopra, both of whom encountered a diverse array of bacterial infections on a daily basis. During the interview, they provided us with a comprehensive overview of the prevalent antibiotic-resistant bacterial strains they routinely encounter, along with insights into the antibiotics that frequently prove ineffective against these isolates.

Notably, our discussion with them led to the emergence of a crucial issue - the patient perspective. Both interviewees expressed deep-seated concerns regarding the doctor-patient relationship and the patient's perception of the proposed therapy. They highlighted the potential for patient rejection of this treatment approach, given that phage therapy essentially involves the deliberate administration of a virus to an individual. This underscores the critical need to equip healthcare practitioners with the skills to effectively communicate the advantages and disadvantages of this therapy, in addition to launching informational campaigns aimed at demystifying phage therapy for the general public, beyond the academic realm.

Furthermore, Sarah advocated for a selective approach, suggesting that the therapy be reserved for vulnerable patients, thereby mitigating the risk of overuse and the consequent emergence of phage-resistant bacterial strains. Lastly, both interviewees expressed apprehension about the actual efficacy of the therapy, with Sarah expressing particular concern about the potential for the therapy's final cost to surpass that of conventional antibiotics.

Erica Solimbergo, MD

• Interview date: 16/08/2023
• Profession: Medical Doctor specialized in Microbiology
• Working at: UOS Trasfusionale Ospedali Riuniti Padova Sud

Dr. Solimbergo conveyed her apprehensions regarding the oral administration of the therapy. While she unequivocally advocates for this method of delivery due to its minimally invasive nature, ease of use, and potential scalability, she also raised pertinent queries about the viability of phages under the harsh conditions of gastric and intestinal fluids. She inquired whether the phages could withstand these conditions, undergo intestinal absorption, and ultimately find their way into the bloodstream. Thanks to Dr. Solimbergo, we dive into the literature about phage therapy, looking for any statistical data about pharmacodynamics of oral phage administration.

Silvia Sangiorgio, Nurse

• Interview date: 14/08/2023
• Profession: Nurse in Orthopedics Department
• Working at: Ospedale di Belluno

Nurse Silvia, employed within the orthopedics and traumatology department, articulated a resolute preference for the oral or intramuscular route of administration. Since the majority of her patients are in the geriatric age, she contends that this approach represents the most straightforward and expeditious means of intervention, affording the capacity to treat a broader patient population, including those for whom venous access may not be feasible.

Indeed, the general frailty of elderly patients, including the vulnerability of their cardiovascular system, makes them more prone to the rupture of vessels subjected to repeated blood draws and accesses, frequently resulting in hematomas. Furthermore, these precarious conditions often lead to the impossibility of utilizing a venous catheter, either due to infection or occlusion, necessitating the placement of an alternative access, which, as a matter of fact, is not always feasible.

Giovanna Romanato, MD

• Interview date: 22/08/2023
• Profession: Medical Doctor specialized in Geriatrics
• Working at: Azienda Ospedaliera Università di Padova

Speaking with Dr. Romanato, we understood which segment of the population is most affected by the problem of antibiotic-resistant infections: the elderly. Dr. Romanato, as a geriatrician specialist, explained to us how in her clinical practice she very often encounter bacterial infections that are difficult to treat in subjects suffering from various comorbidities, including diabetes, obesity, hypertension, immunosuppression, patients hospitalized for several weeks with stable intravenous access. In these cases, rapid and effective therapy is necessary, because the time window to act is often very narrow and the possible therapeutic solutions are often limited by the patient's debilitated general condition.

Prof. Antonella Viola

• Interview date: 30/08/2023
• Profession: Full Professor of General Pathology at the Department of Biomedical Sciences
• Working at: University of Padua

The interview with Professor Viola was notably the lengthiest and most comprehensive of all. At the outset, we provided a thorough overview of the project to Professor Viola, seeking to establish a profound foundation that would enable us to pose more nuanced questions compared to those presented to other healthcare practitioners. The dialogues, in essence, adopted a distinct perspective: the primary goal was not to elucidate the clinical dimensions of antibiotic resistance but rather to conduct an in-depth exploration of the immunological aspects pertaining to our therapeutic system.

It is our duty to emphasize that Professor Viola does not work with bacteriophages. Therefore, all the information she provided has been developed based on her extensive general knowledge and experience. The purpose of the interview, in fact, was not to obtain numerical certainties, as such data are currently unavailable even in the literature. Instead, the aim was to engage in reasoning and logical discussions to draw the most valid observations possible regarding the immunological aspects of our project.

