APPLIZD AND ENVIRONMENTAL MICROBIOLOGY, Nov.
1976,
p. 659-665
Copyright © 1976 American Society for Microbiology
Vol. 32, No. 5 Printed in U.S.A.
Autoflora in the Upper Respiratory Tract of Apollo Astronauts J. GLEE DECELLE AND GERALD R. TAYLOR* Northrop Services, Incorporated, Houston, Texas 77034, and National Aeronautics Administration, L. B. Johnson Space Center, Houston, Texas 77058*
and Space
Received for publication 23 April 1976
The typical microbial inhabitants of the oral and nasal cavities of Apollo astronauts were identified before space flight and generally found to be similar to those previously reported for healthy male adults. Additional analyses of samples collected immediately after return of the Apollo 13, 14, 15, and 16 crew members to earth were performed to evaluate the effects of space travel on the microbial bioburden of the upper respiratory tract. In-flight cross-contamination and buildup of pathogens such as Staphylococcus aureus were noted, although significant increases in nonpathogenic species were absent. Other proposed alterations, such as dysbacteriosis (flooding of the mouth with a single species) and simplification of the autoflora, did not occur. Generally, the incidence and
quantitation of each species after flight was within the preflight range, although the number of viable Haemophilus cells recovered from the mouth decreased significantly after space flight. Except for those minor alterations listed above, the aerobic and anaerobic bacterial components of the upper respiratory autoflora of Apollo astronauts was found to be stable after space flight of up to 295 h.
The behavior of infectious, or potentially in- C. A. Berry, Bacteriol. Proc., p. 16, 1967). This fectious, agents in the upper respiratory tract of could presumably result from the efficiency of astronauts is of importance when considering the spacecraft cabin filtration system and the the habitability of the space flight environ- lack of contaminating sources other than acment. Space cabins are by necessity very small, companying crew members. It has further been completely closed chambers, generally with a suggested that this "simplification," typified as recirculating ventilation system. During space a decrease in the number of different types of flight, crews must live close together for ex- microorganisms composing the microbial poputended periods, with frequent intervals of high lation, could be accompanied by a significant activity. This contributes to opportunities for quantitative increase in a single species (1, 4, 6, cross-infections of the type often observed in the 38). This dysbacteriosis (1) has been theorized confined environments of space flight simula- by Zaloguev and his co-workers to result when microorganisms better fitted for life in the tion chambers (6, 7, 23, 30, 31). In addition, certain aspects of space flight space cabin environment crowd out certain othcreate an unusual situation which also must be ers commonly found in the autoflora of the crew evaluated. For example, it has been proposed members (1, 3, 38). Although some of the works referenced above that because of the reduced gravity, droplets expelled from the upper respiratory tract would involve actual space flight analyses, the fragbe larger and would remain in the space cabin mentary data have as yet been inconclusive. At atmosphere longer, thus increasing the oppor- any rate, these studies are not generally applitunity for exchange between astronauts (Yu cable to the Apollo space flight series because of Nefedov, S. N. Zaloguev, and V. P. Savina, important environmental differences. Such dif24th Cong. Int. Astronaut. Fed., 1973). The ferences include, among other things, atmosresults of studies with space simulation cham- pheric pressure and composition (including parbers, and the rudimentary results from the tial pressures of 02 and C02), environmental space flights of Vostok 1 through 4, have control systems, and crew hygiene regimens. prompted investigators in the United States However, these studies should be accepted as and the Soviet Union to suggest that the auto- indicators that the activity of the upper respiflora of the upper respiratory tract could be- ratory autoflora during space flight are potencome simplified (1, 19, 20, 24, 35, 38; H. 0. tially dangerous to the health of the astronaut Wheeler, W. W. Kemmerer, L. F. Dietlein, and (2). Accordingly, the activity of the upper respi659
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DECELLE AND TAYLOR
APPL. ENVIRON. MICROBIOL.
