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Journal of Clinical Microbiology, September 1998, p. 2565-2570, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Microaerophilic Conditions Promote Growth of
Mycobacterium genavense
L.
Realini,1,*
K.
De Ridder,1
J.-C.
Palomino,1
B.
Hirschel,2 and
F.
Portaels1
Mycobacteriology Unit, Department of
Microbiology, Institute of Tropical Medicine, Antwerp,
Belgium,1 and
Division of Infectious
Diseases, Hôpital Cantonal, Geneva, Switzerland2
Received 10 March 1998/Returned for modification 30 May
1998/Accepted 10 June 1998
 |
ABSTRACT |
Our studies show that microaerophilic conditions promote
the growth of Mycobacterium genavense in semisolid medium.
The growth of M. genavense at 2.5 or 5% oxygen was
superior to that obtained at 21% oxygen in BACTEC primary
cultures (Middlebrook 7H12, pH 6.0, without additives). By using
nondecontaminated specimens, it was possible to detect growth
with very small inocula (25 bacilli/ml) of 12 different M. genavense strains (from nude mice) within 6 weeks of incubation
under low oxygen tension; conversely, with 21% oxygen, no growth of 8 of 12 (66.7%) M. genavense strains was detected (growth
index, <10). The same beneficial effect of 2.5 or 5% oxygen was
observed in primary cultures of a decontaminated clinical specimen. Low
oxygen tension (2.5 or 5%) is recommended for the primary isolation of
M. genavense. Microaerophilic cultivation of other atypical
mycobacteria, especially slow-growing (e.g., Mycobacterium
avium) and difficult-to-grow (e.g., Mycobacterium ulcerans) species, is discussed.
| |
INTRODUCTION |
Mycobacteria are generally
considered obligate aerobes (25) that grow at oxygen
tensions ranging from atmospheric to microaerophilic conditions
(17, 24). Routinely, primary cultures are incubated in
room air or air containing 10% CO2. Increased
CO2 tension produces earlier and more luxuriant growth of
Mycobacterium tuberculosis (18), especially on
agar media. In the BACTEC system, the recommended atmosphere for
mycobacteria is 5 to 10% CO2 in air (39).
Primary cultures of all mycobacteria, including strict pathogens
(e.g., M. tuberculosis), saprophytic mycobacteria other than
M. tuberculosis (e.g., Mycobacterium gordonae),
opportunistic mycobacteria other than M. tuberculosis (e.g., Mycobacterium avium), and even
difficult-to-grow mycobacteria (e.g., Mycobacterium
genavense and Mycobacterium ulcerans), are
incubated under the same conditions (room air or 5 to 10%
CO2 in air). M. genavense is a
fastidious mycobacterium which most commonly causes disease in AIDS
patients (3, 5, 7, 14, 27), but there are recent reports of
M. genavense infections in patients without human
immunodeficiency virus (2, 22). M. genavense also infects birds (15, 16, 31, 32) and dogs (19). Approximately 50% of M. genavense isolates grow in liquid media such as
Middlebrook 7H12 (5). By radiometry, Hoop et al.
(16) cultured M. genavense from 67.6% of
bird tissues (livers and intestines positive for acid-fast bacilli
[AFB] by microscopy). Results from radiometric assays can require 2 months or more (7, 16). Growth may be improved at pH 6.0 (4, 15, 16, 40, 41). We recently demonstrated that culture conditions recommended for M. tuberculosis
(polyoxyethylene stearate and PANTA [polymyxin B-amphotericin
B-nalidixic acid-trimethoprim-azlocillin] at pH 6.8) inhibit primary
isolation of M. genavense in Middlebrook 7H12,
and we recommended the use of medium at pH 6.0 without additives in the BACTEC system (34). Solid media such as
Löwenstein-Jensen and Middlebrook agar are not suitable for
the isolation of M. genavense
(16). Designation of mycobacteria as fastidious may reflect
only, for example, that the culture medium or pH is
inadequate, as demonstrated for M. genavense,
but in addition may involve inappropriate oxygen tensions.