What is the estimated timeframe for the development of antibodies against the virus following administration to the patient?

The timing of this process is contingent upon several factors, chiefly the method of administration. Limited clinical trial data are available on this topic; however, it is established that the immune system mounts an effective response against the virus. Given that our bodies regularly encounter numerous phages in our daily environment, concerns regarding an inflammatory response are generally unfounded. Nevertheless, the pace of immune response development varies and is heavily influenced by the inherent immunogenicity of phage proteins. Phages can be categorized as more or less immunogenic. It is crucial to note that immunity is divided into innate and adaptive components, both of which can be triggered by a phage. It is likely that your primary concern pertains to adaptive immunity, which can be considered the most potent in neutralizing the phage and, therefore, poses the greatest threat to therapy efficacy.

Based on your knowledge and experience, what is the level of safety of our therapy?

What is of concern does not pertain to safety, as the available data suggests that the immune system is not stimulated in a pathological manner. In fact, any concomitant inflammation can potentially aid in the combat against bacterial infections, as it can synergistically enhance the innate response against the pathogen. This presupposes, of course, that the therapeutic preparation is pure, devoid of any residual bacterial production remnants or other molecules that might be potentially toxic or harmful to the human organism. The primary issue, therefore, revolves around effectiveness, with the production of neutralizing antibodies posing a probable constraint on therapy efficacy.

Of paramount importance is the likelihood that the therapy will be most commonly required by elderly patients. For such individuals, there are two potential concerns: a) the development of a neutralizing humoral response, and b) the presence of preexisting antibodies capable of neutralizing the phage, either due to prior exposure to the virus or a closely related variant.

In cases where a humoral response develops, the initial antibodies produced will likely be IgM, appearing over the course of several hours to a few days. Subsequently, with multiple administrations, a robust IgG response may emerge, spanning at least 6-8 days, and potentially compromising the therapy's efficacy.

Of greater apprehension is the scenario wherein neutralizing antibodies are already present in the patient prior to therapy initiation. In such instances, the effectiveness of the therapy will invariably be constrained, likely from the very first administration.

Which response, the humoral or nonspecific response, is more likely to result in the neutralization of phages, specifically in terms of their removal from the reticuloendothelial system of the liver and spleen? Moreover, how might this vary depending on the site and route of inoculation?

Undoubtedly, both components play a role in this process. Elimination by the spleen typically transpires within a couple of days, although this is not the case for immunocompromised individuals. Intravenous administration inherently carries a heightened probability of eliciting a neutralizing immune response. In contrast, the oral route, due to the abundance of phages present in the gastrointestinal tract, may exhibit greater tolerance or tolerogenic properties, consequently resulting in a less intense response. Additionally, a decision must be made concerning the impact on the patient's microbiota; oral administration facilitates the potential influence of the therapy on the microbiota.

It is evident that repeated administration of the same phage to the same patient is unlikely to be feasible without the development of a neutralizing response. This issue is already well-documented in the fields of gene therapy and adenoviral vaccines. While not insurmountable, it necessitates careful consideration.

In other words, if we previously thought it could be an excellent continuous therapy for chronically weak subjects such as those suffering from mucoviscidosis, the conversation with the doctor made us understand the difficult feasibility of this idea and therefore the need to change our point of view.

In the case of immunosuppressed patients the immune response is no longer a problem. However, other questions must be evaluated: how long does the phage remain in circulation? How is it eliminated? Can it cause other types of damage?

In your opinion, how could we reduce the immunogenicity of the phage?

To achieve this aim, you need a comprehensive study to pinpoint the most immunogenic proteins, with a subsequent analysis of possible modifications and concealment of these proteins, all while ensuring the preservation of those associated with tropism. However, the primary concern that warrants attention is the potential existence of preexisting neutralizing antibodies in the patient prior to treatment.

What is the probability of an antibody cross-reaction between a phage antigen and a self antigen?

It certainly cannot be discounted, as evidenced by the experience with the SARS-CoV-2 virus. The most prudent course of action is to harness artificial intelligence programs to assess the immunogenicity of proteins, evaluate the potential for autoimmune cross-reactivity, and anticipate the likelihood of allergic reactions.

Analysis

Engaging in a bidirectional dialogue with healthcare professionals and industry experts has enabled us to make several critical decisions throughout the project's development process. This dialogue has allowed us to tailor our system to the needs of both the recipients (the patients) and the practitioners (the professionals), taking into consideration various ethical, human, and safety-related aspects that we might not have otherwise contemplated.

