WO2000052139A1 - Culture de mycobacteries - Google Patents

Culture de mycobacteries Download PDF

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Publication number
WO2000052139A1
WO2000052139A1 PCT/GB2000/000760 GB0000760W WO0052139A1 WO 2000052139 A1 WO2000052139 A1 WO 2000052139A1 GB 0000760 W GB0000760 W GB 0000760W WO 0052139 A1 WO0052139 A1 WO 0052139A1
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Prior art keywords
culture
mycobacteria
detergent
growth medium
medium
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PCT/GB2000/000760
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English (en)
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Brian William James
Philip Marsh
James S. Chadwick
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Microbiological Research Authority
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Application filed by Microbiological Research Authority filed Critical Microbiological Research Authority
Priority to AU29255/00A priority Critical patent/AU770840B2/en
Priority to CA002364579A priority patent/CA2364579A1/fr
Priority to JP2000602751A priority patent/JP2002537798A/ja
Priority to EP00907778A priority patent/EP1159400A1/fr
Publication of WO2000052139A1 publication Critical patent/WO2000052139A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to a method of culture of mycobacteria, to a growth medium therefor, and to a method of culture of mycobacteriophage.
  • TB vaccine based on an attenuated strain of M.bovis (BCG) has been available for several years, but protection is restricted to particular ethnic groups, for reasons that are unknown. Mycobacteria have also been associated with several other conditions such as Crohn's Disease.
  • a major problem associated with the study and production of pharmaceutical products based on mycobacteria is the difficulty associated with bacterial growth.
  • Conventional methods involve growth on solid agar slopes and, consequently, manufacturing products using this type of approach is both labour intensive and costly. These processes are poorly defined leading to batch variation.
  • Tween ® -albumin medium Media for culture of tubercle bacilli is described by Dupos et al in AM.REV.TUBERC. volume 56, 1947, pp334-345.
  • a growth medium referred to as "Tween ® -albumin” medium was used, containing 0.01-0.05 percent Tween ® 80 and from 0.5-1.0 percent albumin. This growth medium has hitherto been the standard growth medium used in this field.
  • Wayne L.G. in Infection and Immunity, Sept.1977, pp528-530 used this same medium and reported a mean generation time of 17-18 hours for Mycobacterium tuberculosis.
  • Lowrie et al, in Journal of General Microbiology, Volume 110, 1979, pp431 -441 used a concentrated version of the same medium.
  • An objective of the present invention is to provide for batch or continuous culture of mycobacteria, in particular continuous culture that will maintain bacterial virulence (or avirulent bacteria possessing virulent cell surface epitopes), and provide cells of defined and consistent properties. Another objective is to provide a growth medium for culture of mycobacteria.
  • a further objective of the present invention is to provide a method of culturing mycobacteriophage which overcomes/alleviates the prior art poor yield problems.
  • a first aspect of the invention provides a method of culture of mycobacteria, comprising culturing said mycobacteria, in batch or continuous culture, with agitation and in the presence of sufficient detergent so that a substantially homogenous suspension of cells is maintained.
  • the method of the invention comprises growing said mycobacteria in batch or continuous culture, at a temperature of 35°C +/- 10°C, at a dissolved oxygen tension of at least 1.0 percent, at a pH of 6.9 +/- 0.9 and with agitation in the presence of sufficient detergent to maintain a substantially homogenous suspension of single cells.
  • Mycobacteria tuberculosis In use of the present invention, illustrated by specific embodiments described below in more detail, and using Mycobacteria tuberculosis, we have developed a method which allows high yields of bacteria from both batch and continuous culture systems. Further, we have shown that mycobacteria generated using the methods of embodiments of the present inventions are highly virulent as demonstrated in a standard guinea pig infection model of M. tuberculosis. Indeed, potency of Mycobacteria tuberculosis grown using these methods is comparable with M. tuberculosis grown using the solid agar slope method.
  • growth of M. tuberculosis in steady-state continuous culture achieved a biomass yield of 1.2gl "1 cell dry weight.
  • the method of the invention enables growth at increased cell densities and with reduced mean generation times, or doubling times. It is further of advantage that using the method of the present invention expression of virulence determinants has been maintained. Thus, the method is of application for production of mycobacteria such as for incorporation into BCG vaccines.
