Method for classifying the presence or absence of a microorganism in a biological medium

Abstract

Porous sol-gel material essentially consisting of units of one or more first polyalkoxysilanes chosen from the following compounds: (chloromethyl)triethoxysilane; 1,3-dimethyltetramethoxydisiloxane; ethyltrimethoxysilane; triethoxy(ethyl)silane; triethoxymethylsilane; triethoxy(vinyl)silane; trimethoxymethylsilane; trimethoxy(vinyl)silane; tetraethoxysilane or tetramethoxysilane (TMOS) and of units of one or more second polyalkoxysilanes chosen from the following compounds: (N-(3-(trimethoxysilyl)propyl)ethylenediamine; 3-aminopropyltriethoxysilane (APTES) and 3-aminopropyltrimethoxysilane, in a first polyalkoxysilane/second polyalkoxysilane molar ratio of 1/0.01 to 1/1, optionally comprising a probe molecule, method of preparation and applications in the trapping of monocyclic aromatic hydrocarbons and other pollutants or in their detection.

Claims

1. A method for determining the presence or absence of at least one microorganism in a biological medium, said method comprising at least: providing an enclosure containing: a liquid or semi-solid phase formed in whole or in part of said biological medium capable of containing at least one living form of said microorganism, nutritional elements necessary for proliferation of said microorganism, and an enzymatic substrate that is specific to said microorganism and that can be metabolized into at least one Volatile Organic Compound (VOC) volatile metabolite; and a gas phase adjacent to said liquid or semi-solid phase, exposing at least said liquid or semi-solid phase to conditions that are favorable for said microorganism to metabolize said enzymatic substrate into at least one molecule of said VOC volatile metabolite; and determining, by optical transduction, the presence or absence of said VOC metabolite, an indicator of the presence of said microorganism; wherein: said VOC metabolite, if formed, interacts with a nanoporous matrix which has an affinity for said VOC volatile metabolite, said matrix being in a form that is separate from said enzymatic substrate and arranged in the gas phase; the detection by optical transduction of a change in the optical properties of said matrix indicates that said matrix interacts with said metabolite; and VOC is a metabolite specific to the metabolization of the enzymatic substrate and representative of an enzymatic route specific to the microorganism subject of detection.

2. The method of claim 1, wherein said VOC metabolite has intrinsic optical properties.

3. The method of claim 1, wherein said VOC metabolite is distinct from natural metabolites generated during microbial growth.

4. The method of claim 1, wherein said enzymatic substrate is a phenol or naphthol substrate.

5. The method of claim 1, wherein said VOC metabolite is a phenol derivative.

6. The method of claim 5, wherein said VOC metabolite is nitrophenol, cyanophenol, cyanonitrophenol, acetylphenol, a propionylphenol derivative, a thiophenol derivative, a naphthol derivative, an aniline derivative, or a naphthylamine derivative.

7. The method of claim 1, wherein said VOC metabolite is p-cyanophenol, 4-nitrophenol (p-nitrophenol), m-cyano-p-nitrophenol (3-cyano-4-nitrophenol), 2, 6-dichloro-4-nitrophenol, 2,6-dichloro-4-acetylphenol, thiophenol, 2, 6-dichloro-4-propionylphenol, 2,6-difluoro-4-acetylphenol, 2,6-dibromo-4-acetylphenol, 1-naphthol, 2-naphthol, -naphthylamine, -naphthylamine, 4-nitroaniline (p-nitroaniline), umbelliferone, naphthazarin, 4-trifluoromethylumbelliferone, 4-methylumbelliferone, o-cyanophenol, m-cyanophenol, 6-cyano-2-naphthol, 6-hydroxyquinoline-N-oxide, 2-methyl-6-hydroxyquinoline-N-oxide, 6-hydroxyquinoline, 7-hydroxyquinoline, or 8-hydroxyquinoline.

