Difference between revisions of "Team:Grenoble-Alpes/actors partnerships"

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.

TIMC

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).

Les midis MINATEC

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

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

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

Visit the medical analysis laboratory!

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?

This is a current issue. There are a lot of new resistant bacteria. Most of my activities are centered on the subject. It includes antibiotic resistance characterization and antibiotic efficiency prognosis.
“It is not a new issue.”
In the hospital, antibiotics are used a lot and there is an emergence of poly resistant bacteria strains. The industry did not succeed in developing antibiotics to remedy to the resistance issues. The bacteria won.
“There are many strains that became resistant to most of the antibiotics” so “the therapeutic possibilities decreased little by little”.
“It is a real issue for public health” but we know how to treat most of the cases though. “There is an emergence of of therapeutic dead-ends in the hospital, that we we did not see since long time ago. People have infections with zero treatment possible to cure them, that is rare.”
“Each year, new resistances emerge and we see their prevalence increasing. That is a reality.”

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

It is a significant pathogen, even though initially not very virulent. In the hospitals it is an issue for weakened people (immunosuppressed people, or in extended care). It is an hospital pathogen. “This strain has a lot of facilities to acquire new resistances”.

Do you know phage therapy? If so, what do you think?

There are difficulties to find the good bacteriophages, the not pathogenic ones.
Ethical problems persist. Resistance problems still persist. Bacteriophages are an interesting concept but should be used as a last resort.
There are also new alternatives like anti-virulence drugs to prevent the virulence of bacteria but not their growth. “They are less selective molecules that act on virulence factors without killing them”. It can be used as a treatment by itself or to associate with antibiotics.
Some drugs to boost immune defenses also exist.

Is it possible to manipulate phages in your service (ours and generally speaking), are there specific safety rules?

Viruses were used in the past for typing in molecular biology labs in hospitals.
However many regulations are necessary for the drugs and even more for “living” drugs. There is an additional constraint not to infect the patients and the medical staff. There is a fear that viruses could transmit the resistances (horizontal transfer).

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

Maybe special authorisations are needed since they are GMOs. At least a declaration is mandatory.

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 ?

2 main classes of tools are available in my service:
1) Isolation with bacterial culture which allows the identification of bacteria and the characterization (antibiotics activity and efficacy against the strain.)
If it is too complex or if we need to be faster :
2) All that includes Biochemical Methods: PCR (PCR in real time -qPCR-). Research for a pathogen.
About the sensibility, PCR is the less risky technic regarding to the sample contamination (between them). It is the most reliable technic and it is very fast (1 to 2 hours). Sequencing is a bit long because it has to be sent out of the hospital so it requires about 3 days or more.
Example : Isolation + antimicrobial susceptibility testing for Pseudomonas and resistance origin identification. If we want more details (carbapenemase type…) the most adapted will be qPCR. Of course ,the method used must be adapted according to the case complexity and the time available.
He estimates that nowadays in the university hospital center the repartition of the methods used is about:
- 90% of antimicrobial susceptibility testing and phenotypic methods
- 10% of Molecular Biology

If not mentioned previously: Do you have an antibiogram? How long does it take to make a diagnosis?

Isolation and antimicrobial susceptibility testing represent 90% of analyses. They are relatively reliable. If the phenotype does not allow to get all the information needed, we switch to molecular biology methods.
So molecular biology in real time represents 10% of analyses. In particular cases there is not any tool available, if new resistance mechanisms appear, we can obtain false positives due to contamination. Still the reliability of real time molecular biology remains good and adapted to the complexity and the need to act quickly.

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

The two main risks for the system are :
1- Biological risk (for the medical staff), pathogen classification (here P2 but not too serious, in particular for healthy people, so there is no epidemic risk)
2- GMO risk, we have to evaluate the diffusion and dissemination risks for us and find how to treat the contaminated waste.

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

We use Citybac® DASRI to throw the infection-risk waste and then everything is incinerated. Water is treated before being dumped.

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

No, he learned on the job. He never got any specific formation on the subject. Some colleagues got teachings in other formations.

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

YES, the regulation is really constraining in medicine, a formation on bioethical laws should be dispensed at the beginning. Doctors are each day more led to have ethical reflexions but can definitely do better. Some people have the knowledge but the majority is not up to date on the regulation and recent laws.

Read the interview with Pr. Olivier Epaulard

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

No.

Do you often encounter antibiotic resistant bacteria in your department ?