Surprisingly, microaerophilic conditions have seldom been applied
to the primary isolation of mycobacteria. Franzblau (10) incubated Mycobacterium leprae in liquid culture at an
oxygen concentration of 2.5% instead of 21% to maintain its metabolic activity in the BACTEC system. Because it is likely that M. genavense is disseminated from the gut (9), an
excellent site for anaerobic and microaerophilic bacteria, we attempted
to improve the growth of primary cultures of M. genavense by modifying oxygen tensions. We report
here the behavior of M. genavense
in a semisolid medium and the influence of different oxygen
concentrations on its growth in the BACTEC system.
| |
MATERIALS AND METHODS |
M. genavense strains.
Twelve
strains of M. genavense were studied. ITM
(Institute of Tropical Medicine) 95-975, 96-1283, 96-1438, 96-1439, and
96-1799 were isolated from five different AIDS patients living in
Geneva, Switzerland; ITM 96-823 and 97-76 were isolated from AIDS
patients from Iowa City, Iowa; and ITM 97-75 was isolated from an AIDS patient from Seattle, Wash. Four other strains originated from birds
living in the Antwerp Zoo, Antwerp, Belgium: ITM 95-610, from a
cutthroat weaver (Amadina fasciata); ITM 95-614, from a gouldian finch (Chloebia gouldiae); ITM 95-615, from a zebra
finch (Poephila guttata castanotis); and ITM 96-6, from a
turquoise tanager (Tangara mexicana). The organisms were
recovered from lungs and spleens of nude mice (BALB/c; IFFA Credo,
Lyon, France) inoculated intraperitoneally 8 to 9 months previously, as
previously described (34). The AFB were counted by the
method of Shepard and McRae (38). Dilutions of tissue
homogenates were made in phosphate-buffered saline (PBS). No
decontamination was performed.
Clinical specimen.
Liver (ITM BK97-1056) (kept at 
20°C)
from an African silver-bill (Euodice cantans) kept in the
Antwerp Zoo was sent to us because the contributor was unable to obtain
growth on Ogawa and Löwenstein-Jensen media even though the
tissues contained large numbers of AFB. These AFB were identified as
M. genavense by characterization of specific
sequences in the rrn operon (data not shown). The specimen
was thawed, minced, and homogenized with a pestle and mortar in PBS.
The tissue homogenate was centrifuged differentially to remove the
larger tissue debris (100 × g, 5 min, 10°C). The supernatant was then centrifuged at 2,700 × g for 45 min at 10°C, and the resulting pellet containing the AFB was
resuspended in PBS. AFB were counted as described above. The bacterial
suspension was divided into four aliquots, each decontaminated by one
of the following procedures: (i) sodium dodecyl sulfate (SDS)
(35), (ii) N-acetyl-L-cysteine-sodium
hydroxide (NALC) (25), (iii) 4% sodium hydroxide (Petroff
[28]), or (iv) 1 N hydrochloric acid (HCl) followed by
neutralization with 4% sodium hydroxide.
Culture media and gas conditions.
Semisolid medium
(23) was used after adjusting the pH to 6.0 with phosphoric
acid. This medium was inoculated with 0.2 ml of a suspension of
106 AFB/ml, inverted repeatedly to avoid formation of
bubbles and aeration, and incubated at 37°C in air for 2 months.
BACTEC pyrazinamide control medium (PZA) (pH 6.0 ± 0.2), without
polyoxyethylene stearate or PANTA (34), was inoculated in
triplicate with 100 µl of a nondecontaminated M. genavense suspension from nude mouse organs to obtain
102 to 105 AFB/vial and incubated at 37°C.
Each aliquot of the clinical specimen decontaminated by one of the
above methods was inoculated into a single vial for each of the
experimental conditions tested. Readings of the release of
14CO2 in the BACTEC 460 TB instrument were made
twice a week for 6 weeks or until the growth index (GI) reached 999. Three different gas mixtures (Praxair NV, Oevel, Belgium) were used:
(i) 10% CO2 in air (21% O2), (ii) 5%
O2-10% CO2-85% N2, and (iii)
2.5% O2-10% CO2-87.5% N2. When
the GI reached 999, 0.2 ml of the vial was inoculated into
thioglycollate medium, blood agar, and Middlebrook 7H11. Ziehl-Neelsen
staining was performed on the BACTEC vials when the GI reached 999 and
on the semisolid medium.
| |
RESULTS |
Growth in semisolid medium.