The initial survey served to confirm the clinically relevant bacterial species, the primary antibiotics in use, and the prevalent resistance patterns. Additionally, it shed light on the variability of these parameters based on different work environments and educational backgrounds. Moreover, it provided insights into the general awareness of phage therapy and the level of inclination toward its adoption.

Through individual interviews, we delved deeper into various workplace realities, gaining an understanding of how patient and professional needs vary. We came to realize that there is no universally perfect therapy; instead, there exists a system that should be adapted into therapies with varying properties, depending on the patient's clinical, pathological, and physiological characteristics. This concept aligns with the evolving direction of medicine known as personalized medicine, which revolves around the fundamental notion that treatment efficacy significantly varies based on patient characteristics.

Finally, in our discussion with Professor Viola, we delved into the aspect that concerned us most about our system: safety. In this regard, an extensive modeling of possible immune responses has been performed by adopting several bioinformatics tools. This aspect is described in the Modelling page.

Pharmacology

When discussing pharmaceuticals, and in particular new therapeutic approaches it is fundamental to evaluate two aspects: pharmacokinetics and pharmacodynamics. The first refers to the way through which the medication reaches its site of activity whereas the latter refers to how it interacts with the pharmacological target. As we could learn from our interviews with medical professionals these two aspects represent a pivotal point in the choice of a new drug. This stimulated us to dive deeper in the current literature to try and comprehend what may apply to our specific approach. It is worth noting that no comprehensive and detailed study has been already made over the pharmacological properties of bacteriophages as their clinical applications are still limited to a few hundred cases, nevertheless some authors attempted to discuss these themes by merging data gathered from different studies [1,2,3].

Pharmacodynamics

As Dabrowska et al.[1] suggest in their comprehensive literature review the pharmacodynamics of phage therapy strictly depends on the accessibility of the phage-host, and inherently on the phage host range. Thus, in this context, the efficacy of the therapy is dependent on the phage host range and its ability to interact with the target bacteria. Moreover, a clear distinction is reported as phage therapy can be described as “passive”[1] in the case in which the treatment supplies a large number of phage particles, sufficient to target all the bacteria; or “active”[1] where the phage particles are able to reproduce themselves inside the host to spread. In our specific case, since we wanted to enhance the safety and security of the therapeutic approach a non replicative phage particle was chosen. As a direct consequence ours should be a “passive” therapeutic approach. The ability of bacteriophages to reach their target is regarded as fundamental also by Stacey et al.[2]. In their review they analyze the safety and efficacy of phage therapy through an analysis of both recent and past clinical trials and they suggest that the possibility of the phage particle to reach its target is crucial for the efficacy of the therapy.

Pharmacokinetics

Once assessed the aspects relevant to the pharmacodynamics it is crucial to analyze the relevance of pharmacokinetics and specifically the route of administration for phage particles, as from this strictly depends the availability of the therapeutic and its efficacy. In their work, Stacy et al.[2] analyzed various clinical trials with heterogeneous routes of administration. It is now relevant to remark that the trials that have been made, up to now, have never deeply studied a specific route of administration, being limited to a few hundred patients, so the landscape is quite nebulous.

From the different administration routes tested, oral, intramuscular, intravenous and intranasal, none has been proved significantly better then the others but, many clarifications need to be made. There is not an homogeneity of the conditions and tested variables as both phage cocktails and purified particles have been administered as well as the host range could have been previously tested as it couldn’t. Since these are all premises that profoundly influence the outcome and efficacy of the therapy, the results cannot be taken as conclusive.

Though there are still trials in more than 100 years of history that proved the efficacy of phage therapy[3]. The vast majority of clinical cases where phage therapy resulted effective have some common qualities: (a) only virulent phages were used, (b) the quality of the media used to produce them was important, (c) the administration was intravenous (IV) [3]. The IV route proved to be successful in a relatively large number of cases taken in exam with few adverse effects revealed. Although the IV route has proven to be the most effective, the current data are not sufficient to conclude that the oral route cannot be successful. Specifically if proper formulation studies will be made, it can represent a valid option for home-therapy used, for example, as a prophylaxis, as some clinicians suggested us.

Immunogenicity assessment

After engaging with medical professionals and specifically with professor Viola, we were concerned about the immunogenicity of the phage particles, and inherently of their safety. To further understand these aspects, we analyzed with appropriate bioinformatic tools the possible immune responses that could arise from our developed system. This analysis is strictly related to the safety and security chapter of our project but has been deeply analyzed in the modeling section of this wiki.

