Original Article Ind. J. Tub.

2000, 47, 87

ROLE OF SILICON IN MODULATING THE INTERNAL MORPHOLOGY

AND GROWTH OF MYCOBACTERIVM TUBERCULOSIS

Satadal Das1 and U.K. Chattopadhyay2

(Received on 12.7.99, Accepted on 4.12.99)

Summary: Silicon is known to enhance the growth and pathogenicity of Mycobactenum tuberculosis. In this study the pattern
of growth of different strains of M.tuberculosis was studied on a carbon free silicon based culture medium and compared with that
on conventional media. Thirty-two strains of M.tuberculosis from mostly clinical isolates were serially propagated on a carbon free
silicate medium and on different conventional media using standard inocula

There was initial good growth on the silicon based medium in comparison with the conventional media. However, the growth
was considerably reduced after early serial transfers but improved again in late serial cultures.

The initial good growth appears to be due to increased activity of silicon induced fatty acid synthase and the late improvement
due to slow adaptation of M.tuberculosis to the carbon free metaboiism by the formation of silicic acid esterified cell wall and
silicon induced genetic alteration. All these changes were probably responsible for the formation of fibrous, ropelike structures and
dense granules seen under electron microscope.

Since the silicon content of the lung tissue is comparatively higher than many other tissues of the human body, it could play a
role in the pathogenicity of tuberculosis in the lungs

Key words :- Silicon, morphology; growth; M tuberculosis

INTRODUCTION loam soil than in the plain loam soil6. Si is known to

Many organisms are known to be able to utilise
silicon (Si) which is present in plenty in nature
(Fig. 1). There are distinct Si accumulator plants like
Cyperaceae,Graminae, Juncaceae and Moquiles
Spp.;organisms like marine phytoplankton, marine
brown algae, ‘horsetails’, foraminifera and porifera
contain enough Si, in the range of 60,000-4,37,000
mg per kg dry matter (DM). Bacteria contain about
180 mg per Kg DM of Si1-2. Addition of Si in culture
media showed a remarkable growth accelerating
effect on Staphylococcus aureus3; its utilisation4 and
in Nocardioform organisms5.
There is considerable indirect evidence of Si
utilisation by bacteria. Thus, ‘non-typhoidal
Salmonella’, Enterobacter, Klebsiella and
Citrobacter can survive better in Si-riched sandy-
enhance the growth of Mycobactenum tuberculosis
in the lungs where Si is present to a high degree.
Experimentally, Si was found to increase
pathogenicity of BCG, a typical mycobacterium,
M.tuberculosis and M. Leprae7-11.
There is a profound similarity of Si chemistry
and carbon (C) chemistry12. The silicon compounds
have kinetic parameters identical to those of their
carbon analogues13. It is possible that an organism
can utilise Si in a C-deficient environment. In the
present study, a possible role of Si in in vitro growth
ofM.tuberculosis even in the absence of a C source
was studied. As lung is the most important Si
accumulator in the body of man’ and because
mycobacteria can utilise Si4, a possible role of Si in
the pathogenicity of M.tuberculosis was sutdied.

