the effects of simulated altitude on the intestinal flora
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THE EFFECTS OF SIMULATED ALTITUDE ON THE
INTESTINAL FLORA OF GUINEA PIGS
APPROVED:
Major [professor
6%,/ Minor Professor
r
Director of the Department of Biology-
Dean of the Graduate School
THE EFFECTS OF SIMULATED ALTITUDE ON THE
INTESTINAL FLORA OF GUINEA PIGS
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
By
Noel R. Funderburk, B. S.
Denton, Texas
May, 1969
TABLE OP CONTENTS
Page
LIST OP TABLES iv
INTRODUCTION 1
MATERIALS AND METHODS 6
RESULTS 9
DISCUSSION 17
APPENDIX 21
LITERATURE CITED 0
iii
LIST OP TABLES
Table • Page
1 Organisms Frequently Isolated 10
2 Colony Characteristics of the Diphtheroid Organisms . 10
3 Changes in Number of Types of Bacteria at 3^0 mm. Mercury Pressure and 100$ Oxygen Concentration . 12
Ij. Changes in the Flora During the Week After Removal from Chambers at 3®0 ram. Hg. Pressure and 100$ Oxygen 13
5 Changes Produced by Chamber Exposure to 380 mm. Hg. Pressure and Room Air and by Chamber Exposure with Normal Atmospheric Pressure and Air 15
6 Changes in the Flora During the Week After Removal from the Chambers with Atmospheric Pressure and Air and with 3^0 mm* Hg. Pressure and Room Air 16
. INTRODUCTION
When man is placed in space for extended periods of
time, microbiological problems may arise. Potential pro-
blem areas are the possibility of having bacterial pathogens
brought aboard the spacecraft, decreased body resistance
to infections, and the upset of normal ecologic relation-
ships of the bacteria in and on the body. Relatively
little research has been done in these areas. Such stud-
ies are desirable because of the number of factors which
are necessarily altered from man's normal state which
may result in changes in the microbial ecology which
accompanies man in all situations. These factors are a
closed environment, change in altitude, change in atmos-
pheric pressure, and a different oxygen concentration.
When one considers that the disease candidiasis and other
intestinal disturbances frequently follow upset of the
flora by antibiotic treatment (7), the possible ecologic
change in the, flora produced by space flight could become
of great importance. Another potential area of concern is
that in space flights of several months or more, and with
changes in the intestinal flora, the host could be deprived
of essential vitamins normally supplied by organisms of
the intestinal tract. These bacteria are known to syn-
thesize vitamins K, B6, B12, biotin, and folic acid.
In the case of vitamin K, which is e.ssential for hemostasia
in the human, the major source may be the intestinal bac-
teria rather than the diet (12).
Studies to date have failed to indicate that there
is a truly indigenous flora of animals in the absence of
reinoculation (5, 6, 8). The isolation of spacecraft, com-
bined with the natural and possibly accelerated decay rate
of bacteria on surfaces, and efficient waste disposal and
air filtration systems, provide "locked-flora" conditions
similar to those used with germ-free and gnotobiotic
animals.
In 191+1 Nelson (8) performed experiments on the intes-
tinal flora of guinea.pigs, in which the animals were kept
in sterile cages with screen wire floors and isolated from
contamination by food, water, air, or other animals. The
flora of these animals underwent gradual simplification
in types of organisms. After three months isolation, only
six of the original ten types of organisms remained.
After twelve months only two types remained, which were
both gram-positive cocci. It was also noted that diffi-
culty was experienced in maintaining these animals after
simplification of flora because of the development of
avitaminosis, in spite of the administration of vitamins
C, D, B^, and B complex.
According to Luckey (6), these findings of Nelson
were confirmed by using white rats. After the animals had
been isolated for three months it was found that the number
of different bacterial species in the intestinal tract
was greatly reduced.
Luckey {$) has reported that bacteria-free animals
which had become contaminated with one or two bacterial
species became once more bacteriologically sterile when
kept in sterile environments.
In an isolation experiment with humans, Gall and
Riely (6) found a shift in both the aerobic and anaerobic
intestinal flora. Shigella species, enteropathogenic
serotypes of Escherichia coli, and Candida species were
frequently cultured.
Coronado et ail.. (2) studied six men for fifty-six
days in simulated spacecraft conditions with diets similar
to those given astronauts. Their studies indicate an
ecologic change in the intestinal flora with decreased
numbers of enterococci. No pathologic state was produced
by this change, however.
Lechtman (ij.) states that during a symposium on space
microbiology held at the 1966 annual meeting of the American
Society of Microbiology at Los Angeles, California,
Moyer also reported on interchange of intestinal flora with organisms of the Providence group, , .Proteus species, Aerobacter species, and Pseu-domonas' species. There were no apparent oral or intestinal upsets during lij. to 30 days of exposure to simulated space-cabin environments.
ll.
One point of interest made by Gall and Riely was that isolation appeared to reduce the numbers of different kinds of species, which should in-dicate a definite ecological upset.
Ehrlich and Mieszkuc (3) have reported that
Resistance to infections initiated by a respiratory challenge with. Klebsiella pneumoniae or influenza virus appeared to be reduced in mice exposed to a simulated space cabin environment consisting of 5 psi and 100 per cent oxygen atmos-phere. The reduced resistance, manifested by enhanced mortality was observed in mice challenged with the infectious agents 1 hour to seven days before entry into the space cabin environment. Increased mortality rates were also obtained as the result of infectious challenge during exposure to the space cabin environment. However, and adaptation to this stress, in terms of suscep-tibility to influenza virus, appeared to be pre-sent upon 36 day exposure to the 5 psi environ-ment. The time at ground level conditions re-quired for the recovery from the stress of the space cabin environment was related to the duration of the exposure and the infectious agent used for the challenge.