Daily. Every day, all the time. They are the result of antibiotics misuse (about 90% of the cases). He estimates that half of antibiotic treatments are wrongly prescribed. According to a study, half of the doctors antibiotic prescriptions are useless. Antibiotic resistant bacteria are enterobacteria in majority. The resistances are due to penicillinase (and other beta-lactamase / enzymes). The number of infections with resistance keeps increasing.
Although rare, there are cases of infection for which no treatment is possible (no antibiotic is able to kill the bacteria which resistant to everything). Some countries like India, Greece, Turkey and some african countries are at a critical stage. They are not anymore trying to manage the use of antibiotics but they are looking for methods to treat patients infected by bacteria resistant to nearly all the antibiotics.
Resistances appear on strains that were sensible to everything in the past. Antibiotics come from mushrooms (penicillium, ...). They are very old molecules, and that explains why bacteria can have facilities to become resistant. To reverse the process of antibiotics abusive utilisation is very complicated and hard to impulse (in particular concerning the education of the population and of practitioners). → Analogy with the global warming: The majority knows but it requires strong behaviour changes not realized. Is it too late ? Resistance of Gram bacilli - like Pseudomonas - increases.
Nowadays, more and more people are immunosuppressed (But they would be dead before without treatment. Often, the immunosuppression comes from a heavy treatment) or go through treatments exposing them to bacteria for a long or excessive duration. For example immunosuppression for grafts or surgeries are more and more risky since they are open doors to infections. On another hand, exposition to bacteria is more and more common because of the treatments lengthening.

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

Concerning common infections, we meet rarely Pseudomonas aeruginosa in general but it is present in the hospital infections in the operating rooms for patients that have a catheter in a vein, a contact with material, or a urinary infection.

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

Yes. He can not have a precise idea. On paper, he is enthousiast to this idea. Moreover phages seem to be easy to produce. While antibiotics can lead to intestinal flora diseases during their ingestion, phages can avoid that. In the case of chronic infections on prosthesis, to change the material is the only way to cure the infection… The phage therapy could also be a way to clean the medical material.
But he feels like it has been 15 years that he is hearing about phages and that nothing happens. He thinks that there are maybe other problems in the utilization of phages than
just the patent problematic, finally a not so important matter according to him. Indeed, in France modified viruses have been patented, a modified bacterium could be patented, so patenting a living organism is possible,... Hence, why not the phages ? One advantage of the phage is their permanent efficacy on the bacteria. Indeed antibiotics are effective only on growing bacteria. However it is the case for infections such as pneumonia and urinary infections but not long ones like bones infection for which the phage utilization would be very interesting.
Russia and Georgia tell that they produce phages in GMP (good manufacturing practice) but with which purity ? Which vivacity ?
Interesting book: “La Phagothérapie - Des virus pour combattre les infections “ Alain Dublanchet

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

Yes !! No particular problem.

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

Not any problem, everybody manipulate bacteria with plasmid containing resistance/selection genes. The evacuation system is ok and allow to avoid any crossing and to spread bacteria in the environment.

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 tools available in my service are multiplex PCR (not very efficient), targeted PCR, 16S amplified PCR + sequencing (not the most efficient). Else, the most used is the Gram coloration and then the culture. A response can be given in 24h/48h (or longer: 5 days for some bacteria and 3 weeks for mycobacteria).
There are also cultures identified by MALDI TOF (2h of preparation on ion exchanging resin + growing time of the bacteria). Specificity is excellent and the sensitivity is good. There is also the possibility of identification by biochemical method (enzyme, (quite rare nowadays).

If not mentioned previously: Do you have an antibiogram? How long does it take to make a diagnosis?

We use mainly phenotypic antimicrobial susceptibility tests (not genetic in general, more for virology) which need about 48h to 72h.
Else by PCR (Staphylococcus aureus by PCR → Research of methicillin resistance).
Nothing replace a antimicrobial susceptibility test for now, result in 48h - 72h (including the culture time).

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

“Are you doing bacterial culture after the transformation or not ?” - “Nope”
Giving a result in less than 10h is interesting, it is less than a bacterial culture so the delay is performant. But the detection PCR need only 2h to give a result… We have to be careful to only extract the bacterial DNA. He thinks the project is interesting. He is not passionate by the identification side but by the therapy side.

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

They already manage more dangerous waste in the waste treatment process. All the waste is incinerated. Our waste represent nothing problematic for the service.
Example : The universitary hospital center is equipped to manage all the waste from all the infectious and assimilated curing activities. The management of our waste therefore represent no problem for a hospital.

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

A bit but not much. That is more about practice during the job exercise : interactions with bioethical institutions, ethical seminars in medicine with people having ethical formations way more thorough. And of course there is the knowledge of bioethical laws.
He thinks there is not the time to do more during the medicine study program. Anyway doctors do ethics every day (tell or not a disturbing truth, to who let the access to this patient record…). Also in research, every day there are ethics, in the realization of experiences, surveys...