  • the term "batch culture” is used in its conventional sense to refer to a fixed volume of culture medium which is inoculated with a microorganism. After a period of adjustment, termed the lag phase, the organism starts to grow and multiply reaching the maximum growth rate possible in that environment - termed exponential growth. After multiple generations essential nutrients become depleted or toxic metabolites build-up causing growth to slow and eventually cease. This is a closed system and the environment is constantly changing as the organism grows.
  • This type of culture is typically per ormed in shake flasks, 50-500 ml, where only temperature is controlled though in embodiments of the invention temperature, pH and oxygen have been controlled.
  • a further benefit of methods of the invention is that through control of environmental parameters there is reduced batch-to-batch variation, leading to cultures of more consistent composition and less bacterial heterogeneity, which is a significant consideration during production of vaccine components from these cultures.
  • Fermenter culture is similarly used with reference to a type of batch culture operated with more control over the environmental parameters such as pH and aeration. Fermenters are normally used for production, hence the culture volume is larger.
  • continuous culture is used to refer typically to a culture of constant volume to which medium is added continuously and from which there is continuous removal of any overflow culture. By adding growth components in the fresh medium, the organism continues to multiply. When this system reaches equilibrium, cell number and nutrient status remain constant, it is said to be in steady state.
  • the term "chemostat culture” refers to the current most common type of continuous culture device. Two elements are generally used to control the culture, the concentration of an essential nutrient, such as carbon source, and the flow rate. After inoculation the culture grows until an essential nutrient becomes depleted and limits growth, however, the continuous addition of fresh medium containing the limiting nutrient permits continued growth. The cell density is controlled by the concentration of limiting nutrient added. The limiting nutrient can be altered by manipulating the medium formulation. The rate of medium addition controls the growth rate (generation time) of the culture.
  • the culture temperature is maintained at 35°C +/- 10°C, more preferably 35°C +/- 5°C, and in specific embodiments of the invention this preferred temperature has been maintained for in excess of three weeks with continuous mycobacteria growth.
  • the pH of the culture medium, in continuous operation is typically controlled to within +/- 0.9 of pH 6.9, more preferably +/- 0.5 of pH 6.9. pH may be controlled using addition of acid or alkaline solution to the culture medium, according to the pH correction required. In specific embodiments of the invention described below, sodium hydroxide at a concentration of 0.5M and sulphuric acid at a concentration of 0.5M is used.
  • the dissolved oxygen concentration of the culture is typically at an initial level of at least 40% (v/v) air saturation, preferably at least 50%.
  • Detergent is present in the method of the invention as a dispersing agent to maintain a high proportion of the mycobacteria suspended in a substantially homogenous suspension, preferably as single cells or small clumps containing 2 to 10 bacilli, preferably 2 to 5 bacilli. In one embodiment, at least 50%, preferably at least 75%, particularly preferably at least 90% of the total mycobacterial cell weight is suspended as above.
  • cells can grow in an environment enabling higher growth rates under relatively constant and controlled conditions. It is possible, though the applicant does not wish to be bound by any theory, that once mycobacteria form pellicles as in previous culture methods they can not thereafter be dispersed - the invention may thus improve the previous methods by preventing or reducing loss of bacilli into such pellicles.
  • Some detergents in use slowly release toxic components into the culture medium, so the amount of detergent present should not be so high as to risk the detergent or any of its components reaching toxic levels. Similarly, excess detergent can lead to foaming of the culture and should be avoided.
  • the level of detergent may suitably be at least 0.1 % (v/v).
  • Anionic detergents are preferred, in particular esters of sorbitan and derivatives thereof, though it is believed that the advantageous effects of the invention and the results obtained in the specific embodiments may likewise be realised using any of a wide range of detergents. Particularly good results have been obtained using a polyethane-diyl derivative of a sorbitan ester, namely Tween ® 80, other such esters being Tween ® 20, Tween ® 40 and Tween ® 60. Despite the presence of detergent it has been found that albumin may be omitted from the growth medium without slowing mycobacterial growth.
