FIELD OF THE INVENTION

The invention relates to the field of microbiological analysis, and in particular of the identification of the Gram type of a bacterium.

Advantageously, the invention applies to the analysis of a hyperspectral or multispectral image of a bacterial colony having grown in a non-chromogenic, non-fluorogenic and dye-free nutritive medium.

PRIOR ART

The Gram classification of a bacterial strain makes it possible to characterize the wall thereof, for example its peptidoglycan percentage, and is used in the taxonomy of bacteria or for evaluating their sensitivity to antibiotics. Two types of bacterium are thus distinguished, namely the Gram-“positive” bacteria and the Gram-“negative” bacteria. The knowledge of the Gram type of a bacterial strain makes it possible for example to select suitable tests for identifying the strain or carrying out antibiotic sensitivity testing, e.g. the choice of a suitable card for an antibiotic sensitivity test carried out by the Vitek© 2 automated device sold by the applicant.

Historically, the Gram type of a bacterial strain was determined by a manual technique known as “Gram staining”, which comprises a large number of manual steps (fixing, staining, etching, washing, counterstaining, etc.) and therefore takes a long time to implement. Various techniques have therefore been developed for automating the detection of the Gram type of bacteria, in particular in order to process a large number of samples. However, these techniques mostly continue to modify the electromagnetic response of the bacteria or of their medium in order to make their Gram easily observable.

For example, a first type of technique consists in automating the staining of the bacterial membrane on microscope coverslips, but the final step of deciding on the Gram type is still carried out by a technician who looks at the coverslips under a microscope. This type of technique is not therefore entirely automated, and is, moreover, difficult to automate. This is because the difference in colors between Gram-positive bacteria and Gram-negative bacteria may be subtle, explaining why the involvement of a laboratory technician is still required.

A second type of technique consists in placing bacteria in the presence of a substrate which degrades by means of an enzymatic reaction initiated by the peptidoglycans of the bacterial membranes. This reaction produces chromophores or fluorophores, the concentration of which is an indication of the Gram. The term chromogenic or fluorogenic “staining” of bacteria is normally used. While prior art techniques of this type can be automated, for example by measuring the light intensity of the chromophores/fluorophores using a suitable device (e.g. spectrometer/fluorometer) and then by comparing, using a computer program, the intensity measured with predefined threshold values, they nevertheless entail the design of particular chromophore or fluorogenic substrates, which are often expensive.

In addition, regardless of the technique used, the bacteria undergo a modification of their natural state (e.g. they comprise dyes, they have bound chromogenic or fluorescent labels, etc.), and cannot therefore be used for subsequent characterization tests (e.g. determining antibiotic sensitivity).

SUMMARY OF THE INVENTION

The objective of the present invention is to provide a process for determining the Gram type of a strain of bacterium which is automatic and which does not require labeling or staining of the bacterium in order to determine its Gram.

To this effect, a subject of the invention is a process for detecting the Gram type of a bacterial strain, comprising:illumination, in the wavelength range 415 nm-440 nm, of at least one bacterium of said strain having a natural electromagnetic response in said range;acquisition, in the range 415 nm-440 nm, of a light intensity reflected by, or transmitted through, said illuminated bacterium; anddetermination of the Gram type of the bacterial strain as a function of the light intensity acquired in the range 415 nm-440 nm.

For the purposes of the invention, the expression “natural electromagnetic” response is intended to mean that the bacterium is not modified by means of elements (dye, chromogene, fluorogene, etc.) which modify its electromagnetic response to an illumination at least in the wavelength range of interest. For example, a colony of the strain is cultured in a non-colored, non-chromogenic and non-fluorescent nutritive medium and the illumination/acquisition is carried out directly on the colony still present in its medium.

In other words, the inventors have discovered a wavelength range in which a bacterium “naturally” has an electromagnetic signature characteristic of its Gram type. The process according to the invention thus consists in measuring this signature and then in extracting the Gram type therefrom. Thus, it is not necessary to use a chromogenic or fluorogenic substrate or dyes. Moreover, the process according to the invention is fast insofar as it consists in illuminating, measuring a spectrum and carrying out processing, in particular computer processing, of this spectrum.

According to one embodiment, said bacterium also has a natural electromagnetic response in the wavelength range 750 nm-800 nm; and:the illumination of said bacterium is also carried out in the wavelength range 750 nm-800 nm;the acquisition of a light intensity reflected by, or transmitted through, the illuminated bacterium is also carried out in the range 750 nm-800 nm; andthe determination of the Gram type of the bacterial strain is carried out as a function of the light intensities acquired in said ranges 415 nm-440 nm and 750 nm-800 nm.

In other words, the bacterium also comprises a natural electromagnetic signature in the range 750 nm-800 nm characteristic of its Gram. The detection of its Gram can also be carried out in this range. Advantageously, by combining the two wavelength ranges, a better accuracy is obtained in the determination of the Gram type.

According to one embodiment, the determination of the Gram type comprises:calculation of a reflectance or absorption factor of said illuminated bacterium as a function of the light intensity acquired;determination of the Gram type as a function of the reflectance or absorption factor calculated.

