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    • 专利摘要:
    The present invention relates to a novel culture medium for mycobacteria, in particular mycobacteria of the Mycobacterium tubercu / osis complex, which makes it possible to significantly reduce the isolation time by culture and therefore the diagnostic delay of mycobacteriosis, in particular tuberculosis. . A culture medium according to the invention contains lecithin, a red dye and lamb serum preferably decomplemented. The present invention also relates to a method for culturing and identifying mycobacteria and, in particular, bacteria of the Mycobacterium tubercu / osis complex. Even more particularly, the present invention relates to a new method for decontaminating, by chlorhexidine treatment, biological samples in a new isolation medium and culture of mycobacteria. The present invention also relates to a method for the phenotypic determination of the susceptibility of Mycobacteria to antibiotics using a solid culture medium according to the invention.
    公开号:FR3033575A1
    申请号:FR1552007
    申请日:2015-03-11
    公开日:2016-09-16
    发明作者:Didier Raoult；Michel Drancourt；Shady Asmar；Sonia Chatellier
    申请人:Biomerieux SA；Aix Marseille Universite；Fondation Mediterranee Infection；
    IPC主号:
    专利说明:

    [0001] A medium and method of culturing mycobacteria comprising lamb serum. The present invention relates to a new culture medium for mycobacteria, in particular mycobacteria of the Mycobacterium tuberculosis complex, which makes it possible to significantly reduce the isolation time by culture and therefore the diagnostic delay of mycobacteriosis, in particular tuberculosis. Mycobacteria are bacteria classified in the phylum Actinobacteria by the sequencing of the 165 rRNA gene and by so-called "multilocus phylogeny" assays [Mignard S, Flandrois JP. A seven-gene, multilocus, genus-wide approach to the phylogeny of mycobacteria using supertrees. Int 3 Syst Evol Microbiol. 2008; 58: 1432-41] and characterized by the presence of mycolic acids in their wall, which gives them a particular dye affinity demonstrated in particular by Ziehl-Neelsen staining, and by a high G + C chromosome %> 60% [Pfyffer GE. Mycobacterium: general characteristics, laboratory detection, in staning procedures. In: Murray Pr, Baron E3, Jorgensen 31-1, Landry ML, Pfaller MA. Manual of Clinical Microbiology 9th Eds. Amercian Society for Microbiology, Washington DC; 2007, pp. 543-572]. The bacterial genus Mycobacterium comprises more than 100 species, including environmental species isolated from inert environments (soil, water), animal-associated species, and a species primarily associated with humans, Mycobacterium leprae, a leprosy agent [Cole S et al. Massive gene decay in the leprosy bacillus. Nature 2001; 409: 1007-11]. Some environmental species are responsible for opportunistic infections in humans (species of the Mycobacterium aviurn complex and the Mycobacterium abscessus complex for example) and some species are responsible for zoonoses, in particular certain species of Mycobacterium tuberculosis complex, responsible for tuberculosis [ Ghodbane R, Drancourt M. Non-human sources of Mycobacterium tuberculosis. Tuberculosis (Edinb).
    [0002] 2013 Nov; 93 (6): 589-95]. The diagnosis of mycobacteriosis is based on the isolation and culture of one of the Mycobacterium species from a clinical specimen in humans or animals. From this point of view, the genus Mycobacterium contains a non-cultivable species in an axenic medium (Mycobacterium leprae), fast-growing species (Mycobacterium fortuiturn, Mycobacterium abscessus) giving visible colonies in less than seven days of cultivation and species to slow growth giving visible colonies in more than seven days of culture, in routine practice, between 3 and 8 weeks of culture, especially with Middlebrook media. In human medicine, species of the Mycobacterium aviurn complex are detectable after 10-20 days of culture and species of the Mycobacterium tuberculosis complex require 10-100 viable organisms per ml of 3033575 2 sample and 6-8 weeks of culture to obtain 100% of positive samples [Colebunders R, Bastian I. A review of the diagnosis and treatment of smear-negative pulmonary tuberculosis. Int J Tuberc Lung Dis. 2000; 4: 97-107]. In view of the numerical importance and severity of human tuberculosis cases, it is on the diagnosis of tuberculosis that improvements in laboratory techniques are particularly sensitive. In fact, tuberculosis is an infectious disease of man and animals caused by one of the nine species of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis, Mycobacterium bovis (and the BCG strains derived from it), Mycobacterium africanum, Mycobacterium canettii Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedll [Dye C. et al. Prospects for worlwide tuberculosis control under the WHO DOTS strategy. Lancet 1998; 352: 1886-1891; Bos KI, Harkins KM, Herbig A, Coscolla M, Weber N, Comas I, Forrest SA, Bryant JM, Harris SR, Schuenemann VJ, Campbell TJ, Majander K, Wilbur AK, Guichon RA, Wolfe DL Steadman, Cook DC, Niemann S, Behr MA, Zumarraga M, Bastida R, Huson D, Nieselt K, Young D, Parkhill J, Buikstra JE, Gagneux S, Stone AC, Krause J. Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis. Nature 2014; 514: 494-7], Mycobacterium rnungi [Alexander KA, Laver PN, Michel AL, Williams M, van Helden PD, Warren RM, Gey van Pittius NC. Novel Mycobacterium tuberculosis complex pathogen, M. mungi. Emerg Infect Dis. 2010; 16: 12969] and Mycobacterium suricattae [Parsons SD, Drewe JA, Gey van Pittius NC, Warren RM, van Helden PD. Novel cause of tuberculosis in meerkats, South Africa. Emerg Infect Dis. 2013; 19: 2004-7]. The World Health Organization (WHO) estimated that TB was responsible for 9 million new cases and 1.5 million deaths in 2013 [World Health Organization. 2014. Global tuberculosis report 2014: Geneva: World Health Organization]. These figures underline the importance of optimizing the microbiological diagnosis of tuberculosis in order to improve the medical care of patients and their relatives. The isolation and culture of Mycobacterium tuberculosis mycobacteria are made from clinical specimens obtained from a patient with signs and symptoms suggestive of tuberculosis. The most common clinical form, which is also the only contagious clinical form, is pulmonary tuberculosis, which is diagnosed by the isolation and culture of Mycobacterium tuberculosis mycobacterium from a respiratory sample such as sputum, bronchial aspiration, bronchoalveolar lavage fluid obtained on bronchoscopy, or even pulmonary biopsy. In patients not producing sputum, stool can be used as an alternative for the isolation and culture of mycobacteria of the Mycobacterium tuberculosis complex in case of pulmonary tuberculosis [El Khechin A, Henry M, Raoult D, Drancourt M Detection of Mycobacterium tuberculosis complex 3033575 3 organisms in the stools of patients with pulmonary tuberculosis. Microbiology 2009; 155: 2384-9]. There are other clinical forms of tuberculosis, particularly ganglionic tuberculosis, but also bone tuberculosis (Mal de Pott) and digestive tuberculosis. Depending on the clinical form, different clinical samples may be sent to the laboratory to isolate and cultivate Mycobacterium tuberculosis complex mycobacteria and to diagnose extrapulmonary forms. For the isolation and culture of mycobacteria of the Mycobacterium tuberculosis complex, solid media, liquid media, and biphasic media comprising a liquid phase and a solid phase are available. The solid media are made from agar or agar. Whole egg-containing media are very common use and the most used medium is Löwenstein Jensen's medium. This medium, like other egg-containing media, contains malachite green which helps to inhibit the growth of contaminating microorganisms. Several formulations containing varying concentrations of malachite green have been proposed with the constant result that a decrease in malachite green concentration increases the environmental contamination ratio and an increase in malachite green concentration tends to decrease the isolation and Mycobacterial culture of the tuberculosis group. A second category of solid media are agar media, in particular Middlebrook 7H10 medium and 7H11 medium (7H10 medium plus 0.1% hydrolysed casein). Middlebrook's medium contains 2% glycerol, which facilitates mycobacterial culture of the Mycobacterium aviurn complex. The liquid media essentially correspond to Middlebrook 7H9 medium. However, the isolation of mycobacteria from the Mycobacterium tuberculosis complex is slow since all the strains of the various species of the Mycobacterium tuberculosis complex are isolated within a period of between 6 and 8 weeks. Any time savings over this period therefore represents a significant improvement in the laboratory diagnosis of tuberculosis and other mycobacterial infections. Patent EP 2,364,357 describes a formulation of culture media for the faster isolation of mycobacteria from the Mycobacterium tuberculosis complex and other mycobacteria, in particular allowing less visually detectable detection and identification with the naked eye. 15 days, or even 10 days, from clinical specimens. This formulation of EP 2 364 357 allows the isolation of mycobacteria with the ability to visually detect the growing mycobacteria with the naked eye, both in liquid medium suitable for detection automata and in solid medium for manual detection, ( especially the compositions of Examples 1B (liquid medium) and 2B (solid medium) the latter being hereinafter referred to as MOD4). These formulations, because of the presence of lecithin, are moreover compatible with chlorhexidine decontamination in the case of clinical specimens which may include commensal flora bacteria that may inhibit the growth of mycobacteria [Ghodbane R, Raoult D, Drancourt M. Dramatic reduction of culture time of 5 Mycobacterium tuberculosis. Sci Rep. 2014; 4: 4236]. The diagnostic delay based on the culture depends not only on the intrinsic qualities of the culture medium - here the presence of lamb serum in the culture medium according to the invention, but also, the ability to detect mycobacteria complex Mycobacterium tuberculosis growing. More particularly, when using solid media, it is the ability to detect as early as possible the appearance of Mycobacterium tuberculosis colonies. In routine, the colonies are detected by the macroscopic observation of the culture medium with the naked eye corresponding to the detection of colonies of Mycobacterium tuberculosis of a few millimeters, which requires the presence of a contrast between the color of the colonies and that of the culture centre.