  • detergent may be present at from 0.1 to 1.0 % (v/v), more preferably from 0.1 to 0.5 %, most preferably about 0.2 % (v/v).
  • detergent may be present at least 0.1 % (v/v), more preferably at least 0.15 % (v/v), and most preferably about 0.2%, its level further preferably being no more than 1.0%, and usually no more than 0.7%.
  • the rate of introduction expressed as a dilution rate.
  • the culture of the invention can be carried out continuously with a dilution rate of at least 0.02 h ' ⁇ resulting in a high yield of bacteria, and these bacteria have been found to have preserved their virulence.
  • a dilution rate of at least 0.025 h "1 can also be sustained, and in a specific embodiment a dilution rate of about 0.03 h '1 was achieved in continuous culture, representing a mean doubling time of about 24 hours.
  • the invention further provides, in a second aspect, a growth medium for culture of mycobacteria, comprising :- a carbon source; a mitogen; trace elements comprising at least Mg, K, P and S; a nitrogen source.
  • the carbon source is preferably selected from glucose, glycerol and an amino acid, and combinations of these carbon sources.
  • the mitogen is present to induce cell division and is preferably asparagine, though other mitogens from inorganic sources are also suitable.
  • Trace elements in the growth medium are preferably selected from Ca, Mg, Zn, Co, Cu, Mn, Fe, K and mixtures thereof, and the nitrogen source is selected from an amino acid and an ammonium salt.
  • the growth medium optionally further comprises an amino acid component selected from alanine, arginine, asparagine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, phenylalanine, serine, and mixtures thereof.
  • the amino acid component can contribute the nitrogen source in the medium.
  • vitamin/co-factor component selected from:- inositol, thiamine, calcium pantothenate, co-enzyme A, nicotinamide, biotin, DL- thioctic acid, and mixtures thereof, preferably biotin; and one or more components selected from sodium hydroxide, glutathione, glycerol, haemin, sodium pyruvate and ⁇ -ketoglutarate, preferably glycerol and/or pyruvate.
  • a particularly preferred embodiment of the invention provides a method of culture of mycobacteria, comprising culturing said mycobacteria, in batch or continuous culture, with agitation in the presence of sufficient detergent so that a substantially homogenous suspension of single cells is maintained, and in the presence of a growth medium according to combination of the above-described media.
  • the mycobacterial culture methods and media of the present invention are suitable for culture of all members of the Mycobacteria tuberculosis complex (MTC) as well as mutant and recombinant forms thereof.
  • the methods and media are used for culture of M. tuberculosis, but are also suitable for culture of M. bovis and other opportunistic mycobacteria.
  • a method of culture of mycobacteriophage comprising culture of mycobacteria as described above, and contacting said mycobacteria with a mycobacteriophage.
  • the phage may be added directly to the mycobacterial liquid culture.
  • Reference to mycobacteriophage includes mutant and recombinant forms thereof.
  • a mycobacteriophage is any phage which is capable of infecting and replicating in a mycobacterium.
  • the mycobacteriophage need not be specific for the mycobacterium which it infects. However, it may be preferred that the phage exhibits specificity for a given mycobacterial species or even sub-species.
  • the phage culture method is employed to culture a phage capable of infecting M. tuberculosis, M. bovis and/or M. paratuberculosis.
  • the phage culture method is particularly suited to the culture of phage capable of infecting, and which are preferably specific for, M. tuberculosis.
  • the phage to be cultured is selected from the group consisting of D-34 (Accession No. ATCC 4243-B1 ), LG (Accession No. ATCC 25618-B1 ), DS6A (Accession No. ATCC 25618-B2), and D29 (Froman et al. 1954).
  • the preferred mycobacteriophages for use in this aspect of the invention are phages which are capable of causing a lytic infection. This facilitates down ⁇ stream phage harvesting.
  • native or genetically engineered or chemically modified bacteriophage which require susceptible mycobacteria for growth can be generated effectively.
  • mycobacteriophage or component parts or phage nucleic acid having prophylactic or therapeutic use and which may also be used as gene delivery systems in whole or in part, may be grown and manufactured in quantities suitable for clinical and commercial application.
