The present invention relates to methods for culturing and isolating
mycobacteria. More specifically, it relates to the microbiology methods and the culture media
5 that can be used for the detection, identification, isolation and/or analytical study of
mycobacteria, for example those present in biological specimens and samples.
Mycobacteria, which belong to the order Actinomycetales, are thin bacilli
which are straight or sometimes slightly curved in shape. Because they have a lipid-rich wall
I 0 which limits the penetration of dyes, the staining of mycobacteria by conventional techniques
and dyes is difficult. However, once stained, the staining of said mycobacteria withstands
destaining both with acid and with alcohol. For this reason, they are described as "acidalcohol-
resistant" bacteria
According to one elementary classification, the various species of mycobacteria
15 are listed according to two m:Uor categories.
20
The first category groups together the strict pathogenic mycobacteria, which
parasitize both animals and human beings. These mycobacteria are responsible for
tuberculosis (Mycobacterium tuberculosis, };fycobacterium bovis, Mycobacterium africanum,
Mycobacterium microti and Mycobacterium canetti) or for leprosy (Mycobacterium leprae).
The second category groups together the saprophytic or commensal
mycobacteria, which are usually non-pathogenic. The term then used is "atypical"
mycobacteria or "riontuberculous" mycobacteria (NTM). The latter, which are ubiquitous in
our environment, are found to be disseminated just about everywhere in soil, plants, dust,
water and distribution networks, our food, etc. Generally harmless to human beings, some
25 nontuberculous mycobacteria can, in immunodepressed subjects, cause opportunistic
infections which mainly affect the lungs, the skin and the lymphatic system.
Thus, from a clinical point of view but also fi·om a sanitary point of view,
whether they are pathogenic or nontuberculous, the detection and/or identification of
mycobacteria is of considerable importance. Early and specific detection of these bacteria
30 makes it possible to propose a suitable solution, in terms of therapeutic treatment, of sanitary
decontamination, etc. Various techniques exist in this regard, for example gene amplification
2
(by PCR) of specific nucleic sequences, immunodetection of antigens, mass spectrometry,
microbiological detection of enzymatic activities, etc.
Whatever they are, these detection/identification methods generally require the
culturing of the mycobacteria possibly present in the samples/specimens to be analyzed, in
5 order to obtain an amount suitable for their detection. Whatever they are, these
detection/identification methods suffer from the same handicap: the slowness of growth and
development of the mycobacteria.
Indeed, compared with other bacteria, with yeasts and with fungi, both
pathogenic and saprophytic mycobacteria are characterized by relatively slow growth and
I 0 development. Moreover, in biological samples in which the presence of mycobacteria is
investigated, the mycobacteria are rarely isolated; they are usually contaminated with an
accompanying flora, the growth and development of which are much faster. As the cell
division cycles progress, the proportion of mycobacteria in the sample decreases, and the
difficulty in being able to visualize and identify them is accentuated thereby.
15
In order to overcome this slowness of growth and development and/or this
contamination by other microorganisms, many enrichment culture media and broths which are
more or less selective for mycobacteria or for a particular species of mycobacteria have been
developed.
20 Among those most commonly used, the Uiwenstein-Jensen medium is a
coagulated (non-agar) culture medium comprising a nutritive component based on whole egg,
on potato starch, on asparagine, on glycerol and on minerals. This medium also contains
malachite green, for the purpose of inhibiting the accompanying flora (more particularly, the
Gram-negative bacteria). An addition of penicillin and/or of nalidixic acid has also been
25 proposed in order to increase the selectivity thereof with regard to mycobacteria.
The Coletsos agar medium, of similar composition, additionally contains
pyruvate and glutamate, sunflower blue and gelatin.
The Middlebrook 7H9, 7HIO and 7HII media, which have emerged more
recently, are semi-synthetic media which have a nutritive component based on amino acids,
30 fatty acids, pyruvate and mineral salts. These media also contain catalase and bovine albumin
(fraction V), which give the mycobacteria protection against various harmful agents (in
3
pmticular toxic peroxides) possibly present in the biological samples and spectmens. The
Middlebrook 7Hl0 medium differs from the Middlebrook 7H9 medium by the presence of
malachite green. The Middlebrook 7H11 medium differs from the Middlebrook 7HIO medium
in that it is enriched with a pancreatic casein peptone. Many variants to these three
5 Middlebrook media have also been envisioned in order to increase the selectivity thereof with
regard to mycobacteria, for example by adding the following selective agents thereto:
polymyxin, amphotericin B, nalidixic acid, trimethoprim, azlocillin and vancomycin.
Lastly, the BCSA (for "Burkholderia cepacia selective agar") medium, initially
developed for culturing and detecting Burkholderia cepacia, has been described as being
10 particularly suitable for the development and growth of mycobacteria, more particularly
"rapidly growing" mycobacteria (Esther et al. - Journal of Clinical Microbiology; 2011,
1421-1425). This is an agar culture medium formulated from a nutritive component
comprising casein peptone, sugars (lactose and sucrose), a yeast extract and selective agents
such as gentamicin, vancomycin, crystal violet and polymyxin B.
15
The present invention aims to improve the methods and techniques used for
detecting, identifYing and/or isolating the mycobacteria present in a biological sample. To do
this, the present invention is aimed at improving the enrichment broths and culture media used
in these methods and techniques, both in terms of their fertility and in terms of their selectivity
20 with regard to mycobacteria. Likewise, it aims to provide novel compositions of enrichment
broth and/or culture medium, capable of allowing the culture and multiplication of
mycobacteria, and having improved performance levels in terms of fertility and/or of
selectivity with regard to mycobacteria.