8. The method of claim 1, wherein the VOC metabolite is a photoacid or a photobase.

9. The method of claim 1, wherein said VOC metabolite can be detected by absorbance and/or fluorescence.

10. The method of claim 1, wherein said VOC metabolite can be detected by absorbance, and in that the associated nanoporous matrix is transparent or absorbs poorly in the detection zone.

11. The method of claim 1, wherein said VOC metabolite can be detected by fluorescence, and in that the associated nanoporous matrix is void of intrinsic fluorescence or has low intrinsic fluorescence.

12. The method of claim 1, wherein said matrix has a pore size distribution adjusted to the size of said VOC metabolite.

13. The method of claim 12, wherein said matrix has a pore size distribution below 100 nm.

14. The method of claim 1, wherein said matrix has a specific surface area varying from 300 to 1000 m.sup.2.Math.g.sup.1.

15. The method of claim 1, wherein said matrix is constituted of an organic, inorganic or organic-inorganic hybrid substance.

16. The method of claim 1, wherein said matrix derives from the polycondensation of alcoholates having formula
M(X).sub.m(OR.sub.a).sub.n(R.sub.b).sub.p, wherein: M corresponds to silicon, aluminum, tungsten, titanium, zirconium, niobium, vanadium, tantalum, yttrium and cerium; R.sub.a corresponds to a C.sub.1 to C.sub.6 alkyl radical or to a C.sub.5 aryl; R.sub.b corresponds to a C.sub.1 to C.sub.6 alkyl, to a C.sub.5 to C.sub.10 aryl or to a C.sub.3 to C.sub.6 aminoalkyl; n, m and p are integers, such that their sum is equal to the valence of M and n is greater than or equal to 2, where m and p may be equal to 0; and X is a halogen.

17. The method of claim 1, wherein said matrix comprises at least one probe molecule that can amplify the optical transduction signal via its interaction with said VOC metabolite trapped in said matrix.

18. The method of claim 17, wherein said probe is 4-benzoylamino-2,5-diethoxybenzenediazonium chloride (Fast Blue BB), dimethylaminocinnamaldehyde (DMACA), or 5,5-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent).

19. The method of claim 1, wherein said matrix is arranged in the gas phase and circulation of said VOC volatile metabolite towards the gas phase is optimized.

20. The method of claim 19, wherein circulation of said VOC volatile metabolite towards the gas phase is optimized by mechanical stirring or surface nebulization of the liquid or semi-solid phase.

21. The method of claim 20, wherein circulation of said VOC volatile metabolite towards the gas phase is optimized by the liquid phase flowing on a divided inert solid phase to increase the surface area of the liquid-gas interface.

22. The method of claim 1, further defined as comprised in a method of conducting an antibiogram test (AST).

Description

DEVICE

(1) Examples of systems for using the method according to the invention will now be described, in reference to the appended drawing.

(2) In the drawing:

(3) FIG. 1 represents, in a schematic and partial manner, an example of a system for using the method according to the invention,

(4) FIG. 2 represents, in a schematic manner, a detection system,

(5) FIGS. 3 and 4 represent two examples of arrangements that facilitate the extraction of gaseous metabolites of interest in the liquid or semi-solid phase of the culture medium,

(6) FIG. 5 is an example of an arrangement for a culture enclosure and a measuring device,

(7) FIG. 6 shows the possibility of using several enclosures each containing a culture medium in a given system,

(8) FIG. 7 represents enzymatic substrates,

(9) FIGS. 8 and 9 show experimental results,

(10) FIG. 10 represents a waveguide useful for detection,

(11) FIG. 11 shows the propagation of a light ray in the guide of FIG. 10,

(12) FIGS. 12A and 12B show other examples of detection devices,

(13) FIGS. 13A and 13B show the use of the detection device of FIG. 12A, in a gaseous medium and a liquid medium respectively.

(14) System 1 according to the invention, as in FIG. 1, includes an enclosure 2, transparent and preferably without intrinsic fluorescence so as not to disturb the optical reading, containing culture medium M and, in gaseous communication with the liquid or semi-solid phase of the culture medium M, a detection device 3 that will be described in more detail below.