Schmidt (9) found that mice exposed to simulated
spacecraft environment for two weeks prior to cutaneous
inoculation with Staphylococcus aureus had reduced resist-
ance to the production of skin lesions.
In another study (10) Schmidt found that simulated
spacecraft environment increased the susceptibility of
mice to mengo virus. Increased susceptibility to
Pasteurella tularensis infection was found under hypobaric, and
hypoxic or hyperoxic conditions but not with normoxic
conditions.
Because of the relatively little research performed
on the effects of spacecraft environment on the intestinal
flora, studies were undertaken to determine if changes
do occur and to attempt to determine their nature and
specific factors responsible for the changes. In present-
day space vehicles, man is exposed to conditions varying
from the normal state including, reduced barometric
pressure, increased oxygen concentration, and isolation in
small groups. The purpose of this paper is to report
the results of studies on the aerobic, mesophilic intes-
tinal flora of guinea pigs subjected to conditions
similar to those encountered by man in spacecraft.
MATERIALS AND METHODS
Guinea pigs used in these studies were random-bred
Hartley strain, male and female animals, weighing 25>0-500
g *ams, and were obtained from Field Caviary, Griffin,
Georgia. The animals were fed Purina Guinea Pig Chow and
w$.ter ad libitum throughout the experiments. Marking the
animals for identification was done by shaving the hair
from the right flank and tattooing numerals. This was
done by pricking india ink into the skin with a fine pen
w]fiich had been honed to a sharp point. i
Hypobaric chambers were used to simulate a space-
craft environment. The largest of the three chambers was
capable of containing twelve guinea pi;gs housed in two
cages. The other two chambers could accommodate six ani-
mals each. Feeders containing sufficient food for the
animals for one week were placed in the cages. Water was
given in small bowls which could be filled daily by means
of external fittings on the chambers. During these tests
the chamber was maintained at a pressure of 370-380 mm.
mercury (equivalent to 18,000 feet altitude) with oxygen
concentration of 100%, with constant removal of carbon diox-
ide . In addition experiments were performed at atmospheric
pressure and oxygen concentration and also at 370-380 mm.
mercury, with room air being admitted to the chamber
instead of oxygen.
Pans of approximately 2$% sodium hydroxide solution
were placed in the large chamber to absorb carbon dioxide,
and oxygen was admitted at an approximate rate of 1 to l.£
liters per minute. With the smaller chambers oxygen was
introduced at an approximate rate of 0.75 liters per min-
ute. No carbon dioxide absorbant was needed in these
smaller chambers because of a more rapid flushing of the
atmosphere by the incoming oxygen. The concentration of
carbon dioxide was monitored during the tests by placing
in the chamber an open test tpbe containing a solution
of methyl red and bromthymol blue which had been boiled and
titrated to a green color with sodium hydroxide. When this
solution was exposed to concentrations of carbon dioxide
found in the air, the color was yellow-green. When exposed
to exhaled air, the color became yellow, and finally orange,
The animals were kept at these conditions for one week
except for one experiment which was terminated-after five
days due to mechanical failure.
Cultures were made prior to placing the animals in
the chamber, and immediately after removing the animals
from the chamber, and one or two weeks later. Cultures
were prepared by taking rectal swabs on sterile cotton-
tipped applicators and emulsifying the material in tryptic
soy broth (Difco). Pour serial ten-fold dilutions were
prepared from the broth suspension by transferring'0.1 ml.
8
of the suspension into a 9.0 ml. tube of broth, mixing,
and again transferring as before. One tenth ml. of the last
three dilutions was transferred onto the surface of agar
plates and spread with a bent glass rod which had been
sterilized by dipping in alcohol and flaming.
The media used were obtained from Difco Company and
prepared according to directions. Plates of tryptic soy
agar, eosin-methylene blue agar, and mannitol-salt agar
were used for dilutions of 10^ and 10^. Tryptic soy- agar
and mannitol-salt agar were inoculated from the 10 - dilution.
After inoculation, the plates were incubated at 37 Celsius
for forty-eight hours. Ths growth was then counted and
identified by standard methods such as colonial morphology,
gram-reaction, and other tests as indicated by Bergey's
Manual (1).
The percentage of the flora represented by each type
of bacteria was then calculated.
RESULTS
The guinea pigs used in these experiments were divided
into four groups in order to test the effects of various
conditions on the intestinal flora. One group was placed
in hypobaric chambers at a pressure of 380 mm. mercury
and 100$ oxygen concentration. Another group of animals
was also placed in the chamber at 38O mm. mercury but with
normal atmospheric concentrations of oxygen. A third group;
of animals was also placed in'the chamber but at normal
atmospheric pressure and oxygen concentration. The last
group was kept in ordinary housing in open cages to serve
as untreated controls.
The aerobic intestinal flora of the guinea pig was
found to be normally composed of gram-positive cocci and
bacilli in predominance, with gram-negative organisms
rarely found. A list of the organisms frequently isolated
is given in Table 1. This is in agreement with the findings
of other workers (8, 11).