25 Before presenting the invention, the following definitions are given in order to
allow better understanding of the invention.
The term "culture medium" is intended to mean a medium comprising all the
elements required for the expression of a metabolism and/or for the growth of
microorganisms. The culture medium may be solid, semisolid or liquid. The term "solid
30 medium" is intended to mean for example a gelled medium or a coagulated medium. Agar is
4
the conventional gelling agent in microbiology for culturing microorganisms, but it is possible
to use gelatin, agarose or other natural or artificial gelling agents.
The term "enrichment broth" denotes more specifically a liquid culture
medium.
5 A culture medium is described as a minimum medium when its composition
10
15
20
comprises only the chemical elements strictly required for the growth and multiplication of the
microorganisms to be cultured. The following are conventionally found therein:
water (generally distilled or deionized water);
a nutritive component general! y comprising:
- a carbon-based source of energy (generally glucose);
- a calcium source (for example CaCh);
- a nitrogen source (for example (Nl4)2S04);
- a sulfur source (for example (Nl4)2S04);
- a magnesium source (for example MgClz);
- an iron source (for example iron citrate);
- a source of trace elements (for example salts ofCu, Zn, Co, Ni, B, Ti);
optionally a pH buffer for maintaining the medium at a suitable pH; and
for the solid or semi-solid media, optionally a gelling agent (for example agar, gelatin or
agarose).
The addition of particular growth factors and/or of various nutrients makes it
possible to reinforce the attributes of the nutritive component and to increase the fertilizing
capacity of the medium. The term "enriched" medium is then used. The addition of these
growth factors and/or of these nutrients can be carried out by means of chemically defined
compounds or compositions, or else by means of complex compositions (for example, fresh
25 blood, sera, yeast extract, whole egg, egg yolk, peptones, etc.).
A culture medium is said to be selective when it comprises at least one selective
agent which allows said medium to promote the growth of a target microorganism or of a
target group of microorganisms, rather than that of the accompanying flora. These selective
agents are essentially compounds with antibiotic and/or antifungal effects, which have a
30 specificity of toxicity (lower toxicity for the target microorganisms than for the accompanying
flora).
5
The term "biological sample" is intended to mean a clinical sample from a
specimen of human or animal origin, or a food sample from any type of food. This biological
sample may be liquid or solid and mention may be made, in a nonlimiting manner, of a
clinical sample of blood, plasma, urine or feces, or of nose, throat, skin, wound, cerebrospinal
5 fluid, bronchoalveolar fluid or expectoration specimens, a food sample from water, from
drinks such as milk or fruit juice, from yoghurt, from meat, from eggs, from vegetables, from
mayonnaise, from cheese, from fish, etc., a food sample derived from an animal feed, .such as
in particular a sample derived from animal meals. For the purposes of simplifying the
vocabulary, use will be made without distinction of the expressions "biological sample" and
10 "biological specimen".
The present invention thus relates to a process (a method) for the enrichment
and selective culture of mycobacteria contained in a biological sample, wherein all or part of
said sample is inoculated in/on a culture medium comprising a nutritive component suitable
15 for the development and growth of mycobacteria, characterized in that said culture medium
also comprises, as selective agents, at least 9-chloro-9-(4'-diethylamino)phenyl-9,10-dihydro-
1 0-phenylacridine hydrochloride and an agent capable of inhibiting Pseudomonas aeruginosa
bacteria.
The present invention is based on the general principle of incorporating
20 9-chloro-9-(4' -diethylamino )phenyl-9, 1 0-dihydro-10-phenylacridine into compositions of
culture media envisioned for culturing mycobacteria, in order to increase the selectivity of said
media with regard to mycobacteria. This selective agent is also known under the generic name
C-390 (CAS 77769-31-4). Up until now essentially used for the purposes of the selective
culture of Pseudomonas aeruginosa (US 4 263 398 and Campbell eta/. -Journal of Clinical
25 Microbiology; 1988, 1910-1912) or the isolation of genomovars of the Burkholderia cepacia
complex (Vermis eta/., - Systematic and Applied Microbiology; 2003 (26) 595-600), the
inventors have demonstrated that the use of such a compound in the culture media can also
result in a good isolation of the mycobacteria and a good elimination of the accompanying
flora, in particular of the microorganisms belonging to the genera Pseudomonas,
30 Enterobacteriaceae, Streptococcus, Burkholderia, Stenotrophomonas, Staphylococcus,
6
Enterococcus, Achromobacter, Acinetobacter, Pandoraea, Bacillus, Elizabethkingia miricola,
Haemophilus, Moraxella, Neisseria, Aspergillus, Scedosporium, Candida and Geosmithia.
The inventors have also noted that C-390 also confers on the culture media a
slight additional bluish tint which can notably improve the visualization and visual detection
5 of the colonies growing on these media.
Advantageously and according to the invention, the C-390 concentration of the
culture medium used to carry out the process for enrichment and selective culture according to
the invention is between 2 mg/1 and 1000 mg/1, preferably between about 4 mg/1 and about
10 256 mg/1, and even more preferentially between about 8 mg/1 and about 128 mg/1.
By way of examples of agents capable of inhibiting Pseudomonas aeruginosa
bacteria, mention may in particular be made of colistin methanesulfonate, fosfomycin,
nalidixic acid, aztreonam, cefsulodine and mangrolide A.