Briefly we started by researching if there were any known linear epitopes of any M13 deposited protein in the database. We carried out this work utilizing the Immune Epitope Database & Tools (IEDP)[4] and the epitope prediction algorithm associated. The IEDB database had only G3P and G8P proteins registered, thus we could not assess the immunogenicity of the other proteins. The MHC class I complex is present on the surface of almost any human cell, except for immune cells. It binds intracellular pathogen peptides and exposes them to CD8+ T cells. Although MHC-I analysis is not much relevant for our project since the phage does not infect any human cell, we decided to look for MHC-I epitopes anyway because of the higher accuracy of those predictions, compared to the MHC-II epitopes predictions.

The MHC class II complex is present on the surface of antigen presenting cells. It binds internalized pathogen peptides and exposes them to CD4+ T cells. Since the phage tropism spares with high confidence any human cells, MHC-II antigen processing and binding affinity is the focus of our interest.

We then used another software to assess what linear epitopes of the phage proteins are immunogenic for T cells. For this purpose, we used the CD4 T cell immunogenicity prediction software[5, 6], as it allows us to assess the immunogenicity for all seven most common alleles in the population[7], for each query. The software requires the protein sequence as input; the user can predict the T cell immunogenicity using the 7-allele method[7], immunogenicity method and combined method (IEDB recommended). The combined method predicts the final score that combines the predictions from the 7-allele method and immunogenicity method. In order to reach the highest reliability possible, we then compared the results of the two softwares (MHC-Il binding prediction tool and CD4 T cell immunogenicity prediction tool), and thus the predicted epitope through their respective parameter (MHC-II binding prediction and T-cell immunogenicity prediction).Our assumption was that the two software evaluate two different processes of the human immune response. On one hand we have MHC processing, on the other hand we have the binding affinity between epitopes and T cell receptors, thus the grade of probability to activate the T cells. In fact, it was our belief that a comparative analysis between the results of two software could lead to a more reliable prediction.

We used two different softwares to predict B cell epitopes namely DiscoTope and Elliscope. Whereas the former predicts only three dimensional epitopes, with the latter we were able to predict linear epitopes too.

References

1. armacodynamic and Pharmacokinetic Obstacles to Phage Antibacterial Action in Animal and Human Bodies’, Microbiol. Mol. Biol. Rev., vol. 83, no. 4, pp. e00012-19, Nov. 2019, doi: 10.1128/MMBR.00012-19.
2. H. J. Stacey, S. De Soir, and J. D. Jones, ‘The Safety and Efficacy of Phage Therapy: A Systematic Review of Clinical and Safety Trials’, Antibiotics, vol. 11, no. 10, p. 1340, Sep. 2022, doi: 10.3390/antibiotics11101340.
3. P. Speck and A. Smithyman, ‘Safety and efficacy of phage therapy via the intravenous route’, FEMS Microbiol. Lett., vol. 363, no. 3, p. fnv242, Feb. 2016, doi: 10.1093/femsle/fnv242.
4. Larsen M.V., Lundegaard C., Kasper Lamberth, Buus S,. Brunak S., Lund O., and Nielsen M. 2005. An integrative approach to CTL epitope prediction: A combined algorithm integrating MHC-I binding, TAP transport efficiency, and proteasomal cleavage predictions. European Journal of Immunology 35(8):2295-303. PMID: 15997466.
5. Sidney, J., Peters, B., Frahm, N. et al. HLA class I supertypes: a revised and updated classification. BMC Immunol 9, 1 (2008). https://doi.org/10.1186/1471-2172-9-1.
6. Dhanda et. al.: Prediction of HLA CD4 immunogenicity in human populations, Frontiers in Immunology, 2018, 9, 1369.
7. Paul et. al: Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes, Journal of Immunological Methods, 2015, 422, 28-34.

Beyond Human Therapy

While we were going deeper into the antibiotic resistance issue related to the therapeutic use of antibiotics in the human healthcare system, it was impossible not to consider the bigger picture in which humans are to be contemplated as part of an interconnected environment. Animals and plants can be infected by bacteria and are involved in the antimicrobial resistance cycle as well as human beings.

In the perspective of scaling up our project, we thought we could adapt our tool for use on animals and plants. Therefore, we decided to carry out some research on antibiotic use in the zootechnical and agricultural sectors and what is done to check the spread of antibiotic resistance. Furthermore, we prepared two surveys, one specific for zootechnical and veterinary experts while the other intended for agri-food professionals, and submitted them to experts in those fields to have an actual picture of the issue that could help anyone interested in scaling up the project.