(15) Detection device 3 comprises a nanoporous matrix as described previously and calls, for example, as shown, for optical detection that involves an incident light beam I whose intensity variation gives information on the presence of a VOC metabolite in the culture medium.

(16) The detection may use a detection system 5, represented schematically in FIG. 2, where a signal s is compared to a reference signal s.sub.rf then useful information is delivered based on this comparison.

(17) The detection uses a nanoporous matrix, as described previously, with which the VOC metabolite may come into contact and which has at least one optical property change as a function of the quantity of VOC metabolite to which the matrix has been exposed. The change in this optical property causes a change in the output light signal I.sub.s, after propagation within the matrix. This output light signal may be detected by any suitable sensor, for example a photoelectric cell, which provides signal s.

(18) The reference signal s.sub.rf may be the signal obtained with, for a given incident light signal I.sub.i, an output light signal I.sub.s after crossing the nanoporous matrix before exposure to VOC metabolites.

(19) Signal treatment delivered by the sensors may occur using any suitable treatment device 10, for example a computer equipped with suitable interfaces for digital-analog conversion.

(20) The information provided by the treatment circuit 10 may be in any form, for example in the form of an alphanumeric message.

(21) In FIG. 1, detection device 3 is shown schematically above the culture medium M, being enclosed in the same enclosure 2 that prevents gaseous exchanges with the exterior.

(22) Any gaseous communication between the enclosure in which the culture medium is located and the enclosure in which the detection may be envisaged, for example using conduit(s) allowing gases to circulate between the liquid or semi-solid phase of the culture medium and the enclosure in which the detection occurs. Gaseous circulation may occur naturally or, as a variant be forced, for example by using a pump or a piston of any mechanism for stirring gas.

(23) A solution to favor the extraction of VOC metabolites from the liquid phase of the culture medium may consist, as shown in FIG. 3, in bubbling the gas by bringing it into the culture medium using a conduit 12, this conduit being able to exit to a immersed diffuser 13, preferably near the base of the enclosure containing the culture medium.

(24) The enclosure contains, preferably, as shown in FIG. 3, a mixer 15, for example with bars, that stirs the liquid phase of a culture medium.

(25) The extraction of the gaseous medium above the enclosure may occur via the intermediate of a conduit 16 that exits above the surface 17 of the culture medium.

(26) Another way to increase the gaseous exchange between the culture medium and the gaseous environment above the culture medium may consist in, as FIG. 4 shows, pumping the culture medium then backing up this culture medium above its surface 17, by making it flow on any element 19 suited to increasing the exchange surface area, for example arranged on a grid 20 above the surface 17 of the culture medium M.

(27) FIG. 5 shows a variant in which a container that contains the enclosure containing the culture medium holds both this and the detection device.

(28) The culture medium M is for example contained as shown in a tube 25, which is arranged above a nanoporous matrix 30 intended to react optically with any VOCs of interest.

(29) The matrix is presented for example in the form of a block that is arranged to be crossed by a beam of incident light I.sub.i.

(30) Gaseous communication between the matrix 30 and the culture medium M may occur for example through a space 33 arranged between the enclosure and the wall of the container 2.

(31) Within a system according to the invention several enclosures 2 each containing a culture medium, and each communicating with an associated detection device may advantageously be used, for example as illustrated in FIG. 6, to allow phenotype identification by putting it in the presence of a microbial strain and different enzymatic substrates all emitting the same volatile metabolite, but targeting different enzymes.

(32) A device to count by MPN (most-probable number) and a device for AST (antibiotic susceptibility testing) can also be used.

(33) The most-probable number (MPN) method is used to count discrete entities that are easy to detect, but difficult to count (for example bacteria in a sample of sol, blood, or in a food matrix).

(34) The method of counting microorganisms by MPN includes the steps consisting in:

(35) 1) Taking original sample and subdividing it into sub-volumes by orders of magnitude (most often powers of 10 or 2). The microorganisms must be split into sub-volumes randomly; this therefore implies even cell distribution in the starting sample just before subdivision.