The organisms listed as diphtheroid bacilli were small
gram-positive rod-shaped organisms which contained meta-
chromatic granules and were commonly found in arrangements!
resembling Chinese letters or palisades. Five different
colony types on tryptic soy agar were found and were desig-
nated by Roman numerals. The characteristics of each type
10
are given in Table 2.
TABLE 1.. ORGANISMS FREQUENTLY ISOLATED
Staphylococcus species, Mannitol Fermenting
Staphylococcus species, Mannitol Non-Fermenting Streptococcus species, Fecal Types Diphtheroid bacilli Micrococcus species Gaffkya species Bacillus species
Those animals which were placed at simulated altitude
with 100$ oxygen concentration showed a definite change
in flora. This change was seen as decreased numbers of
different types of bacteria or by change in the types of
TABLE 2. COLONY CHARACTERISTICS OF THE DIPHTHEROID ORGANISMS
Type I Colonies 3-4- nun* in diameter, flat, and granular in appearance with irregular edges, ivory in color, and dry and friable in consistency.
Type II Colonies 3-]| mm. in diameter, umbonate and granu-lar in appearance with irregular edges, orange in color and dry and friable in consistency.
Type III Colonies 2-3 in diameter, convex, and smooth in appearance with entire edges, white in color and butyraceous in consistency.
Type IV Colonies 2-3 mm. in diameter, convex, and smooth in appearance with entire edges, deep yellow to orange in color and butyraceous in consistency. ,
Type V Colonies 0.8 - 1 mm. in diameter, effuse, and finely granular in appearance with fimbriate edges, white in color, and adherent to the agar surface.
11
bacteria. Of this group, thirteen out of eighteen animals
(13/18) or 72.2% lost one or more types from the flora,; as
compared with the control group, in which 3/18, or 37.%%,
of the animals showed a decreased in numbers of types. In
addition, 8/13,, or t h e animals in this, test group
whose flora became simplified in types, changed by loss of
two or more types, whereas none of the control animals'
flora decreased by two organisms. The animals in this group
exposed to simulated spacecraft environment in which the .
number of types of bacteria did not change during expos-
ure made up the remaining five animals, or 21.8%. The con-
trol group of animals whose flora did not change in num-
ber of types constituted 3/8, or 37«5$> of the group.
None of the animals exposed to simulated spacecraft condi-
tions were found to have gained numbers of types of bacteria
during the exposure, but two out of eight, or 2$%, of the
control animals increased in numbers of different types
during this period of tjrne. These data are summarized in
Table 3-
A comparison of the flora of the animals exposed to
simulated spacecraft environment, with that of the control
animals reveals that 11/18, or 61%, of the test groups and
5/8, or 62.$%, of the control animals had a change in the
predominant type of organisms during the test period.
There is essentially no change found in this aspectrof
the flora by exposure to the test conditions.
12
An attempt was made to determine if the total numbers
of bacteria in the intestinal tract was also changed by
the simulated spacecraft conditions. The average number
of bacteria per milliliter of the broth suspensions pre-
pared from rectal swabs was determined from Table V of the
Appendix. The average number of bacteria per milliliter •
of the cultures taken before exposure was x^lO?.
After exposure at 380 *>im« mercury pressure and 100$ oxygen
concentration for one week, the average number increased
to 1.30 x 10® organisms per milliliter. One week after
the animals had been returned to normal housing the number
had decreased to I4..I8 x 10^ per milliliter. Even though
rectal swabs are not we'll suited to quantitative determi-
nations, this apparent increase appears to be significant.
TABLE 3. CHANGES IN NUMBER OP TYPES OP BACTERIA AT 380 MM. MERCURY PRESSURE AND
100$ OXYGEN CONCENTRATION
Text Conditions Increased in
Number of Types Kept Same
Number of Types Decreased in Number of Types
Animals exposed in chamber to 380 mm. Hg. and 100$ oxygen 0 27.8fo 72.2$
Control animals kept in normal hous ing 2£.0$ . . 37-5 37.5
13
An examination of the cultures taken immediately after
the animals were removed from the altitude chamber and
compared with those taken one week after the animals had
been returned to ordinary housing conditions shows that
the intestinal flora had increased in types of organisms
one week later, indicating that the changes produced by the
altered environment were being reversed. The flora increased
in numbers of different types in 9/lIj., or 61$, of the animals,
remained the same in J4./II4., or 29%, and decreased in I/II4., or
7$. The control animals whose flora increased in complex-
ity were 1/7, or 11}..2$, of the group. Those which kept the
same number of types were J4./7> or 67«3$» and 28.$% had a
decrease in types. One death occurred in this group due
to an unknown cause. These results are presented in
Table 1}..
TABLE ij.. CHANGE IN THE FLORA DURING THE WEEK AFTER REMOVAL FROM CHAMBERS AT 38O MM. HG. PRESSURE AND 100$ OXYGEN
Increased in Kept Same Decreased in Group Number of Types Number of Types Number of Types
(0 Test Group 61}..Ofo 29.0% 7«0;
Control Group 11}..2 67*3 28.$
In order to determine whether reduced barometric
pressure, isolation, altered oxygen concentration, or a
combination of these factors was responsible for the changes
found in the intestinal flora, other groups were tested.
lij-
under conditions varying in one or more of the above
factors.
One groups of five animals was placed in the altitude
chamber' for one week with 380 mm. mercury pressure, but
with atmospheric air instead of 100$ oxygen in the chamber.
Under these conditions, 2/5, or of the animals' flora
decreased in numbers of different types of bacteria, the
flora of an equal number animals retained the same number
of types, and the flora of the other animals increased in
number of types.