ratory tract autoflora was studied during the throat-mouth gargle were maintained at + 277°K series of Apollo space flights, with special em- (+4°C) during transportation to the L. B. Johnson phasis on the Apollo 13 through 16 missions. Space Center. Storage time was standardized at 10 h The results are evaluated in terms of the effects for all samples collected. The nasal samples in TSB were diluted 10' to of space flight on the quantitative bioburden 10-4 TSB for quantitation of aerobic microorgaand on the activity of microorganisms fre- nisms.in Those samples in VIB were diluted to 10-4 in quently isolated from the nasal and oral cavi- VIB for anaerobic quantitation. A 0.1-cm3 sample ties of the crew members. from each TSB sample and dilution tube was placed on blood agar (Trypticase soy agar with 5% sheep MATERIALS AND METHODS added) and Staphylococcus 110 agar (S110Sample collection schedule. The time periods blood and spread across the surface of the medium during which the upper respiratory tract areas of BBL) with a sterile glass rod. The VIB samples and diluApollo astronauts were sampled are illustrated in tion tubes were plated on anaerobic blood agar (conTable 1. Data from four sampling periods are reTrypticase soy agar, 0.5% yeast extract ported for each Apollo flight. The Apollo 13 and 14 taining crew members were sampled 30 days before, 2 weeks [Difco], 5% sheep blood, and 1.0% vitamin K-hemin solution). The undiluted samples and the 10-1 dilubefore, and immediately preceding flight, as well tions were also plated on paromomycin-vancomycinas immediately after return of the Apollo from space menadione agar. Rogosa agar (Difco) was plated flight. For Apollo 15 and 16, a sampling period 5 with 0.1-cm3 portions from each of the undiluted days before launch was substituted for the 2-week tubes, 10-1, and 10-2 dilution tubes. preflight sampling period of Apollo 13 and 14. Be- sample The gargle samples were diluted 10-1 to 10-5 in cause one crew position was substituted 3 days before launch (8), only the two permanent subjects TSB for aerobic quantitation and in VIB for anaerowere evaluated for the Apollo 13 mission. Three bic quantitation. A 0.1-cm3 sample from each sample and TSB dilution tube was plated on blood agar, astronauts were studied with all of the other re- S110 agar, and mitis-salivarius agar (Difco). Samported missions. from the Apollo 15 and 16 astronauts were also Sample collection procedure. Each crew member ples inoculated onto bacitracin chocolate agar for quantiwas cautioned not to brush the teeth, use a mouth- tation of Haemophilus species. The gargle samples wash, eat, drink, or smoke before sample collection. A pair of calcium alginate swabs (Wilson Diagnos- and VIB dilution tubes were plated on anaerobic tics, Inc., Glenwood, Ill.) moistened with sterile 0.3 blood agar, paromomycin-vancomycin-menadione, mM phosphate buffer was used to sample a 2-cm2 and Rogosa agar. The aerobic plates were incubated at +308°K surface area in each of the two nasal chambers of the Apollo 13 and 14 astronauts. The nasal passages of (+35°C) for 48 h, after which each morphologically colony was quantitated and picked to a the Apollo 15 and 16 crew members were not sam- different pled. To obtain a throat-mouth gargle, all 11 of the tube of TSB, incubated at +308°K, and allowed to until turbid. The resulting broth suspension subject astronauts gargled with 60 cm3 of a 0.3 mM grow phosphate buffer solution which was rinsed through was used to make a Gram stain and to streak onto a the oral cavity three times. All preflight samples blood agar plate to check the purity of the culture. If were taken upon arising, whereas postflight sam- the specimen was shown to be a pure culture, bioples were taken within 1 h after the astronauts were chemical media were inoculated for identification of aboard the recovery ship and before any hygiene the microorganism. The anaerobic plates were incuregimen was initiated. One of each pair of swabs bated at +308°K (+35°C) in an atmosphere of hydrowas placed in a tube containing 5 cm3 of Trypticase gen and carbon dioxide generated by the GasPak soy broth (TSB; BBL), and the remaining swab was (BBL) method. After incubation for 96 h, each morplaced in a tube containing 5 cm3 of veal infusion phologically different colony was quantitated and broth (VIB; BBL). These tubes and containers of picked to an anaerobic broth. An aerobic blood agar plate was also streaked to verify that the picked colony was not capable of growth under aerobic conTABLE 1. Time periods for collection of Apollo ditions. Each broth culture was incubated at +3080K astronaut upper respiratory tract samples (+35°C) until turbid. Gram stains were made and biochemical identification media were subsequently Days before Apollo Crew launch Recov- inoculated from the anaerobic broth. mis- member Sample type ain 5~~Ofl no.o ~~30 15 5 0 ery day RESULTS Recovery of anaerobic bacteria from nasal 13 and 1, 2, 3, 4, Nasal swab +b + -c + + 14 passages of Apollo 13 and 14 crew members. + + _ + + 5 Gargle An average of 1.2 species of anaerobic bacteria . 