The ATP measurements were refined to a point where the growth potential of microscopic samples of host-dependent microbes can be measured, even before an increase in microbial numbers has occurred. This tool differs from other biochemical indicators of physiologic states in its sensitivity and in the fact that it can be exploited with unwashed host-dependent and host-grown organisms

Dhople, A. M.

Hanks, J. H.

English

NASA Technical Reports Server

ENERGETICS OF THE HOST-DEPENDENT 
MYCOBACTERIUM LEPRAEMURIUM 
DURING TRANSITION TO A CAPACITY 
FOR EXTRACELLULAR GROWTH 
A. M. Dhople and J. H. Hanks 
Johns Hopkins-Leonard Wood Memorial Leprosy Research Labomtory 
Baltimore, Maryland 
The possibility of cultivating so-called hostdependent microbes (HDM) is 
fascinating. We now focus on Mycobacterium Zepraemurium, the previously 
"obligate intracellular agent" of rat leprosy. Since this organism, until re- 
cently, had been noncultivable for 70 years, it is a model from which one can 
derive principles applicable to  the urgently needed cultivation of M. leprae- 
rnunum. Metabolic or biochemical monitors of the physiologic state of the 
experimental materia are a sine qua non because (1) such methods measure 
inhibitory conditions as readily as favorable ones, and (2) the lack of normal 
enzyme content makes host-dependent organisms particularly susceptible to 
inhibition. 
In order to develop a biologically significant biochemical tool for quantitating 
the energetics and growth potential of unwashed, host-grown microbes during 
the progression, regression, and therapy of diseases such as leprosy, we have 
taken advantage of the fact that ATP can be measured in picogram amounts; 
that it is the source of energy for biosynthesis; and that ATP data can be 
interpreted either in terms of functional biomass or growth potential as 
follo ws: 
6 ATP pools are extremely labile, either used rapidly for bio- 
synthesis, exchanged with related nucleotides, or in damaged 
cells, degraded rapidly by ATPase. 
e Under constant conditions (for example, in vivo), the ATP pools 
within bacterial cells are controlled by the net balance between 
rates of generating energy and rates of biosynthesis. ATP per 
aliquot or per culture measures functional biomass or cell numbers. 
6 Minimal levels of ATP suffice for energy of maintenance; maximal 
levels promote maximum growth rates. Thus, ATP per bacterial 
cell can rank suspensions of a given species in terms of growth 
potential. 
https://ntrs.nasa.gov/search.jsp?R=19760008458 2020-03-22T18:05:49+00:00Z
Because ATP data afford a means for distinguishing between genuine and 
spurious microscopic growth, the ATP system was utilized to investigate two 
reports of the microscopic growth of Mycobacterium lepraemunum in extra- 
cellular environments. In order to  standardize conditions for incubations 
both in vitro and in mice, all inoculums consisted of a few million M. leprae- 
murium cells enclosed in Rightsel-Ito type diffusion chambers. 
Oiwa in 1967 reported slow growth of M. lepraemun'um when adsorbed onto 
siliconed slides and supplied daily with a medium containing 10 percent serum 
and filtered mouse brain extract. All components were prepared and handled 
as for mammalian cell cultures. In procedure 1 it is seen that this medium 
provided redundant sources of nitrogen and vitamins and practically every- 
thing required by fastidious microbes or mammalian cell cultures. 
In the Rightsel system, the chambers were implanted in the mouse peritoneal 
cavity for 50 days before being analyzed. The results obtained in these two 
types of experiments are presented in figure 1. 
IN MICE 
............................................................................ 1OO0/o 
t IN VlTRO 
, 
e\, 
, 
td BAC COUNTS 
*--a ATP 
I I I I I I 
10 20 30 40 50 
TIME (days) 
Figure 1. Bacteria counts and ATP in extracellular M, lepraemurium 
cells incubated in mice and in vitro. 
In the Oiwa medium (see figure I), irrespective of incubation system, modifica- 
tions of brain extracts, or frequency of renewals, the energetics of the 
Procedure 1 
Preparation of Basal Medium and Brain Extract 
Ingredients 
KH, PO, 
Na2HP0,, 12H20 
Sodium glutamate 
Succinic acid 
Magnesium sulphate 
Fe Ammonium citrate 
Sodium citrate 
LAsparagine 
LGlutamine 
Casamino acids 
Glucose 
Sucrose 
Yeast extract 
Cytochrome - c 