All M. genavense strains were microaerophilic in semisolid
medium: growth appeared as a 3- to 5-mm zone, with the superior limit
remaining 3 to 7 mm under the surface of the culture medium. Figure 1 shows the microaerophilic
natures of four different strains of M. genavense.
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FIG. 1.
Microaerophilic nature of M. genavense primary cultures in Marks semisolid
medium. Vials: 1, ITM 97-75; 2, ITM 95-975; 3, ITM 96-6; 4, ITM
95-610.
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|
Growth in the BACTEC system.
Table
1 shows the number of strains reaching a
GI of 999 in the BACTEC system within 6 weeks of incubation under the
three different oxygen concentrations: 2.5, 5, and 21%. The 12 strains reached a GI of 999 within 6 weeks (the time routinely required to
retain the vials [39]) under low oxygen
concentrations, even with small inocula (102 AFB/vial). By
comparison, when air is used to gas the vials, growth of M. genavense is inhibited. This is most obvious with smaller
inocula: for example, at 102 AFB/vial, only 2 of 12 (16.7%) strains reached a GI of 999 within 6 weeks.
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TABLE 1.
Influence of oxygen concentration on growth of 12 strains of M. genavense (from nude mice) in
the BACTEC systema
|
|
An illustrative example from Table
1 is shown in Fig.
2, demonstrating bacterial growth
with inocula of 10
5, 10
4, 10
3, and
10
2 AFB/vial of strain ITM 96-1283 in the BACTEC system in
the presence
of 21, 5, or 2.5% oxygen in the gas mixture. The impact
of lower
oxygen tension on growth can be better appreciated with
smaller
inocula. Growth of
M. genavense ITM
96-1283 was detected earlier
with 5 or 2.5% oxygen than with 21%
oxygen; for example, with
an inoculum of 10
4 AFB/vial, the
GI reached 999 in 13 days under 2.5 or 5% O
2 versus
23 days with 21% O
2. Moreover, even with smaller inocula, of
10
2 and 10
3 AFB/vial, a difference was observed
between 2.5 and 5% O
2. With
an inoculum of 10
2
AFB/vial, the growth of
M. genavense ITM
96-1283, based on the
time needed to reach a GI of 999, was higher in
the presence of
2.5% O
2 (23 days) than 5% O
2
(34 days).
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FIG. 2.
Effects of oxygen on growth of different inocula of
M. genavense ITM 96-1283 in the BACTEC system
(Middlebrook 7H12, pH 6.0, without additives).  , 2.5%
O2;  , 5% O2;  , 21% O2.
|
|
Figure
3 shows the growth of the 12 strains with the three different gas mixtures following inoculation of
10
2 AFB/vial in the BACTEC system. For all strains tested,
earlier
growth was detected under 5 or 2.5% O
2 than under
21% O
2. For
8 of the 12 strains, there was no growth after
6 weeks of incubation
(42 days) in the presence of 21% O
2.
There are some differences
between the strains: strains ITM 95-975, 96-823, and 96-1438 grew
better at 5% O
2 and strains ITM
95-614, 96-1283, and 97-75 had
higher growth rates in the BACTEC system
with 2.5% O
2, while the
growth curves for strains
ITM 95-610, 95-615, 96-6, 96-1439, 96-1799,
and 97-76 at 5 and 2.5%
O
2 were similar. Multiplication of all
M. genavense strains was detected in the BACTEC system
in less
than 6 weeks under the lower oxygen concentrations.
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FIG. 3.
Effects of different oxygen concentrations on growth of
12 M. genavense strains (102 AFB in
PZA) in BACTEC primary cultures within 6 weeks of incubation. ,
2.5% O2; , 5% O2; , 21%
O2.
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|
The positive impact of low oxygen concentrations on
M. genavense growth in the BACTEC system was observed with
the clinical
specimen. Figure
4 shows
growth curves obtained with the three
different gas mixtures when the
vials were inoculated with a specimen
(10
6
AFB/vial) decontaminated by SDS, NALC, the Petroff method, or
HCl.