In other words, the reflectance or absorption factor reflects more faithfully the natural electromagnetic behavior of the bacterium, since this corrects the measured signal with respect to the spectral behavior of the illumination and the sensor (e.g. the white point, the black point, the non-uniformity of the spectrum of the illumination in the range(s) of interest, etc.) and thus allows a more accurate detection.

In particular, the determination of the Gram type comprises:calculation of a first derivative in the wavelength of the reflectance or absorption factor calculated;determination of the Gram type as a function of the first derivative calculated.

In other words, the inventors have noted that the electromagnetic signature characteristic of the Gram is more discriminating when deriving the light intensity or the reflectance/absorption factor, which allows a more accurate detection.

According to one embodiment, the determination of the Gram type is carried out as a function of the light intensity at a single wavelength of the range 415 nm-440 nm, in particular the wavelength 420 nm. More particularly, the determination of the Gram type comprises:comparison of a value equal to the light intensity at 420 nm, or calculated therefrom, to a predetermined first threshold and a predetermined second threshold separating the Gram-positive straining from the Gram-negative staining, the first threshold being greater than the second threshold;determination of negative Gram if said value is above the first threshold and determination of positive Gram if said value is below the second threshold.

In other words, a good level of accuracy in the detection is obtained even with a single wavelength, in particular at 420 nm. In particular, a simple thresholding makes it possible to have a minimum accuracy of 80%. This makes it possible to simplify the computer calculations and/or the illumination and the sensor. The determination can be carried out on the basis of the light intensity itself or after a preprocessing thereof (e.g. filtering of the noise, removal of an offset, passage to a first or second derivative, etc.).

According to one embodiment, the determination of the Gram type comprises the application of a predetermined linear prediction linking the light intensity acquired, or a value calculated therefrom, to the Gram type, followed by thresholding of the result of the linear prediction applied.

As a variant, the determination of the Gram type comprises the application of a two-class classification of SVM type to the light intensity acquired, or to a value calculated therefrom.

According to one embodiment, the illumination and the acquisition are carried out directly on a sample comprising a colony of the bacterial strain and a nutritive medium in which said colony has grown.

In other words, the invention advantageously directly applies to a sample usually produced in the microbial work flow, namely colony growth, for example on Petri dishes, such that the invention does not involve the provision of a specific sample preparation in order to be carried out. Moreover, a colony comprises a very large amount of bacteria of the strain. Thus, measuring the intensity reflected by, or transmitted through, a colony makes it possible to naturally increase the degree of reliability of the detection, the intensity being naturally averaged for example. However, it will be noted that the invention applies to the individual detection of a bacterium, it being possible for a statistical study to be carried out when several bacteria are observed. Likewise, the invention obviously applies to media other than those normally used in Petri dishes (usually agars), for instance liquid media in which the bacteria are in suspension.

In particular, the determination of the Gram type is carried out as a function of said nutritive medium. In other words, the nutritive medium also has its own “electromagnetic signature” in the wavelength ranges of interest, and can therefore disrupt the detection of the Gram type. By taking the nutritive medium into account, either during the processing, or during the choice of the medium used for the growth of the colony, a better detection accuracy is obtained.

In particular, an opaque substrate at the surface of which a colony has grown, said substrate preferably having a reflectance p of less than or equal to 10%, preferably less than or equal to 5%. In particular, in the context of a detection based on the reflected light intensity, a better accuracy is obtained.

According to one embodiment, the acquisition of the light intensity comprises the acquisition of a hyperspectral or multispectral image of a colony of bacteria of the strain, and the light intensity is determined as a function of at least one pixel of said image corresponding to the colony. In particular, the light intensity is equal to the mean pixels of said image corresponding to the colony.

In other words, the hyperspectral or multispectral imaging makes it possible to simultaneously acquire the two ranges 415 nm-440 nm and 750 nm-800 nm by means of a single sensor, and also makes it possible to obtain a spatial mean of each wavelength acquired over several pixels, thereby increasing the accuracy of the detection. As is known per se, the term “hyperspectral” corresponds to the acquisition of a wavelength range as a whole (to within a sampling spacing), whereas the term “multispectral” generally refers to the acquisition in distinct ranges.

A subject of the invention is also a system for carrying out the process which has just been described. In particular, a subject of the invention is a system for detecting the Gram type of a bacterial strain, comprising:an illumination configured to illuminate, in the wavelength range 415 nm-440 nm, at least one bacterium of the strain;a sensor configured to acquire, in the range 415 nm-440 nm, a light intensity reflected by, or transmitted through, said illuminated bacterium; anda computer unit configured to determine the Gram type of the bacterial strain as a function of the light intensity acquired in the range 415 nm-440 nm.

According to one embodiment:the illumination is configured to illuminate said bacterium in the wavelength range 750 nm-800 nm;the sensor is configured to acquire, in the wavelength range 750 nm-800 nm, a light intensity reflected by, or transmitted through, said illuminated bacterium; andthe computer unit is configured to determine the Gram type of the bacterial strain as a function of the light intensity acquired in said ranges 415 nm-440 nm and 750 nm-800 nm.

According to one embodiment, the system is configured to illuminate, and to acquire the image of, a sample comprising a colony of bacteria of said strain and a nutritive medium in which said colony has grown, in particular a Petri dish.

