Antibiotic Resistance

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Antibiotic resistant bacteria affect individuals independent of age, socio-economic status and country of residence and thus pose one of the biggest threats to global health today. It is an inevitable evolutionary response to antimicrobial usage. Susceptible pathogens are being replaced by more resistant variants by the process of natural selection [2]. However, despite the fact that this is a natural process, it is greatly accelerated by the misuse of antibiotics in humans and livestock [3][4]. An increasing number of infections such as pneumonia, tuberculosis and food borne diseases are becoming harder to treat as antibiotics become less effective against particular bacterial strains. Standard procedures such as organ transplantations, chemotherapy and routine surgeries like caesarean sections will involve more risk in the future if no alternative methods are developed. The length of hospitalizations will increase as a consequence, leading to higher medical costs and potentially increased mortality rates [1]. Therefore, the treatment of multi-drug resistant bacteria is highly relevant.

         Dr. med. Andreas Kronenberg

         Member of scientific board at the Swiss Center for Antibiotics Resistance 

         Group Leader at Institute for Infectious Diseases, University of Bern

• Andreas Kronenberg gave us a broad overview about the current situation in Switzerland, predictions for the future and available resources on this topic
• He sees the importance of our project and names phage therapy as one of many solving approach
• Challenges in phage therapy contain the high selectivity for specific pathogens, which makes an empiric therapy difficult and its standardization
• Current phage therapies have the disadvantage that the bacterium needs to be identified, which is not always possible in a clinical setting, and if so, identification needs 24 to 48 hours
• The immune system might degrade phages.

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         Karin Waefler

         Technical Officer at World Health Organization

• Although WHO has not published any official guidelines or recommendations regarding phage therapy, they provided us with general information concerning its potential and a statement about its use from the Federal Council of Switzerland
• Furthermore, WHO provided us the global list with bacterial strains that should be targeted with priority
• She points out the problem, that phage therapy has a reduced potential for low- and middle-income countries as their infrastructure is often poorly developed and shipped phages as well as the patients bacteria could have evolved multiple times in the time of shipment

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[1] Fact sheet on antibiotic resistance, WHO, https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance, Accessed: 12.06.2019
[2] Clinical Impact and relevance of antibiotics resistance, French GL, Advanced Drug Delivery Reviews, 57(10), pp. 1514-1527, 2005
[3] Prevalence of Inappropriate Antibiotic Prescriptions Among US Ambulatory Care Visits, 2010-2011, Fleming-Dutra KE et al., JAMA: The Journal of the American Medical Association, 315(17), pp. 1864-1873, 2016
[4] Antibiotics Overuse in Animal Agriculture: A Call to Action for Health Care Providers, Martin MJ, American Journal for Public Health, 105(12), pp. 2409 - 2410, 2015

Clinical Application

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For the clinical use of phages, the following considerations are important: product availability, production, formulation and administration, dosage and evaluation. The first condition for the use of phage therapy is simply to have bacteriophages available for treatment. This implies having access to phages that are both biologically active against the patient’s bacterial isolate and satisfy regulatory requirements. As there are no registered products to date in western medicine, this is currently a bottleneck to which we aim to make a contribution with our project. It would be desirable that an organized phage bank would exist that facilitates exchanges.
Furthermore, production of phages needs to have a high level of purity for clinical use. As phage preparations are viewed as medicinal products, they are subject to Good Manufacturing Practice (GMP) compliance. The stability and consistency of a phages is harder to guarantee and control than that of a chemical however, which puts a strain on the clinical development of phage therapy.
The administration and dosage of treatment must be considered as well for optimal treatment, as circulating phages are sequestered by the reticuloendothelial system in the spleen and liver. The presence of bacterial targets also influences the pharmacokinetics, making it hard to estimate in a patient.
Finally, therapeutic evaluation is necessary to improve therapy further [1].