(36) 2) Testing each of the sub-volumes to determine whether the microorganism is present or absent. This step consists, in our case, in determining whether there is a non-negligible quantity, or not, of target VOC metabolite.

(37) 3) Knowing the number of sub-volumes and the results (+ or ) in each of them, Poisson's law can be used to find the most-probable value for the microorganism concentration in the original sample, and a confidence interval for the concentration measurement.

(38) For the AST test (Antibiotic Susceptibility Testing), the steps consist in:

(39) 1) Subdividing the original sample into sub-samples of identical volumes.

(40) 2) Next incubating each of them in the presence of antibiotic. Various antibiotics are tested, generally one per antibiotic family (macrolides, beta-lactams, fluoroquinolones, etc.). Additionally, for a given antibiotic, different concentrations are tested.

(41) 3) Testing each sub-sample again to determine if there has been microbial growth or not. The result consists in revealing the antibiotics to which the tested species is sensitive, and the minimum concentration of antibiotic necessary to inhibit the growth (MIC, Minimum Inhibitory Concentration; 1 MIC value per antibiotic).

(42) Each detection device 3, which is in gaseous communication with the corresponding culture medium, can detect whether there is degradation of the enzymatic substrate or not.

(43) The nanoporous matrix 30 may be made in the form of a waveguide, as shown in FIGS. 10 and 11.

(44) According to this embodiment, the nanoporous matrix preferably has a cylindrical shape, and its refractive index is greater than that of the external medium, so that it behaves as a waveguide. This matrix is illuminated by a light beam of intensity I.sub.0, or incident beam. The light intensity I transmitted by the waveguide is then measured.

(45) The interaction of the VOC produces a light absorption from the waveguide, which can be measured by comparing the incident intensity and the transmitted intensity. For example, one comparison is determining log(I.sub.0/I)=e L c where e is the molar extinction coefficient (Mol.sup.1.Math.l.Math.cm.sup.1), L the optical path (cm), and c the concentration of the absorbent species created by the interaction of the VOC in the porous matrix (mol.Math.l.sup.1).

(46) I.sub.0 may be determined before absorption, or by an independent measurement method, and therefore, in a general manner, by a reference measurement.

(47) In these conditions, from the measurement for 1 (intensity of the transmitted beam) c can be estimated, since e and L are known.

(48) A surface forming a mirror 50 can be arranged at one end of the guide, as shown in FIGS. 12A and 12B, opposite the input face through which the signal of intensity I.sub.0 enters. The mirror is either flat as shown in FIG. 12A, or round as shown in FIG. 12B. This has the effect of doubling the optical pathway compared to the configuration in FIG. 10. The sensitivity of the measurement is then increased because for a given concentration c, the attenuation measured, quantified by log I.sub.0/I, is higher.

(49) In the configurations in FIGS. 12A and 12B, just like that in FIG. 10, it is the variation in the time of the return light signal that allows detection: there is no reference beam.

(50) The reference I.sub.i denotes the incident signal and I.sub.r the return signal. In the example of FIG. 12A, the mirror 50 is for example made of silicon. In FIG. 12B, the round end 51 that reflects light is for example prepared by molding the matrix 30 in a round-based tube. The light may be reflected given the laws of refraction, due to the difference in index.

(51) Thus, according to a preferred embodiment, optical detection is conducted after an incident beam passes back and forth in a waveguide formed by the matrix or after a waveguide formed by the matrix passes, the waveguide being for example equipped, in the case of the incident beam passing back and forth, with a mirror (50), preferably made of silicon, or of a round end (51), preferably a hemisphere.

(52) The nanoporous matrix 30 may be arranged in the gas phase such as is shown in FIG. 13A or in the nutrient medium as shown in FIG. 13B.