  • the medium may be modified by the incorporation of an agent which is capable of promoting and/or assisting phage adsorption on the mycobacteria cell surface.
  • Preferred agents include bovine serum albumin and other molecules having cell surface adsorption-promoting properties such as cations. In use, the latter are typically employed at a concentration of approximately 0.015 M.
  • an agent is incorporated at a final concentration of between 0.01 and 1 % w/v, preferably between 0.05% and 0.5% w/v.
  • a typical final concentration is approximately 0.1 % w/v.
  • the agent is preferably added to the mycobacteria culture medium prior to or substantially at the same time as inoculation of the mycobacteriophage.
  • the mycobacteria culture medium of the present invention includes a detergent
  • the use of an agent may help alleviate potential detergent-related problems.
  • the detergent concentration of the medium may be increased, for example to substantially the same concentration as prior to phage inoculation, thereby providing optimal mycobacterial growth conditions once again.
  • the infectious phage seed culture ie. inoculum
  • seed bank stocks stored at high tighter suspension (eg. 10 9 -10 10 pfu/ml '1 ) in, for example, phosphate buffer saline solution at -20°C.
  • Infectious phage seed culture may take the form of purified, semi-purified phage, or a mixture of phage and phage-infected mycobacteria which has been generated in, for example, shake flasks or smaller culture vessels.
  • the time of phage seed inoculation may vary according to the mycobacterium, medium, phage, and the multiplicity of infectious dose (MOI) employed.
  • MOI multiplicity of infectious dose
  • the MOI is in the range of one phage to 10 mycobacteria, but may be varied in accordance with the system employed.
  • Phage inoculation may occur at any point during the mycobacterial growth cycle. Preferably, inoculation occurs approximately 25-35 hours following initiation of bacterial logarithmic growth. Typically, inoculation occurs 30 hours following initiation of bacterial logarithmic growth.
  • Culture conditions are preferably monitored and maintained for a further 24 hour period to allow the desired number of bacteriophage replication cycles to occur.
  • Purification of phage may be achieved using conventional chromatography methods, such as immunoaffinity purification for phage which has been engineered to express an appropriate ligand, or centrifugation of phage from filtered culture supernatant followed by resuspension in an appropriate buffer.
  • infected cells which have yet to undergo phage-induced lysis may be harvested through conventional filtration methods and lysed mechanically or chemically or by ultrasound to release contained phage which may then be further purified as described above.
  • FIG. 1 shows a schematic view of continuous culture apparatus according to the invention
  • Fig. 2 shows a graph of optical density at 540 before and after initiation of continuous culture
  • Fig. 3 shows viable M. tuberculosis in guinea pig lungs following aerosol challenge with bacteria grown using the medium of the invention
  • Fig. 4 shows viable M. tuberculosis in guinea pig spleens following aerosol challenge with bacteria grown using the medium of the invention.
  • a medium reservoir 1 is attached via medium addition pump and line 2 to culture vessel 6.
  • the glass culture vessel 6 comprises a titanium top plate through which are connected temperature probe 7, oxygen electrode 8, air inlet and sparger 9, vent 10, pH electrode 11 , alkali addition line 12 and acid addition line 13. Samples of the content of the culture vessel may be taken through sample port 14 and effluent from the culture vessel drains into or is pumped into effluent reservoir 15.
  • the remaining features in Fig. 1 are: a magnetic stirrer unit 3; a heating pad 4; and a magnetic bar 5.
  • This continuous culture apparatus is used for continuous culture of mycobacteria as described in examples below.
  • Fig. 2 illustrates the continuous culture of M. tuberculosis. After inoculation, the culture was operated in batch for 4 days. Medium addition was then initiated in fed-batch mode. Continuous medium addition was started at 300 h.
  • Fig. 3 illustrates the viable M. tuberculosis in guinea pig lungs following aerosol challenge (error bars + standard deviation are shown) and compares the influence of culture mode on the virulence of M. tuberculosis.
  • the virulence of chemostat grown cells was compared with cells grown to mid-exponential batch phase in ABCD ModTB medium and on Middlebrook agar.