Analyzing the issue

Antibiotics have been used in food production since the 1930s, when sulphonamides were marketed for use in animals, and had a rapid increase in their use during wartime when milk production was fundamental, and penicillin was tested for udder infections in cows. [1] In the 1950s, streptomycin was considered a valid treatment for bacterial disease in plants. [2]

Antimicrobials are used in agriculture both for treating and preventing diseases in plants and animals to ensure food security and to prevent the spread of diseases. In past times, low concentrations of antibiotics were commonly added to animal feed as growth and production promoters and although this practice is now discouraged it still occurs in some areas. [3]

The Food and Agriculture Organization (FAO) has estimated from 63.000 tonnes/yr to over 240.000 tonnes/yr of antibiotic consumption in agricultural and zootechnical sectors globally. The variety of the data is due to poor surveillance and data collection in many countries. However, only 0,2-0,4% of antibiotics used in agriculture are employed in crop fields while the majority are used in livestock. [3]

Nowadays, copper-based bactericides and antibiotics are the main treatments for bacterial infections in plants. However, copper agrochemicals tend to accumulate in the soil, becoming toxic for both plants and animals and as well as antibiotics, its intense use has led to resistance phenomena. Resistance to streptomycin has been reported in several bacterial species, such as Xanthomonas campestris pv. Vesicatoria, which infects tomatoes and peppers, and Erwinia amylovora, an apple pathogen. [2]

A broad variety of antibiotics are used in the zootechnical sector such as aminoglycosides, β-lactams, and tetracyclines, that is used on cattle, poultries, sheep, and swine, macrolides and sulfonamides, which are used on cattle, poultries and swine, ionophores, used on cattle, poultry and sheep, polypeptides, used on poultry and swine, and fluoroquinolones used on cattle and poultry. Most of these antibiotics are used in humans too and their misuse has caused the raising of antibiotic resistant bacteria affecting both animals and humans[4]. An analysis made by European Antimicrobial Susceptibility Surveillance in Animals on healthy cattle, pigs, and chickens raised attention on isolated resistant Enterococcus faecium and Enterococcus faecalis to tetracycline and erythromycin[5]. Fuoroquinolone resistant Campylobacter is raising concerns as contaminated poultry serves as a vehicle for transmission of campylobacter to humans and it has been observed a temporal link between approved use of fluoroquinolone in food producing animals and the increased prevalence of resistant Campylobacter in both animal reservoirs and humans. [6]

What is done to stem the issue?

In the agricultural sector, it has been observed an increased interest in research for the use of phages against plant pathogens. For instance, a phage cocktail has been tested on 41 Pseudomonas syringae pv. Porri strains with success in order to prevent bacterial blight of leeks. [7] In another successful research, a mix of 6 phages combined with a plant activator has been tested against Xanthomonas campestris pv. Vesicatoria in field experiments, showing better containment of bacterial spot on tomatoes than copper treated or untreated controls. [8]

Also in the zootechnic end veterinary sector, phage therapy has captured scientists' attention and research is currently going on. As an example, anti-Salmonella phage cocktails were administered to market-weight pigs, and they have shown a reduction in the Salmonella concentration in the ileum after exposure to the bacteria. [9]

Moreover, some political actions are made. In Europe, resolution 2018/2858(RSP) prohibits the preventive use of antibiotics on livestock and in animal feed and is limiting the use of antibiotics on an entire group of animals only in case of a high risk of spread of infections verified by a vet and in absence of alternatives.

Survey for expert in veterinary and zootechnical sector

Our survey reached 9 professionals: 3 Animal Science professors, 1 vet and 1 veterinary assistant, and 3 livestock professionals. Although most of the interviewees obtained a master’s degree or a higher qualification, the survey was meant to be submitted to any worker of the veterinary and zootechnical sector then some people of the cross-section have a lower degree.

Professionals were asked how many bacterial infections they have treated in their professional career: 30% of the interviewed have treated bacterial infections over 100 times while 40% of them have never treated one. People who have treated bacterial infections at least once were asked how many times bacterial infections required a change of antibiotic therapy, and most of them answered less than 25% of times they treated bacterial infections while the others have never had to change antibiotics.

We asked the professionals which antibiotic they mostly use in their job and the majority of them answered amoxicillin.

When asked about their knowledge of antibiotic resistance, all the experts were at least superficially aware of it. They were also asked if they had never heard about phage therapy in their professional sector, and most of them had never heard of it.