1. In-Charge Department of Laboratory Medicine, Peerless Hospital & B.K. Roy Reseaich Centre, Calcutta
2. Associate Professor, Department of Micorbiology All India Institute of Hygiene and Public Health, Calcutta
Conesspondence. Dr. U.K Chattopadhyay, Deplt. of Microbiolog) All India Institute of Hygiene and Public Health 110, Chittaranjan
Avenue, Calcutta-700 073
 88 SATADAL DAS AND U.K. CHATTOPADHYAY
MATERIAL AND METHODS
M.tuberculosis strains - The strains used were
H37Rv.H37,Ra and 30 clinical isolates. Pure
cultures
were maintained on Lowenstein-Jensen Medium
(LJM) and were checked for purity4.
Culture Media - Lowenstein Jensen Medium
was prepared by usual procedure14. The Kirschner
silicate medium (KSM), in each MacCartney bot-
tle, comprised 3 ml of Kirschner Medium15 concen-
trated 2.5x, plus 3 ml Na2Sio3 (Fluka, Switzerland)
11.8 g/dl used as a solidifying base, with addition
of H3 PO4 (1.5 ml. of 16% V/V aqueous solution).
The system of preparation of media utilising Na2
Sio3 and H3 PO4 was described previously by many
workers4,5,16 but the ratio of the chemicals and tech-
nique used in this experiment were thoroughtly al-
tered. As a result, the medium became transparent
like glass; adjustment of pH was easier, release of
water from the medium was very little and stability
was longer (6 months). The silicate minimal me-
dium (SMM) comprising Na2HPO4,0.750g;KH2PO4
1 .0g; MgSO4,0.150g, NH4NO3, -1.250g and distilled
water 100 ml was sterilised by passing through
sintered glass filter lacking any C source, and so-
lidified with a mixture of Na2 SiO3 and H3 PO4,as
described for KSM above. All the chemicals were
of the Analar or equivalent grade and only double
distilled water was used.
Cultivation Proceduers - The inocula were
prepared by transferring 6-7 colonies from the stock
culture media to glass homogenisers followed by
grinding and suspension in 5 ml phosphate buffer
(pH 7.4) saline and homogenising by shaking with
glass beads. The step was followed by low speed
centrifugation (10 min) to give a uniform
homogeneous suspension. These suspensions were
then matched and standardised with reference to
Macfarland’s standard (1%) and 0.02 ml of these
standardised suspensions was used as inoculum. In
our previous experiment4, we had estimated viable
counts of these inocula; each produced 100-200
isolated colonies on LJM. Repeated subculture was
done simultaneously on LJM, KSM and SMM. The
time taken for growth on a particular medium at
37°C, its increase with the duration of incubation,
colonial morphology, effect’of serial transfers on
growth, microscopic morphology with different
types of strains and acid fastness were noted. The
growth characteristics were also examined with
respect to the capacity for reversion to the original
colonial and morphological (microscopic)
characters, when returned to the LJM. The original,
as well as the KSM and the SMM propagated
cultures were checked for authenticity and purity
by requisite tests4 and for their mycobacterial
characters at different stages of the study.
Electron Microscopy (EM) Study -Electron
microscopy observations of the mycobacteria grown
 SILICON MODULATION OF GROWTH OF M. TUBERCULOSIS
on silicate and conventional media were done with
the help of Jeol JEM-200 CX electron microscope
(Jeol Ltd. Tokyo, Japan) after staining with 1%
solution of uranyl acetate on a carbon coated copper grid.
FINDINGS AND DISCUSSION
Two types of media in which Si was an
important elemental part, with or without any evident
source of C (in KSM and SMM media respectively),
were used to detect if M. tuberculosis could use Si,
at least partially, in exchange for carbon. The results
of the study [Graph 1] showed that colonies of M
tuberculosis (H37RV,H137 Ra, 30 clinical isolates)
appeared earlier on KSM as compared with LJM.
But on KSM, the growths remained uniform for
about 25 days after which they increased in a
significant way t i l l they became confluent. On
subsequent passages, the growth of all the strains
on KSM showed a relative diminution in the final
amount and their maximum attainable growths were
no more than those on the LJM.
A significant observation seems to be that
growth of different strains of M. tuberculosis did
not improve on the C-free, inorganic salt-based
minimal medium (SMM) as compared with C-
containing KSM medium; all the cultures which
could grow on the KSM were also able to grow on
the SMM and they grew in a more satisfactory
manner after repeated (4-6) serial passages on this