  • Guinea pig challenge with plate-grown cells produced a classical disease process with exponential multiplication in guinea pig lungs up to three weeks post-infection, when lung counts reached 10 6 to 10 7 c.f.u. per lung. After 3 weeks the lung counts declined marginally.
  • Low numbers of bacilli were detected in spleen tissues 2 weeks post- infection followed by an exponential increase up to day 21. Infection with both batch and chemostat grown cells produced a comparable disease process demonstrating that culture virulence was retained.
  • Fig. 4 illustrates viable M. tuberculosis in guinea pig spleens following aerosol challenge as in Fig. 3 (error bars + standard deviation are shown).
  • M. tuberculosis strain H37Rv (NCTC cat. no. 7416) - a representative strain of M. tuberculosis.
  • Stock cultures were grown on Middlebrook 7H10 + OADC for 3 weeks at 37 ⁇ 2°C harvested and stored at - 70°C as a dense suspension in deionised water.
  • CAMR Mycobacterial Medium A chemically defined culture medium was developed, and was designated CAMR Mycobacterial Medium (see Appendix 1 below).
  • the medium was prepared with high quality water from a Millepore water purification system and filter sterilised by passage through a 0.07 ⁇ m pore size cellulose acetate membrane filter capsule (Sartorius Ltd).
  • Middlebrook 7H10 + OADC agar was used to prepare inoculum cultures, enumerate the number of culturable bacteria in chemostat samples, and to assess culture purity.
  • Culture experiments were performed in a one litre glass vessel operated at a working volume of 500 ml.
  • the culture was agitated by a magnetic bar placed in the culture vessel coupled to a magnetic stirrer positioned beneath the vessel.
  • Culture conditions were continuously monitored and controlled by an Anglicon Microlab Fermentation System (Brighton Systems, Newhaven), linked to sensor probes inserted into the culture through sealed ports in the top plate.
  • the oxygen concentration was monitored with a galvanic oxygen electrode (Uniprobe, Cambridge) and was controlled through feedback control of the agitation rate.
  • Temperature was monitored by an Anglicon temperature probe, and maintained by a heating pad positioned beneath the culture vessel.
  • Culture pH was measured using an Ingold pH electrode (Mettler-Toledo, Leicester) and controlled by automatic addition of either sodium hydroxide (0.5 M) or sulphuric acid (0.5 M).
  • the culture system was operated by controlling nutrient addition from the medium reservoir and a constant culture volume was maintained by an overflow tube fitted to the side of the vessel.
  • the vessel was filled with 350 ml of sterile culture medium and parameters were allowed to stabilise at 37°C ⁇ 2°C, pH 6.9 ⁇ 0.2 and a dissolved oxygen tension of approximately 70% air saturation.
  • a dense inoculum suspension was prepared by resuspending Middlebrook agar cultures, grown at 37 ⁇ 2°C for 3 week, in sterile deionised water.
  • the inoculum was aseptically transferred to the culture vessel, to provide an initial culture turbidity of approximately 0.25 at 540 nm. After inoculation the culture was allowed to establish for approximately 50 h. As the culture entered exponential growth, a further 100 ml medium was added and batch growth was monitored by optical density and viable count determination.
  • the culture was inoculated and allowed to establish for approximately 50 h as detailed.
  • the culture was then operated in fed batch mode for 48 h with medium addition (approx. 100 ml) as the culture entered exponential growth and 24 h later.
  • Continuous culture was then initiated at a dilution rate of 0.03 h "1 [equivalent to a mean generation time (MGT) of 24 h].
  • Culture parameters were maintained at a dissolved oxygen tension of 50 % (v/v) air saturation at 37 ⁇ 2°C and pH 6.9 ⁇ 0.2. Growth was monitored by optical density, dry weight and viable count determination.
  • Culture analyses The optical density of culture samples was recorded at 540 nm (OD 540 ) in a UV- 260 spectrophotometer (Pye Unicam) against a water reference. Culture biomass was determined by dry weight analysis. Samples were treated with 4% (v/v) formaldehyde for at least 24 h and filtered through a pre-dried, pre-weighed, 0.45 ⁇ m pore sized, nylon membrane filter (Gelman Sciences), under vacuum. The membrane was rinsed with 10 ml of deionised water, before re-drying to a constant weight, and re-weighing.