Advantageously and according to the invention, use may also be made, in said
15 culture medium, of an agent capable of inhibiting Burkholderia cepacia bacteria,
preferentially an agent chosen from fosfomycin, aztreonam and mangrolide A.
20
25
30
According to one preferred embodiment of the invention, said culture medium
also comprises at least one additional selective agent chosen from:
amphotericin B, in particular at a concentration preferentially between I mg/1 and
200 mg/1, and even more preferentially between 2 mg/1 and 50 mg/1;
nalidixic acid, in particular at a concentration preferentially between 5 mg/1 and
200 mg/1, and even more preferentially between 15 mg/1 and I 00 mg/1;
vancomycin, in particular at a concentration preferentially between I mg/1 and 50 mg/1,
and even more preferentially between 2 mg/1 and 20 mg/1;
colistin methanesulfonate, in particular at a concentration preferentially between
10 mg/1 and 200 mg/1, and even more preferentially between 15 mg/1 and 150 mg/1;
fosfomycin, in particular at a concentration preferentially between I 00 mg/1 and
3000 mg/1, and even more preferentially between 200 mg/1 and 2000 mg/1; and
malachite green, in particular at a concentration preferentially between 0.05 mg/1 and
2 mg/1, and even more preferentially between 0.2 mg/1 and I mg/1.
5
7
According to one preferred mode of the invention, said culture medium
comprises, as additional selective agents:
amphotericin B, at a concentration of between 2 mg/1 and 50 mg/1,
colistin methanesulfonate, at a concentration of between 15 mg/1 and 150 mg/1,
fosfomycin, at a concentration of between 200 mg/1 and 2000 mg/1.
According to one preferred mode of the invention, in addition to the
fosfomycin, said culture medium advantageously comprises glucose-6-phosphate, in particular
10 at a concentration preferentially between 10 mg/1 and 50 mg/1.
According to one particular aspect, the present invention is aimed at improving
the methods for culturing and/or isolating mycobacteria which are currently in use, by
intervening with regard to the composition of the usual culture media (such as Middlebrook
15 media, the Ulwenstein-Jensen medium, Columbia medium, BCSA medium) so as to increase
the selectivity thereof by inhibiting the non-mycobacteria. To this effect, the present invention
proposes to perfect the composition of these culture media by introducing C-390, and
optionally other selective agents as previously stated. Likewise, the present invention proposes
to improve the fertility thereof through the provision of various nutrients such as: glycerol, a
20 supplement based on oleic acid, on albumin, dextrose and on catalase (or OADC supplement),
active carbon, fresh blood, a yeast extract, alpha-ketoglutarate, casein, pyruvate, peptones,
amaranth, egg yolk, RNA, polyoxyethylene stearate, or tyloxapol.
In this context, the process for the enrichment and selective culture of
mycobacteria according to the invention is advantageously can·ied out with a culture medium
25 of which the nutritive component reproduces that of a culture medium chosen from:
30
a Middlebrook medium (in particular one of the Middlebrook media 7H9, 7H1 0 and
7Hl1) optionally supplemented with glycerol and/or an OADC supplement;
a Uiwenstein-Jensen medium;
a Columbia medium, optionally supplemented with an OADC supplement and/or fresh
blood, in particular horse blood; and
a BCSA medium.
5
10
8
According to one even more preferred mode, the process for the enrichment and
selective culture of mycobacteria according to the invention is carried out with a culture
medium comprising a nutritive component reproducing that of a Middlebrook medium,
supplemented with:
glycerol, in particular at a concentration preferentially between 2 ml/1 and 20 mill,
an OADC supplement, in particular at a concentration preferentially between 50 ml/1
and 200 ml/1, and optionally
one or more nutrients chosen from: fresh blood, a yeast extract, alpha-ketoglutarate,
casein, pyruvate, peptones, amaranth, egg yolk, RNA, polyoxyethylene stearate and
tyloxapol.
Advantageously and according to the invention, said OADC supplement is an
aqueous composition comprising 50 g/1 of bovine albumin, 20 g/1 of glucose, 0.04 g/1 of
catalase and 0.5 g/1 of oleic acid.
Advantageously and according to this preferred mode of the invention, the
15 process for the enrichment and selective culture of mycobacteria according to the invention is
carried out with a culture medium comprising a nutritive component reproducing that of a
Middlebrook medium, supplemented with:
20
glycerol, in particular at a concentration preferentially between 2 ml/1 and 20 mill,
an OADC supplement, in particular at a concentration preferentially between 50 ml/1
and 200 mill, and
a yeast extract, in particular at a concentration preferentially between 0. I g/1 and 20 g/1.
Advantageously and according to the invention, said process applies more
particularly to the enrichment and to the selective culture of mycobacteria belonging to the
25 species M abscessus, M bolletii, M massiliense, M chelonae, M immunogenum,
M salmoniphilum, M avium, A£. tuberculosis, M intracellulare, M malmoense, M gordmwe,
M kansasii and M fortuitum.
Advantageously and according to the invention, the colonies of mycobacteria
developing on said culture medium are visually pinpointed using chromogenic and/or
30 fluorogenic synthetic substrates specific for enzymatic activities expressed by the
mycobacteria being sought. To this effect, mention may be made of 4-methylumbelliferyl9
be/a-D-glucoside which allows the labeling of the colonies expressmg a beta-glucosidase
activity.
The present invention also extends to a culture medium suitable for carrying out
5 a process for the enrichment and selective culture of mycobacteria according to the invention.