    [0003] More specifically, in EP 2,364,357, mycobacterial culture media include growth factors of mycobacteria and, preferably, antibiotics without activity against mycobacteria, characterized in that they comprise the additional components. following: - lecithin, and - defibrinated blood, and - decomplemented fetal calf serum. However, the implementation of blood, makes these formulations difficult to handle during its industrial manufacture and limits the expiration period of said medium because the blood must be implemented within less than 24 hours and stored at 4 ° C to remain fresh. In addition, the blood must be taken in the presence of anticoagulants to prevent its coagulation these anticoagulants may have a negative effect on the growth of mycobacteria. Therefore, there is interest in developing a medium retaining the characteristics and performance of the media described above, but not including blood for the purpose of the present invention.
    [0004] The inventors have found that replacing fetal calf serum with lamb serum combined with a synthetic dye without activity against mycobacteria results in detectable growth results in the absence of blood. n, at least 3033575 identical or even improved allowing the rapid isolation of mycobacteria complex Mycobacteriurn tuberculosis and other mycobacteria, in particular allowing detection and visually detectable identification with the naked eye in less than 15 days, or even in 10 days, from clinical specimen samples. This combination of lamb serum combined with a synthetic dye compensates for the nutrient properties of blood for the growth of mycobacteria and the contrasting agent for visual detection capability with the naked eye. This contrasting agent makes it possible to identify the colonies with the naked eye and then isolate them and analyze if necessary by Ziehl-Neelsen or Kinyoun staining or by maldi-tof-type mass spectrometry as described below.
    [0005] More specifically, the present invention provides a mycobacteria culture medium, comprising lecithin, mycobacteria growth factors and, preferably, at least one antibiotic compound with no activity against mycobacteria, and at least one dye. without antimycobacterial activity, which further comprises at least the following additional components: - lamb serum, preferably decomplemented, and - at least one dye without activity against mycobacteria, to contrast the color of the culture medium relative to colonies of mycobacteria. Lamb serum is not a known ingredient for the isolation of extracellular bacteria.
    [0006] Preferably, the lamb serum is in a volume proportion of 2.5 to 25%, preferably at least 15%. The lamb serum is preferably decomplemented, that is to say, it has removed, in known manner, all the proteins called "serum complement" by heating, in particular at 56 ° C for one hour, this serum complement may have antibacterial activity. In the medium according to the invention, in the absence of contrasting agent, it is very difficult to differentiate the cream color of the colonies with respect to the light yellow color of the medium by the presence of lecithin and others. In the absence of contrasting agent, it is necessary to use a low magnification inverted microscope instrument to observe colonies of about one millimeter size. Advantageously, the detection of colonies can be done by autofluorescence Mycobacterium Mycobacterium tuberculosis complex illuminated under ultraviolet light and an appropriate length, can detect colonies with a size between 0.25 and 1 mm.
    [0007] Preferably, the dye is a red dye preferably at a concentration of 10 to 100 mg / L. Preferably, the red dye is chosen from azorubine and Ponceau red 4R, preferably at a concentration of 30 to 50 mg / l. As shown later in the examples, these dyes do not affect the growth of mycobacteria and exhibit high stability over time, especially at least 5 weeks. More preferably, the dye is a mixture of azurobine and Ponceau red 4R, preferably in a proportion of 50/30 concentrations. More preferably, the dye is a mixture of 50mg / L azorubine and Ponceau 4R red at 30mg / L. More particularly, the culture medium of mycobacteria according to the invention consists of a basic culture medium, known for the cultivation of mycobacteria, including in particular mineral salts, sugars, amino acids, proteins and vitamins, said culture medium base being supplemented with said additional components mentioned above. Preferably, the mycobacteria culture medium according to the invention contains at least one antioxidant compound, preferably ascorbic acid, preferably at a concentration of at least 0.1 g / l. This antioxidant compound makes it possible to overcome the control of the oxygen tension of less than 5% and to carry out a culture in an atmosphere of 5% CO 2 or even in the open air and ambient atmosphere at the same temperature of 37 ° C. More particularly, the lecithin is in a proportion by weight of 0.1 to 5%, preferably 0.5 to 1%. More particularly, lecithin is lecithin of egg yolk. More particularly, a mycobacteria culture medium according to the invention comprises a basic mycobacteria culture medium comprising, in addition to distilled water, the following components: ammonium sulfate, magnesium sulfate, copper sulfate, sulfate zinc, sodium citrate, ferric ammonium citrate, sodium chloride, calcium chloride, monopotassium phosphate, disodium phosphate, L-glutamic acid, biotin and pyridoxine. These components are, in particular, the components contained in mycobacteria culture media known as Middlebrook media.
    [0008] In practice, this culture medium is initially in the form of a lyophilizate of said components listed above, intended to be diluted in distilled water to form the culture medium according to the invention. Even more particularly, the antibiotic compound without antimycobacterial activity is taken from the group consisting of polymyxins, nalidixic acid, trimethoprim, azlocillin and vancomycin. Advantageously, the culture medium further comprises an antifungal agent which is preferably amphotericin B. The combination consisting of the antibiotics and antifungal agents mentioned above, with the exception of vancomycin, constitutes the so-called known assembly. those skilled in the art under the name PANTA. Said culture medium is advantageously a solid culture medium containing a gelling agent preferably chosen from agar and agar, preferably in a weight proportion of 0.5 to 5%, more preferably 1 to 2%. Preferably, it is a Middlebrook solid medium of reference 7H10, added with said gelling product and additional growth factors of additional mycobacteria: oleic acid, bovine albumin, preferably the fraction V of the bovine albumin, dextrose, catalase and glycerol. The malachite green, contained in the 7H10 medium, is a growth inhibitor of bacteria other than mycobacteria. The additional mycobacteria growth factors above, other than glycerol, are known as OADC factors. The present invention also provides a method for detecting by culturing a mycobacterium using a mycobacteria culture medium according to the invention to incubate a sample which may contain a mycobacterium, in said mycobacteria culture medium, at a temperature of 30 to 37 ° C, suitable for culturing the species of mycobacterium contained in the sample. The present invention further provides a method for determining the viability of a mycobacterium previously detected in a sample by an appropriate method such as, but not limited to, a microscopic observation method, a method for detecting a sequence of DNA, or an antigen detection method. The present invention finally provides a method for determining the sensitivity and resistance of a mycobacterium to antibiotics or more generally, any substance which can be contacted with the mycobacterium in culture in the medium according to the invention.
    [0009] Most mycobacteria are cultured at 37 ° C. However, certain mycobacteria, such as Mycobacterium ringarinum, Mycobacterium haernophilum, Mycobacterium ulcerans, are cultured at a temperature of 28 ° C to 30 ° C. Mycobacterium szulgai can be grown between 25 ° C and 37 ° C, Mycobacterium consplcuurn is grown between 22 ° C and 31 ° C. The optimum growth temperature of Mycobacterium xenopi and Mycobacterium shirnoidei is 45 ° C. The bacteria Mycobacterium genavense, Mycobacterium kansae Mycobacterium fortuiturn, Mycobacterium avlurn complex bacteria, namely Mycobacterium avlurn, Mycobacterirrn Intracellulare, Mycobacterium chirnaera and Mycobacterium colornblense are cultured at 37 ° C.