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

No. In reality they do bioethics all the time. Consents, palliative care, accompanying someone's death, bioethical laws: they face ethics every day. The training is clearly not empty on the subject, whether in class or in practice.

WARNING: this includes images some readers may find disturbing (OPEN WOUNDS)

Necrotic wound (black tissues)before phage treatment	Healing wound (pink tissues) after phage treatment

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 has been 13 years since I have the infection. 6 months ago it was two feet.

Were you treated early on right foot?

Yes, I had a lot of surgery, then it did a perforating ache (dermatological lesion) and they treated me with antibiotics. The last time I had surgery, I had 7 months of treatement with antibiotics, and there was no result.

Do you know which antibiotic you have been treated with?

No, but at the last operation my doctor told me that the last option was to cut the leg.

When did the antibiotics stop working?

It has been 3 years since antibiotics are no longer effective.

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

No, at home I took the antibiotics but one day my face was gray, and meanwhile I went for X-ray in Meylan and a lady said to me "if you want I know someone who has been treated with the phages, do you want to see him?“
I said there was no problem because the doctor wanted to cut my legs. So I went to see the person being treated by the phages, and he was able to walk. Then I contacted a doctor from Nîmes, Mr. Riche. I took an appointment at Nîmes to see him. The doctor knew of what bacteria I was infected so he told me "you have to take such phages and put them on the wound.

Was it a liquid to put?

Yes, and it was not enough, I had to have surgery. I asked my doctor at the CHU de Grenoble (University Hospital of Grenoble) whether during the operation he could put the phages on me but he refused. So Mr. Riche advised me someone in Clermont-Ferrand who operates with phages. So I took appointment with the doctor from Clermont-Ferrand and he operated on me with the phages. Then I continued to put phages on the wound and I drank too.

Is it really better? Do you feel it?

Yes

Did you notice side effects because of the treatment with phages?

No and I saw the person who was treated with the phages and he was fine.

How long did it need until you saw an evolution after you started to use phages?

6 months, even less. In addition to taking phages there is a diet to do, and take blood tests (18ml). The blood tests must be re-injected intramuscularly but the nurses do not have the right to do it so it was my wife who did it.

And you were not afraid at all even after that?

No because otherwise I was cut off the leg

Yes it is true that you prefer to try rather than lose your leg

Yes. There is a blood test today and then 4 days later then 8 days later and there are 8 sessions to be done.

Do you put the phages everyday?

Yes since 1 year and the wound is almost healed. But with my job (farmer) I had to go back to work, so they plastered me with a plate underneath, so I could work.But the wound remained and the infection appeared on the left foot too.

Is it the same wounds on both feet?

Yes, but the left is because of the shoes. I have a foot sensitivity disorder so it rubbed, it made a sore so the nurses tried to treat me but the problem is that toes have broken. I asked to operate with phages but they refused and suddenly I asked if after the nurses could put me phages on my toes and they told me no problem.

Who told you about Mr. Riche?

I did it myself, I inquired. Mr. Riche advised me to put more on my wounds of hydrogen peroxide and we saw that after a day the wound became pink when before it was black.

Do You know what bacteria infected You?

Staphylococcus and en another. So, Mr. Riches, seeing that said to me that such phages I needed.

Do you know if it is a phage cocktail or a phage in particular?

One for Staphylococcus and one for the other bacterium.

Phage therapy therapy is more developed in Eastern countries than in France, right?

Yes phages come from Ukraine. It is Monsieur Riche who makes them come. But now when it arrives at the customs, they break the flacons. So now it passes by another name.

With the research you have done, have you found other doctors who practice phage therapy ?

No, just Monsieur Riche and the doctor from CLermont-Ferrand. The other day I saw him and said, "See, I still have my leg”

It proves that it works well.

Yes, and Monsieur Riche has written a book, it can be found on the internet and he has made ointment, it is a healing ointment that I put on my wounds.

You think we can reach Mr. Riche for more information?

Yes Yes ! It will make him happy

How much does a treatment cost?

It costs nothing, it is flasks, it costs 45 euros with all inclusive for 6 bottles!

Do you still take AB anyway?

Yes

And once you've taken phage treatment, do you feel really better?

Yes, not the first shot but afterwards as I was in shape. And he cared for cancer too, from a woman who lives in Meylan. Thus she was saved.

How much time do you still have?

It is necessary to wait until the wound is completely closed

And how long have you been treating yourself to phages?

5 years

Did the treatment hurt you?

No, we feel nothing at all, it is just put on wounds and swallow (morning fasting) and also I pay attention to what I eat, wholemeal bread, whole rice no potato no red meat, no coffee, no sugar .. it is for healing especially