         Prof. Dr. med. Jürg Hafner 

         Department of Dermatology, University Hospital Zurich

• Chronic wounds are very common as 1% of the population is expected to deal with a chronic wound in his or her lifetime. It is mainly caused by diabetes and insufficient blood flow in arteries and vein. Roughly 80% of these wounds are populated by bacteria, whereas in up to 1/3 of cases pathogens like Methicillin-resistant Staphylococcus aureaus (MRSA), Streptococcus pyogenes and Pseudomonas aeruginosa are found, which are not part of the natural microbiome.
• From literature it is hard to verify the influence of bacteria on wound healing as no randomized clinical trails have been done to study this phenomenon yet. However, empirical data suggests, that bacteria can build biofilms on wounds, which presumably influence wound healing in a negative way. Thus, it is likely that bacterial infections are relevant for wound healing and therefore, they should be studied more extensively.
• The current treatment of chronic wounds includes periodical debridement of the biofilm and topical disinfection using octenilin for example. Moreover, flanking measures are applied to remove the cause of the disease including the decrease of venous congestion, the release of water from the leg and the use of catheters to improve blood flow. In case of non-healing wounds, surgical procedures are necessary. The application of antibiotics is not desirable as it poses the risk to select for antibiotic resistant bacteria.
• Currently there is no application of phages to chronic wounds. However it is a promising strategy to fight bacterial infections as no antibiotics are needed to be used in the process. Moreover, further application could involve system application of phage therapy for various other conditions.

         Désirée Schwendimann

         Assistant doctor in anesthesia, Inselspital Bern 

• Mrs. Schwendimann gives us an insight about how antibiotic resistance affects her work as an anesthetist.
• Profound cleaning is necessary whenever a hospital has a patient with resistant bacteria and various safety measures have to be followed.
• In case of a surgery, the whole operation room has to be deep cleaned, before it can be used again.
• Furthermore, she mentions that hospitals in the Netherlands have a system that limits the occurrence of antibiotic resistances. Nurses have less patients, which leads to less mistakes with cleaning hands between patients, surgeries are performed less on elderly people and they always have immunologists in their teams that check the bacterial situation.

[1] Clinical Indications and Compassionate Use of Phage Therapy: Personal Experience and Literature Review with a Focus on Osteoarticular Infections. Patey O. McCallin S. Mazure H. Liddle M. Smithyman A. Dublanchet A. Viruses, Vol. 11, No. 18, 2019

Patient Safety

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A review looked at 30 studies to make conclusions about the safety of phage therapies. Among these studies, various routes of administration were considered. Most phages were safe as no patient developed side effects or toxicity directly associated with phage administration. However, certain precautionary measures are recommended. Purification and appropriate dilution of phages increase safety, as this eliminates crude bacterial lysates and bacterial toxins, thereby avoiding potential immune responses and constitutional symptoms. In addition, phages that carry virulent or drug-resistant genes must be avoided. Therefore, the use of strictly-lytic phages is necessary [1].

         Dr. Grégory Resch

         Department of Fundamental Microbiology (DMF), University of Lausanne

• No major side effect from phage therapy was observed in clinical trials to date and endotoxin shock from the lysis of the infectious bacteria has never been reported as well.
• In a case in France, a patient was treated with an intravenously applied phage cocktail for over six weeks and no adverse effects occurred.
  
• However, when a non-purified phage solution is applied, that has a high level of endotoxins from phage amplification in bacterial host, inflammation can occur. Therefore, purification is necessary and can be done by the Queen Astrid Military Hospital in Belgium, Pherecydes Pharma in France as well as at USC San Diego and Adaptive Phage Therapeutics in the USA.
• Concerning interactions with the microbiome in case of oral application, a study from Nestle targeting patients with diarrhea found that phages might not be as aggressive against commensal bacteria as antibiotics,. However results were influenced by the fact, that many phages were likely already killed by the stomach acid and didn't reach the intestine.
• A prophages is DNA sequence of phage origin that got included into the genome of bacteria. Temperate phages will be silent in the host, but can be reactivated and be an indirect trigger for various human diseases. The software PHASTER can be used to check whether a bacterium has a phage sequence in its genome.

         Dr. Jean-Paul Pirnay

         Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital Brussels 

• Purification of the phage solution is necessary before applications in patients, otherwise the endotoxin produced by bacteria during phage amplification can cause severe complications especially in intra venous applications. However, purification will always lead to the reduction of variability, as some phages stick to the filtration column and will be removed.
• Moreover, the whole phage genome has to be sequenced to ensure the absence of toxic genes.
• To improve patient safety and efficacy of the treatment, aims should be made to better understand the role of the immune system on the one hand and the synergy or disynergy between phages and antibiotics or between multiple phages on the other hand. Furthermore, phage resistance needs to be studied as well.

