Technical Field

The present invention relates to a device and a method for detecting and/or monitoring the presence and/or the activity of bacteria of interest in a sample, and more particularly to a device and a method for rapidly, easily and reliably detecting and/or monitoring the activity of bacteria in a sample and determining an inhibiting agent or a nutrient capable of affecting the detected bacteria.

Background of the art Nowadays, one of the major problem regarding the use of antibiotic resides in the fact that there is a clear abusive use of them. Indeed, the use of generic antibiotics to treat any type of bacteria, when unidentified, causes the bacteria to develop new resistances to antibiotics.

It is of common knowledge that bacteria are capable to mute and might become more and more resistant to the current antibiotics and that the abusive use of antibiotics promotes this mutation and will, in the end, cause a strong comeback of the bacterial infections. As a matter of fact, the proliferation of antibiotic resistant bacteria is already now one of the main health hazard and will get even worse in the foreseeable future. Fast and simple antibiotic testing methods are therefore of prime importance. In other words, bacteria which are resistant to antibiotics are proliferating in all countries. Number of deaths is increasing and even small surgical interventions may lead to hospital-acquired infection with resistant bacteria. In general, surgery rooms are periodically closing for extended periods of time to allow for chemical disinfection. As mentioned above, development of these bacteria is driven mostly through inappropriate usage of antibiotic, especially in developing countries. There are already bacterial strains totally resistant to all known antibiotics, leading to fatal consequences, a problem referred to as AMR (antimicrobial resistance) in the literature. In a World Health Organization report published in 2014, the number of deaths attributed to AMR was estimated at 25Ό00 per year in Europe and 23Ό00 in US. For 2050, the estimation is over 10 million death globally. In the future many patients may postpone even banal surgeries fearing AMR bacteria in hospitals.

One of the main reason regarding the abusive use of antibiotic is the very long time which is needed to detect the presence and the type of bacteria which can last from a few hours to forty days in some cases. In such cases, the patient can not wait and generic antibiotic are delivered to him even without exactly knowing what kind of bacteria is present, if any, thereby provoking the bacteria mutation.

Indeed, classical methods for testing the effects of antibiotics on bacteria, such as AST (antimicrobial susceptibility testing), are expensive and time consuming, since one have to wait until bacteria are dividing to a sufficiently large culture to be usable (e.g. tuberculosis, ca. 30 days). The most popular test is the Kirby-Bauer test, which consists of the visual observation in a Petri Dish of the bacterial development in presence of antibiotics. Its main drawbacks are: the long duration of the test (crippling in many clinical cases), the requirement of a skilled laboratory staff, and the cost of quicker setup such as cytometry or micro-calorimetry. New methods are reviewed in Karan Syal et al. , “Current and emerging techniques for antibiotic susceptibility tests”, Theranostics, 7, 1795 (2017) and H. Leonard et al., “Recent advances in the race to design a rapid diagnostic test for antimicrobial resistance” ACS Sensors, 3, 2202 (2018), but they basically suffer from the same drawbacks.

More promising is a technique which has been recently published which uses nanodetectors to first detect the presence of bacteria and then determine, within a short amount of time, if an antibiotic is effective against the detected bacteria even without knowing the type of bacteria. This technique is described in S. Kasas et al., “Detecting nanoscale vibrations as signature of life” PNAS, 112, 378 (2015) and C. Lissandrello et al., “Nanomechanical motion of Escherichia Coli adhered to a surface” Applied Physics Letters, 105, 113701 (2014), which uses Atomic Force Microscope (AFM) cantilevers covered with the studied bacteria. Living bacteria are applying surface stress, leading to important (ca. 10 nm) deflections of the cantilever. More particularly, in this publication, one explains that the method consists in first depositing a sample onto an end of a silicon micro-lever which is then immerged in a liquid. Since the bacteria are alive, they will move on the micro-lever or at least produce vibrations (through their cilia) on this micro-lever and these vibrations are detected through the use of a laser. Once detected, one applies an antibiotic on the bacteria, if the vibrations diminish, then this means that the antibiotic is effective and killed the bacteria, or at least part of them, if not, then the antibiotic is not effective. This solution permits a rapid, i.e. within few minutes, and efficient determination of a specific antibiotic to use, even without knowing exactly the nature of the concerned bacteria upfront.