Then we asked questions about what is best to communicate both to the breeder/owner of the animals and to the consumers of the animal products: in the first case, environmental protection was the most chosen answer followed by the cost of the treatment, while in the second case side effects for the consumers was the most popular answer.

Survey for expert in agricultural sector

Our survey reached 15 professionals: 4 professors, 3 farmers, 3 agronomists, 2 researchers, and 3 other experts. Although most of the interviewees obtained a master’s degree or a higher qualification, the survey was meant to be submitted to any worker in the agricultural sector, then some people of the cross-section have a lower degree.

Professionals were asked how many bacterial infections they have treated in their professional career: 47% of them have never treated a bacterial infection.

The professionals who have treated bacterial infections before were asked which bacteria they have treated the most. To make the survey more accessible, each bacterium was followed with the most common disease it is associated with.

Since in Italy it is not allowed to use antibiotics in field crops, we asked the interviewed that had treated bacterial infections before if they had ever used antibiotics in their job.

Then we asked the ones who have treated bacterial infections but have never used antibiotics which alternative method was used. The most popular alternative method to treat infections is the use of cupric agrochemicals, but some professionals use resistant plant varieties, plant protection products, or a mix of active substances.

People who have treated bacterial infections at least once were asked how many times bacterial infections required a change of antibiotic therapy, and most of them answered they had never needed to change antibiotics.

When asked about their knowledge of antibiotic resistance, all the experts were at least superficially aware of it. They were also asked if they had never heard about phage therapy in their professional sector

We asked some questions about the use of our tool P.A.S.T.A. in their sectors after a brief explanation of its characteristics. In particular, our tool would be adapted for an eventual use on plants according to the specific problem that needs to be faced.All the professionals were inclined to use our tool.

Then we asked questions about what is best to communicate both to the farmers and to the consumers of the vegetal products: in the first case, efficacy of the treatment, while in the second case side effects for the consumers was the most popular answer.

References

1. Kirchhelle, C. Pharming animals: a global history of antibiotics in food production (1935–2017). Palgrave Commun 4, 96 (2018). https://doi.org/10.1057/s41599-018-0152-2.
2. Sieiro C, Areal-Hermida L, Pichardo-Gallardo Á, Almuiña-González R, de Miguel T, Sánchez S, Sánchez-Pérez Á, Villa TG. A Hundred Years of Bacteriophages: Can Phages Replace Antibiotics in Agriculture and Aquaculture? Antibiotics (Basel). 2020 Aug 7;9(8):493. doi: 10.3390/antibiotics9080493. PMID: 32784768; PMCID: PMC7460141.
3. FAO. What is it? | Antimicrobial Resistance. Food and Agriculture Organization (FAO) website, visited on 2nd of October 2023.
4. Charles W. Schmidt. Antibiotic resistance in Livestock: More at Steak than Steak. Focus Volume 110 Number 7. July 2002.
5. de Jong A, Simjee S, Garch FE, Moyaert H, Rose M, Youala M, Dry M; EASSA Study Group. Antimicrobial susceptibility of enterococci recovered from healthy cattle, pigs and chickens in nine EU countries (EASSA Study) to critically important antibiotics. Vet Microbiol. 2018 Mar;216:168-175. doi: 10.1016/j.vetmic.2018.02.010. Epub 2018 Feb 10. PMID: 29519512.
6. Zhang, Q., Beyi, A.F. & Yin, Y. Zoonotic and antibiotic-resistant Campylobacter: a view through the One Health lens. One Health Adv. 1, 4 (2023). https://doi.org/10.1186/s44280-023-00003-1.
7. Rombouts, S.; Volckaert, A.; Venneman, S.; Declercq, B.; Vandenheuvel, D.; Allonsius, C.N.; Van Malderghem, C.; Jang, H.B.; Briers, Y.; Noben, J.P.; et al. Characterization of novel bacteriophages for bio-control of bacterial blight in leek caused by Pseudomonas syringae pv. porri. Front. Microbiol. 2016.
8. Obradovic, A.; Jones, J.B.; Momol, M.T.; Balogh, B.; Olson, S.M. Management of tomato bacterial spot in the field by foliar applications of bacteriophages and SAR inducers. Plant. Dis. 2004.
9. Wall, S.K.; Zhang, J.; Rostagno, M.H.; Ebner, P.D. Phage Therapy to Reduce Preprocessing Salmonella Infections in Market-Weight Swine. Appl. Environ. Microbiol. 2010.