(53) In the example of FIGS. 13A and 13B, the excitation signal is brought by at least one optic fiber and the signal after crossing though the nanoporous matrix 30 is sent by at least one fiber. The fibers cross a septum 31, which seals a flask 2 defining the enclosure containing the culture medium M.

(54) The waveguide is for example oriented vertically. The optic fiber(s) used to carry the incident and return signals are for example stuck to one end of the waveguide, for example the upper end as shown.

(55) Preferably, the nanoporous matrix is only submerged in the liquid phase if:

(56) a) the colored or diffusing compounds of the liquid phase do not unduly disturb the optical measurement in the matrix;

(57) b) other solutes (pH buffers for example), that could enter into the nanopores, do not unduly disturb the optical measurement;

(58) c) the optionally immobilized probe in the matrix does not leave the nanopores to diffuse in the liquid phase.

(59) According to one of its features, the invention further relates to a system dedicated to the detection of a microorganism in a biological medium including an enclosure to receive a culture medium, a detection device for a VOC metabolite released by the microorganism metabolizing an enzymatic substrate, this detection system being in gaseous communication or directly in contact with said culture medium.

(60) The enclosure containing the culture medium may have sufficiently high volume to also hold the detection system at least partially.

(61) In an example of implementation of the invention, the culture medium is contained in a recipient that is located above a nanoporous matrix exposed to VOC metabolites, and whose optical properties are sensitive to the presence of the VOC metabolite.

(62) This nanoporous matrix is contained in an enclosure that can be crossed by an incident light beam so that absorbance over time can be measured, as a function of the quantity of VOC metabolite having reacted with the matrix.

(63) The nanoporous matrix may be presented in the form of a waveguide. The nanoporous matrix may be run through by a beam of incident light moving back and forth, this beam being able to reflect on a reflective, flat or concave surface. The reflective surface may be defined by a silicon mirror.

(64) The system may include gas circulation with sampling above the culture medium and reinjection in the culture medium, preferably using a diffuser.

(65) In another embodiment of the invention, the system includes culture medium circulation with culture medium sampling and rejection above at least one element suited to increasing the surface area of gaseous exchanges between the culture medium and the gaseous environment, preferably a bed of elements, such as beads on which the culture medium is poured.

(66) A device in accordance with the invention may comprise: a glass flask (non-absorbent, non-fluorescent substance); a septum for injecting the sample into the nutrient medium; a nutrient medium containing:

(67) a C source

(68) an N source

(69) mineral salts

(70) oligoelements

(71) one or more enzymatic substrates

(72) a pH buffer

(73) optionally, adsorbents to capture residual antibiotics

(74) optionally growth inhibitors to make the medium specific one or more nanoporous sensors, placed in the gas phase, for the capture and optical detection of VOC volatile metabolite(s) emitted by the microorganisms.

(75) This sensor includes, or even is constituted, by a nanoporous matrix, preferably containing silicon oxide, with a size distribution suited to the specific capture of the VOC volatile metabolite. Any porous substance created by polycondensation of alcoholates having formula M(X).sub.m(OR.sub.a).sub.n(R.sub.b).sub.p may also be envisaged, in which: M corresponds to a metal chosen from silicon, aluminum, tungsten, titanium, zirconium, niobium, vanadium, tantalum, yttrium and cerium, R.sub.a corresponds to a C.sub.1 to C.sub.6 alkyl radical or to a C.sub.5 aryl, R.sub.b corresponds to a C.sub.1 to C.sub.6 alkyl radical, to a C.sub.5 to C.sub.10 aryl or to a C.sub.3 to C.sub.6 aminoalkyl, n, m and p are integers, such that their sum is equal to the valence of M and n is greater than or equal to 2, where m and p may be equal to 0, and X is a halogen, preferably chlorine.