    [0010] More particularly, in a method of culturing a mycobacterium according to the invention, a sample containing a bacterium of the Mycobacterium tuberculosis complex is cultured at a temperature of 37 ° C. in a said culture medium of mycobacteria. A method of culturing mycobacteria according to the invention may also relate to Mycobacterium tuberculosis out-complex mycobacteria, such as those mentioned above, in particular Mycobacterirrn rnarinurn, species of the Mycobacterrirrn aviurn complex, Mycobacterium haernophilurn, Mycobacterium xenopi, species of the complex. Mycobacterium abscessus, Mycobacterirrn genavense, Mycobacterirrn kansasli, Mycobacterium ulcerans and Mycobacterium fortulturn. The term "bacteria of the Mycobacterirrn tuberculosis complex" is intended to mean bacteria of the species Mycobactericir tuberculosis, Mycobactericirn bovis and its clones or subspecies BCG, Mycobacterium africanum, Mycobacterirrn canettli, Mycobacterium caprae, Mycobacterium microti, Mycobacterium mycobacterirrnumungi and Mycobacterium susicattae. Advantageously, in a method for culturing a mycobacterium according to the invention, the following four steps are carried out: a sample of biological sample which may contain mycobacteria is cultured until a growth of bacteria is detectable, and - it is recognized that the bacterium detected is a bacterium of the mycobacterium genus by a staining test, preferably a Ziehl-Neelsen or Kinyoun staining test, and 30 - where appropriate, the species of said mycobacterium by means of molecular analysis, preferably by analysis of the molecular mass of bacterial proteins by mass spectrometry.
    [0011] The molecular analysis means can be means for analyzing bacterial proteins, in particular by determining their molecular mass by mass spectrometry. However, alternatively, conventional molecular genomic identification (DNA or RNA) methods may be used with probes and / or amplification primers specific for the different species of mycobacteria, in particular as described in application WO 2008 / 050,064. In particular, for the detection of mycobacteria of the Mycobacterium tuberculosis complex, it is possible to use a system called multi spacer sequence typing (hereinafter abbreviated to MST) consisting of a series of nucleic acid fragments of non-coding intergenic zones of the genome of the bacteria of the Mycobacterium tuberculosis complex, said fragments constituting genetic markers allowing the identification of the different species of the Mycobacterium tuberculosis complex and the genotyping of the isolates of the same species of the Mycobacterium tuberculosis complex and, in particular of the species Mycobacterium tuberculosis, by analyzing the sequences of said fragments of areas called MSTs.
    [0012] Advantageously, bacterial growth of the Mycobacterium tuberculosis complex is detected within 15 days, preferably within 10 days. The culture media according to the invention also make it possible to detect 50% of all the different main species of mycobacteria in less than one week, namely Mycobacterium marine plain, Mycobacterium aviurn, the bacteria of the species Mycobacterium tuberculosis, Mycobacterium bovis and its clones or subspecies BCG, Mycobacterium africanum, Mycobacterium canee Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipeck Mycobacterium mungiet Mycobacterium supicattae In a preferred embodiment in which a culture of a biological sample of contain a mycobacteria and contaminating bacteria whose growth can inhibit the growth of mycobacteria, the method is characterized in that: 1 / a preliminary step of initial decontamination of said sample in said culture medium with chlorhexidine is carried out for a period limited, so to limit the action of chlorhexidine to activity against bacteria other than mycobacteria, preferably about 15 minutes of treatment in 1% chlorhexidine solution with stirring, and 2 / chlorhexidine is removed by washing the sample and treated in step 1 / with a neutral buffer, and the bacterial pellet containing the contaminating bacteria thus inactivated and the non-inactivated mycobacteria, which are inoculated on a said mycobacterial culture medium, are centrifuged and recovered. The use of chlorhexidine as a decontaminant was previously limited to the decontamination of samples in solid culture media because chlorhexidine causes precipitation of the liquid culture media. In addition, chlorhexidine is, in principle, known to be toxic to mycobacteria. However, it has previously been found that the presence of egg lecithin (but not soy lecithin) in the culture medium makes it possible to avoid or defer the precipitation of the liquid culture medium, on the one hand, and, d on the other hand, the time-limited action of chlorhexidine, by carrying out said washing step 2, makes it possible to inhibit the posterior activity of chlorhexidine with regard to the mycobacteria. Chlorhexidine, however, retains its primary activity against commensal flora bacteria whose growth can inhibit the growth of mycobacteria, referred to above as contaminating bacteria. In particular, this prior decontamination step is carried out for the biological samples known to be carrying a commensal flora, such as sputum samples, skin biopsies or stool samples, which represent 95% of the biological samples available. for an analysis of mycobacteria. On the other hand, for biological samples such as from blood culture or lymph node biopsy or lung biopsy or bone biopsy, such decontamination is not required. The above method is also particularly advantageous when said biological sample is taken from stool. Said culture medium is a solid culture medium and a growth of said mycobacteria is detected with the naked eye when it is possible to perceive the formation of a colony of bacteria on said solid culture medium. In general, observing the formation of a bacterial colony corresponding to a bacteria concentration of 106 bacteria / ml. In a preferred embodiment of a method according to the invention, the species of bacteria of the mycobacterium genus is identified by means of molecular analysis consisting of an analysis of the protein profile obtained by mass spectrometry, by comparing with a series of protein profile spectra obtained with reference mycobacterial strain samples of different species cultured under the same culture conditions.
    [0013] Here, the term "same culture conditions" means that the same culture medium is used, the same culture temperature for the same species of mycobacterium. A solid culture medium according to the present invention makes it possible to reduce the growth time of M. tuberculosis colonies in the presence of antibiotic and thus rapidly obtain antibiogram results. Furthermore, it has been found according to the present invention that it is possible to advantageously use the E-test method for carrying out antibiograms of M. tuberculosis. The E-test (AB Biodisk, Solna, Sweden, bioMérieux, Marcy-l'Etoile, France) consists of a paper strip impregnated with a concentration gradient of a studied antibiotic, allowing a direct reading of the defective concentration. inhibition of growth of the bacteria (Minimal Inhibitory Concentration, MIC). The E-test was validated for M. tuberculosis on reference media different from that according to the invention [Esteban J. et al. Eur 3 Clin Microbiol Infect Dis 2005; 24: 856-857] and the characteristics of the solid culture medium according to the invention did not make it possible to predict whether it would be suitable for carrying out the E-tests.
    [0014] It has been demonstrated according to the present invention that the solid culture medium according to the invention also allows faster realization and easier reading of a phenotypic test of antibiotic susceptibility and resistance of the Mycobacterium tuberculosis complex than standard media. The present invention thus makes it possible to use the invention of a medium for the rapid isolation and growth of the Mycobacterium tuberculosis complex, for rapid phenotypic susceptibility testing of Mycobacterium tuberculosis complex with antituberculous antibiotics as illustrated in FIG. 'example. More specifically, the present invention provides a culture method for rapid phenotypic determination of mycobacterial susceptibility to antibiotics, wherein steps are performed in which: i) a culture of a mycobacterium is cultured; Preferably the Mycobacterium tuberculosis complex, on said solid culture medium in the presence of at least one given antibiotic, at different known concentrations, and ii / - the lowest concentration of antibiotic, preferably selected from rifampicin, is determined, isoniazid, ethambutol, pyrazinamide and streptomycin, which inhibits any visible growth of said bacterium (MIC: minimum inhibitory concentration). Thus, in known manner, for each pair of mycobacterium-antibiotic, a minimum inhibitory concentration or MIC can be determined and compared to the critical antibiotic concentrations that a patient can safely receive and which blocks the growth of the strain. bacterial involved. The susceptibility or resistance of the bacterium to the antibiotic is then determined as follows: if the MIC is lower than the lower critical concentration, the bacterium is sensitive to the antibiotic, and if the MIC is greater than the concentration Superior criticism, the bacterium is resistant to the antibiotic involved. In a preferred embodiment according to the invention, in step i / -, at least one strip of paper impregnated with said antibiotic is deposited on said solid culture medium at different concentrations along a gradient of concentration along said strip. More particularly, a petri dish comprising said solid culture medium according to the invention is seeded at a concentration of at least 104 colonies / ml, preferably 106 colonies / ml, and then a strip is deposited on said solid culture medium. , In particular an E-test strip which comprises a continuous gradient in increasing concentrations of antibiotic from one end to the other of said strip, said antibiotic concentrations being written directly on the strip, and incubation is carried out the box on which the strip rests and read the MIC at the intersection of the bacterial growth inhibition disk with the strip.