This implementation however is expensive and delicate in preparation. It asks for skills of precise cantilever handling, optical alignment, etc... not really suitable in the hospital environment. Moreover, despite the numerous advantages of this technique, there are some efficiency problems in that possible artefacts are likely to happen in case of external vibration and in that in order to obtain reliable results a simultaneous assay must be carried out on a bacteria-free nanodetector.

Conceptually similar idea was implemented using high frequency (5 MHz) crystal, and measuring its phase noise as described in W.L. Johnson et al. , “Sensing bacterial vibrations and early response to antibiotics with phase noise of a resonant crystal” Scientific Reports, 7, 12138 (2017). Obtained results using bacteria, are similar to those measured with AFM cantilevers but at the expense of further complexity and cost.

There is therefore a clear need for a method and a device permitting a bacteria detection as well as a related effective antibiotic determination within a short period of time, i.e. few minutes maximum.

In this regard, a primary object of the invention is to solve the above-mentioned problems and more particularly to provide a system and a method providing a reliable detection of a bacteria and determination of an effective antibiotic specific to the bacteria of interest within a short period of time and easily repeatable without a long calibration/installation time.

Another object of the invention is providing a rapid, robust and inexpensive device to study bacterial antibiotics resistance.

Summary of the invention The above problems are solved by the present invention.

A first aspect of the invention is a bacteria activity detection system comprising a nano-detector onto which a bacteria sample to be analyzed is to be deposited, a fluidic cell filled with fluid for housing said nano-detector, and a light source adapted to illuminate said nano-detector, characterized in that the system further comprises an interferometer microscope and an imaging device adapted to capture images of the activity of the bacteria in the bacteria sample through said interferometer microscope when a bacteria sample to be analyzed is deposited on said nano-detector.

According to a preferred embodiment of the present invention, the bacteria detection system further comprises a light source intensity averaging module disposed along the light source path and adapted to reduce the speckle pattern of the captured image.

Advantageously, the light source intensity averaging module comprises a rotating ground glass element which is crossed by the light ray. Preferably, the rotating ground glass element is a rotating ground glass disc.

According to a preferred embodiment of the present invention, the rotating ground glass element is rotated at a speed of 680 rpm.

Advantageously, the light source intensity averaging module comprises an optical fiber disposed so as to guide said light ray and which is vibrated through the use of a piezoelectric element.

Preferably, the light source is a laser, preferably a high intensity laser. This permits to obtain a low spectral width in liquid. Preferably, the laser has over 10 mV.

According to a preferred embodiment of the present invention, the imaging device is a camera adapted to acquire high-velocity images of the activity of the bacteria in the bacteria sample onto the nano-detectors.

Advantageously, the nano-detectors are nano-oscillators.

Preferably, the nano-oscillators consist in Silicone micro-levers.

According to a preferred embodiment of the present invention, the interferometer microscope is a Mirau Interferometer. It is therefore possible to have an image enlargement of from 10x to 50x so as to be able to clearly see the micro-levers.

A second aspect of the invention is bacteria activity detection method adapted to monitor and determine an activity of a bacteria within a sample of interest in a bacteria detection system of the furs aspect comprising the steps of depositing the sample onto the nano-detectors, placing the nano-detectors within the microfluidic cell, illuminating the nano-detectors with the light source and capturing images of the activity of the bacteria in the bacteria sample disposed on the nano-detectors, through a interferometer microscope.