[1] A Systemic and Critical Review of Bacteriophage Therapy Against Multidrug-resistnt ESKAPE Organisms in Humans. Haddad LE. Harb MA. Stibich MA. Chemaly RF. Clinical Infectious Diseases, Vol. 69, pp. 167 – 178, 2019

Biosafety

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After injecting their DNA into a host, phages either multiply and induce lysis of the host cell or they are stabilized as prophages either integrated into the host chromosome (temperate phages) or as a free plasmid molecule. Temperate phages can mediate horizontal gene transfer that occurs in bacteria through transduction. Transduction is a mechanism by which a segment of the bacterial genome is inserted in a newly formed viral particle instead of the viral genome which can therefore be transferred to other bacteria and can recombine with the host’s genome by homologous recombination. From a biosafety point of view, the introduction of new genetic material from a recombinant viral particle into the bacterial gene pool may have positive, negative or neutral outcomes depending on the genetic marker introduced [1].
The survival of bacteriophages outside a host depends on the nature of the phage itself and is further influenced by the surrounding environmental conditions. Even if free phages can survive a rather long time in the environment, they need to infect susceptible bacterial hosts to replicate and propagate themselves. Many phages are known to be highly specific for their receptors and are therefore characterized by a narrow host range. However, some phages that are able to infect a large range of bacterial species have also been identified. These broad-host-range phages may promote genetic diversity and genetic exchange among a wider range of bacterial populations. Determination of the host range, virulence, resistance to environmental factors or the potential for gene transfer of the manipulated bacteriophage is therefore an important step in the risk assessment process to evaluate the probability of the phage’s propagation in a particular environment and its potential role in global gene transfer [1].

         Dr. Ursula Jenal

         Jenal & Partners Biosafety Consulting

• T7 phage is categorized as BSL-1. Its narrow host range among bacterial strains, incapability to infect eukaryotic cells and decreased efficiency of DNA packaging under UV light reduces its capacity to survive in nature and disbalance the ecosystem in case of an accidental release from the laboratory.
• If we randomize the tail fiber protein of T7 however, the situation has to be analyzed anew, as no laws are in place that regulates this case. Therefore, a risk assessment is necessary.

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         Dr. Grégory Resch

         Department of Fundamental Microbiology (DMF), University of Lausanne

• Evolved phages most probably already exist in nature, but haven't been find and characterized yet. Therefore, they should not be considered to be GMO and most likely do not affect environment.
• However, if a super phage is created, that can infect and lyse many bacterial strains, it can only be used in a highly controlled environment to prevent destabilization of the ecosystem.

[1] Contained Use of Bacteriophages: Risk Assessment and Biosafety Recommendations. Verheust C. Pauwels K. Mahillon J. Helsinki DR. Herman P. Applied Biosafety, Vol. 15, No. 1, pp. 32 – 44, 2010

Regulatory Process

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Phages are currently classified as Medicinal Products (MP) under European Union (EU) legislation and as a drug by the Food and Drug Administration (FDA) in the United Stated, which necessitate that phages be produced under Good Manufacturing Practice (GMP) guidelines and require clinical trials. The few formal experimental clinical trials that have been completed to date have produced inconclusive results on the efficacy of phage therapy which contradicts the many successful treatment outcomes observed in recent individual case reports. Therefore, phages continue to lack any market approval in Western medicine as a recognized drug but are nevertheless increasingly used as an experimental therapy for the compassionate treatment of patients experiencing antibiotic failure [1].

         Pia Stadelmann

         Swissmedic

• Swissmedic explained us the legal framework for the use of bacteriophages
• Phages are considered as medicinal products and therefore the provisions of the Therapeutic Product Act (HMG) are used
• They must have a production authorization granted by Swissmedic and comply with Good Manufacturing Practice (GMP)
• For clinical trials a license from the ethics commission and Swissmedic is required
• The usage of the product under a temporary license (“compassionate use”) is only possible, if the product is already in clinical trial

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         Dr. Jean-Paul Pirnay

         Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital Brussels

• Regulatory issues include the declaration of phages as medicinal products, which presume Good Manufacturing Practice, preclinical studies, phase I, II and III clinical trials and centralized marketing authorization. This is too expensive as phages are biological entities that cannot be patented. Moreover, this process is also not feasibly for an evolving therapy.
• In Belgium phages were used earlier under the WMA declaration of Helsinki, Article 37, which enables their use for desperate cases without the need to perform clinical trials and work under GMP conditions. While demonstration of the efficiency of phage therapy is not feasible from compassionate use cases only, the generated data suggests that phage therapy is safe, as no adverse effects were detected. The main problem with this approach is however, that too much time is lost in case of critically ill patients.
• The Dedicated PT framework is a new concept elaborated by scientists in cooperation with Belgian Authorities. It includes the characterization of phages, their storage in a banking system, the production of a single phage (active pharmaceutical ingredient) using a suitable host according to a monograph and external quality testing by a "Belgian Approved Laboratory". Then approved phages can be bought in a pharmacy upon prescription as magistral preparations.

         Dr. Grégory Resch

         Department of Fundamental Microbiology (DMF), University of Lausanne

• The phage from which the library is derived needs to be well characterized and has to undergo a clinical trial
• The evolved phages don't have to undergo this procedure again, as they underwent only an accelerated natural selection process. Instead they only have to be sequences to exclude the existence of dangerous sequences
• A physic with no other treatment option can contact Swissmedic via a special demand formular. Phages that were authorized in another country with similar standards can be used in this case

[1] Clinical Indications and Compassionate Use of Phage Therapy: Personal Experience and Literature Review with a Focus on Osteoarticular Infections. Patey O. McCallin S. Mazure H. Liddle M. Smithyman A. Dublanchet A. Viruses, Vol. 11, No. 18, 2019

Project Feedback

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         Dr. med. Andreas Kronenberg

         Member of scientific board at the Swiss Center for Antibiotics Resistance 

         Group Leader at Institute for Infectious Diseases, University of Bern

• Andreas Kronenberg highlights the importance of our project.
• Identification of phages needs to be quicker than current method, which takes 24 - 48h.
• Our therapy needs to account for the problem, that each case is very individual. Therefore, a solution that serves all cases is necessary.

         Dr. Jean-Paul Pirnay

         Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital Brussels

• Efficacy of infection doesn’t depend on tail fiber protein alone but includes many parameters like attachment, replication in high copy numbers, quick infection, no induction of resistance response and optimal working temperature of 37 °C.
• Therefore, mutations in the whole genome would be necessary for maximal efficacy.
• For clinical applications a concentration of 10^7 PFU/ml is necessary.
• Whole genome sequencing is necessary to ensure the absence of toxic genes within the genome.
• Stability of phages has to be tested after amplification.

         Prof. Dr. med. Jürg Hafner 

         Department of Dermatology, University Hospital Zurich

• A biological tool to kill bacteria would be very useful as antibiotics should be used limited in order to avoid the generation of resistant bacteria.
• The first application of our product would be in chronic dermal wounds, which could be tested easily. However, he sees further application in a systemic intra-venous treatment for various conditions.

         Dr. Grégory Resch

         Department of Fundamental Microbiology (DMF), University of Lausanne

• He likes, that our strategy is adaptable in the course of the treatment and new phages can be identified all the time to account for potential emergence of resistance.
• Use T3 sequence as a positive control to verify that the system is working.
• Compare the results with classical phage training.
• Changing only the tail fiber sequence might not lead to the maximum efficacy of infection.
• Phages might aggregate before application. This can be avoided by leaving the phages on different plates and mix only shortly prior to application.
• Control the stability of phages via titer measurement.

Public Engagement

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         Scientifica

Scientifica is a fair that aims at educating the broad public about the newest scientific achievements. It includes special activities for children as well as a specific program for politicians and economists. Scientists have the opportunity to explain their projects and raise enthusiasm for science and get valuable feedback from a very diverse audience:

• The project was received well by the public and people were open to the concept to use phages to treat bacterial infections.
• People were particularly interested in the price of phage therapy, comparison to naturally occurring phages, biosafety issues as well as efficacy of treatment.

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         Presentation at the Intra-Departmental Seminar

The Seminar is a series of talks that allows for optimal interaction and sharing of expertise with people from different scientific backgrounds.

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