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Original Article Ind. J. Tub.

2000, 47, 87



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

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

MATERIAL AND METHODS Cultivation Proceduers - The inocula were

prepared by transferring 6-7 colonies from the stock
M.tuberculosis strains - The strains used were culture media to glass homogenisers followed by
H37Rv.H37,Ra and 30 clinical isolates. Pure grinding and suspension in 5 ml phosphate buffer
cultures (pH 7.4) saline and homogenising by shaking with
were maintained on Lowenstein-Jensen Medium glass beads. The step was followed by low speed
(LJM) and were checked for purity4. centrifugation (10 min) to give a uniform
homogeneous suspension. These suspensions were
Culture Media - Lowenstein Jensen Medium then matched and standardised with reference to
was prepared by usual procedure14. The Kirschner Macfarland’s standard (1%) and 0.02 ml of these
silicate medium (KSM), in each MacCartney bot- standardised suspensions was used as inoculum. In
tle, comprised 3 ml of Kirschner Medium15 concen- our previous experiment4, we had estimated viable
trated 2.5x, plus 3 ml Na2Sio3 (Fluka, Switzerland) counts of these inocula; each produced 100-200
11.8 g/dl used as a solidifying base, with addition isolated colonies on LJM. Repeated subculture was
of H3 PO4 (1.5 ml. of 16% V/V aqueous solution). done simultaneously on LJM, KSM and SMM. The
The system of preparation of media utilising Na2 time taken for growth on a particular medium at
Sio3 and H3 PO4 was described previously by many 37°C, its increase with the duration of incubation,
workers4,5,16 but the ratio of the chemicals and tech- colonial morphology, effect’of serial transfers on
nique used in this experiment were thoroughtly al- growth, microscopic morphology with different
tered. As a result, the medium became transparent types of strains and acid fastness were noted. The
like glass; adjustment of pH was easier, release of growth characteristics were also examined with
water from the medium was very little and stability respect to the capacity for reversion to the original
was longer (6 months). The silicate minimal me- colonial and morphological (microscopic)
dium (SMM) comprising Na2HPO4,0.750g;KH2PO4 characters, when returned to the LJM. The original,
1 .0g; MgSO4,0.150g, NH4NO3, -1.250g and distilled as well as the KSM and the SMM propagated
water 100 ml was sterilised by passing through cultures were checked for authenticity and purity
sintered glass filter lacking any C source, and so- by requisite tests4 and for their mycobacterial
lidified with a mixture of Na2 SiO3 and H3 PO4,as characters at different stages of the study.
described for KSM above. All the chemicals were Electron Microscopy (EM) Study -Electron
of the Analar or equivalent grade and only double microscopy observations of the mycobacteria grown
distilled water was used.

