the effects of simulated altitude on the intestinal flora

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


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


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%


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




^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 !



Enterococcus

Coliform bacillus Miscellaneous

27

TABLE I - Continued

ANIMAL 13

ORGANISM 12/13/68* 12/20/68 12/27/68

Died Pneumonia




*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$


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



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#


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



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$


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



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




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



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 %


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




-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




*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



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



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




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


Non-MannitcTl Fermenting Staphylococcus


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




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


ANIMAL 33

ORGANISM 12/13/68 12/20/68 12/27/68



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*


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


ANIMAL 35

38

ORGANISM 2/19/69 2/27/69 3/10/69

Marmitol Fermenting Staphylococcus



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




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.

the effects of simulated altitude on the intestinal flora

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