CE19 - Technologies pour la santé

Bacterial Universal Sensing – BacUS

Bacus

Universal detection of bacteria

Objectives and main issues

The Bacus project proposes new alternatives for the rapid detection of pathogenic bacteria. The aim is to monitor bacterial growth in real time during the enrichment step thanks to the detection of bacteria monitored on biochips (1 cm2) functionalized with universal peptide probes. In this context, «universal« means that these ligands bind any bacteria, whatever its nature or physiological state, the presence or absence of peptidoglycan, etc. An original aspect of Bacus is therefore based on the use of antimicrobial peptides (AMPs) as ligands for pathogenic bacteria. Another strength of Bacus is the combination of broad-spectrum probes based on MPAs with other sets of highly specific probes of ESKAPEE pathogens. Thus, a single device will allow the evaluation of Yes/No sterility as well as the identification of the ESKAPEE pathogen in case of a positive response.<br />This project will be carried out thanks to a close collaboration between a chemical partner (synthesis and characterization of MPAs), a technological partner (functionalization of biochips and specific instrument development), and a hospital partner (implementation of the technology developed in quasi-real conditions).

The detection and identification of bacteria in the blood and the determination of their sensitivity to antibiotics are essential for the management of patients. In this context, the faster the laboratory response, the higher the chances of survival. In order to improve this control of analysis time and cost, we propose to develop devices allowing a faster detection thanks to a biochip reading by Surface Plasmon Resonance Imaging (SPRi) DURING THE SAMPLE ENRICHMENT STAGE. This coupling of the bacterial multiplication phase with the detection phase makes it possible to drastically reduce the delay between the sampling and the transfer of the information to the physician. In a very original way, we propose to implement A NEW TYPE OF LARGE SPECTRE LINES ON THE SURFACE OF BIOPUCES (antimicrobial peptides), in order to detect the presence of bacteria using a small number of different probes (less than a dozen). Finally, a last important aspect of the project is based on the incorporation, on these same biochips, of SPECIFIC PROBES (antibodies, phages and/or aptamers) TARGETING THE PATHOGENS OF THE «ESKAPEE« GROUP, which includes the bacteria most frequently isolated in bacteremia and potentially the most resistant such as certain enterobacteria or Staphylococcus aureus. Appropriate management must be chosen if their presence is confirmed. In this way, the BacUS project responds to various important demands related to the medical diagnosis of bacteremia, namely: a faster response on the presence of a bacterial pathogen, a clear identification of a pathogen from the «ESKAPEE« group and a controlled implementation cost compared to currently available solutions.

The results achieved so far are almost in line with those expected and described in the initial planning (see the Gantt chart of the project as submitted and the table in section B above).
In terms of project coordination, the kick-off and biannual monitoring meetings have taken place as planned and have been recorded in minutes. D0-D3
Concerning the progress of the WPs, peptide syntheses could be carried out, i.e. on about ten different sequences, possible by solid syntheses on about forty different resins. Purifications proved to be more laborious than expected for different sequences, which explains why this milestone has been partially achieved so far. These purifications will be completed as soon as possible. D7-D8
Nevertheless, even if the totality of the synthesized sequences could not be purified to date, we were able to prepare the first series of SPR biochips functionalized by this set of antimicrobial peptides. M1. This allowed the realization of first experiments for the detection of bacteria under model conditions, which led to a publication in the journal Talanta in May 2019. doi:10.1016/j.talanta.2019.05.062
Early on, we were able to carry out the first series of peptide stability tests under model conditions similar to the real conditions (with the difference that the blood samples came from healthy, not sick patients). These results were presented in the thesis work of Mr. Eric Pardoux, PhD student involved in the first year of the Bacus project. D11
Finally, we were able to achieve an exhaustive synthesis of recent publications describing the implementation of anti-microbial peptides for biosensor applications, which led to the publication of a review in the journal Molecules in April 2020. doi:10.3390/molecules25081998

The outlook remains in line with that set out in the initial document.