medium. The ‘carry over’ of C from the original
medium via the inoculum and the very small amount
of impurities in the chemicals of Analar grade
GROWTH PATTERNS OF H R ON DIFFERENT MEDIA
89
reagents could provide very little C for growth. That
probably resulted in considerably reduced growth
after initial improvement which could improve again
after slow adaptation to the C-free metabolism, after
repeated passages. These findings suggest that M
tuberculosis strains are able to utilise Si facultatively
in the absence of C, either partly or wholly. In one
of our previous experiments, mycobacterial strain
(H37Ra) was shown to contain more silicon by
electron microanalyser when grown on SMM
(24.90%) than when grown on LJM (0.84%)5.
Additional characteristics noted in the present
experiment were the penetration of SMM,
development of branching of the bacillary bodies,
increased beading, presence of coccoid bodies and
minimised acid fastness. One important finding was
that the addition of 0.04 g/d 1 ferric citrate and 0.002
g/dl pyridoxine in the final stage of SMM produced
mycelial forms of mycobactejia. Electron
microscopy observations showed plentiful fibrous
rope like structures and dense granules in the
mycobacteria grown on silicate media. Additional
findings were the decreased size and less lipoidal
bodies in comparison with the mycobacteria grown
on conventional media.
The findings of this study, thus, indicate that Si
utilisation is probably an important marker of M.
tuberculosis pathogen!city. There is plenty of Si in
the lung tissue. Si can, thus, promote growth of M
Tuberculosis in the lungs, either by local action or
by systemic effect. The local action of Si on
M.tuberculosis is either a direct one or via indirect
means [Fig.2], Indirectly, Si can help in the
Graph 1
90 SATADAL DAS AND U.K. CHATTOPADHYAY
Fig. 2
ROLE OF Si IN TUBERCULOSIS
multiplication of M. tuberculosis in monocytes and
macrophages by increasing the permeability of
phagolysosomal membrane leading to diffusion of
enzymes, release of tumour necrosis factor, increased
prostaglandins, decreased succinic dehydrogenase17
and complement fractions18. Si, as a hapten, also
causes immunogenic cell proliferation leading to
formation of antigen/antibody complexes.
Systemically, Si level of more than 3.9 mg/d 1 in
whole blood causes decrease of superoxide
dismutase, catalase and glutathione19 which are more
prbminent in tropical countries due to 50 times more
(about 1000 mg/day) intake of Si than elsewhere.
All these alterations become prominent in silkosis
and lead to peribronchial and perivascular
flbroblastic reaction in the lungs with formation
of fibrous silicose nodules proceeding to
conglomerate silicosis, which always are conclusive
evidence of tuberculosis. However, in this paper,
our main point of discussion is the direct effect
of Si on M. tuberculosis. The uniform early
appearance of colonies of M.tuberculosis on the
silicate media, on the 3rd day of culture occurs
simultaneously with the increased fatty acid
synthase activity which also becomes maximum
on the 3rd day in Si activated cells20. This increased
enzymatic activity probably causes rapid
multiplication of cells leading to the early formation
of colonies on KSM and SMM media in comparison
with the LJM. In this early phase, there was also no
significant alteration of the bacterial morphology.
After the initial rapid multiplication phase, there is
a ‘stationary’ phase, with absent fatty acid synthase
activity, which diminishes after 5 days. Multiplication
of M. tuberculosis then comes to a halt, but after about
20 days or after repeated subcultures, there is
improvement in growth again, probably due to Si
utilisation as indicated by the formation of silicic acid
esterified cell wall21, also known as’wall bound silicate’
which is very difficult to remove from the bacteria22.
The cell multiplication is helped by Si-induced genetic
alterations e.g., by Si-guided nucleic acid synthase23-24,
Si-induced gene expression before translation25 and by
transformation26. Peculiarly, in this late phase, many
bacteria become branched, and after the addition of
small quantities of ferric citrate and pyridoxine, a
typical conversion to mycelial forms occurs due to
some unknown mechanisms. The Si-induced growth
of M. tuberculosis, therefore, may signify a new
aspect of mycobacterial metabolism with, perhaps,
its pathogenicity linked to it.
 SILICON MODULA I ION OF GROWTH OF M. TUBERCULOSIS
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