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ACTORS AND PARTNERSHIPS

It’s impossible to use bacteriophages and to develop a detection device if we don’t know what are the needs and the constraints in hospitals and in medical analysis laboratory. So we need to discuss our project with physicians, specialists and even with the whole population. We also developed partnerships with local research laboratories and companies, to borrow equipment, recover bacteria and bacteriophages, and, most important, ask them for advice.

We discussed with a lot of people to develop our project, to better understand what is useful to implement, create and integrate into our project from their remarks.

GROUND FLOOR: We established partnerships with local companies like Biomérieux and local laboratories like TIMC in order to benefit from their advice/expertise and we had the opportunity to get some of their biological material. Our engineers and researcher partners gave us a lot of recommendations all along the summer and helped us to get an external point of view on our project.

FIRST FLOOR: We visited a medical analysis laboratory and we met the laboratory manager. We discussed with him about the detection system used in his laboratory and about the advantages and limits of our system, if it was used in this kind of laboratory.

SECOND FLOOR: We met with doctors and patients who talked with us about bacteriophages and antibiotic resistance. We discussed with them to know if our detection system could be used in hospitals and what modification we should make in order to improve it. You can have a look at them on the third floor (see our schematic below).

THIRD FLOOR: All the societies must be implicated in our project. But it is essential for us to be aware of what people know about phage therapy and antimicrobial resistance. It’s important to evaluate people's knowledge and then produce an appropriate outreach.

CLICK ON THE PEOPLE YOU WANT TO HEAR FROM ! Everybody has something interesting to share with you.

GROUND FLOOR:

Learn how partnerships had influenced our project !

BIOMERIEUX

According to their advice we have:
- used a lysis buffer as an efficient positive control
- planned to add a cover to protect the contents of the tubes
- placed a ball bearing in the axis of the pipette
- understand that hybridization will happen even without mutations, rendering false positives. A possible idea to fix this problem is to do as for the resistance probe design on Staphylococcus aureus: it is necessary to find an enzyme that cuts at the place of the mutation (butt or cohesive) and a plasmid that is cut in the same place. In this case even if there is hybridization with the two ends cut it does not reform the plasmid, so it is not transformed if there is no mutation.
But the plasmid must be in very large excess to decrease the effect of competition and it is unlikely to find an enzyme that cuts at the level of mutation. This problem might be solved by using CrisPr-Cas9 system.

Béatrice advised us to make dilutions of the positive control for the freeze-drying of bacteria rather than comparing with the frozen equivalent each time. She also advised us to compare the processing efficiency after freeze-drying with the conventional laboratory freezing method (number of colonies similar to a given dilution).

Cécile Breton's conference on phage therapy, antibiotic resistance, hyper-efficiency and specificity of phages led us to choose the subject of our project.

Avalun offered us SMD camera fluorescence detection and advised us to work with STmicroelectronics.

FIRST FLOOR:

INTERVIEW WITH PIERRE-ALAIN FALCONNET
Medical Analysis Laboratory Manager
ORIADE NOVIALE
38240 MEYLAN

We presented our system to Pierre Alain Falconnet to see if it could work in the context of a medical analysis laboratory. The system seems totally adapted to the laboratory use. According to him, the operating principles are good. It is still necessary to make specificity and sensitivity tests and to know the processing time of a sample.
The most important is to have an accreditation (COFRAC, CEIVD) because without this, the analysis laboratories will probably not want to have the system.
If the use of phages is regulated by a standard P2 and not P3 or P4, he could even use the machine in his own laboratory.
He offered to help us by giving us test samples.

Read the interview with Pierre-Alain Falconnet

Did you know iGEM before I told you about it?

No

Do you often encounter antibiotic resistant bacteria in your department ?

"YES, all the time, and we sometimes even encounter bacteria resistant to all antibiotics and this occurs more and more."

Do cases of Pseudomonas aeruginosa infections represent a large percentage of infections ?

This does not represent a large percentage of infections in the case of city medicine. In hospitals, there are many more, but it is also not the most prevalent infection.
It may be responsible for cystic fibrosis in immunosuppressed patients, urinary tract infections, respiratory infections, intubated infections (clinical care).

Do you know Phage Therapy? If so, what do you think?

He heard about it "Antibiotic therapy we see these limits [..] We see resistances appearing, we see that in some cases doctors can not bring solutions of treatment. The new antibiotic molecules do not come out every day and they do not bring much new stuff and it is very toxic. " There is no new class of antibiotics. He had a scientific monitoring about the bacteriophages, “maybe tomorrow we will have phagograms” he said. A former colleague of him is working on aromatograms.

Is it possible to manipulate phages in your service (ours + in general), are there specific safety rules?

He does not know enough about the virulence of the phage, and its potential to be harmful. The hospital has a P3 laboratory (tuberculosis research) P4 in virology. There are some private labs with P3 and P4 (biomnis in Lyon).
If phage use follows P2 safety rules then no problem.

Is it possible to handle modified E.coli in your department (problem of horizontal gene transfer)?

Yes it is possible, we just need to apply adequate security rules for staff.

What are the diagnostic tools available in your service to detect pathogenic bacteria (and for Pseudomonas especially) ? How much time is needed to provide a diagnostic ? With which reliability (specificity and sensibility) ? What is the prices of those devices (price of the machine and price of diagnostic per patient) ? According to you, what are the qualities of a good diagnostic device adapted to the hospitals ?

- The Bactec: hypersensitive, relatively specific
- Real-time PCR: more sensitive than anything previously done, more specific than before : now we even see vibrios that we did not see
- Antibiogram. Can quickly make nonsense, problems of resistance.
In this laboratory, there is no system using GMOs.