Another group of five animals was also placed in the
altitude chamber for one week, but both* the barometric
pressure and atmospheric composition were kept at ground
level. In this group the flora decreased in numbers of
types in 2/5, or k-0%, of the animals, and remained the same
in 3/5 > or 60%. Table 5 presents these data.
It can be seen that more pronounced changes in the
flora occurred in the group of animals tested with both
decreased barometric pressure and 100$ oxygen concentration,
than .in any other group of animals. In comparison with the
control animals, it is noted that changes were produced by
isolation in the chamber and by decreased barometric pres-
sure. Neither of these factors nor both combined could pro-
duce changes equal to those produced by all three factors
under consideration.
In both of these groups, 60$ of the animals' flora
underwent a change in predominant type of organism during
the test period. This is in agreement with the control
group and with the group exposed to 100/£ oxygen and pres —
sure of 380 mm, mercury.
TABLE $. CHANGES PRODUCED BY CHAMBER EXPOSURE TO 380 MM". HG. PRESSURE AND ROOM AIR AND BY CHAMBER EXPOSURE WITH NORMAL ATMOSPHERIC PRESSURE AND AIR
Increased in Kept Same Decreased in Test Conditions Number of Types Number of Types Number of Types
Animals exposed in chamber to 380 mm. Hg. pressure with ^ room air 20.0$ i}.0.0$ lj.0.0$
Animals exposed in chamber to normal atmos-pheric pres- . _ sure and air 0 60.0 4O.O
The cultures taken from the animals in the chamber at
760 mm. mercury and room air one week after removal from
the chambers are not found to change to a great extent.
A decrease in number of types by one type only was found
in 2/5, ov 1+0$, of the animals. Another 2/5, or ij.0$, were
found to have kept the same number of types and 1/5, or 20$,
was found to have increased by one type.
The flora of V 5 , or 80$, of the animals exposed to
38O mm. mercury and 21$ oxygen concentration was found
16
to have increased in numbers of types one week after
return to normal housing. The remaining animal in this
group was found to have kept the same number of types.
These data may be found in Table 6.
TABLE 6. CHANGES IN THE FLORA DURING THE WEEK AFTER REMOVAL FROM THE CHAMBERS WITH ATMOSPHERIC PRESSURE AND AIR AND
WITH 380 MM. HG. PRESSURE AND ROOM AIR
. Increased in Kept Same Decreased in Test Conditions Number of Types Number of Types Number of Types
Animals exposed in chamber to 380 mm. Hg. pressure with room air 80.0$ 20.0fo 0
Animals exposed in chamber to atmospheric pressure and air 20.0 40.0 14.0.0 fo
During the week after removal of the animals from
the chamber, 3/18> or 16.Q%, of the animals which had been
kept at 380 mm. mercury and 100$ oxygen concentration
were found dead. The cause of death in two of these ani-
mals was found to be pneumonia, and that of the third
undetermined. No deaths were recorded among the other
two groups of animals placed in the chambers without increased
oxygen concentration. One de&th due to unknown cause occur-
red among the control animals.
. DISCUSSION
The results of these experiments indicate that an
ecologic change in the aerobic, mesophilic flora of the
intestinal tract is produced by simulated spacecraft en-
vironment. It is reasonable to assume that the anaerobic
and fungal populations will also be affected under these
conditions, if not directly by the environment, then by
the change" in that portion of the flora which was studied
or by physiologic changes in the host, although, this work
did not deal with any anaerobic organisms.
These findings that conditions of isolation, decreased
barometric pressure, and increased oxygen concentration
produce a-simplification in the intestinal flora in terms
of numbers of different types cannot be due to the effects
of a "locked-flora" system alone. The changes found here
occurred much more rapidly than those reported by others
with such systems (5,8), and control animals which were
kept in isolation did not produce the same changes as were
obtained under simulated spacecraft environment. Neither
can the effects of hypobaric pressure account for the
changes, as was shown by the group of animals placed -under 1
atmospheric concentrations of oxygen and 380 mm. mercury
pressure. The combination of isolation, hypobaric pres-
sure, and increased oxygen concentration must act additively.
17
18
These changes are of considerable importance to future
space flights. If similar changes occur in the flora of
humans under these conditions, man might be expected to
experience intestinal upsets, nausea, and perhaps more
severe disease as a result. This could prevent completion
of assigned tasks during the flight. Luckey (£) has re-
ported that bacteria-free guinea pigs or animals with only
one or two bacterial types die of bacterial shock when
placed in ordinary open cages with other animals. If man
does not suffer immediately from the simplification of
the flora and the space flight is of months or longer
duration, the crew could possibly suffer disease upon
return to earth, through reinoculation of the intestinal
tract.
Man may not experience changes in either type or
degree similar to these found in guinea pigs. The intes-
tinal flora of man is greatly different and this flora may
not have the same susceptibility to these conditions. In
addition, the volume of the intestinal tract of man is
many times larger than that of the test animals. There-
fore a longer period of time may be required for loss of
any one type of bacterium, due to the massive numbers pre- '
sent in man. The diets fed the astronauts also may be a
factor because those used at the present time are not
sterilized and are likely to contain some bacteria indigenous
to man. This could effect a frequent reinoculation of the
men. By another me<i.:.,j the diets now being used could result
19
in increased possibility of intestinal upset. Changes in •
the diet have long been recognized as affecting the intes-
tinal flora. These changes when combined with changes pro-
duced by the environment could enhance intestinal disturbances
In addition, the food could serve as a vector for recognized
pathogenic bacteria. If the flora of the astronauts were
to be reduced in amount or type and they then became infected
with organisms such as Salmonella or Shigella species, the
resulting"disease might be much more severe due to lack
of normal intestinal flora, and also to possibly reduced
defense mechanisms.
