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 which basically relates to a bacterial activity detection device and method, preferably using thin film piezoelectric transducers with an appropriate amplification and audible acoustic analysis. The proposed method and device has the advantage to be inexpensive and easy to use without complex setup or preparation. Furthermore, its sensitivity is high enough to perform the test using a very limited quantity of bacteria directly extracted from body liquids.

In other words, the system comprises a direct piezoelectric detection of bacteria induced motion. The main advantages of the present invention consist in that it provides an improved simplicity of use since it requires no laser/optics alignment, improved robustness thanks to a simple experimental protocol, no calibration, no artifact coming from laser illumination and very low price.

A first aspect of the invention is a bacteria activity detection system comprising a nano-oscillator adapted to receive a bacteria sample to be analyzed on it and adapted to generate current upon activity of said bacteria, at least one pair of electrodes disposed on said nano-oscillator and adapted to collect the current generated by said nano oscillator, wherein said electrodes are connected to a current amplifier adapted to amplify said generated current and to send it to an acoustic loudspeaker, characterized in that said loudspeaker is configured to reproduce an acoustic signal representative of the bacteria activity. It therefore possible to obtain a direct sound signal of the bacteria activity.

Advantageously, the nano-oscillator is a piezoelectric membrane. Thus, it is possible to obtain direct signal through the membrane itself.

According to a preferred embodiment of the present invention, the piezoelectric membrane is a gold coated piezoelectric membrane. Therefore the quality is improved and the membrane is reusable.

Preferably, the piezoelectric membrane is a piezopolymeric membrane. Therefore the price is cheaper.

According to a preferred embodiment of the present invention, the nano-oscillator presents a chitosan treated surface.

Preferably, the bacteria detection system further comprises a fluidic cell filled with fluid for housing said nano-oscillator and the electrodes.

Preferably, the electrodes are gold coated copper electrodes.

Advantageously, the electrodes are clamping said nano-oscillator.

According to a preferred embodiment of the present invention, the amplifier is also connected to a digital signal analyzer so as to transmit said amplified current to it. It is therefore possible, in addition to obtain the sound, to collect a digital signal, preferably in the form of its Fourier Transformation, so as to be able to process it digitally.

A second aspect of the invention is a bacteria activity detection method adapted to monitor and determine an activity of a bacteria within a sample of interest in a bacteria activity detection system of the first aspect, comprising the steps of depositing the sample onto the nano-oscillator, collecting the current through the electrodes and amplifying it in a current amplifier, sending the amplified current to an acoustic loudspeaker and/or a digital signal analyzer, and determining activity of a bacteria based on the sound emitted by the loudspeaker and/or the result of the digital signal analyzer.

Advantageously, the bacteria activity detection method further comprises the step of placing the piezoelectric membrane with the sample within a microfluidic cell, Preferably, the bacteria activity detection method further comprises the step of adding a bacteria activity modulating agent on the sample and continuing the current collecting and amplifying step.

Advantageously, the bacteria activity modulating agent is a nutrient or an inhibiting agent on the sample and continuing the current collecting and amplifying step.

A third 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.

A third aspect of the invention is an inhibiting agent susceptibility determination method comprising the inhibiting agent efficiency determination method of the third aspect wherein the step of adding a inhibiting agent consists in repeated injection steps each step injecting a predetermined quantity of inhibiting agent and being followed by a bacteria activity determining step so as to determine a minimum inhibitor concentration able to kill the bacteria.

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 Bacteria activity detecting device according to the present invention

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.

The bacteria activity detection system comprises a nano-oscillator adapted to receive a bacteria sample to be analyzed on it and adapted to generate current upon activity of the bacteria, at least one pair of electrodes, preferably gold coated copper electrodes preferably clamping the nano-oscillator, disposed on the nano-oscillator and adapted to collect the current generated by the nano-oscillator, and a fluidic cell filled with fluid, preferably containing nutriments for the bacteria, for housing the nano-oscillator and the electrodes, wherein the electrodes are connected to a current amplifier adapted to amplify the generated current and to send it to an acoustic loudspeaker, which is configured to reproduce an acoustic signal representative of the bacteria activity.

Ideally, the nano-oscillator is a piezoelectric membrane, preferably a gold coated piezoelectric membrane, even more preferably a piezopolymeric membrane which preferably presents a chitosan treated surface.

As we can see, the membrane is clamped with rings, preferably copper rings, even more preferably gold coated copper rings which are intended to work as electrodes. The resulting collected current is amplified with an amplificatory, preferably a current preamp and then sent to both an acoustic loudspeaker, which is therefore able to render the live of the bacteria audible and to a digital signal analyzer.

In addition, the amplifier is also connected to a digital signal analyzer so as to transmit said amplified current to it.

In order to be able to reuse said membrane, it is preferable that said membrane is made of a material which can be washed so as to “reset” the surface properly in order to perform successive experiments such material can be, for example, gold or an alloy of it.

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 the bacteria activity detection system described above, the method comprising the steps of depositing the sample onto the nano-oscillator, preferably placing the piezoelectric membrane with the sample within a microfluidic cell, collecting the current through the electrodes and amplifying it in a current amplifier, sending the amplified current to an acoustic loudspeaker and/or a digital signal analyzer, and determining activity of a bacteria based on the sound emitted by the loudspeaker and/or the result of the digital signal analyzer.

Once in place, the method can comprise the step of adding a bacteria activity modulating agent, such as a nutrient or an inhibiting agent, on the sample and continuing the current collecting and amplifying step and 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.

As a preferred embodiment, one can also add the inhibiting agent in repeated injections each injection consisting in a predetermined quantity of inhibiting agent and being followed by a bacteria activity determining step so as to determine a minimum inhibitor concentration able to kill the bacteria.

With the present invention, in order to measure the nanometric vibration of the thin membrane sensitive to bacteria activity, the system must be calibrated at preferably DC-10 kHz.

In order to determine the antibiotic efficiency on a bacteria of interest (which is unknown) the method consists in monitoring the current tendency when an antibiotics is added to the solution comprising the antibiotic sample adhered to the membrane.

This provides a robust protocol to measure and interpret bacteria activity with the acoustic system, to be able in particular to tell if a given antibiotic is efficient on a very limited number of bacteria.

This method permits to find in the acoustic signature how bacteria reacts to antibiotics, also in very limited quantity. Then, it is possible to measure the antibiotic susceptibility, that is, the minimum amount of antibiotic to kill the bacteria, also called as minimum inhibitor concentration (MIC), which is an information of prime importance for the medical community.

Furthermore, this method provides knowledge of the bacterial state and activity (beyond alive/dead) through the acoustic signature, as well as MIC for several antibiotic/bacteria couples.

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.