In your laboratory, do you realize the accreditations imposed by the COFRAC (French Accreditation Committee)?

CEIVD (european conformity in vitro diagnostic medical device) reagent labeling: reagent studies -> repeatability, reproducibility, sensitivity, specificity, accuracy, detection limits, limit of quantification, analytical interferences .... We do not need to repeat the tests.
"You come to see a lab like us, you present a reagent that is not marked CEIVD, many will say no, we are not interested."

System overview. Do you think at first sight that it meets the security criteria of the service? Other remarks?

The system is fine, there are no special remarks.
Biomérieux, buys startups that have developed systems: time-saving research, turnkey technologies at the laboratory.
You have to validate the techniques before using them. To make an antibiogram in the day would of course be ideal.
Avalun: capillary reader. Test work is done in a laboratory.
The machine can be tested without any problems in the laboratory if accreditation is obtained.
Comex, and the general assembly of the laboratory, ethically accept whether the new technology can be purchased.
Image problem with the use of GMOs "We can help by donating strains"

How are biological waste managed in your service? Is the waste management of our kit manageable in your service? In the hospital?

Use of American bins for each waste from each room. Subcontracting to Veolia who comes to collect the bins.
So no worries for our waste

Did you have any bioethics training during your studies or after your studies?

No

If no, do you think that bioethics training is missing in your learning?

Yes

SECOND FLOOR:

INTERVIEW WITH PR. MAX MAURIN

Dr. Max Maurin notes that in the last few years, he had to face new therapeutic dead-ends caused by the antibiotic resistance that did not exist before. The issue of antibiotic resistance is, in fact, a major issue that we should urgently address. Pseudomonas is a pathogen that he encounters quite often in his department. It is not too virulent but it has numerous resistance mechanisms. According to Dr. Maurin, phage therapy is a very interesting alternative even if it arises some ethical unsolved issues and can also cause a resistance of the bacteria toward phages. The use of bacteriophages in a medical establishment seems feasible because a few years ago, viruses were already used in the biological laboratory of hospitals.

Overall, Dr. Maurin thinks that our system is very interesting and answers well the given problem. However, some biological risks have to be taken into account such as the dissemination of the system components. If these aspects are well evaluated and handled, the system is very promising. Yet, Dr. Morin thinks that it will be very difficult to play on the sensitivity and the specificity of our system to compete with today’s methods in molecular biology. Among all the different specificities of our system, it is mostly the bacteriophages selection that caught his attention the most.

At last, Dr. Morin thinks that physicians do not have enough ethical formations and are not aware enough of new bioethical laws.

Read the interview with Pr. Max Maurin

Can you present yourself ? Is it okay to take a picture of you ?

Yes. Originally a medical doctor, I chose to pursue an internship in medical biology with a specialization in microbiology. I also did a Master and a thesis in a university hospital (double degree)? After my internship, I was an assistant in the university hospital then I became master of conferences and practitioner at the hospital. Nowadays, I am in charge of the bacteriology department in the Grenoble UHC.

Did you know iGEM before I told you about it?

Not in the details but I have already heard about it thanks to my colleagues (some of them even helped the previous Grenoble iGEM team).

Do you often encounter antibiotic-resistant bacteria in your department?

INTERVIEW WITH PR. OLIVIER EPAULARD

Each day, Pr. Olivier Epaulard is confronted with patients suffering from diseases caused by resistant bacteria. According to him, a misuse of antibiotics is the cause of 90% of the resistances and one antibiotic treatment out of two is wrongly prescribed. The number of antibiotic resistances does not cease to grow, like the number of immunosuppressed patients. Therefore, antibiotic resistance is a major issue of our century. Pseudomonas is also a pathogen that he encounters quite often in his department. Pr. Epaulard cannot make up his mind on the subject of phage therapy because he does not understand why it is not more developed. The positive aspect of this alternative is that bacteriophages destroy all bacteria - even the inactive ones -, something that antibiotics cannot do. According to him, the use of bacteriophages inside his department would not cause any issue.

Pr. Epaulard thinks that the project is very interesting. He is not overly passionate about the identification aspect but rather by the therapy aspect of the project. The diagnostic tools he uses have already very good specificity and sensitivity. It is going to be hard to compete with them. He verified many aspects of the biology in the system and was pleased with our answers.

Pr. Epaulard thinks that one can learn about bioethics through practice. Therefore, there is no need for more theoretical courses on ethics in a physician formation.

Read the interview with Pr. Olivier Epaulard

Did you know about iGEM before I told you about it?

INTERVIEW WITH H.G, PATIENT TREATED WITH PHAGE THERAPY

After getting infected feet wounds, H.G was treated with antibiotics but the infection was not cured. No antibiotic worked and the infection spread. The only way to treat this patient was to amputate the two legs. H.G had heard about phage therapy and contacted a doctor specialized on the subject. He chose to try phage therapy to cure his wounds. Bacteriophages were applied for a year on the wounds and he is now almost cured.

On the pictures, you can see the state of his left foot before and after the phage therapy.

Read the interview with H.G

Where did the infection come from?

A wound, I have hammer legs (claw feet/toes) (deformation of the toe), the toes do not touch the ground and in the shoe it made a light bulb. One evening a piece left and it made a sore and the infection came. It's been 13 years since I have the infection. 6 months ago it was two feet.

Were you treated early on right foot?

Do you know which antibiotic you have been treated with?

When did the antibiotics stop working?

Do you know if it was always the same, or it was an antibiotic cocktail?

Was it a liquid to put?

THIRD FLOOR:

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