    [0015] According to other known embodiments, the strip may be replaced by a blotter disk impregnated with the critical concentration of antibiotic, in order to verify the existence of a zone of inhibition around the disc to establish that the strain is sensitive to the antibiotic. According to another known embodiment, the antibiotic can be incorporated directly into the mass of said solid culture medium at the said critical concentration and the growth of the strain can be compared to the said solid culture medium impregnated with the said antibiotic, with the growth of the same strain on the same solid culture medium containing no antibiotic, to check the presence or absence of bacterial growth in the culture medium with and, respectively, without antibiotic and to deduce that the strain is sensitive to the antibiotic if there is less growth of the strain in the solid culture medium containing said antibiotic. Other features and advantages of the present invention will become more apparent on reading the nonlimiting examples which follow in connection with the drawings in which: FIG. 1 is a graphical representation of the detection time t (in hours) of Mycobacterium tuberculosis on the medium according to the invention in comparison with the medium Colestos, using inocula of different inoculated concentrations of 102, 103, 104 cfu / ml.
    [0016] FIG. 2 is a graphical representation of the detection time t (in hours) of Mycobacterium tuberculosis on the medium according to the invention in comparison with the Löwenstein-Jensen medium ("L-3"), using inocula of different concentrations. inoculated with 102, 103, 104 cfu / ml. EXAMPLE 1: DETERMINATION OF THE COMPOSITION OF THE MEDIUM ACCORDING TO THE INVENTION 1) In order to eliminate the blood present in the solid medium MOD-5% similar to that of Example 2C of EP 2 364 357, the inventors - after many unsuccessful attempts - successfully substituted sheep blood with an equivalent concentration (5%) of lamb serum to define MOD-L5 medium (MOD-Lamb Serum). The performances of this MOD-L5 medium were compared with those of the MOD medium and MOD MOD4-WB MOD4 medium without MOD-without blood blood. The composition of said MOD-5% medium is given below for 1000 ml: Sterile distilled water: 841.3 ml 1- Middlebrook medium 7H10: 20 Ammonium sulphate: 0.50 g Monopotassium phosphate: 1.50 g Disodium phosphate: 1.50 g Sodium citrate: 0.4 g Magnesium sulphate: 0.05 g Calcium chloride: 0.00050 g Zinc sulphate: 0.0010 g Copper sulphate: 0.0010 g L-glutamic acid: 0.50 g Ferric ammonium citrate: 0.04 g Pyridoxine hydrochloride: 0.0010 g Biotin: 0.00050 g Malachite green: 0.000250 g Agar (Agar): 13.5 g 2- Additional growth factors: Bovine albumin: 7.5 g 3033575 14 Dextrose: 3 g Catalase: 0.0045 g Oleic acid: 0.09 g Bovine serum albumin: 1.65 g Nicotinamide Adenine Dinucleotide (NAD): 0.022 g Vinylpyrrolidine Copolymer: 1.67 mL Citric acid: 0.0842 g Hemin 330 pg 0.00033 g Tween 80: 2 mL 10 Yeast extract: 1g Pancreatic digest of casein: 1g Glucose: 2g Glycerol: 5 mL 3- Antibiotics without antimyc activity Bacterial and antifungal: Polymyxin: 20,000 units Amphothericin B: 0.002 g Nalidixic acid: 0.008 g Trimethoprim: 0.002 g Azlocillin: 0.002 g Vancomycin: 0.005 g 4- Additional elements: Fetal calf serum: 50 ml Defibrinated sheep blood: 50 lecithin: 5 g For MOD-LS-5%, the composition is identical for the components of points 1 to 3 and has the following additional elements: 4- Additional elements: lamb serum: 50 ml Lecithin: 5 g. For MOD-WS-5%, the composition is identical for the components of points 1 to 3 and has the following additional elements: 4- Additional elements: fetal calf serum: 50 ml Lecithin: 5 g Five strains of Mycobacterium tuberculosis The reference strain Mycobacteriurn tuberculosisH37Ry and four clinical strains, including a strain of the family 3033575 from Beijing, were used. Concentration suspensions equal to 105 CFU / mL were prepared from these strains and 100 μL of each suspension were inoculated triplicate on all three media. The media were incubated in parallel at 37 ° C under an atmosphere containing 5% CO2 and micro aerophilic (oxygen tension between 2.5% and 5%). The mean time to onset of the first colonies was 4 ± 0.3 days on MOD, 3.8 ± 0.4 days on MOD-LS, and 5.1 ± 0.6 days on MOD-WS. There was no significant difference in the onset of colonies on MOD compared to MOD-LS (P = 0.1413). In contrast, comparing MOD-WS medium with MOD medium and MOD-LS medium, the difference was more significant (P <0.05). These results indicate that it is advantageous to substitute lamb serum for blood to obtain slightly improved growth results. In a second step, the inventors have optimized the concentration and the type of lamb serum to be introduced into the medium. A variety of media were prepared: 15-MOD-15% -HIBS as MOD-WB-5% but with 15% (150mL) decomplemented bovine serum instead of 5% non-decomplemented bovine serum-MOD-15% BS: 15% (150mL) undecompleted bovine serum - MOD-15% -HILS: 15% (150mL) unbound lamb serum - MOD-15% -LS: 15% (150mL) non-decompleted lamb serum 20 - MOD-10% -LS: 10% (100mL) undecompleted lamb serum - MOD-5% -LS: 5% (50mL) non-decompleted lamb serum - MOD-15% -HIBS-5% -LS: 15% (150mL) decomplemented bovine serum and 5% (50mL) undecompleted lamb serum - MOD-15% -HIBS-5% -HILS: 15% (150mL) unbound bovine serum and 5% (50mL) serum d lamb uncomplemented - MOD-15% -BS-5% -LS: 15% (150mL) undecomplemented bovine serum and 5% (50mL) non-decompleted lamb serum The performance of these media was measured by cultivating five Mycobacterium tuberculosis with the reference strain Mycobacterium tuberculosis H37Rv 30 and four clinical strains including a strain of the Beijing family. Suspensions of concentration equal to 105 CFU / ml were prepared from these strains and 100 μl of each suspension were inoculated into triplicate on the various media.
    [0017] Two criteria were taken as a basis for the comparison of the performances of these different culture media: detection time of the first colonies, density and richness of the colonies after 7 days of incubation. Following a visual inspection, a gradient from 1 (lower) to 5 (higher) is given to each Petri dish to measure the richness of the crop. The results are reproduced in Table 1 below (in Petri dishes). Table 1 MOD- MOD- MOD- MOD- MOD- MOD- MOD- 15% - HIBS- 5% - LS MOD- 15% - HIBS- 5% - HILS MOD15% BS5% LS 15% - 15% - BS 15% - 15% - LS 10% - LS 5% - LS HIBS HILS Times of 101 ± 102 ± 12 87 ± 88 ± 91 ± 98 ± 89 ± 90 ± 95 ± detection 10.8 13.6 14.7 14.8 10 13 17 12 (hours) Density / Wealth 2 ± 2.2 ± 3.5 ± 2.8 ± 2.3 ± 2 ± 3 ± 3 ± 3 ± 0.8 0.7 1.4 1.1 0.8 0.8 1.1 1.3 1.3 Comparing media containing 15% bovine serum and 5% lamb serum 10 decomplemented or not decomplemented (MOD-15% -HIBS-5% -HILS, MOD-15% HIBS-5% -LS and MOD-15% -BS-5% -LS), no significant difference was observed in detection time colonies or the wealth of colonies. In addition, no significant effect of bovine serum decomplementation (MOD-15% -HIBS versus MOD-15% -BS) was observed on the detection time (P = 0.8321), nor on the 15 wealth of colonies (P = 0.6030). There is also no significant effect of lamb serum decomplementation (MOD-15% -HILS versus MOD-15% -LS) on the detection time (P = 0.8649) of colonies. On the other hand, one observes a highest score of richness on medium MOD-15% HILS.