Advantageously, the bacteria activity detection method further comprises the step of adding a bacteria activity modulating agent within the microfluidic cell and continuing the image capturing step.

According to a preferred embodiment of the present invention, the bacteria activity modulating agent is a nutrient or an inhibiting agent.

A second aspect of the invention is an inhibiting agent efficiency determination method comprising the bacteria activity detection method of the second aspect and further comprising the steps of judging that an added inhibiting agent is efficient if the detected bacteria activity lowers after addition and that an added inhibiting agent is inefficient if the detected bacteria activity rises or stays constant.

Brief description of the drawings

Further particular advantages and features of the invention will become more apparent from the following non-limitative description of at least one embodiment of the invention which will refer to the accompanying drawings, wherein

Figure 1 represents a schematic representation of a preferred embodiment of the present invention

Figure 2 represents an example of the nanodetectors - Figure 3 shows a microscope image of the nanodetectors upon use.

Detailed description of the invention

The present detailed description is intended to illustrate the invention in a non- limitative manner since any feature of an embodiment may be combined with any other feature of a different embodiment in an advantageous manner. Figure 1 shows the first aspect of the invention which is the system of the present invention which comprises Bacteria activity detection system comprising a nano-detector, onto which a bacteria sample to be analyzed is to be deposited, wherein the nano-detectors are nano-oscillators, such as silicone micro-levers which are shown in figure 2 and more precisely figure 3, a fluidic cell filled with fluid for housing said nano-detector, and a light source, which is a laser, preferably a high intensity laser, adapted to illuminate said nano-detector, characterized in that the system further comprises an interferometer microscope, preferably a Mirau Interferometer, and an imaging device adapted to capture images of the activity of the bacteria in the bacteria sample through said interferometer microscope when a bacteria sample to be analyzed is deposited on said nano-detector.

According to the preferred embodiment, the imaging device is a camera adapted to acquire high-velocity images of the activity of the bacteria in the bacteria sample onto the nano-detectors. In this manner, it is possible to visually monitor in real time the bacteria activity. In order to reduce the speckle pattern of the captured image, the bacteria detection system further comprises a light source intensity averaging module disposed along the light source path and preferably consists in a rotating ground glass, preferably a rotating ground glass disc element, which is crossed by the light ray. The experiments have shown that a rotating ground glass element rotated at a speed of 680 rpm gives the best results. Alternatively, the light source intensity averaging module comprises an optical fiber disposed so as to guide said light ray and which is vibrated, preferably at a frequency of 200Hz, through the use of a piezoelectric element.

A second aspect of the invention relates to a bacteria activity detection method adapted to monitor and determine an activity of a bacteria within a sample of interest in a bacteria detection system described above.

Such a method comprises the steps of depositing the sample onto the nano detectors, placing the nano-detectors within the microfluidic cell, illuminating the nano detectors with the light source and capturing images of the activity of the bacteria in the bacteria sample disposed on the nano-detectors, through an interferometer microscope.

Then, it is possible to carry out a step of adding a bacteria activity modulating agent, such as a nutrient or an inhibiting agent (an antibiotic for example), within the microfluidic cell and continuing the image capturing step.

It is therefore possible to conduct a modulating agent efficiency determination method comprising the bacteria activity detection method explained above and further comprising the steps of judging that an added modulating agent such as an inhibiting agent is efficient if the detected bacteria activity lowers after addition and that an added inhibiting agent is inefficient if the detected bacteria activity rises or stays constant.

Figure 3, for example shows an image of such process where the vibration are clearly identifiable through the wavelength of the waves observed on the levers.

While the embodiments have been described in conjunction with a number of embodiments, it is evident that many alternatives, modifications and variations would be or are apparent to those of ordinary skill in the applicable arts. Accordingly, this disclosure is intended to embrace all such alternatives, modifications, equivalents and variations that are within the scope of this disclosure. This for example particularly the case regarding the different apparatuses which can be used.