The phenomenon of "bacterial-flooding" has been re-
ported by Luckey (5) in animals living with reduced intes-
tinal flora. Flooding is the displacement of bacteria from
the intestinal tract to other areas of the body, most
commonly the oral and respiratory tract. It is of interest.
to note that three of the animals which had been exposed
to simulated spacecraft atmosphere and whose flora had
been reduced in types, died during the following week.
Escherichia coli and fecal streptococci were isolated from
the lungs of these animals. Apparently this could be another
hazard of space travel.
It was found that the predominant organism remaining :
after the flora became simplified was frequently not the
same organism which was predominant before the animal
was subjected to the test conditions. In addition it was
20
not possible to identify any particular types as becoming
dominant in a majority of the animals. This suggests
that a number of variable factors are important in deter-
mining which organisms survive and which do not, besides
the virulence of the organism and the number of that type
in the intestinal tract.
It was noted that there was an apparent increase in
numbers of bacteria, coinciding with decrease in number of
types. It seems possible that with decreased interaction
between different types, those which remained were allotted
to increase in actual numbers. This information, however,
is only apparent because of the techniques used. Further
studies would have to be done using quantitative fecal sam-
ples to state unequivocally that such changes do occur.
These studies have produced evidence that the com-
biantion of hypobaric pressure, isolation, and increased
oxygen concentration can produce definite ecologic changes
in the intestinal flora of guinea pigs. Whether similar
changes may occur in man over long periods of time is yet
to be learned, but the need for such,studies has been in-
dicated by these experiments.
APPENDIX
TABLE I. RESULTS OP CULTURES TAKEN PROM GUINEA PIGS EXPOSED TO 380 MM.MERCURY PRESSURE AND 100$ OXYGEN
CONCENTRATION CONFINED IN CHAMBER
ANIMAL 1
ORGANISM 12/13/68* 12/20/68 12/27/68
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
•Ji-Animal in chamber from 12/13/65 to 12/20/66.
i|.8?
7.0 11.2
76.7 0.1|
6.0$
0.1 3.8 lk.$
75.6
2.6$ 6.6 1.0
•1.8 88.0
ANIMAL 2
ORGANISM 10/16/68# 10/23/68 10/30/68
97.3?
2.7
Type Type Type
I II III IV'
99.9$
0 .1
Died Cardiac Puncture
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcu3
Enterococcus* Diphtheroid bacillus Type Diphtheroid bacillus Diphtheroid, bacillus Diphtheroid bacillus Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
-::-Anima 1 in chamber from 10/16/65 to 10/23/68'
21
22
TABLE I - Continued
ANIMAL 3
ORGANISM 10/16/68* 10/23/68 10/30/68
Mannitol Fermenting Staphylococcus 11}.. 8$ 99.0$ 16.7$
Non-Mannitol Fermenting Staphylococcus 68 ,l\. 68 ,i|.
Enterococcus 16.7 1.0 5*8 Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV 0.1 9.1 Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
Animal in chamber from 10/16/68 to 10/23/65.
ANIMAL l\.
ORGANISM 12/13/68* 12/20/68 12/27/68
Mannitol Fermenting •
Staphylococcus 1.2$ 0.1$ 33.3$ Non-Mannitol Fermenting
28.2 Staphylococcus 1.1 28.2 Enterococcus 7.2 30.8 Diphtheroid bacillus Type I
30.8
Diphtheroid bacillus Type II Diphtheroid bacillus Type III 79.9 Diphtheroid bacillus Type IV 10.6 7-7 Diphtheroid bacillus Type V 99 U Coliform bacillus 0.1 i Miscellaneous
*Animal in chamber from 12/13/65 to 12/20/68/
23
TABLE I - Continued
ANIMAL 5
ORGANISM 10/16/68# 10/23/68 10/30/68
Mannitol Fermenting Staphylococcus 13.0$ 98.7% 2.0j
Non-Mannitol Fermenting Staphylococcus
Enterococcus ' 70.0 Diphtheroid bacillus Type I 87.0 1.3 28.0 Diphtheroid bacillus Type II Diphtheroid Bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
•{{•Animal in chamber from 10/16/65 to 10/23/6ST
ANIMAL 6
ORGANISM 10/16/68* 10/23/68 10/30/68
Mannitol Fermenting Staphylococcus 99.1$ - 99.8$ ^0.0%
Non-Mannitol Fermenting Staphylococcus
Enterococcus 0.1 32.0 Diphtheroid bacillus Type I 0.8 Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV 0.1 Diphtheroid bacillus Type V Micrococcus 18.0 Co] Mi£
.iform bacillus icellaneous
0.1
s-Animal in chamber from 10/16/65 to 10/23/68.
2l\.
TABLE I - Continued
ANIMAL 7
ORGANISM 10/16/68* 10/23/68 10/30/68
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
^Animal in chamber from 10/16/65 to 10/23/68.