In the case in point, a culture medium according to the invention comprises a nutritive
component suitable for the development and growth of mycobacteria, and is characterized in
that it also comprises, as selective agents, 9-chloro-9-( 4' -diethylamine )phenyl-9, I 0-dihydro-
1 0-phenylacridine hydrochloride and an agent capable of inhibiting Pseudomonas aeruginosa
I 0 bacteria.
According to one particular embodiment, said culture medium is free of crystal
violet.
Advantageously and according to the invention, said culture medium comprises
a nutritive component suitable for the development and growth of mycobacteria and, as
15 selective agents, 9-chloro-9-(4' -diethylamino )phenyl-9, I 0-dihydro-1 0-phenylacridine
20
25
hydrochloride and an agent capable of inhibiting Pseudomonas aeruginosa bacteria. Said
culture medium is also characterized by all or some of the following technical characteristics:
the C-390 concentration is between 2 mg/1 and 1000 mg/1, preferentially between about
4 mg/1 and about 256 mg/1, and even more preferentially between about 8 mg/1 and
about 128 mg/1;
said culture medium comprises at least one additional selective agent (other than C-390
and the agent chosen for inhibiting Pseudomonas aeruginosa), chosen from:
amphotericin B, in particular at a concentration preferentially between
I mg/1 and 200 mg/1, and even more preferentially between 2 mg/1 and
50 mg/1;
nalidixic acid, in particular at a concentration preferentially between 5 mg/1
and 200 mg/1, and even more preferentially between 15 mg/1 and I 00 mg/1;
vancomycin, in particular at a concentration preferentially between 1 mg/1
and 50 mg/1, and even more preferentially between 2 mg/1 and 20 mg/1;
5
10
15
20
25
30
10
colistin methanesulfonate, in particular at a concentration preferentially
between I 0 mg/1 and 200 mg/1, and even more preferentially between
15 mg/1 and 150 mg/1; and
fosfomycin, in patticular at a concentration preferentially between I 00 mg/1
and 3000 mg/1, even more preferentially between 200 mg/1 and 2000 mg/1,
and optionally glucose-6-phosphate, in particular at a concentration
preferentially between I 0 mg/1 and 50 mg/1;
said culture medium comprises, as additional selective agents:
amphotericin B, at a concentration of between 2 mg/1 and 50 mg/1;
colistin methanesulfonate, at a concentration of between 15 mg/1 and
!50 mg/1;
fosfomycin, at a concentration of between 200 mg/1 and 2000 mg/1, and
glucose-6-phosphate, in particular at a concentration preferentially between
I 0 mg/1 and 50 mg/1;
said culture medium comprises a nutritive component reproducing that of a culture
medium chosen from:
a Middlebrook medium, optionally supplemented with glycerol and/or an
OADC supplement;
a Uiwenstein-Jensen medium;
a Columbia medium, optionally supplemented with an OADC supplement
and/or fresh blood, in particular horse blood; and
a BCSA medium;
said culture medium comprises a nutritive component reproducing that of a
Middlebrook medium, supplemented with:
glycerol, in particular at a concentration preferentially between 2 ml/1 and
20 ml/1,
an OADC supplement, in particular at a concentration preferentially between
50 ml/1 and 200 ml/1, and optionally
one or more nutrients chosen from: fresh blood, a yeast extract, alphaketoglutarate,
casein, pyruvate, peptones, amaranth, egg yolk, RNA,
polyoxyethylene stearate and tyloxapol.
5
10
II
Advantageously, the present invention relates to a culture medium comprising a
nutritive component consisting of Middlebrook 7H9 medium, supplemented with:
glycerol, at a concentration of between 2 ml/1 and 20 ml/1,
an OADC supplement, at a concentration of between 50 mill and 200 ml/1, and
a yeast extract, at a concentration of between 0.1 g/1 and 20 g/1;
and comprising, as selective agents:
C-390, at a concentration of between 2 mg/1 and 50 mg/1,
amphotericin B, at a concentration of between 2 mg/1 and 50 mg/1,
colistin methanesulfonate, at a concentration of between 15 mg/1 and 150 mg/1,
fosfomycin, at a concentration of between 200 mg/1 and 2000 mg/1, accompanied by
glucose-6-phosphate, at a concentration of between 10 mg/1 and 50 mg/1.
According to one advantageous embodiment of the invention, the composition
15 of said culture medium can include one or more markers capable of allowing detection and/or
visual identification of the mycobacteria developing in or on said medium. They may in
particular be synthetic enzymatic substrates, with chromogenic and/or fluorogenic properties.
The invention also relates to a method, a process for the enrichment and
20 selective culture of mycobacteria, to the use of C-390 for the purposes of culturing and/or
isolating mycobacteria, and also to a culture medium, characterized by all or some of the
technical characteristics set out above and below.
Other objectives, characteristics and advantages of the invention will emerge in
25 the light of the description which follows and of the examples developed below, the objective
of which is to facilitate the understanding of the invention and the use thereof. These examples
are given by way of explanation and cannot limit the scope of the invention.