(76) The pore size is less than 100 nm, preferably varies from 3 to 100 . The specific surface area may vary from 300 to 1000 m.sup.2/g, preferably from 300 to 900 m.sup.2/g, and more preferably from 400 to 900 m.sup.2/g; a mechanical stirring device, with a bubbling or nebulization or glass bead system: any method accelerating the mass transfer of the VOC volatile metabolite from the liquid phase to the gas phase; a membrane impermeable to liquids but not gases, to protect the sensor from liquid projections (due to the stirring); a UV-visible absorbance spectrophotometer or a spectrofluorimeter, outside the flask.

(77) Advantageously, steps 1) to 3) of a method of the invention may be conducted in a flask, for example a blood culture flask.

(78) A device where different enzymes are tested on a given strain to identify a phenotype of a microorganism can also be envisaged. Different cultures containing different enzymatic substrates generating the same volatile compound are cultured with the same strain. A sensor is placed in the atmosphere of each culture to detect whether or not there is enzymatic activity. Such a device is particularly advantageous for multi-target microorganism assay.

(79) The presence of several enzymatic substrates targeting the main enzymatic routes present in mycobacteria may advantageously allow these microorganisms to be detected in the gas phase. Two technological solutions present themselves: either the substrates all lead to the same VOC volatile metabolite: then a simple detection is carried out; or the substrates form different VOC volatile metabolites with distinct optical properties: if the number of substrates is sufficient, the mycobacteria detected can then be identified.

(80) In the case for example of blood culture, a mixture of several enzymatic substrates targeting the most common enzymatic routes can be used, to be able to detect any of the pathogens found in blood culture.

(81) All the pathogenic species of bacteria that can infect the blood system must present at least one of the targeted enzymatic routes.

(82) The drawing in FIG. 7 describes the example of three enzymatic phenol substrates all emitting the same VOC volatile metabolite, m-cyano-p-nitrophenol. From mixing these 3 substrates in a culture medium 3 enzymes can be targeted: -galactosidase, esterase and alkaline phosphatase. Therefore, any species of bacteria that presents at least one of these 3 enzymes can be detected.

(83) In the sense of the invention, unless otherwise indicated, one means at least one and the expression comprised between . . . and . . . includes the limits of the interval being defined.

(84) The examples indicated below are given to illustrate the invention, without being limiting.

EXAMPLES

Example 1

Synthesis of a Nanoporous Monolith with an Affinity for p-nitrophenol, pNP

(85) This example shows the synthesis of a mixed basic Sol matrix Si(OMe).sub.4-NH.sub.2TEOS. The synthetic protocol selected is the sol-gel process for a nanoporous matrix for the capture of monocyclic aromatic species, shown in application WO 2010/004225.

(86) Starting Reagents:

(87) Precursors:

(88) TMOS (tetramethoxysilane) (Si(OMe).sub.4)

(89) APTES, Si(C.sub.3H.sub.6NH.sub.2)(OC.sub.2H.sub.5).sub.3 ((3-aminopropyl)triethoxysilane)

(90) Solvent:

(91) MeOH (methanol)

(92) Hydrolysis with ultrapure water

(93) Molar Ratios:

(94) Alkoxides/methanol/water:1/5/4

(95) TMOS/APTES: 0.97/0.03

(96) Proportions for about 5 mL of 3% NH.sub.2TEOS sol

(97) TMOS 1.786 mL

(98) MeOH 2.43 mL

(99) APTES 0.084 mL

(100) Water 0.864 mL

(101) Protocol:

(102) TMOS and MeOH are mixed with magnetic stirring for 2 min in a Pyrex beaker placed in a bath at 25 C. (ethanol and liquid nitrogen).

(103) APTES is added to the micropipette and the whole is stirred for 2 min.

(104) The ultrapure water is added and the whole is stirred for 30 s.

(105) The matrices form quickly because the sol freezes quickly (1 mL in a tank, where the tanks have been pretreated at 50 C. for 24 h by degassing the oven at least three times during this period).

(106) The tanks are closed with their stopper and left for 24 hours. The stoppers are then removed and replaced by a microporous film to let the solvents evaporate.