88 .Qffo
8.9 0.1
1.0
99J
0.1
0.1
Died Pneumonia 10/28/68
ANIMAL 8
ORGANISM ll/Hj/68* 11/19/68 11/26/68
Mannitdl Fermenting 1.2$ Staphylococcus 0.1% 1.2$
Non-Mannitol Fermenting Staphylococcus 0.2 0.7
Enterococcus 0.1 Diphtheroid bacillus Type I 99.5 98.0 Diphtheroid bacillus Type II 0.1 Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus 0.1 Miscellaneous
100$
ic-Anima 1 in chamber from 11/11 /6 to 11/19/68.
25
TABLE I - Continued
ANIMAL 9
ORGANISM 11/3V68* 11/19/68 11/26/68
Mannitol Fermenting Staphylococcus 1.8$ 3.2$ 22.2"/
Non-Mannitol Fermenting ' Staphylococcus 2 ,l\. )|J|
Enterococcus 0.9 1.1 33-3 Diphtheroid bacillus Type I 91]..8 Diphtheroid "bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus 0.1 0.3 Miscellaneous (Streptococcus) 95'k
^^.^ai in chamber from ll/llj/68 to 11/19/6ST
ANIMAL 10
ORGANISM 11/1V68# 11/19/68 11/26/68
Mannitol Fermenting Staphylococcus 1.0%
Non-Mannitol Fermenting Staphylococcus 98.9
Enterococcus Diphtheroid bacillus Type I 91.3$ 20.0°, Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV 8.5 80.0 Diphtheroid bacillus Type V Coliform bacillus 0.1 0.2 Miscellaneous
•*Animal in chamber from 11/11)768 to 11/19/68.
26
TABLE I - Continued.
ANIMAL.! ,11
ORGANISM 12/13/68# 12/20/68 12/27/68
6.0
1+.0
20.0
70.0
8.1$
10.6
81.3
20.0% Maruiitol Fermenting
Staphylococcus Non-Mannitol Fermenting
Staphylococcus Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous : _ r^_
x-Animal in chamber from 12/13/68 to 12/20/68.
60.0 20.0
ANIMAL 12
ORGANISM 12/13/68# 12/20/68 12/27/68
30/.l$ 6.0% 11.6:
26 .Ij. • 33-8 18.3 0.1
I 6.1 76.6 14-7-2 II III 18.3 IV 0.5 7.3 V
17 4 !
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus
Coliform bacillus Miscellaneous
27
TABLE I - Continued
ANIMAL 13
ORGANISM 12/13/68* 12/20/68 12/27/68
Died Pneumonia
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
*Animal in chamber from 12/13/68 to 12/20/68.
0.9?
1.1 21 .5
73 4 0.1
8.3 i
834 8.3
ANIMAL 1J+
ORGANISM 2/18/69* 2/25/69 3/10/69
100$
Mannitol Fermenting Staphylococcus
N on -Ma nn 11 ol Ferme nt ing Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous (Gaffkya)
-;c-Animal in chamber from 2/lti/69 to 2/2$/69.
3.0$
3.0
1|2.0
52.0
Hj.,0$
.lj.8.0
21}.. 0,
li|.0
28
TABLE I - Continued
ANIMAL 15
ORGANISM 2/18/69*- 2/25/69 3/10/69
30.0^
30.0
13.0
27.0
£0.0$
50.0
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous (Micrococcus)
-«-Animal in chamber from 2/18/69 to 2/25/69.
23.0%
8.0 66.0
3-0
ANIMAL 16
ORGANISM 2/18/69* 2/25/69 3/10/69
Mannitol Fermenting 0.1# Staphylococcus 0.1# 0.1# 0.1#
Non-Mannitol Fermenting Staphylococcus 0.1
Enterococcus 0.1 Diphtheroid bacillus Type I 99.6 99.9 93-9 Diphtheroid bacillus Type II 0.1 6.0 Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellane ous
-x-Animal in chamber from 2/18/69 to 2/25/69.
29
TABLE I - Continued
ANIMAL 17'
ORGANISM 2/18/69* 2/25/69 3/10/69
Mannitol Fermenting Staphylococcus 77-0% 10.9%
Non-Mannitol Fermenting Staphylococcus 2.0 ' 89.1
Enterococcus Died Diphtheroid, bacillus Type I 21.0 3/5/69 Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous •
•s:-Animal in chamber from 2/18/69 to 2/25/69.
ANIMAL 18
ORGANISM 2/18/69* 2/25/69 3/10/69
Mannitol Fermenting Staphylococcus 0.1$ 2.0% 3•
Non-Mannitol Fermenting Staphylococcus 0.1 1.1
T T - n A ^ f > r ) Q
Diphtheroid bacillus Type I 99.8 98.0 11.2 Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V 8i|.3 Coliform bacillus Miscellaneous
- -Animal in chamber from 2/18/69 to 2/25/69.
30
TABLE II. RESULTS OP CULTURES TAKEN PROM GUINEA PIGS EXPOSED TO 380 MM. MERCURY PRESSURE AND ATMOSPHERIC
OXYGEN CONCENTRATION CONFINED IN CHAMBER
ANIMAL 19
ORGANISM 3/18/69* 3/26/69 lj/2/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus 0.7? Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V 100$ 100$ 99.3 Coliform bacillus T Miscellaneous
-::-Anima 1 in chamber from 3/19/69 to 3/26/69.
ANIMAL 20
ORGANISM 3/18/69* 3/26/69 I4./2/69
Mannitol Fermenting Staphylococcus 12.0$ 2.0$ 3«5$
Non-Mannitol Fermenting Staphylococcus
Enterococcus 76.0 2.0 6J4..2 Diphtheroid bacillus Type I Diphtheroid bacillus. Type II Diphtheroid bacillus Type III 96.0 10.9 Diphtheroid bacillus Type IV Diphtheroid bacillus Type V 21 .lj. Coliform bacillus Miscellaneous (Bacillus) 12.0
tf-Animal in chamber from 3/19/69 to 3/26/69.