Ca-pantathonate 
ATP 
Distilled water to make 
pH 
Modified 
Original (Oiwa) (Basal Medium) 
Sterilization Seitz Seitz 
Add aseptically: 
Penicillin (1 0,000 ylml) 1.0 ml 1.0 ml 
Bovine serum 100 ml 100 ml 
Brain Extract 
Remove brains from young mice; prepare 10 percent (wlv) extract 
in the above medium; centrifuge for 60 min in cold at 
2,000 X g; supernate turbid. 
Centrifuge the supernate for 2 hours in cold at 18,000 X g; 
supernate clear. 
Portion of the above brain extract pasteurized by heating at 
333 K (60°C) for 30 minutes. 
Keep frozen until ready to use. 
M. lepraemunum cells declined steadily and uniformly. After 17 days of in- 
cubation, 72 percent of the original number of bacilli was recovered. Mean- 
while, the ATP per cell had decreased to 9 percent of the original values, 
leaving no possibility that genuine growth could occur. 
Very different results were obtained'from chambers incubated by Rightsel 
in the peritoneal cavities of mice. After 50 days, the bacterial biomass had 
increased 2.7-fold and the ATP per culture 2.5-fold. Since ATP per cell was 
93 percent of the original, the Rightsel system is regarded as the first to 
permit extracellular growth of a so-called "obligate intracellular microbe." 
It follows that M. lepraemunum is capable of being cultivated in bacteriologic 
media. 
In 1972, Nakamura (see procedure 2) from Japan reported that M. leprae- 
murium undergoes microscopic growth in a nonconventional system, charac- 
terized by restricted air volume and the inclusion of 1-cysteine in the medium. 
The autoclaved base, to be used as a control medium, is adequate for the 
growth of tubercle bacilli. Tht, filtered supplements for the complete medium, 
NC-5, are listed in the right-hand column of procedure 2. 
Procedure 2 
The Nakamura System 
A. Physicochemical: 6 ml of medium in #13 S.C. tubes 
leaves 34 percent air space. 
7 mg percent of 1-cysteine, HCL. 
B. Components: 
EK = Base NC-5=Base+ 
salts + % supplements below % 
Na-glutamate 0.90 a-ketoglutaric acid 0.120 
Na-pyruvate 0.30 Cytochrome C 0.012 
Glucose 0.25 Hemin 0.003 
Glycerol 2.00 1-cysteine, HCL 0.007 
Ca-pantothenate 0.006 Serum lO.O/vol 
pH = 7.0 pH of all = 7.0 
Autoclave 12 1 " - 15 min. All filtered through 
pH = 7.0 Millipore MF, 0.22 pm 
Inoculate and immediately distribute 6 ml/tube. 
All percentages = final concentrations in the medium. 
Figure 2A demonstrates why quantitation of functional biomass (F Bm) is 
more instructive than microscopic counts or, indeed, plate counts of microbial 
species that possess a high plating efficiency. When the in vivo grown cells of 
M. lepraemurium are inoculated in the Kirchner semisynthetic base (EK), the 
F Bm of M. lepraemurium fell to 50 percent of the original at the third day 
F 
NC-5 Medium 
5 
F. Calf Serum 
3 20 40 60 
lncubat ion (Days) 
A 
F 
NC-5 Medium 
F. Calf Serum 
Incubation (Days) 
B 
Figure 2. A-ATP per culture. B-Biomass per culture. 
and declined progressively thereafter. This curve duplicates the slopes shown 
earlier with the mouse brain extract medium. Meanwhile, in the presence of 
the physicochemical conditions and catalysts provided by the supplements 
in the Nc-5 medium, the F Bm falls only to 75 percent of the original, that 
is, one half as far. Thus, within three days, it was known that the critical 
conditions and catalysts of the Nakamura system resided in the supplements, 
not in the base. By the 10th day in the complete medium (Nc-5 curve), the 
cultures had reestablished the energy levels utilized by M. lepraemurium for 
growth in mouse tissues, that is, the cells had successfully reconstructed the 
leaky in vivo type membranes into those which are competent for extra- 
cellular growth. On the NC curve between the 4th and the 10th day, M. 
lepraemurium already had spent 6 days in expanding its metabolic equipment 
and membrane activity and possibly had accomplished increases in cell volume. 
Within 20 days the rate of expanding F Bm had improved slightly. After 20 
days fetal calf serum is shown to be superior to goat serum. Thus, the goals 
of such experiments had been accomplished within three weeks. 
After 10 days of lag (figure 2B), microscopic measurements of total biomass 
(cell numbers X their average length) increased in parallel with the ATP curves, 
but the cultures had to be incubated at least twice as long to guarantee that 