    [0018] A significantly lower colony detection delay was observed on media incorporating lamb serum (MOD-15% -HILS and MOD-15% -LS) than that observed on media incorporating bovine serum (P <0.05).
    [0019] More particularly, MOD-15% -HILS medium has a density and richness score that is significantly higher than those exhibited by bovine serum-based media. There is no statistically significant difference in colony detection time or colony richness score by comparing MOD-15% -HILS and MOD-15 ° / 01-IIBS-5 ° / 01 -1ILS. By gradually decreasing the concentration of lamb serum (MOD-15% -LS, MOD-10% -LS and MOD-5% -LS), there is a gradual increase in the detection time and a gradual decrease in the density score and wealth of the colonies.
    [0020] The colonies were detected in parallel by naked eye or by auto fluorescence using a binocular equipped with fluorescence and a monochrome camera (MZ1OF stereomicroscope, Leica Microsystems SAS, Nanterre, France) equipped with a GF1 filter. From these results, the inventors decided to use the medium MOD-15% -HILS for the further development of the visual colony detection capacity of the colonies with the naked eye. 2) The inventors then worked to contrast the white-beige color of the colonies of Mycobacterium tuberculosis on the yellow background of MOD culture media above to increase the capacity of visual detection to the naked eye of the growth of mycobacteria. Different lots of MOD-15% -HILS medium were prepared and stained using different neutral dyes of different colors and concentrations. To do this, the inventors first used food colorants for their safety and availability in order to assess which of these dyes would make it possible to obtain a satisfactory contrast between the colonies of mycobacteria and the agar base. The following food colorants were incorporated in the medium according to the invention to obtain a range of colors. Blue-sky: 0.4% blue dye Blue-pale: 0.2% blue dye Yellow: 0.4% yellow dye Salmon: 0.04% red dye 30 Orange: 0.3% yellow dye + 0.1% red dye Orange-reddish: 0.2% red dye + 0.2 % yellow dye Light red: 0.3% red dye + 0.1% yellow dye 3033575 18 Red: 0.4% red dye (Color chosen for MOD9) Red-burgundy: 0.3% red dye + 0.1% blue dye Reddish-purple: 0.2% red dye + 0.2% blue dye Violet: 0.3% red dye + 0.1% yellow dye 5 Yellowish green: 0.3% yellow dye + 0.1% blue dye Pistachio green: 0.2% yellow dye + 0.2% blue dye Green: 0.3% blue dye + 0.1% yellow dye Maron: 0.2% blue dye + 0.1% yellow dye + 0.1% red dye Finally, it is the red color that has been retained. The inventors then determined that this food coloring contained azorubine and Ponceau Rouge. Azorubine or carmoisine is a red food color identified as E122. This nitrogen compound is a disodium salt of 1-hydroxy-1- (4-sulfo-naphthylazo) -naphthalenesulfonic acid. Ponceau red is a synthetic azo dye (C201-111N2Na301153) also known as CI 16255, Cochineal Red A, used as food coloring (E1242 also known as E124 additive) The combination of Azorubine 50mg / L and Ponceau 4R 30mg / The dye in red MOD-15% -HILS medium exhibited the best colony / agar contrast.The MOD-15% -HILS medium in which sheep blood (5%) and decomplemented bovine serum (15%) have been substituted by the decomplemented lamb serum (15%) and the mixture of azorubine (50 mg / L) and Ponceau 4R (30 mg / L) is called "MOD9" The composition of said MOD9 medium according to the invention is given below: MOD9 Solid Media for 1000 mL: Sterile Distilled Water: 841.3 mL 1- Middlebrook Medium 7H10: Ammonium Sulfate: 0.50 g Monopotassium Phosphate: 1.50 g Disodium Phosphate: 1.50 g Citrate sodium: 0.4 g Magnesium sulphate: 0.05 g Calcium chloride: 0.00050 g Zinc sulphate: 0.0010 g 3033575 1 9 Copper sulphate: 0.0010 g L-glutamic acid: 0.50 g Ferric ammonium citrate: 0.04 g Pyridoxine hydrochloride: 0.0010 g 5 Biotin: 0.00050 g Malachite green: 0.000250 g Agar (agar): 13.5 g 2- Additional growth factors: Bovine albumin: 7.5 g Dextrose: 3 g Catalase: 0.0045 g Oleic acid: 0.09 g Bovine serum albumin: 1.65 g Nicotinamide Adenine Dinucleotide (NAD): 0.022 g Vinylpyrrolidine Copolymer: 1.67 mL Citric acid: 0.0842 g Hemin 0.00033 gTween 80: 2 mL Yeast extract: 1g Pancreatic digest of casein: 1g 20 Glucose: 2g Glycerol: 5 mL 3- Antibiotics without antimycobacterial and antifungal activity: Polymyxin: 20,000 units Amphothericin B : 0.002 g 25 nalidixic acid: 0.008 g Trimethoprim: 0.002 g Azlocillin: 0.002 g Vancomycin: 0.005 g 4- Additional elements of the invention: Decomplemented lamb serum: 150 mL Lecithin: 5 g Azorubine: 0.050 g Ponceau 4R: 0.030 g In an embodiment variant, one added 0.1 g of ascorbic acid which made it possible to overcome the control of the oxygen tension of less than 5% and to achieve a culture in atmospheric of 5% of CO2, even in the open air and ambient atmosphere at the same temperature of 37 ° C.
    [0021] EXAMPLE 2: INFLUENCE OF THE RED COLOR OF MOD9 MEDIUM ON THE GROWTH OF M. TUBERCULOSIS USING CHEMICALLY DEFINED DYES. In this example, the inventors compared the red color of the MOD9 medium obtained by adding azorubine alone, Ponceau red alone, or a mixture of the two dyes; and the influence of these three formulations on the growth of M. tubercu / asis a) Media: -MOD9-A: The dye is azorubine 50mg / L -MOD9-P: The dye is Ponceau 4R 30mg / L 10 -MOD9-A / P: The dye is a mixture of azorubine 50mg / L and red Ponceau 4R (30mg / L) Two experiments were conducted: -On the various media cast in Petri dishes (P-MOD9) -On different media poured into tubes (bioMérieux) (T-MOD9) 15 b) Strains: The tests carried out on media in Petri dish format are carried out with five strains of Mycobacterium tuberculosis, the reference strain H37Rv and four clinical strains. , susceptible to anti-TB drugs, one of which is Beijing; whereas the tests carried out on the media in tube format are carried out with two strains of M. tuberculosis / asis, the reference strain H37Rv and a Beijing strain. c) Protocol: The strains maintained in culture on MOD9 medium are calibrated at 106 cfu / ml by microscopic observation after Ziehl-Neelsen staining. In addition to the more dilute suspensions, 104 cfu / mL and 102 cfu / mL are prepared.
    [0022] The experiments are carried out in triplicate for tests launched on petri dish format and in duplicate on tube format (limited in relation to the number of tubes). The suspensions prepared are inoculated at a rate of 100 μL for the Petri dishes and 200 μL for the tubes and incubated in a 37 ° C., 5% CO2 incubator (with the semi-open caps for the tube format).
    [0023] After inoculation, the tubes are placed in a horizontal position for 3 days, then straightened in an upright position until the end of the test. Unseeded media used as negative controls are incubated in parallel.
    [0024] A reading with the naked eye of the media is performed every 12 hours for the detection of colonies. The identification of the colonies is subsequently confirmed by ZiehlNeelsen staining. d) Results - Color: the inventors observed a difference in the intensity of the red color of the MOD9 culture medium, with increasing intensity going from the colored medium by the red Ponceau alone, the azorubine alone, and the mixture red Ponceau more azorubine. - Growth of M. tubercu / asis: The results (mean and standard deviation) of the detection time of the first colony of the different strains of M. tubercu / asis on the different media are presented in Tables 2 and 3 below.