Better growth was obtained with 2.5 or 5% O
2 than with
21%
O
2 for each of the four decontamination methods. The
GI reached
999 with all methods at low oxygen concentrations but
never reached
999 at 21% oxygen when SDS, the Petroff method, or
HCl were applied.
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FIG. 4.
Influence of oxygen concentration on primary cultures of
an M. genavense clinical specimen
(106 AFB in PZA) decontaminated by SDS, NALC, the Petroff
method, or HCl in the BACTEC system. , 2.5% O2; ,
5% O2; , 21% O2.
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|
In Fig.
5, the four decontamination
methods were compared at 2.5% oxygen. Earlier detection of growth was
observed when the
specimen was decontaminated with NALC or SDS than by
the Petroff
method or HCl.
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FIG. 5.
Comparison of decontamination methods (SDS, NALC,
Petroff, and HCl) for recovery of M. genavense
from a clinical specimen in BACTEC vials gassed with 2.5%
O2, 10% CO2, and 87.5% N2.  ,
SDS;  , NALC; , Petroff; ×, HCl.
|
|
Thioglycollate, blood agar, and Middlebrook 7H11 subcultures from
BACTEC vials showed all vials to be free of contaminating
organisms.
| |
DISCUSSION |
Most aerobic bacteria may in fact be microaerophilic, requiring
oxygen at 1,000 to 3,000 Pa, (37), but cannot tolerate
oxygen at the ordinary partial pressure in air (20,000 Pa).
Lebek (21) differentiated M. tuberculosis from Mycobacterium bovis by the microaerophilic nature of the latter, and Grange and Yates
(12) recommended this test to differentiate species within
the M. tuberculosis complex. Marks (23)
applied this approach in his identification scheme to
differentiate aerobic Mycobacterium species
(M. tuberculosis, M. chelonae,
M. flavescens, and M. fortuitum)
from microaerophilic species (M. bovis,
M. ulcerans, M. gordonae,
M. terrae, M. avium, and M. intracellulare). Respiratory assays on mycobacteria were performed
during the early 1900s, mainly with M. tuberculosis (6, 8, 26, 36, 43). The investigators conducting these assays concluded that M. tuberculosis is strictly
aerobic but that an atmosphere enriched with 5 to 10% CO2
enhances growth, especially on agar media such as Middlebrook 7H10
(1). CO2 seems to be essential for growth of
mycobacteria. An appreciable proportion of carbon in mycobacteria
arises from CO2 fixation through a variety of carboxylation
reactions (33). As Beam and Kubica reported, "It seems
advisable to incubate all primary isolation cultures for mycobacteria
in an atmosphere of 5-10% CO2 in air" (1).
Our results show the microaerophilic nature of M. genavense, as shown by growth under the surface in
semisolid medium, similar to Campylobacter sp.
(20) and some other species of mycobacteria (e.g.,
M. bovis) described by Marks (23) in his
identification scheme. We initially used 5% O2 (10%
CO2 and 85% N2) in the BACTEC system,
the oxygen concentration commonly used for isolation of Campylobacter jejuni (20). Franzblau and
Harris (11), however, demonstrated in a study of the
metabolism of M. leprae that ATP maintenance is optimal
at 2.5 to 10% O2, and they proposed that a gas
mixture of 2.5% O2, 10% CO2, and 87.5%
N2 could be used in the BACTEC system
(10) for drug susceptibility testing. For M. genavense, earlier and better growth
can be detected in the BACTEC system at low oxygen concentrations.
Growth of the 12 different strains of M. genavense was detected with 2.5 or 5% oxygen within 6 weeks, even when bacillary concentrations were low. Conversely, at 21%
oxygen, there was no detectable growth of 8 of the 12 strains (66.7%).
The differences observed between the strains
some preferring 2.5%
oxygen and others 5% oxygencould not be correlated with the origins
of the strains.