significant growth had occurred. Experience has shown that microscopic 
quantitations of the total Bm of M. lepraemurium cultures is exceptionally 
tedious. M. lepmemurium is one of the mycobacterial species that elongate 
during the early stages of growth. If the work required to  enumerate bacterial 
cells by the pin head method is regarded as one unit, determination of the 
average length of the cells requires three units of effort. In short, valid micro- 
scopic measurements of the biomass of M. lepraemurium per culture requires 
not one, but four units of labor. 
Physiochemically, M. lepraemurium hitherto has proven to be noncultivable 
in many thousands of experiments of dozens of investigators. Any system 
which permits the growth of such an organism in vitro will be nonconvention- 
al in some definable respect. In the Nakamura system (see table 1): 
0 The unique feature is in filling 66 percent of the tube space with 
a medium containing cysteine. 
In the lower right section of table 1 is shown that, when the ratio 
of air in the tube was increased, the onset of growth was severely 
repressed. 
The left section of table 1 ranks the contribution made by each compound in 
the medium. 
In order to facilitate investigations with host-grown microbes we outline two 
propositions : 
1. Metabolic or biochemical indicators of physiologic states are more 
significant than microscopic or plate counts, because (a) such 
methods measure inhibitory conditions as readily as favorable 
ones, (b) they help to study the physiologic sag and thus the 
essentials can be learned without waiting for growth to occur, 
and (c) the lack of normal enzyme versatility makes host-dependent 
organisms particularly susceptible to inhibitions. 
2.  Given useful sources of both nitrogen and carbon and relatively 
constant conditions, ATP per culture measures F Bm, the param- 
eter that should correlate with plate and microscopic counts. 
Physiologic monitors are required to analyze the transition of in vivo-grown 
microbial cells from noncultivable to cultivable state. Even assuming the in- 
vestigation of any microbe with high plating efficiency, plate or microscope 
counts cannot analyze the crucially important events which are fundamental 
to the adaptation of a host-dependent microbe to existance in vitro. Thus, 
the physiologic sag of the host-dependent microbe has been defined as analo- 
gous to, but far more severe than, the sag or lag which occurs when minimal 
inoculums of cultivable microbes are transferred to a new medium. The 
observations here demonstrated that measurement of F Bm produces informa- 
tion of the type that cannot be gained by plate or microscopic counts, also 
that significant results can be obtained during half the incubation time required 
to obtain the earliest information by means of biologic methods. 
In conclusion, I wish to summarize two points. 
1. It appears that ATP measurements have been refined to  a point 
where the growth potential of microscopic samples of so-called 
Table 1 
The Merits of Conditions and Components 
of the Nakamura System 
Method: Starting with the complete NC-5, each item was 
deleted in turn. 
Item 
NC-5 
a-Ketoglutaric 
Hemin 
Cytochrome C 
FCS 
1-cysteine, HCI 
Na-pyruvate 
Ca-pantothenate 
Na-glutamate 
% of NC-5 = the percentage gain when each item was restored 
to  the deficient medium. 
host-dependent microbes can be measured, even before an increase 
in microbial numbers has occurred. 
% 
NC-5 
100 
62 
59 
41 
40 
2 9 
24 
18 
15 
2. This tool differs from other biochemical indicators of physiologic 
states in its sensitivity and in the fact that it can be exploited with 
unwashed host-dependent and host-grown organisms. We trust 
that the present studies have created new means of investigating 
the energetic integrity and the biosynthetic potential in M. leprae 
during experiments of the type demonstrated and also during the 
progression, regression, and therapy of the disease in humans. 
Rank 
2 
3 .  
5 
6 
- -  Item 
Glycerol 
Glucose 
Depth of 
medium 
6 rnl 
5ml  
3 m l  
1 rnl 
%of 
,NC-5 
6 
5 
% of air 
space 
34 
45 
67 
89 
Rank 
7 
%of 
NC-5 
100 
88 
5 2 
3 2 


Energetics of the host-dependent Mycobacterium Lepraemurium during transition to a capacity for extracellular growth

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server