EXAMPLES: Production and evaluation of selective media according to the invention
30
II - Selection of a "basic" composition for the culture of mycobacteria
12
Various compositions of culture media, known from the literature as being
fertile to mycobacteria, were tested and compared for their capacity to make mycobacteria
grow. Some were enriched beforehand. The media tested are the following:
- medium A: Middlebrook agar,
5 - medium B: Middlebrook agar enriched with glycerol (4 ml/1),
- medium C: Middlebrook agar with Tween 80 (1 g/1),
- medium D: Middlebrook agar enriched both with glycerol ( 4 ml/1) and with an OADC
supplement (1 00 ml/1),
- medium E: Middlebrook agar supplemented/enriched both with Tween 80 and with the
10 OADC supplement,
15
- medium F: Columbia agar ( oxoid),
- medium G: Columbia agar (oxoid), enriched with the OADC supplement,
- medium H: Columbia agar with blood,
- medium I: BCSA medium (bioMerieux, France).
a) Preparation of media A toE
The media referenced A to E were prepared from a stock solution consisting of
a ten-times concentrated Middlebrook 7H9 culture broth (1 OX Middlebrook 7H9), having the
composition:
20 ammonium sulfate (5 g/1),
L-glutamic acid (5 g/1),
25
30
disodium phosphate (25 g/1),
monopotassium phosphate (I 0 g/1),
sodium citrate (I g/1),
magnesium sulfate (0.5 g/1),
calcium chloride (0.005 g/1),
zinc sulfate (0.0 I g/1),
copper sulfate (0.01 g/1),
ammonium ferric citrate (0.4 g/1),
pyridoxine (0.0 I g/1), and
biotin (0.005 g/1).
13
The pH of this broth was adjusted to 6.6 ± 0.2. The broth was then sterilized in
an autoclave at ll6°C for I 0 minutes. It is stored in a refrigerator until used.
To obtain medium A, 50 ml of broth were added to 450 ml of deionized water.
5 g of bacteriological agar are added to the whole mixture to obtain a Middlebrook agar. The
5 whole mixture was then sterilized in an autoclave at ll6°C, for I 0 minutes.
10
15
20
25
Media B, C, D and E were prepared similarly to medium A, and contain in
addition:
or else:
glycerol ( 4 mill) and optionally a supplement based on oleic acid, on albumin, on
dextrose and on catalase (OADC supplement; I 00 ml/1),
Tween 80 (I g/1) and optionally an OADC supplement (100 ml/1).
b) Media F to I
Media F, Hand I are commercially available media.
Medium G was prepared similarly to medium F, and contains in addition the
OADC supplement (I 00 ml/1).
c) Evaluation of the fertility of media A to I with regard to the mycobacteria.
The fertility of each of media A to I was evaluated by means of 20
mycobacterial strains:
12 strains of Afycobacterium abscessus,
3 strains of Mycobacterium bolletii,
5 strains of Mycobacterium massiliense.
These strains, initially stored at -20°C in glycerol, are thawed and cultured,
firstly, on Columbia agar with horse blood (5%). Each strain is then suspended in I ml of
saline solution (0.85%) until it reaches a turbidity equivalent to the McFarland 0.5 standard
(equivalent to approximately 1.5 x I 08 CFU/ml).
For the mycobacteria which have a tendency to agglutinate, the cells are
30 dispersed with a vortex, in a tube containing 3 glass beads 3 mm in diameter, for I 0 minutes.
5
10
14
An aliquot of I Jll of each suspension of mycobacteria is inoculated onto each
of the culture media to be evacuated, and plated out so as to obtain isolated colonies. The
cultures are incubated at 30°C. The growth of the colonies is observed for 7 days.
The results obtained are summarized in table 1 below.
Medium Medium MediumC: MediumD: Medium E:
A: B: Middlebrook Middlebrook Middlebrook
Middlebrook Middlebrook +Tween 80 +glycerol. +Tween 80
+glycerol. +OADC +OADC
Number of strains showing 0/20 0/20 0/20 11/20 0/20
growth (55%)
Medium F: MediumG: MediumH: Medium I:
Columbia Columbia Columbia with BCSA
+OADC blood
0/20 3/20 1/20 6/20
(15%) (5%) (30%)
Table 1
2/ - Evaluation of the effect of various nutrients on the growth of the mycobacteria
On the basis of the formulation of medium D previously tested, the effects of
various nutrients on the culture of the mycobacteria were evaluated: active carbon, horse
blood, yeast extract, alpha-ketoglutarate, casein, pyruvate, peptones, amaranth, egg yolk
15 emulsion; RNA (Sigma, ref. R6625-25G), polyoxyethylene stearate, tyloxapol.
In a manner very similar to medium D, the following media (denoted J to V)
were prepared:
- medium J: medium D +active carbon (2 g/1),
- medium K: medium D + 5% horse blood (50 ml/1),
20 - medium L: medium D +yeast extract ( 4 g/1),
- medium M: medium D + alpha-ketoglutarate (I 5 g/1),
- medium N: medium D +casein (I g/1),
- medium 0: medium D +sodium pyruvate (7 g/1),
- medium P: medium D +peptone (10 gil),
25 - medium K: medium D +amaranth (0.04 g/1),
15
medium R: medium D + egg yolk emulsion (33 ml/1),
mediumS: medium D +RNA (0.05 g/1),
- medium T: medium D + polyoxyethylene stearate (0.1 g/1),
- medium U: medium D + tyloxapol (5 ml/1),
5 - medium V: medium D +egg yolk emulsion (33 ml/1) +active carbon (4 g/1).
The fertility of these media is evaluated by means of the same 20 mycobacterial
strains used in the test previously described, and treated identically before inoculation.
The cultures are incubated at 30°C. The growth of the colonies is monitored for
10 7 days. Table 2 below summarizes the observations made on the 3rd and 7th days of culture.