TABLE II - Continued
ANIMAL 21
31
ORGANISM 3/18/69* 3/26/69 4/2/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type Diphtheroid .bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Coliform bacillus Miscellaneous (Bacillus)
I . II III IV V
7.0%
56.0 50.0
37.0 50.0
^-Animal in chamber from 3/19/69 to 3/26/69.
19> 0?
76.0
5.0
ANIMAL 22
ORGANISM 3/18/69* 3/26/69 lj/2/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous (Micrococcus)
l.i
1.0 98.0
23.0$
7.0
16.0$ 27.0 81}.. 0 ij.1.0 (Bacillus.) 2.0
«-Animal in chamber from 3/19/69 to 3/26/69.
32
TABLE II - Continued
ANIMAL 23
ORGANISM 3/18/69* 3/26/69 i f /2 /69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Ent'erococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous (Bacillus)
^Animal in chamber from 3/19/69 to 3/26/69.
k-W
96.0
ij-.o?
96.0
TABLE III. RESULTS OF CULTURES TAKEN FROM GUINEA PIGS EXPOSED TO ATMOSPHERIC PRESSURE AND OXYGEN
CONCENTRATION CONFINED IN CHAMBER
ANIMAL 2I4.
ORGANISM 3/18/69* 3/26/69 V 2 / 6 9
Mannitol Fermenting Staphylococcus 0.31o 11.3# 0.6 %
Non-Mannitol Fermenting Staphylococcus
Enterococcus• 1.2 1.0 1 .1 Diphtheroid bacillus Type I Diphtheroid bacillus Type II
0.8 0.8 Diphtheroid bacillus Type III 0.8 • 2 . 1 0.8 Diphtheroid bacillus Type IV
97.5 85.5 Diphtheroid bacillus Type V 97.5 85.5 97.0 ; Coliform bacillus 0.5 Miscellaneous (Bacillus) 0.2 0 . 1
-^Animal in chamber from 3/18/69 to 3 /26/69.
TABLE III - Continued
ANIMAL 25
33
ORGANISM 3/18/69* 3/26/69 J+/2/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous (Micrococcus)
-x-Aniraal in chamber from 3/19/69 to 3/26/69.
0.
30.8
15.1
53-1
0.3
0.6?
3.2
31+.2
62.0
7.0$
57.0
36.0
ANIMAL 26
ORGANISM 3/18/69* 3/26/69 J+/2/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
*Ahimal in chamber from 3/19/69 to 3/26/69.
O.i
76.0
20.3
•3.5
0.1$
91+.5
36.2%
63.8
TABLE III - Continued
ANIMAL 2?
3k
ORGANISM 3/18/69* 3/26/69 1^2/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous ^
-::-Animal in chamber from 3/19/69 to 3/26/69.
0.1JI
2 .0
£5.6
ij-2.3
13.3?
66.7
16.0
I4..O
$1.0
ij.0.0
3.8
ANIMAL 28
ORGANISM 3/18/69* 3/26/69 i t /2/69
Mannitol Fermenting 8 . # Staphylococcus 0.1%, 3.1% 8 . #
Non-Mannitol Fermenting 12/. 6 Staphylococcus 0.1 12/. 6
2^/6 Enterococcus 2.3 . 0.9 2^/6 Diphtheroid bacillus Type I Diphtheroid bacillus T^pe II
52.5 6.J+ Diphtheroid bacillus Type III 52.5 1.3 6.J+ Diphtheroid bacillus Type IV
k$-0 82.1 Diphtheroid bacillus Type V k$-0 82.1 Coliform bacillus Miscellaneous (Bacillus) I4..0
*Animal in chamber from 3/19/69 to 3/26/69.
35
TABLE IV. CONTROL ANIMAL KEPT IN NORMAL HOUSING
ANIMAL 29
ORGANISM 11/20/68 11/21+/68 12/3/68
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Coliform bacillus Miscellaneous
l.% 0.8% 21.8%
0.7 0.7 8.2 0.1 0.1 0.1
I 97.1 98.0 V7.2 II 0.2
V7.2
III 7-3 IV 0.2 0.2 0.9 V
11}-.5
ANIMAL 30
ORGANISM 12/13/68 12/20/68 12/27/68
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
11.7?
1.6 7-8
77-3 1.6
51.7?
32.1 11.3
1+ • 9
56.2%
31.5 5.6
0.7
TABLE IV - Continued
ANIMAL 31
36
ORGANISM 12/13/68 12/20/68 12/27/68
Mannitol Fermenting Staphylococcus
Non-MannitcTl Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
61.1#
23.8 0.1).
9.6 S.i
69.5#
15.3 10.9
1.9 2.1|
21.2JS
25.0 28'.8 21.1
3.8
ANIMAL 32
ORGANISM 12/13/68 12/20/68 12/27/68
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Coliform bacillus Miscellaneous
I II III IV V
58.1$
25 .0 7.5
14..1 5.o
30.6^
8 .2 1|2.8
18 .j|
26.3$
52.6 15.8
5.3
37
TABLE IV - Continued
ANIMAL 33
ORGANISM 12/13/68 12/20/68 12/27/68
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type
Coliform bacillus Miscellaneous
20.5% 0.1
2.2 32.2 0.1 1.6 11.7 99.8
I II III 1 8.7 IV 27.0 0.3 V
ANIMAL 3k
ORGANISM 2/18/69 2/27/69 3/10/69
Mannitol Fermenting Staphylococcus 29. Of*
Non-Mannitol Fermenting Staphylococcus 62.0
Enterococcus Diphtheroid bacillus Type I 3.0 Diphtheroid bacillus Type II 6.0 Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
65.9??