    [0025] Detection Time 106 CFU / mL 104 CFU / mL 102 CFU / mL (hours) P-MOD9-A / P 32 ± 8.7 82.4 ± 20.2 125.6 ± 21.2 P-MOD9-A 32.8 ± 10.6 79.2 ± 22.1 124.8 ± 23.9 P -MOD9-P 33.6 ± 11.3 81.6 ± 20.8 136 ± 19.6 Table 2: Detection time of the first colony of the different M. tubercu / asis strains on the different media in the Petri dish Detection time 106 CFU / mL 104 CFU / mL 102 CFU / mL (hours) T-MOD9-A / P 51 ± 18 105 ± 15.1 162 ± 37.3 T-MOD9-A 63 ± 11.5 114 ± 12 171 ± 24.7 T-MOD9-P 60 ± 9.8 107.2 ± 9.9 ± 20.8 Table 3: Detection time of the first colony of the different strains of M. tubercu / osis on the different media in tubes The inventors did not observe any significant difference in the detection time between the different dyes and combinations used. to color the MOD9 medium in red.
    [0026] In total, all the results indicate that the MOD9 medium should be stained red with an azorubine mixture at 50 mg / L plus red Ponceau at 30 mg / L. Example 3: STABILITY OF THE RED COLOR OF MOD9 MEDIUM In this example, the inventors endeavored to check the stability over time of the raspberry red color of MOD9 medium on agar, appreciated by its appearance to the eye under the conditions usual storage at 4 ° C and use. For this purpose, the inventors have prepared a first batch of 6 MOD9 agars in a 55 mm petri dish, left in a temperature-controlled cold room at 4 ° C. corresponding to the usual storage temperature of the media containing organic compounds. The color of the dishes was observed on day 0, then at 37, 14, 21, 30, 345 and then two months, three months and six months after incubation. A digital photograph of the agar group was recorded at each observation. Under these conditions, the inventors did not observe any modification of the red color of the MOD9 medium under any of the experimental conditions, indicating the high staining stability of the MOD9 medium in the composition indicated in Example 1, kept at 4 ° C. C. A second batch of 6 petri dishes containing MOD9 was incubated in ambient air at laboratory temperature between 18 ° C and 22 ° C for 45 days and observed under the same conditions. Under these conditions, the inventors have not observed any change in the red color, indicating the possibility of storage in the laboratory environment, more particularly in laboratories that do not have sufficient cold resources. A third batch of 6 petri dishes containing MOD9 was incubated at 37 ° C in an incubator containing 5% CO2 for 45 days and observed under the same conditions. Under these conditions, the inventors did not observe any change in the red color, indicating the possibility of incubating the MOD9 medium according to the international recommendations for the detection of mycobacterial colonies, more particularly colonies of Mycobacterium tuberculosis. In parallel, the inventors conducted a second series of experiments in a second laboratory at the same temperatures and for 5 weeks during which they observed the same raspberry red coloration and the sterility of the MOD9 medium. These results are consistent with those conducted in the first laboratory. EXAMPLE 4 COMPARATIVE PERFORMANCE OF THE MOD9 ENVIRONMENT AND THE COLETSOS MEDIUM FOR THE GROWTH OF M. TUBERCULOSIS STRAINS In this example, the inventors compared the performance of the MOD9 medium in 55 mm Petri dishes with those of the Coletsos medium in tubes. of glass. Coletsos medium is an egg medium comprising sodium pyruvate. Suspensions of 15 strains of Mycobacterium tuberculosis prepared at different concentrations were used. The 15 strains of Mycobacterium tuberculosis used include the Mycobacterium tuberculosis H37Rv reference strain and 14 clinical isolates including two isolates from the Beijing family. Suspensions of 104, 103 and 102 CFU / mL were prepared from these strains. The 2 media were inoculated in triplicate with 100 μl of each suspension, and then incubated at 37 ° C. in an atmosphere containing 5% CO 2, the results were read every 12 hours to determine the detection time of the first colonies of M tuberculosis. The detection times of the first colonies on each medium are shown in Table 4 below: Detection time (days) MOD9 (Petri dish) Coletsos (Tube) 104 CFU / mL 2.5 ± 0.4 * 6.46 ± 1.1 103 CFU / mL 3.4 ± 0.37 * 7.96 ± 1,102 CFU / mL 4.2 ± 0.87 * 9.2 ± 1.87 TABLE 4 Significantly lower colony detection time was observed on MOD9 medium in Petri dishes compared to Coletsos medium in tubes, regardless of size. the concentration tested. These results are illustrated in FIG. 1. EXAMPLE 5 COMPARATIVE PERFORMANCE OF THE MOD9 ENVIRONMENT AND THE LOWENSTEIN-JENSEN MEDIUM FOR THE GROWTH OF M. TUBERCULOSIS STRAINS The performances of the MOD9 medium in a 55 mm petri dish were compared with those of middle Lôwenstein-Jensen (bioMérieux, La Balme-les-Grottes, France) in glass tubes. A total of 21 strains of Mycobacterium tuberculosis were used, including the Mycobacterium tuberculosis H37Rv reference strain and 20 clinical isolates including two belonging to the Beijing family. Suspensions of 106, 104 and 102 CFU / mL were prepared from these strains. Both media were inoculated into triplicate with 100 μL of each suspension and incubated at 37 ° C in an atmosphere containing 5% CO 2. Observation of culture media is done every 12 h to determine the detection time of the first colonies of Mycobacterium tuberculosis. The detection times of the first colonies on each of the media are shown in Table 5 below: Detection time (days) MOD9 (Petri dish) Lôwenstein-Jensen (Tube) 106 CFU / mL 1.5 ± 0.4 * 5.7 ± 1.5 104 CFU / mL 3.5 ± 0.6 * 7.6 ± 0.8 102 CFU / mL 4.9 ± 1 * 10.8 ± 1.7 TABLE 5 A significantly lower detection time of colonies was observed on MOD9 medium in Petri dishes compared to Löwenstein-Jensen medium in tube, regardless of the concentration tested. These results are illustrated in FIG. 2. EXAMPLE 6 COMPARATIVE PERFORMANCE OF THE MOD9 ENVIRONMENT AND THE LOWENSTEIN-JENSEN MEDIUM FOR THE ISOLATION OF THE MYCOBACTERIA STRAINS In this example, the inventors observed the performances of the MOD9 medium in isolation. mycobacteria from clinical samples. A total of 250 samples including 203 (81.2%) respiratory specimens [(173 sputum (69.2%), 27 bronchial aspirations (10.8%) and three bronchoalveolar lavages (1.2%)] and 47 (18.8%) non-respiratory specimens [36 stools (14.4%) and 11 urine (4.4%)] collected from 145 patients were decontaminated using chlorhexidine (0.7%) (Asmar, 2014) and then cultured (100 μL inoculum) in parallel on MOD9 ( Petri dish 55 mm) and Lôwenstein-Jensen tubes (bioMérieux, FRANCE) A total of 40 mycobacteria were isolated from samples collected from 13 different patients, identified as M. tuberculis / Asis and 2 M. The number of isolates obtained on MOD9 [38 isolates (36 M. tubercu / asis and 2 M. 25 abscessus)] from 26 sputum samples, 11 stool specimens and one bronchial aspiration sample was greater than 32 isolates obtained on Löwenstein-Jensen medium (30 M. tubercu / asis and 2 M. abscessus) from 27 sputum and 5 stool samples (this difference is significant, Fisher's statistical test with a P value <0.5195). In total, 8 isolates of M. tubercu / asis were obtained only on 3033575 MOD9 medium and 2 isolates of M. tuberculosis were obtained only on Löwenstein-Jensen medium. Mean detection time was 9.9 ± 4 (4-16) days for MOD9 versus 17.5 ± 6.6 (8-35) days for Löwenstein-Jensen (P <0.05). The average detection time of Mycobacterium tuberculosis colonies is 10.3 ± 3.9 (5-18) days on MOD9 versus 18.1 ± 6.4 (10-35) days on Lowenstein-Jensen (P <0.05). The average detection time of colonies isolated on both media is 9.6 ± 4 (4-18) days on MOD9 versus 16.8 ± 6.2 (8-35) days on Löwenstein-Jensen (P <0.05). The average time to detect Mycobacterium tuberculosis colonies isolated on both media was 9.5 ± 3.9 (5-18) days on MOD9 10 versus 17.4 ± 5.9 (10-35) days on Löwenstein-Jensen (P <0.05). The contamination rate on MOD9 is 1.6% (3 stools and 1 sputum) and 4.4% (8 stools, 2 sputum and 1 bronchial aspiration) on Löwenstein-Jensen (P = 0.1129). EXAMPLE 7 COMPARATIVE PERFORMANCE OF THE MOD9 ENVIRONMENT AND THE BACTECTM MEDIUM FOR THE ISOLATION OF THE MYCOBACTERIA STRAINS The inventors compared a protocol combining a decontamination of the contaminated clinical samples with chlorhexidine (0.7%) and inoculation of the MOD9 medium with the protocol of reference combining the decontamination of clinical samples contaminated with NALC-NaOH and liquid seeding BACTECTm-MGITTm (Becton Dickinson) for the isolation of mycobacteria from clinical samples.