15
MediumD MediumJ: MediumK: Mediuml:
3rd day: Number.of strains
shol!fillg groWth
0
11
Medium D
+carbon
0
9
Medium D
+yeast extract
7th day: Number of strains
showing growth (55%} (45%)
17
(85%}
19
(95%)
20
(100%}
MediumM: Medium N: MediumO: Medium P: MediumQ: Medium R:
Medium D Medium D Medium D Medium D Medium 0 Medium 0
+pyruvate +peptone +egg yolk
9 3 13 2
{45%} {15%) (1;5%) • (10%)
14 19 17 19 17 19
(70%} (95%) (85%) (95%} (85%} (95%}
MediumS: MediumT: MediumU: MediumV:
Medium D Medium D Medium D Medium D
+RNA + polyoxy. st. + tyloxapol +egg yolk
+carbon
1 1
0 0
(5%} (5%)
17 18 15 18
(85%} (90%) (75%) (90%}
Table 2
3/- Evaluation of the effect of various agents that are selective with regard to mycobacteria
On the basis of the formulation of medium L previously developed and tested,
the effects of various selective agents were evaluated for their level of selectivity with regard
16
to mycobacteria: amphotericin B (antifungal agent), nalidixic acid, vancomycm, colistin
methanesulfonate, C-390 (Biosynth, Switzerland), fosfomycin and malachite green, at various
concentrations.
Table 3 below summarizes the results of this evaluation, carried out on the 20
5 mycobacterial strains used in the tests previously described, and on 95 strains of
microorganisms not belonging to the Mycobacterium genus, after 7 days of incubation.
Among the 95 strains mentioned are:
10
15
20
25
30
50 strains of Pseudomonas aeruginosa,
I 0 other strains of non-fermentative bacteria:
I strain of Achromobacter xylosoxidans,
1 strain of Acinetobacter sp.,
1 strain of Burkholderia multivorans,
I strain of Burkholderia cenocepacia,
I strain of Burkholderia cepacia,
1 strain of Burkholderia contaminans,
I strain of Pandoraea apista,
I strain of Pandoraea pnomenusa,
2 strains of Stenotrophomonas maltophilia,
7 strains of enterobacteria:
I strain of Escherichia coli,
I strain of Klebsiella pneumoniae,
I strain of Providentia rettgeri,
I strain of Enterobacter cloacae,
I strain of Enterobacter aerogenes,
I strain of Serratia marcescens,
I strain of Citrobacter fi'eundii,
I 0 strains of species of Gram-positive bacteria:
2 strains of Staphylococcus aureus,
I strain of Staphylococcus epidermidis,
I strain of Streptococcus pyogenes,
I strain of Streptococcus pneumoniae,
5
10
15
20
17
I strain of Streptococcus salivarius,
I strain of Streptococcus gordonii,
I strain of Enterococcus faecium,
I strain of Enterococcus faecalis, and
I strain of Bacillus subtilis.
8 strains of fungi/yeasts:
I strain of Candida albicans,
I strain of Candida glabrata,
2 strains of Aspergillus fumigatus,
I strain of Aspergillus terreus,
I strain of Scedosporium apiospermwn,
I strain of Scedosporium prolificans,
I strain of Rasamsonia (Geosmithia) argillacea.
In this table 3, the results are presented as number and as percentage of strains
having shown growth on the medium in question after 7 days of culture. The notations NS and
NT mean respectively "not significant" (inhibition of bacteria by amphotericin B) and "not
tested" (action of the antibacterial agents on the yeasts and fungi).
Medium L Medium L Medium l
+amphotericin B +nalidixic acid
5 mg/1 10mg/l 20 mg/1 16 mg/1 32 mg/1 64 mg/1
Mycobacteria 20 20 20 20 20 20 20
(100%) (100%) . J100%) (100%) (100%) (100%) (100%)
fs_e_udomoni:Is·aeruginosa 50 NS NS NS 50 43 35
{100%) {100%) (86%) (70%)
Other non-fermentative 10 NS NS NS 10 10 10
bacteria (100%) (100%) (100%) (100%)
Enterobacteria 7 NS NS NS 4 0 0
(100%) {57%)
Gram-positive bacteria 10 NS NS NS 9 8 7
(100%) (90%) {80%) {70%)
~ungifyeasts
8 6 4 3 NT NT NT
(100%) (75%) (50%) (37.5%)
Medium L
+vancomycin
2.5 mg/1 5.0 mg/1 10 mg/1
19 19 19
(95%) (95%) (95%)
50 5.0 50
(100%) (1.00%). (1.00%)
9 9 9
(90%) (90%) (90%)
7. . 7_ 7
(100%) (100%) (10Q%)
0 0 0
NT NT NT
18
Medium L
+colistin methanesulfonate
32 mg/1 64 mg/1 128 mg/1
20 20 20
(100%)_ {100%) (100%)
5 .5 5
(1.0%) (10%) (10%)
10 10 10 (100%) (100%) (100%) I
3_ 3 3 I
(43%) (43%) 7 6 I
(70%) (60%)
NT- -NT_,
Mediuml
+ fosfomycin
400 mg/1 800 mg/1 1600 mg/1
20
_(1(){)%)
15.