7.i+ 26 .ij. 0.3
Died 3/9/69
TABLE IV - Continued
ANIMAL 35
38
ORGANISM 2/19/69 2/27/69 3/10/69
Marmitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Diphtheroid bacillus Type Coliform bacillus Miscellaneous
I II III IV V
l.i
61.8 37-2
li+.Q?
2.0
8I4..O
3 2 . <
32.6 32.6
2.2
ANIMAL 36
ORGANISM 2/19/69 2/27/69 3/10/69
Mannitol Fermenting Staphylococcus
Non-Mannitol Fermenting Staphylococcus
Enterococcus Diphtheroid bacillus Type I Diphtheroid bacillus Type II Diphtheroid bacillus Type III Diphtheroid bacillus Type IV Diphtheroid bacillus Type V Coliform bacillus Miscellaneous
0.1# k$'0fo
0 . 5 0 . 6 6 . 0 90 .8 , k-7 1 . 1
7 2 . 6 12 .3 1 .6 2 3 5 . 6 7 . 0
13.8
39
TABLE V. NUMBERS OP BACTERIA PER MILLILITER OP BROTH SUSPEN-SION PREPARED PROM RECTAL SWABS TAKEN PROM ANIMALS EXPOSED
TO 380 MM. HG. PRESSURE AND 100$ OXYGEN
Animal Number Pre-Exposure Post-Exposure 1 Week
Post-Exposure
1 2 3 Ij. 5. 6 7 8 9
10 11 12 •13 lif. 15 16 17 18
Average
1.70 Ij-.H
3 45 1.33 5-60 5-69 3.31 dXk 6 . 0 6 5.00 i .93 5.31 9 .60 3.30 1.1+3 5.20 1 .60
x 10§ x 10' X 102 x 10" x 10; x 10/ X lOn X 10® x io£ x 10, x 10? x ioy x 10P x lof X 10!+ x 10/ x 10?-X 107
1.32 1 .00 5.7 '3.11*-5.00 1 .00 1:00 1 .02 943 2.96 1.23 $.7k i . 2 0 5.00 1.60 1.00 1.01 8.12
X 10Z x 107 x io!+ x 10Z x lol X 10o x 10° x 10® x 10? x 10S x 10? x 10., x 107 x 10° x lOS x 10; x 10° x 10 b
2.27 x 10? (died)/
6.58 x loH l . l j .1 x 10X 3.9 x 10? 5.9 x 10^
(died,) 6.00 x 10£
x 10? 5.00 x 10^ I4..OO x 10^ 1134 x 10'
(died )i. 2 . 7 0 x 10, 2.60 x 103 i+.31 X 1CK
(died), 1.78x 10b
k>l$ x 107 1.30 x 108 Ij.,18 x 10*
LITERATURE CITED
1. Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957 •" Bergey's manual of determinative bacteriology. 7th ed. The Williams and Wilkins Co., Baltimore.
2. Cordaro, J. T., W. M. Sellers, R. J. Ball, and J. P. . Schmidt. 1966. Study of man during 56-day exposure to an oxygen-helium atmosphere at 258 mm. hg. total pressure. X. Enteric microbial flora. Aerospace Medicine, 36:59l+-596.
3» Ehrlich, R., and B. J. Mieszkuc. 1969. Resistance to experimental bacterial pneumonia and influenza infection in space cabin environment. Aerospace Medicine, 1.0 (2): 176-179.
if. Lechtman, M. D., and R. Nachum. 1967. Microbiological aspects of space flight. American Journal of Medical Technology, 33 (6):5l5-523.
5. Luckey, T. D. 1963* Germfree life and gnotobiology. Academic Press, New York.
6. Luckey, T. D. 1966. Potential microbic shock in manned aerospace systems. Aerospace Medicine, 36:1223-1228.
7. McCoy, E. I96J4.. Changes in the host flora induced by chemotheraputic agents. Annual Review of Microbiology, 8:257-272.
8. Nelson, R. C. 1914-1. Progressive changes in the flora, of the intestinal tract of guinea pigs from birth to maturity. M. S. Thesis. University of Notre Dame, Notre Dame, Indiana.
9. Sc3pip.idt, J. P., J.'T. Cordaro, and R. J, Ball. 1967. Effect of environment on staphylococcal lesions in mice, Applied Microbiology, 15 (6):Il4.65~li4.67 .
10. Schmidt, J. P. Resistance to infectious disease versus exposure to hypobaric pressure and hypoxic, normoxic or hyperoxic atmospheres. Infectious Diseases Branch, Biosciences Division, U.S.A.P. School of Aerospace Medicine, Brooks Air Force Base, Texas
ij.1
11. Smith, H. W., and W. E. Crabb. 1961. The faecal bacterial flora of animals and man: its develop-ment in the young. Journal of Pathogenic Bacteriology, 82:JP3~66.
12. White, A., P. Handler, and E. L. Smith. 1968. Prin-ciples of biochemistry, i th ed. McGraw-Hill Co., New York.
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