    [0027] A total of 300 samples comprising 238 (79.3%) respiratory samples [(203 sputum (67.6%), 31 bronchial aspirations (10.3%) and 4 bronchoalveolar lavages (1.3%)] and 62 (20.6%) [43 stool (14.3%), 14 urine (4.6%), and 5 lymph nodes] collected from 161 patients were used in this study and all samples (except lymph nodes) were decontaminated using the NALC-25 reference method. NaOH and then cultured (500 μl of inoculum) in a MGITTm vial (Becton Dickinson) after addition of 800 μl of the OADC supplement and 500 μl of PANTA antibiotic mixture, and then incubated in the BACTECT ™ (Becton Dickinson) automaton. An amount of between 0.1 ml and 1 ml of remaining biological material (depending on availability) was decontaminated with chlorhexidine (0.7%) (Asmar, 2014) and then inoculated (100 μl of inoculum) onto MOD9 medium (box). Petri dish 55 mm) The lymph nodes were homogenized and s in culture directly without decontamination on MOD9 and MGITTm. A total of 46 mycobacteria were isolated from clinical specimens collected from 17 different patients, these strains were identified as 44 Mycobacterium tuberculosis and 2 Mycobacterium abscessus. The number of isolates obtained on MOD9 [43 3033575 26 isolates (41 M. tuberculosis and 2 M. abscessus)] from 29 sputum samples, 11 stool samples, 1 bronchial aspiration sample and 3 ganglia, was significantly greater than the 31 isolates obtained on MGITTm (30 M. tuberculosis and 1 M. abscessus) from 23 sputum, 3 stools, 1 bronchial aspiration and 5 lymph nodes (P <5 0.05). In total, 15 isolates (14 M. tubercu / asis and 1 M. abscessus) were obtained only on MOD9 versus 3 strains of M. tuberculosis obtained solely on MGITTm. The mean detection time is 9.8 ± 3.8 (4-18) days for MOD9 versus 14 ± 8 (3-32) days for MGITTm (P <0.05). The average time to detect colonies of Mycobacterium tuberculosis is 10 ± 3.7 (5-18) days on MOD9 versus 14.2 ± 8 (4-32) days on MGITTm (P <0.05). The average time to detect colonies that grew on both media was 8.7 ± 3.5 (4-18) days on MOD9 versus 13.3 ± 7 (4-25) days on MGITTm (P <0.05). The mean time to detect colonies of Mycobacterium tuberculosis that grew on both media was 8.9 ± 3.5 (5-18) days on MOD9 versus 13.6 ± 7 (4-25) days on MGITTm (P <0.05). The contamination rate observed by the chlorhexidine-0.7 ° / o / MOD9 protocol was 1.33% (3 stools and 1 sputum) and significantly lower than that observed by NALC-NaOH / MGITTm (4.67%, 7 stools, 6 sputum and Bronchial aspiration) (P <0.05). EXAMPLE 8: PHENOTYPIC DETERMINATION OF M. TUBERCULOSIS SUSCEPTIBILITY TO ANTIBIOTICS a / - Methods: The inventors tested six clinical strains of the species Mycobacterium tuberculosis isolated from respiratory samples during routine diagnosis in their clinical microbiology laboratory. These six strains were identified from the species Mycobacterium tuberculosis by their characteristic cord morphology after Ziehl staining, and then by PCR amplification and sequencing of intergenic spacers [Djelouadji Z et al. A Single-Step Sequencing Method for the Identification of Mycobacterium tuberculosis Complex Species. PLoS Negl Trop Dis.
    [0028] 2008 Jun 18; 2 (6): e253]. These six strains were tested susceptible to first-line antituberculous antibiotics using a solid reference phenotypic test testing only the so-called critical concentrations for each of the four antibiotics tested: isoniazid, 0.2 μg / ml; rifampicin, 1 μg / ml; ethambutol, 5 μg / ml and streptomycin, 5 μg / ml. [Woods GL, Warren NG, Inderlind CB. Susceptibility test methods: Mycobacteria, Nocardia, and other Actinornycetes in: Manual of Clinical Microbiology, 9th Edition (Nurray PR, Baren EJ, 3033575 Jorgensen JH, Bry NL, Pfaller MA, American Society for Microbiology, 2007, 12231247). tested in parallel a tuberculosis resistant clinical strain (minimal inhibitory concentration of rifampicin> 1 μg / ml) as determined by the reference method in solid medium [Woods GL, Warren NG, Inderlind CB.Susceptibility test methods: Mycobacteria, Nocardia, and over actinomycetes in: Manual of Clinical Microbiology, 9th Edition (Nurray PR, Baren EJ, Jorgensen JH, Bry NL, Pfaller MA, American Society for Microbiology, 2007, page 1223-1247) .These strains were suspended in sterile phosphate buffer at the concentration of 106 colonies / ml.
    [0029] This suspension served to inundate in parallel a sterile petri dish containing the standard reference medium: Middlebrook 7H10 supplemented with a mixture of oleic acid, albumin, dextrose and catalase (OADC), a sterile petri dish containing the medium. MOD-15% HIBS and a sterile Petri dish containing the MOD9 solid medium according to the invention of Example 1 above.
    [0030] The inventors used the system called E-test® (Biomérieux, France) to determine the susceptibility of the strains to the various antibiotics. This system consists of a paper strip impregnated with antibiotic (isoniazid or ethambutol) according to a continuous concentration gradient along the strip. This device allows a reading with the naked eye of the concentration from which there is no longer observed growth of Mycobacterium tuberculosis thus defining the minimum inhibitory concentration of the antibiotic (isoniazid or ethambutol) vis-à-vis of the strain of Mycobacterium tuberculosis that is tested. The Petri dishes were incubated at 37 ° C under an atmosphere containing 5% CO 2 for two weeks with daily observation from the sixth day of incubation. The colonies were confirmed as Mycobacterium tuberculosis on the basis of their appearance and after Ziehl staining showing cords of acid-fast bacilli. A bacterial growth inhibition disk is formed around the strip in areas containing an antibiotic concentration greater than the minimum inhibitory concentration, it being understood that this is determined by the intersection of the inhibition disk with the strip. Results: The inventors observed that: (1) colonies of Mycobacterium tuberculosis appeared after 7 days of incubation on MOD9 medium according to the invention as on comparative medium MOD15% HIBS. (2) the reading of the E-test is possible under these conditions after 7 days of incubation on the medium according to the invention because there is a sufficient number of colonies allowing the reading and a very good contrast between the colonies which appear gray on the red background of the medium MOD 9 according to the invention; as on the MOD-15% HIBS medium and better than on the reference medium, due to the incomplete growth of the colonies on the reference medium and a poor contrast between the colony color (translucent) and the medium 10 reference number, (3) the results of the isoniazid and ethambutol E-tests obtained on the MOD9 medium according to the invention are similar to those obtained beforehand with MOD-15% HIBS-and are more accurate than those of the reference tests, allowing the determination of a true minimal inhibitory concentration.
    [0031] This experiment demonstrates that, by using a solid medium according to the invention, performing phenotypic susceptibility tests of Mycobacterium tuberculosis to antituberculous drugs according to the E-test method is as quick and easy as performing E-test. on a solid reference medium. A petri dish containing the reference medium and the medium according to the invention incubated after inoculation of the same strain of the species Mycobacterium tuberculosis, shows the presence of colonies of Mycobacterium tuberculosis easily visible after 7 days on the medium according to the invention. invention and hardly visible on the reference medium after 11 days, allowing easy reading of the inhibitory concentration of isoniazid (IZ) on a first strip and ethambutol (ET) on a second strip.