430%)
6
20
(100%)
g
(16%)
6
(60%)
2
129%)
0
NT
Table 3
20
(_100%)
6
(12%)
5
(50%)
2
{29%)
0
NT
Medium L
+ C-390
32 mg/1 64 mg/1 128 mg/1
19 19 19
(95%) (95%) ;- (95%)
46 45 35
(92%) (90%) (70%)
7 4 2
(70%) {40%) _(20%)
o· 0 0
0 0 0
NT NT NT
Mediuml
+ malachite green
0.125 mg/1 0.25 mg/1 0.5 mg/1
16 20 20
(80%) -· (_100%) (100%)
50 50 r 50
{ioo%) (100%) {100%)
10 10 10
(100%) (100%) I (100%) r 7 7 -! 7
'1100%1 {100%) (lOci%)
10 10 9
(100%) (100%) (90%)
NT NT -1 NT
5 4/ - Optimization of the media selective for mycobacteria according to the invention
In the light of the previous data, two further selective culture media, denoted
medium W and medium X, were developed. These media were developed from the
composition of medium L, which is an enriched culture medium as previously described, to
which a particular combination of selective agents was added. The composition of said
10 selective media is summarized as follows:
- medium W: medium L +colistin methanesulfonate (32 mg/1)
+ fosfomycin ( 400 mg/1) + glucose-6-phosphate (25 mg/1)
+amphotericin B (5 mg/1)
- medium X: medium L + colistin methanesulfonate (32 mg/1)
15 + fosfomycin ( 400 mg/1) + glucose-6-phosphate (25 mg/1)
19
+ amphotericin B (5 mg/1)
+ C-390 (32 mg/1).
These two media were tested on 188 strains of microorganisms:
5 - 98 strains of nontuberculous mycobacteria:
10
15
20
25
30
52 strains of Mycobacterium abscessus,
3 strains of Mycobacterium bolletii,
34 strains of Mycobacterium chelonae,
I strain of Mycobacterium immunogenum,
6 strains of Mycobacterium massiliense,
2 strains of Mycobacterium salmoniphilum,
- 94 strains of microorganisms not belonging to the Mycobacterium genus:
54 strains of Pseudomonas aeruginosa,
15 other strains of non-fermentative bacteria:
1 strain of Achromobacter spl,
I strain of Achromobacter xylosoxidans,
I strain of Acinetobacter sp.,
I strain of Acinetobacter baumannii,
I strain of Burkholderia cepacia,
1 strain of Burkholderia contaminans,
I strain of Burkholderia multivorans,
I strain of Burkholderia stabilis,
1 strain of Elizabethkingia miricola,
I strain of Pandoraea apista,
I strain of Pandoraea pnomenusa,
4 strains of Stenotrophomonas maltophilia,
7 strains of enterobacteria (the same as those previously mentioned),
10 strains of Gram-positive bacteria (the same as those previously mentioned),
8 strains of fungi/yeasts (the same as those previously mentioned).
5
20
In this table 4, the results are presented as number and as percentage of strains
having shown growth on the medium in question and after 7 days of culture.
Medium L MediumW: Medium X:
Medium l Medium L
+colistin +colistin
methanesulfonate methanesulfonate
(32 mg/1) (32 mg/1)
+ fosfomycin (400 mg/1) + fosfomycin {400 mg/i)
+ G6P (25 mg/1) + G6P (25 mg/1)
+Amp B (5 mg/1) +Amp B (5 mg/1)
+ C-390 (32 mg/lt
M. abscessus (52) 52 52 51 ---
M. boletti (3) 3 3 3
-------·-------·· ·····------------- ----··-----·-------·--· ..... ···-·-·-·----
M. chelonae (34) 34 34 34
--·--··--- ··--
M. immonogenum (1) 1 1 1
-·· ·--··-·-···- -·---··------------··---···
M. massi/iense {6) 6 6 ---- 6 !!.!: salmoniphilum (2) 2 2 2
Total mycobacteria (98): 98 (100%) 98 (100%) 97 (99%)
P. aeruginosa (54) 54 0 0 ··-----·-- ----·--·-----·
Other non-fermentative bacteria
14 9 6
(15) . - -··-------··
Enterobacteria {7) 7 2 0
Gram-positive bacteria (10) 10 0 0 --
_£tJ_ngi/ye~'!S (8) 8 3 0
Total non-mycobacteria (94): 93 (99%) 14 (15%) 6 (6%)
Table 4
51- Validation of the composition of medium X for the culture and selection of
Mycobacterium tuberculosis
On the basis of the composition of medium X above, a liquid selective culture
10 medium was formulated. This liquid medium X (without agar) was tested for the detection of
mycobacteria, in particular of Mycobacterium tuberculosis, in the context of an analysis of
sputum samples originating from 32 patients not suffering from cystic fibrosis and from 56
patients suffering from cystic fibrosis.
To do this, the BacT/ALERT® 3D automated microbial detection system
15 (bioMerieux, France) was used. Flasks containing the liquid medium X (or broth X) were
incubated in the automated detection device for at least 28 days, and compared with the
BacT/ALERT® MP reagent conventionally recommended for the detection of mycobacteria.
5
21
The composition of this reagent corresponds schematically to a Middlebrook
7H9 medium supplemented with oleic acid, glycerol, bovine serum albumin, amaranth and a
cocktail of antibiotics (amphotericin B, azlocillin, nalidixic acid, polymyxin B, trimethoprim
and vancomycin).

CLAIMS
1) A process for the enrichment and selective culture of mycobacteria
5 contained in a biological sample, wherein all or part of said sample is inoculated in/on a
culture medium comprising a nutritive component suitable for the development and growth of
mycobacteria,
characterized in that said culture medium also comprises, as selective agents, at least 9-chloro-
9-( 4' -diethylamino )phenyl-9, 1 0-dihydro-1 0-phenylacridine hydrochloride (or C-390) and an
10 agent capable of inhibiting Pseudomonas aeruginosa bacteria.