on silicate and conventional media were done with reagents could provide very little C for growth. That
the help of Jeol JEM-200 CX electron microscope probably resulted in considerably reduced growth
(Jeol Ltd. Tokyo, Japan) after staining with 1% after initial improvement which could improve again
solution of uranyl acetate on a carbon coated copper grid. after slow adaptation to the C-free metabolism, after
repeated passages. These findings suggest that M
FINDINGS AND DISCUSSION tuberculosis strains are able to utilise Si facultatively
in the absence of C, either partly or wholly. In one
Two types of media in which Si was an of our previous experiments, mycobacterial strain
important elemental part, with or without any evident (H37Ra) was shown to contain more silicon by
source of C (in KSM and SMM media respectively), electron microanalyser when grown on SMM
were used to detect if M. tuberculosis could use Si, (24.90%) than when grown on LJM (0.84%)5.
at least partially, in exchange for carbon. The results Additional characteristics noted in the present
of the study [Graph 1] showed that colonies of M experiment were the penetration of SMM,
tuberculosis (H37RV,H137 Ra, 30 clinical isolates) development of branching of the bacillary bodies,
appeared earlier on KSM as compared with LJM. increased beading, presence of coccoid bodies and
But on KSM, the growths remained uniform for minimised acid fastness. One important finding was
about 25 days after which they increased in a that the addition of 0.04 g/d 1 ferric citrate and 0.002
significant way t i l l they became confluent. On g/dl pyridoxine in the final stage of SMM produced
subsequent passages, the growth of all the strains mycelial forms of mycobactejia. Electron
on KSM showed a relative diminution in the final microscopy observations showed plentiful fibrous
amount and their maximum attainable growths were rope like structures and dense granules in the
no more than those on the LJM. mycobacteria grown on silicate media. Additional
findings were the decreased size and less lipoidal
A significant observation seems to be that bodies in comparison with the mycobacteria grown
growth of different strains of M. tuberculosis did on conventional media.
not improve on the C-free, inorganic salt-based
The findings of this study, thus, indicate that Si
minimal medium (SMM) as compared with C-
utilisation is probably an important marker of M.
containing KSM medium; all the cultures which
tuberculosis pathogen!city. There is plenty of Si in
could grow on the KSM were also able to grow on
the lung tissue. Si can, thus, promote growth of M
the SMM and they grew in a more satisfactory
Tuberculosis in the lungs, either by local action or
manner after repeated (4-6) serial passages on this by systemic effect. The local action of Si on
medium. The ‘carry over’ of C from the original M.tuberculosis is either a direct one or via indirect
medium via the inoculum and the very small amount means [Fig.2], Indirectly, Si can help in the
of impurities in the chemicals of Analar grade
Graph 1

Fig. 2


multiplication of M. tuberculosis in monocytes and enzymatic activity probably causes rapid

multiplication of cells leading to the early formation
macrophages by increasing the permeability of
of colonies on KSM and SMM media in comparison
phagolysosomal membrane leading to diffusion of with the LJM. In this early phase, there was also no
enzymes, release of tumour necrosis factor, increased significant alteration of the bacterial morphology.
prostaglandins, decreased succinic dehydrogenase17
and complement fractions18. Si, as a hapten, also After the initial rapid multiplication phase, there is
causes immunogenic cell proliferation leading to a ‘stationary’ phase, with absent fatty acid synthase
formation of antigen/antibody complexes. activity, which diminishes after 5 days. Multiplication
Systemically, Si level of more than 3.9 mg/d 1 in of M. tuberculosis then comes to a halt, but after about
whole blood causes decrease of superoxide 20 days or after repeated subcultures, there is
dismutase, catalase and glutathione19 which are more improvement in growth again, probably due to Si
prbminent in tropical countries due to 50 times more utilisation as indicated by the formation of silicic acid
(about 1000 mg/day) intake of Si than elsewhere. esterified cell wall21, also known as’wall bound silicate’
All these alterations become prominent in silkosis which is very difficult to remove from the bacteria22.
and lead to peribronchial and perivascular
The cell multiplication is helped by Si-induced genetic
flbroblastic reaction in the lungs with formation
alterations e.g., by Si-guided nucleic acid synthase23-24,
of fibrous silicose nodules proceeding to
Si-induced gene expression before translation25 and by
conglomerate silicosis, which always are conclusive
transformation26. Peculiarly, in this late phase, many
evidence of tuberculosis. However, in this paper,
bacteria become branched, and after the addition of
our main point of discussion is the direct effect
of Si on M. tuberculosis. The uniform early small quantities of ferric citrate and pyridoxine, a
appearance of colonies of M.tuberculosis on the typical conversion to mycelial forms occurs due to
silicate media, on the 3rd day of culture occurs some unknown mechanisms. The Si-induced growth
simultaneously with the increased fatty acid of M. tuberculosis, therefore, may signify a new
synthase activity which also becomes maximum aspect of mycobacterial metabolism with, perhaps,
on the 3rd day in Si activated cells20. This increased its pathogenicity linked to it.

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