    [0032] Around the strips are zones of inhibition of ovoid-shaped bacterial growth around the portions of the strip containing an antibiotic inhibitory concentration, so that the intersection of the lower limit of said inhibitory zones with the strips make it possible to read on the dipstick the minimum inhibitory concentration of the antibiotic. 30
    权利要求:
    Claims (20)
    [0001]
    REVENDICATIONS1. Mycobacteria culture medium, comprising lecithin, growth factors of mycobacteria and, preferably, antibiotics without activity against mycobacteria, characterized in that it further comprises at least the following additional components: lamb serum, preferably decomplemented lamb serum, and at least one dye with no activity against mycobacteria, which makes it possible to contrast the color of the culture medium with respect to the colonies of mycobacteria.
    [0002]
    2. mycobacteria culture medium according to claim 1, characterized in that the lamb serum is in a volume proportion of 2.5 to 25%, preferably at least 15%.
    [0003]
    3. mycobacteria culture medium according to claim 2, characterized in that the dye is a red dye, preferably in a concentration of 10 to 100 mg / l.
    [0004]
    4. mycobacteria culture medium according to claim 3, characterized in that the dye is selected from azurobine and Ponceau red 4R preferably in a concentration of 30 to 50mg / L each.
    [0005]
    5. mycobacteria culture medium according to claim 4, characterized in that the coTorant is a mixture of azurobine and Ponceau red 4R, preferably in a proportion in concentrations of 50/30. 20
    [0006]
    6. mycobacteria culture medium according to one of claims 1 to 5 characterized in that it contains at least one antioxidant compound, preferably ascorbic acid, preferably at a concentration of at least 0.1g / l.
    [0007]
    7. mycobacteria culture medium according to one of claims 1 to 6 wherein the lecithin is in a weight proportion of 0.1 to 5%, preferably 0.5 to 1%, preferably lecithin being from lecithin of egg yolk.
    [0008]
    8. Mycobacteria culture medium according to one of claims 1 to 7, characterized in that it comprises a base mycobacteria culture medium comprising, in addition to distilled water, the following components: ammonium sulfate, magnesium sulphate, copper sulphate, zinc sulphate, sodium citrate, ferric ammonium citrate, sodium chloride, calcium chloride, monopotassium phosphate, disodium phosphate, L-glutamic acid, biotin and pyridoxine. 3033575 30
    [0009]
    9. mycobacteria culture medium according to one of claims 1 to 8, characterized in that said antibiotics without antimycobacterial activity are polymyxin, amphotericin B, nalidixic acid, trimethoprim, azlocillin and vancomycin, and the culture medium comprises, in addition, an antifungal agent, preferably amphotericin B.
    [0010]
    10. Culture medium of mycobacteria according to one of claims 1 to 9, characterized in that said culture medium is a solid culture medium containing a gelling agent preferably selected from agar and agar, preferably in a weight ratio from 0.5 to 5%, more preferably from 1 to 2%.
    [0011]
    11. Mycobacteria culture medium according to claim 10, characterized in that it is a Middlebrook type solid medium of reference 7H10, supplemented with said gelling product and the following additional growth factors of mycobacteria: oleic acid bovine albumin, preferably fraction V of bovine albumin, dextrose, catalase and glycerol.
    [0012]
    12. A method of culturing a mycobacterium using a mycobacteria culture medium according to one of claims 1 to 11, characterized in that a sample containing a said mycobacterium is heated in a said medium. mycobacteria culture at a temperature of 30 ° C to 37 ° C, suitable for culturing the species of mycobacterium contained in the sample.
    [0013]
    13. The method of culturing a mycobacterium according to claim 12, characterized in that a sample containing a bacterium of the Mycobacterium tuberculostus complex is cultivated at a temperature of 37 ° C in a said mycobacteria culture medium.
    [0014]
    14. A method of culturing a mycobacterium according to claim 12 or 13, characterized in that the following steps are carried out: - the culture of a biological sample sample which may contain mycobacteria is carried out until bacterial growth is detectable, and it is recognized that the bacterium detected is a bacterium of the mycobacterium genus by a staining test, and if appropriate, the species of said mycobacterium is identified by means of molecular analysis, preferably by mass spectrometry. 30
    [0015]
    A method of culturing a mycobacterium according to one of claims 12 to 14, wherein a culture of a biological sample is made which can contain a mycobacterium and contaminating bacteria whose growth can inhibit the growth of mycobacteria, characterized in that: 1 / a preliminary step of initial decontamination of said sample in said culture medium with chlorhexidine is carried out for a limited period, in order to limit the action of chlorhexidine to an activity against bacteria other than mycobacteria, preferably about 15 minutes of treatment in a 1% chlorhexidine solution with stirring, and 2 / chlorhexidine is removed by washing the thus treated sample from step 1 / with a neutral buffer, and centrifuged and recover the bacterial pellet containing the contaminating bacteria thus inactivated and the non-inactivated mycobacteria, which are ocule on a said culture medium of mycobacteria.
    [0016]
    16. A method of culturing a mycobacterium according to one of claims 12 to 15, characterized in that one carries out a culture of a sample of biological sample, said biological sample being made from stool. 15
    [0017]
    17. A method of culturing a mycobacterium according to one of claims 12 to 16, characterized in that said culture medium is a solid culture medium and detects a growth of said mycobacteria to the naked eye when one can see the formation of a colony of bacteria on said solid culture medium.
    [0018]
    18. A method of culturing a mycobacterium according to claim 17, wherein a solid culture medium is used, characterized in that the steps are carried out in which: i / - a culture of a so-called mycobacterium, preferably Mycobacterklm tuberculosis complex, on a said solid culture medium in the presence of at least one given antibiotic, at different known concentrations, and ii / - the lowest concentration of antibiotic, preferably selected from the rifampicin, isoniazid, ethambutol, pyrazinamide and streptomycin, which inhibits any visible growth of said bacterium.
    [0019]
    19. A method of culturing a mycobacterium according to claim 18, characterized in that at the step -i, is deposited on said solid culture medium, at least one strip of paper impregnated with a said antibiotic to different concentrations according to a concentration gradient along said strip. 3033575 32
    [0020]
    20. A method for detecting the growth of a bacterium of the Mycrobacterium Tuberculosis complex in less than 15 days, preferably not more than 10 days, enabling the detection of 50% of mycobacteria in less than a week, characterized in that a culture method according to one of claims 12 to 19.
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    同族专利:
    公开号 | 公开日
    EP3268463B1|2018-12-19|
    FR3033575B1|2019-05-24|
    EP3268463A1|2018-01-17|
    WO2016142626A1|2016-09-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3360440A|1966-04-13|1967-12-26|Haab Walter|Cold water reconstitutable microbiological medium, process for preparation and use, ad product|
    WO2010063911A1|2008-12-05|2010-06-10|Universite De La Mediterranee |Mycobacteria culture medium and method including mycobacteria of mycobacterium tuberculosis complex|EP3732300A4|2017-12-29|2022-01-12|Akdeniz Univ|A novel medium for growth and antibiotic susceptibility test of mycobacteria|FR2907793A1|2006-10-26|2008-05-02|Univ Aix Marseille Ii|Identifying a bacterium of the Mycobacterium tuberculosis complex comprises amplifying a nucleic acid fragment from one of 16 intergenic zones in the genome of the bacterium|FR3061912A1|2017-01-13|2018-07-20|Fondation Mediterranee Infection|TRANSPORT AND / OR CULTURE MEDIA FOR MYCOBACTERIUM TUBERCULOSIS|
    KR102274809B1|2020-06-25|2021-07-08|어게인비에스|Medium composition for detecting drug resistant mycobacterium tuberculosis and kit for detecting drug resistant mycobacterium using it|
    法律状态:
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    2016-09-16| PLSC| Search report ready|Effective date: 20160916 |
    2017-02-10| PLFP| Fee payment|Year of fee payment: 3 |
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    2020-12-18| ST| Notification of lapse|Effective date: 20201110 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1552007A|FR3033575B1|2015-03-11|2015-03-11|MEDIUM AND METHOD FOR CULTURING MYCOBACTERIA COMPRISING LAMB SERUM|
    FR1552007|2015-03-11|FR1552007A| FR3033575B1|2015-03-11|2015-03-11|MEDIUM AND METHOD FOR CULTURING MYCOBACTERIA COMPRISING LAMB SERUM|
    PCT/FR2016/050539| WO2016142626A1|2015-03-11|2016-03-09|Medium and method for culturing mycobacteria comprising lamb serum|
    EP16712964.2A| EP3268463B1|2015-03-11|2016-03-09|Medium and method for culturing mycobacteria comprising lamb serum|
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