The decontaminated clinical specimen showed earlier growth
of M. genavense in BACTEC vials gassed
with low oxygen tension. In a comparison of the growth results
following the different decontamination methods and incubation under
low oxygen concentrations, better growth was obtained when the clinical
material was decontaminated with NALC or SDS than by the Petroff method
or HCl. At 2.5% oxygen, the GI reached 999 in 13 days with an inoculum
of 106 AFB decontaminated with NALC or SDS; by comparison,
only 104 AFB from nondecontaminated material were necessary
to reach a GI of 999 in 13 days under the same gaseous conditions (Fig.
2). M. genavense appears to be very sensitive
to decontamination procedures, and this may also explain the
difficulties encountered in its in vitro growth.
In 1996, Hoop et al. (16) reported cultivation of
M. genavense in 67.6% of bird tissues
inoculated in BACTEC vials. It is important to note that the 32.4%
negative cultures were inoculated with AFB-positive tissues by
Ziehl-Neelsen staining. Moreover, these specimens were
decontaminated by the SDS method (the same technique we used
[35]) and inoculated in BACTEC vials supplemented with
PANTA. We previously demonstrated that PANTA inhibits growth of
M. genavense (34). Although this was
not specified by Hoop et al. (15, 16), we presume that
the vials were gassed as recommended by the manufacturerair
(21% oxygen) with 5 to 10% CO2. Among the factors
affecting primary cultures of M. genavense, the
medium, the decontamination method, and the oxygen tension have to be
taken into account.
Recently, Wayne and Hayes (42) described experiments with
cultures of M. tuberculosis in a deep liquid medium
under conditions of known oxygen depletion. Two stages of
nonreplicating persistence were observed, corresponding to
microaerophilic and anaerobic conditions, possibly explaining
the ability of M. tuberculosis to remain dormant
in the host for long periods of time. By contrast, for atypical
mycobacteria, which are not strict pathogens and are believed to have
their reservoirs in the environment (29), we conjecture that
microaerophilic conditions (2.5 or 5% O2) do not lead to
persistence but rather improve growth. As suggested by Jenkins et al.
(17), factors such as aeration and carbon dioxide
concentration may not have always been optimal for isolation of
mycobacteria from the environment; in particular, M. ulcerans thus far has not been isolated from the environment
despite numerous attempts (29, 30). As early as 1965, Hanks
(13) proposed that it might be wise to conduct experiments
at minimal concentrations of oxygen for cultivation of M. leprae and Mycobacterium lepraemurium, as these species
universally produce nonpulmonary lesions.
Our experiments demonstrate the microaerophilic nature of M. genavense in primary cultures and suggest potential
applications in routine laboratories which use the BACTEC system.
Further studies are necessary to determine optimal carbon dioxide
concentrations, as well as to test lower concentrations of oxygen. For
isolation of atypical mycobacteria, e.g., M. avium or
M. ulcerans, from clinical or environmental specimens,
the possible microaerophilic nature of the organism should be
taken into consideration, especially when difficult-to-grow species
are suspected.
| |
ACKNOWLEDGMENTS |
This study was supported by the Damien Foundation, Brussels,
Belgium, and by the IWT project, grant 95-0129. Laurence Realini was supported by a grant from the Fonds National Suisse de la Recherche
Scientifique (grant 3139-039166).
We thank Becton Dickinson for use of a BACTEC 460 TB instrument and all
vials needed for this research. We are very grateful to P.-A. Fonteyne
for critical remarks and to S. R. Pattyn and W. M. Meyers for reviewing the manuscript. We are grateful to R. A. Clark and L. S. Schlesinger, Iowa City, Iowa, for strains ITM
96-823 and 97-76 and to M. B. Coyle, Seattle, Wash., for
strain ITM 97-75. We are also grateful to W. De Meurichy and L. Bauwens, Zoo of Antwerp, Antwerp, Belgium, for bird organs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Tropical Medicine, Department of Microbiology, Nationalestraat 155, B-2000 Antwerp, Belgium. Phone: 32 3 247 63 24. Fax: 32 3 247 63 33. E-mail: realini{at}microbiol.itg.be.
| |
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Journal of Clinical Microbiology, September 1998, p. 2565-2570, Vol. 36, No. 9
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