  • Total viable counts were performed by preparing a 10-fold dilution series of the sample in sterile deionised water, and plating 100 ⁇ l aliquots of appropriate dilutions onto Middlebrook 7H10 plates in triplicate. The plates were incubated at 37°C for 3 weeks before enumerating the number of colonies formed. Culture purity was checked by plating neat samples onto Middlebrook 7H10 and Blood agar and incubating at 37°C.
  • M. tuberculosis strain H37Rv was established in CAMR Mycobacteria Medium supplemented with 0.2% Tween ® 80. After inoculation the culture followed typical batch growth kinetics with a lag phase of approximately 50 hours before entering exponential growth. A minimum doubling time of approximately 14 h was recorded. Cultures were predominantly single cell suspensions.
  • the virulence of batch and chemostat grown ceils was compared with cells grown on Middlebrook agar.
  • Guinea pigs challenged with plate-grown cells produced a classical disease process with exponential multiplication in guinea pig lungs up to three weeks post infection, when lung counts reached 10 6 to 10 7 c.f.u. per lung (fig. 3).
  • Low numbers of bacilli were detected in spleen tissues 2 weeks post- infection followed by an exponential increase up to day 21 , after which growth rate declined (fig. 4).
  • Infection with both batch and chemostat grown cells produced a comparable disease process demonstrating that culture virulence was retained.
  • the invention thus provides methods for batch and continuous culture of dispersed mycobacteria in high yield and without loss of virulence, and also provides a growth medium therefor.
  • large-scale and consistent production of vaccine components is enabled, for manufacture eg. of bacterial subunits, whole bacilli for vaccine uses and whole bacilli for immune therapies.
  • Mycobacteriophage e.g. D-34 (Accession No. ATCC 4243-B1 ) is specific for M. tuberculosis.
  • Stock bacteriophage is prepared from liquid culture or soft agar overlay and is stored as a high titre suspension 10 9 - 10 10 pfu ml "1 in PBS at - 20°C.
  • M. tuberculosis strain H37Rv is performed in the controlled culture system using the CAMR Mycobacterium Medium as detailed previously in Example 1.
  • the CAMR Mycobacterium Medium is modified by the incorporation of 0.1 % (w/v) bovine serum albumin (BSA). After inoculation, the culture is allowed to establish and turbidity at 540 nm is monitored to determine the onset of exponential growth. Stock bacteriophage suspension is slowly thawed and added to the culture 30 h after the initiation of logarithmic growth.
  • BSA bovine serum albumin
  • a multiplicity of infection of 1 phage to 10 bacilli is used. Culture conditions are continuously monitored and maintained for a further 24 h or until two cycles of phage propagation have occurred. Bacteriophage replication can be followed by monitoring the change in culture turbidity and oxygen utilisation.
  • the culture is pelleted by centrifugation at 10,000 g for 15 min and the supernatant containing the bacteriophage is retained.
  • the bacteriophage is concentrated by ultrafiltration, washed with phosphate buffered saline and filter sterilized by passage through a 0.45 ⁇ m cellulose acetate membrane filter.
  • the titre of the concentrated bacteriophage suspension is determined against M. tuberculosis using the conventional soft overlay method.