15
2) The process as claimed in claim 1, characterized in that the C-390
concentration of said culture medium is between 2 mg/1 and 1000 mg/1.
3) The process as claimed in claim 1 or 2, characterized in that said agent
capable of inhibiting Pseudomonas aeruginosa bacteria is chosen from: colistin
methanesulfonate, fosfomycin, nalidixic acid, aztreonam, cefsulodine and mangrolide A.
4) The process as claimed in any one of claims 1 to 3, characterized in that said
20 culture medium also comprises an agent capable of inhibiting Burkholderia cepacia bacteria,
preferentialy chosen from fosfomycin, aztreonam and mangrolide A.
5) The process as claimed in any one of claims 1 to 4, characterized in that said
culture medium also comprises at least one additional selective agent chosen from:
25 amphotericin B, nalidixic acid, vancomycin, colistin methanesulfonate, fosfomycin and
malachite green.
6) The process as claimed in claim 5, characterized in that said culture medium
comprises, as additional selective agents:
30 amphotericin B, at a concentration of between 2 mg/1 and 50 mg/1;
colistin methanesulfonate, at a concentration of between 15 mg/1 and 150 mg/1;
fosfomycin, at a concentration of between 200 mg/1 and 2000 mg/1.
5
10
28
7) The process as claimed in claim 6, characterized in that said culture medium
also compnses, in addition to the fosfomycin, glucose-6-phosphate, at a concentration of
between I 0 mg/1 and 50 mg/1.
8) The process as claimed in any one of claims I to 7, characterized in that said
culture medium comprises a nutritive component reproducing that of a culture medium chosen
from:
a Middlebrook culture medium,
a Uiwenstein-Jensen medium,
a Columbia medium,
a BCSA medium.
9) The process as claimed in any one of claims 1 to 8, characterized in that said
15 culture medium comprises a nutritive component reproducing that of a Middlebrook medium,
20
supplemented with:
glycerol,
an OADC supplement,
one or more nutrients chosen from: fresh blood, a yeast extract, alpha-ketoglutarate,
casein, pyruvate, peptones, amaranth, egg yolk, RNA, polyoxyethylene stearate and
tyloxapol.
10) The process as claimed in claim 9, characterized in that said culture medium
comprises a nutritive component reproducing that of a Middlebrook medium, supplemented
25 with:
glycerol, at a concentration of between 2 ml/1 and 20 ml/1,
an OADC supplement, at a concentration of between 50 ml/1 and 200 ml/1, and
a yeast extract, at a concentration of between 0.1 g/1 and 20 g/1.
30 11) A culture medium comprising a nutritive component suitable for the
development and growth of mycobacteria, characterized in that it also comprises, as selective
29
agents, 9-chloro-9-( 4' -diethylamino )phenyl-9, l 0-dihydro-1 0-phenylacridine hydrochloride (or
C-390) and an agent capable of inhibiting Pseudomonas aeruginosa bacteria.
12) The culture medium as claimed in claim 11, characterized m that it
5 comprises C-390 at a concentration of between 2 mg/1 and 1000 mg/l.
10
15
13) The culture medium as claimed in claim 11 or 12, characterized in that said
agent capable of inhibiting Pseudomonas aeruginosa bacteria is chosen from: colistin
methanesulfonate, fosfomycin, nalidixic acid, aztreonam, cefsulodine and mangrolide A.
14) The culture medium as claimed in any one of claims 11 to 13, characterized
in that it also comprises an agent capable of inhibiting Burkholderia cepacia bacteria, said
agent being preferentially chosen from fosfomycin, aztreonam and mangrolide A.
15) The culture medium as claimed in any one of claims 11 to 14, characterized
in that it comprises at least one additional selective agent chosen from amphotericin B,
nalidixic acid, vancomycin, colistin methanesulfonate and fosfomycin.
16) The culture medium as claimed in any one of claims 11 to 15, characterized
20 in that it comprises, as additional selective agents:
25
30
amphotericin B, at a concentration of between 2 mg/1 and 50 mg/1;
colistin methanesulfonate, at a concentration of between 15 mg/1 and 150 mg/1;
fosfomycin, at a concentration of between 200 mg/1 and 2000 mg/l, and glucose-6-
phosphate, at a concentration of between 1 0 mg/1 and 50 mg/l.
17) The culture medium as claimed in any one of claims 11 to 16, characterized
in that it comprises a nutritive component reproducing that of a culture medium chosen from:
a Middlebrook medium,
a Uiwenstein-Jensen medium,
a Columbia medium,
a BCSA medium.
30
18) The culture medium as claimed m claim II, compnsmg a nutritive
component consisting of Middlebrook 7H9 medium, supplemented with:
glycerol, at a concentration of between 2 ml/1 and 20 ml/1,
an OADC supplement, at a concentration of between 50 ml/1 and 200 ml/1, and
5 a yeast extract, at a concentration of between 0.1 g/1 and 20 g/1;
and comprising, as selective agents:
C-390, at a concentration of between 2 mg/1 and 1000 mg/1,
amphotericin B, at a concentration of between 2 mg/1 and 50 mg/1,
colistin methanesulfonate, at a concentration of between 15 mg/1 and 150 mg/1,
I 0 fosfomycin, at a concentration of between 200 mg/1 and 2000 mg/1, accompanied by
glucose-6-phosphate, at a concentration of between 10 mg/1 and 50 mg/l.