Example 2

(107) -Glucuronidase activity is targeted on two biological models, i.e. Escherichia coli ATCC 11775 (-glucuronidase positive) and Hafnia alvei ATCC 13337 (-glucuronidase negative).

(108) The VOC volatile metabolite chosen is p-nitrophenol (pK.sub.a=7.15).

(109) The matrix selected is that described in example 1. It allows pNP to accumulate in pNP.sup. form.

(110) The table below details the composition of the MES-pNPG culture medium that generates p-nitrophenol by -glucuronidase activity.

(111) This culture medium is called MES-pNPG because MES is its pH buffer, and pNPG (4-nitrophenyl--D-glucuronide) is its enzymatic substrate.

(112) The pH is checked after calibration (3 points: 4, 7 and 10): it is equal to 6.09.

(113) TABLE-US-00001 Component Concentration Nutrient medium (with bio-soyase 2 g/L mineral salts and trace yeast extracts 2 g/L elements) NaCl 5 g/L (86 mM) MgSO.sub.4 250 mg/L (2.08 mM) pH buffer MES sodium salt 16.292 g .Math. L.sup.1 (75 mM) MES acid monohydrate 15.994 g .Math. L.sup.1 (75 mM) -D-glucuronidase sodium glucuronate 108 mg .Math. L.sup.1 enzymatic activity monohydrate (461 M) inducers methyl -D-glucuronide 55.2 mg .Math. L.sup.1 (240 M) Enzymatic substrate pNPG (4-nitrophenyl--D- 35.6 mg .Math. L.sup.1 of -D-glucuronidase glucuronide) (113 M) enzyme according to the invention

(114) To impose on a mainly acid form of paranitrophenol, the culture medium is buffered to pK.sub.a1, i.e. 6.15, with MES (4-morpholinoethanesulfonic acid).

(115) 2.1Reference Control for Enzymatic Activity

(116) The first stage is controlling enzymatic activity and bacterial growth in the liquid phase constituted by the culture medium.

(117) The purpose is to confirm in advance, in the liquid phase, the effective formation of the VOC volatile metabolite that absorbs pNP and to quantify it (M) by absorbance spectroscopy at 400 nm. The bacterial population is also followed (cfu/mL) over time, to determine the generation time for the bacterial model in the culture medium prepared (MES-pNPG).

(118) The preculture is a colony of Escherichia coli 5 (Escherichia coli ATCC 11775) on a TSA plate that is suspended in 4 mL of LB culture medium (Lysogenic Broth), with stirring.

(119) The preculture is held at 30 C. static for 14 h.

(120) The culture is put in a LB culture medium (Lysogenic Broth), then in MES-pNPG medium as described previously.

(121) The most concentrated preculture tube has a degree of absorbance (DO) of: DO(x)=0.5685, i.e. an effective degree of absorbance of 1.137.

(122) Inoculation is based on 1/50th for a degree of absorbance of 0.7 therefore: (1.1137/0.7)50=81.2. Therefore inoculation is at 1/81st, i.e. 49 L for a total volume of 4 mL.

(123) Therefore 2 tubes of 4 mL of LB are inoculated at 37 C., at 250 rpm for 2 hours and 25 minutes. DO550(x)=0.35 is measured, i.e. 0.7 effective degree of absorbance.

(124) The buffer MES-pNPG is inoculated (t=0) to 1/700th: 20 L EC5 culture in LB at 0.7 degree of absorbance for 14 mL of MES-pNPG.

(125) The incubation is at 37 C., at 250 rpm.

(126) For each sample: 0.5 mL is sampled for a cascade dilution in physiological serum and plated on LB agar; 1.5 mL is used to measure the bacterial concentration with DensiCheck; After measuring with DensiCheck, the 1.5 mL used is filtered on Acrodisc 0.2 m and poured into a plastic cell for the UV-vis spectrophotometry (sweep: 200 nm/min, from 600 to 300 nm, with Milli-Q water cell for reference): the first spectrum is run; 80 L of 5M NaOH is added to neutralize the MES buffer and turn all the pNP into phenolates anions pNP: then a second spectrum is run (the pNP peak will be shifted to high wavelengths so it is easier to quantify the absorbance; and the molar extinction of pNP is higher).