  • Solution 4 FeSO 4 • 7H 2 O 1.0 DL-thioctic acid 1.0 Cone. HCI 0.5 ml ethanol 950 ml
  • ACES buffer N-[Carbamoylmethyl]-2-aminoethanesulfonic acid Stock solution formulations
  • ACES buffer 10.0 g KH 2 PO 4 0.22 g Millipore water 500 ml Solution 1 10 ml Solution 2 10 ml Solution 3 100 ml Solution 4 10 ml Solution 6 10 ml NaHCO3 0.042 g Glycerol 2 ml Solution 5 10 ml

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Abstract

La présente invention concerne une méthode de culture de mycobactéries consistant à mettre en culture les mycobactéries, dans une culture soit par lots soit en continu, avec une agitation et en la présence de suffisamment de détergent pour maintenir une suspension sensiblement homogène des cellules mycobactériennes. Selon une méthode préférée, les mycobactéries sont mises en culture dans une culture continue, à une température de 35 °C +/- 10 °C, à une tension en oxygène dissous d'au moins 1 % à un pH de 6,9 +/- 0,9, à un taux de dilution d'au moins 0,02 h-1 et avec une agitation en la présence de suffisamment de détergent pour maintenir une suspension sensiblement homogène de cellules individuelles. La présente invention concerne également un milieu de croissance pour la culture des mycobactéries, contenant une source de carbone, un mitogène, des éléments à l'état de trace comprenant au moins Mg, K, P et S, ainsi qu'une source d'azote. Un autre aspect de l'invention a trait à une méthode de culture d'un mycobactériophage, comprenant une culture de mycobactéries telle que décrite ci-dessus et consistant à mettre lesdites mycobactéries en contact avec un mycobactériophage.
PCT/GB2000/000760 1999-03-02 2000-03-02 Culture de mycobacteries WO2000052139A1 (fr)

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AU29255/00A AU770840B2 (en) 1999-03-02 2000-03-02 Culture of mycobacteria
CA002364579A CA2364579A1 (fr) 1999-03-02 2000-03-02 Culture de mycobacteries
JP2000602751A JP2002537798A (ja) 1999-03-02 2000-03-02 マイコバクテリアの培養
EP00907778A EP1159400A1 (fr) 1999-03-02 2000-03-02 Culture de mycobacteries

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WO2011002860A1 (fr) * 2009-07-01 2011-01-06 Biomerieux, Inc. Procédé et milieu de culture pour une détection améliorée de mycobactéries
US8017753B2 (en) 2001-06-22 2011-09-13 Health Protection Agency Mycobacterial antigens expressed under low oxygen tension
US8389268B2 (en) 2009-07-01 2013-03-05 BIOMéRIEUX, INC. Method and culture medium for enhanced detection of mycobacterium
US8932851B2 (en) 2009-07-01 2015-01-13 bioMērieux, Inc. Method and culture medium for enhanced detection of Mycobacterium
WO2019041015A1 (fr) * 2017-09-01 2019-03-07 União Brasiliense De Educação E Cultura - Ubec Milieu de culture mmp pour la production de lipopeptides antimicrobiens à partir de la culture de bactéries, procédé de production de lipopeptides antimicrobiens et utilisation de celui-ci

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8404826B2 (en) 2001-06-22 2013-03-26 Health Protection Agency Mycobacterial antigens expressed under low oxygen tension
US8017753B2 (en) 2001-06-22 2011-09-13 Health Protection Agency Mycobacterial antigens expressed under low oxygen tension
WO2003004520A3 (fr) * 2001-07-04 2003-10-16 Health Prot Agency Antigenes mycobacteriens exprimes pendant la latence
US7393540B2 (en) 2001-07-04 2008-07-01 Health Protection Agency Mycobacterial antigens expressed during latency
EP2196473A1 (fr) 2001-07-04 2010-06-16 Health Protection Agency Antigènes mycobacteriens exprimes pendant la phase de latence
US8003776B2 (en) 2001-07-04 2011-08-23 Health Protection Agency Mycobacterial antigens expressed during latency
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WO2011002860A1 (fr) * 2009-07-01 2011-01-06 Biomerieux, Inc. Procédé et milieu de culture pour une détection améliorée de mycobactéries
US8617871B2 (en) 2009-07-01 2013-12-31 Biomerieux, Inc. Method and culture medium for enhanced detection of Mycobacterium
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CN102471756B (zh) * 2009-07-01 2016-10-12 生物梅里埃有限公司 用于分枝杆菌的增强的检测的方法和培养基
CN107043802A (zh) * 2009-07-01 2017-08-15 生物梅里埃有限公司 用于分枝杆菌的增强的检测的方法和培养基
CN107043802B (zh) * 2009-07-01 2020-11-17 生物梅里埃有限公司 用于分枝杆菌的增强的检测的方法和培养基
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CA2364579A1 (fr) 2000-09-08
AU2925500A (en) 2000-09-21
JP2002537798A (ja) 2002-11-12

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