1. Talanta. doi:10.1016/j.talanta.2019.05.062
2. Molecules. doi:10.3390/molecules25081998

A bacteremia is caused by the presence of bacteria in the bloodstream, which is a sterile fluid in healthy people. This is one of the most common causes of death among hospitalized patients in intensive care units. Nowadays, in case of bacteremia suspicion, some blood is drawn from the patient and then "cultured", usually overnight, in experimental conditions favorable for bacteria multiplication in the vial. Then, and only after this enrichment step is completed, the bacterial identification can be launched. This latter step may last from few minutes and up to several days, depending on the nature of the bacterial contaminants, and also depending on the acceptable cost for its identification (the faster solutions, like PCR, are also the most expensive assays for the bacterial analysis). The assessment of the presence of bacteria, followed by their identification along with their susceptibility to antibiotics is the most important issue when facing a bacteremia. In this context, the fastest medical response, the higher survival chances.
In order to better control both the critical time-lapse and cost issues, we propose to develop devices enabling a much faster response by using Surface Plasmon Resonance imaging (SPRi) of biochips DURING THE ENRICHMENT PHASE. The coupling of the multiplication phase with the bacteria detection into a single step will then significantly decrease the delay between the blood sampling and the response given to the doctor. One original aspect of our project is the use of A NEW KIND OF WIDE-SPECTRUM LIGANDS (THE ANTI-MICROBIAL PEPTIDES) FOR THE FUNCTIONALIZATION OF THE BIOCHIPS. The objective will be the use of a very small set of ligands (less than ten) for the arraying of biochips enabling the detection of the presence of any bacteria. Last but not least, an other important aspect of our project will be the inclusion, on the microarray, of another set of ligands (antibodies, phages and/or aptamers) HIGHLY SPECIFIC TO EACH MEMBER OF THE ESKAPEE PATHOGEN GROUP. This class of pathogen may lead to quite dramatic situations, and thus deserves a specific detection to drive the doctor to the best decision. Then, the BacUS project addresses several issues directly linked to a better fosterage of the patients in case of bacteremia, namely: a faster answer on the presence/absence of bacteria; the identification of any ESKAPEE pathogen if present in the sample; and cost-effective solution in regards to the current methods used so far.
The BacUS project gathers three complementary partners: partner 1 (who is also coordinating the project) has a well-known experience in the operation of SPRi instruments for small molecules, bacteria or cell analysis as well in biochip microarraying with different ligands (proteins, peptides and aptamers); partner 2 is expert in the synthesis and characterization of peptide derivatives and also in the use of peptides for diagnostic purposes; partner 3 is a joint laboratory between the University and the Grenoble General Hospital and completes, every day, about 500 analysis, this partner is also expert in the qualification of new methods for bacteremia diagnostics.
Besides a work package focused on the project supervision and coordination, the BacUS projects is composed of four other work packages. These tasks are increasing in complexity and will merge at the end of the project in order to test our method on real human blood samples. The overall risk taken by our project should be limited as BacUS is based on very encouraging preliminary results acquired by the three partners.
We also wish to underline the fact that BacUS perfectly matches the objectives of the Défi 'Vie, santé et bien-être", and in particular of the axes 9 dedicated to the "Technologies pour la santé. On a more general aspect, this project aims at developing efficient and cost-effective solutions for diagnostics, which also meets the challenges defined by the national health authorities.

Project coordination

Yoann Roupioz (Systèmes Moléculaires et nano Matériaux pour l'Energie et la Santé)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

DCM DEPARTEMENT DE CHIMIE MOLECULAIRE
SyMMES Systèmes Moléculaires et nano Matériaux pour l'Energie et la Santé
Laboratoire de Bactériologie

Help of the ANR 324,523 euros
Beginning and duration of the scientific project: - 36 Months

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