(127) The initial degree of absorbance of 0.7, which is then diluted at t=0 to 1/700th, corresponds to 1.54.Math.10.sup.8 cfu/mL.

(128) The MES-pNPG buffer therefore includes 2.2.Math.10.sup.5 cfu/mL at t=0.

(129) FIG. 8 shows growth and monitoring for -glucuronidase enzymatic activity for a culture of Escherichia coli inoculated at 2.Math.10.sup.5 cfu/mL (at 37 C.) in the MES-pNPG buffer. The generation time for the exponential phase is 24 minutes.

(130) 2.2Using the Method According to the Invention to Characterize the Presence of the Microorganism Escherichia coli in the Culture Medium

(131) The purpose is here to detect, in the gas phase, the VOC coming from a culture in medium MES-pNPG using a nanoporous monolith.

(132) The device selected means absorbance of the monolith can be followed over time, between 300 and 600 nm (UVIKON 933, Double Beam UV/Vis Spectrophotometer, KONTRON Instruments) without opening the closed container in which the culture and the monolith are present together. This device is shown in FIG. 5.

(133) The preculture is a colony of Escherichia coli 5 (Escherichia coli ATCC 11775) on a TSA plate (plate 100802_CT4 of EB) that is suspended in 4 mL of LB with ampicillin (2 tubes), with stirring.

(134) The preculture is held at 30 C. static for 13.5 hours.

(135) The culture is put in LB, then in MES-pNPG buffer.

(136) The most concentrated preculture tube has a degree of absorbance of DO(x)=0.52, i.e. an effective DO of 1.04.

(137) Inoculation is based on 1/50th for a degree of absorbance of 0.7 therefore: (1.04/0.7)50=74. Therefore inoculation is at 1/74th, i.e. 54 L for a total volume of 4 mL.

(138) Therefore 2 tubes of 4 mL of LB are inoculated without ampicillin.

(139) Incubation lasts 1.5 hours at 37 C., at 250 rpm.

(140) A degree of absorbance of DO550(1)=0.30 is measured (i.e. 6.Math.10.sup.7 cfu/mL).

(141) 0.5 mL is sampled for a cascade dilution to 10.sup.5 to do a plate count: 6.2.Math.10.sup.7 cfu/mL.

(142) The MES-pNPG buffer is inoculated at 1/600th: 8.5 L of Escherichia coli 5 culture in LB at 0.3 of DO for 5 mL of MES-pNPG.

(143) 1.5 mL of this culture goes in the closed set-up intended for spectroscopic measurements; the rest is incubated in a classic 15 mL tube.

(144) Bacterium Escherichia coli 5 is cultured in the enzymatic culture medium in a closed tube in a sealed enclosure in the presence of a basic nanoporous monolith. The absorbance kinetics of the monolith are monitored through the wall of the enclosure (a closed spectrophotometry cell). Therefore an empty cell is placed on the reference beam, to compensate for the potential absorbance of the plastic in the cell.

(145) The incubation is at 37 C., at 150 rpm (t=0).

(146) The monolith used is indeed transparent and colorless before the start of the experiment. The degree of absorbance of the monolith at 375 nm at t=0 is 0.25.

(147) At t=9.25 h, the device is removed from the oven to put it in the spectrophotometerours (300 to 600 nm, data interval 1 nm, scan speed 200 nm/min; 10 cycles, cycle time 80 min (1 h20), 1 sample).

(148) In parallel, a plate count is conducted on the tube, which contained the same inoculum and was incubated in parallel, which means the population can be estimated.

(149) The graph in FIG. 9 shows the increase in absorbance at 375 nm of the nanoporous monolith exposed to pNP vapor (Escherichia coli 5 culture at 37 C. in MES-pNPG, inoculated at t=0 with 10.sup.5 efu/mL).