15 and 6, 7, 8, 9, Nasal swab with a mean quantitation of 2,520 cells/cm2 of 16 10, 11 Gargle + _ + + surface area was recovered from the nasal pasa sages of each astronaut at each sample time Crew member numbers: Apollo 13 = 1, 2; Apollo before flight. The average recovery was 1.0 spe14 = 3, 4, 5; Apollo 15 = 6, 7, 8; Apollo 16 = 9, 10, 11. cies, with a mean quantitation of 41,700 cells/ +, Sample collected. c -, Sample not collected. cm2 the day of return from space flight. Al-
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though there was a slight reduction in the number of different isolates and a slight increase in the total load of viable anaerobic cells, these changes were not statistically significant (P < 0.05). Although representatives of the genera Peptococcus and Peptostreptococcus were infrequently recovered from the nasal passages, only the genus Propionibacterium was isolated frequently enough to be considered a component of the nasal autoflora. Accordingly, only that genus is evaluated here. The preflight recovery pattern of this genus, outlined in Table 2, demonstrates no statistically significant (P < 0.05) difference between the five crew members, indicating that propionibacteria regularly inhabited each crew member at about the same quantitative level. Such regularity provided an opportunity to evaluate the effect of short-duration (Apollo 13, 143 h; Apollo 14, 217 h) space flight on the stable portion of the nasal autoflora. The data shown in Table 2 indicate that the recovery of viable cells, belonging to the genus Propionibacterium, from crew members immediately upon their return from space flight was not significantly (P < 0.05) different than for any preflight sample collection period, except in the case of astronaut 4. This value was higher than the values obtained from the other astronauts postflight and was also higher than the preflight values for this crew member. Because this postflight elevation was not duplicated by the other crew members, it cannot be considered a typical response to space flight. Recovery of aerobic bacteria from nasal passages of Apollo 13 and 14 crew members. The qualitative and quantitative recoveries of
aerobic bacteria from the nasal passages of the five subject crew members at various times before and after space flight are shown in Table 3. When compared to preflight recovery, there is a marked decrease in the number of times the bacilli, corynebacteria, gram-negative rods, and streptococci were isolated immediately after flight. Simultaneous with this decrease, the incidence of the potentially pathogenic Staphylococcus aureus increased from a maximum of two isolates during any preflight period to a maximum of five postflight, being transferred to all of the crew members. Quantitatively, the total nasal population increased significantly (P < 0.05) postflight (Table 3), due to the accession of S. aureus mentioned above as well as an increase in quantitation of corynebacteria and Micrococcaceae. Other microorganisms were isolated too infrequently to establish any pattern. Recovery of anaerobic bacteria from the oral cavity of crew members from Apollo 13 through 16. The quantitative and qualitative recovery of anaerobic bacterial genera from the oral cavity of the 11 subject astronauts is presented in Table 4 in decreasing frequency of isolation. Analysis of the generic recovery frequency data shows no major difference between the occurrence of any particular genus before and after flight. Similarly, quantitative analysis of the mean number of viable cells present belonging to any particular genus indicates that, in general, the postflight load was within the preflight range. Recovery of aerobic bacteria from the oral cavity of crew members from Apollo 13 through 16 crew members. A summary of the
TABLE 2. Quantitationa of the genus Propionibacterium cultured from nasal passages of five Apollo astronauts (Apollo 13 and 14)
TABLE 3. Recovery of aerobic bacteria from the nasal passages offive Apollo astronauts (Apollo 13 and 14)
Days before
Crew member Apollo launch
Day
1
2 3 4 5
14
3.48 1.88 0 2.80 4.00 2.40 3.55 3.58 3.40 0
0
2.58 2.40 2.88 2.80 2.40
ryc
mean
viation
recovery
2.00 3.10 0
5.31b 2.96
2.49 2.08 2.32 3.81 2.19
2.89 2.13 2.61
2.67
2.58
1.63 1.34 0.22 Standard deviation
1.92
1.35
Geometric
Group
Apollo Geomet dard-
no.
30
Days before launch
Stan-
of
+0.73 ±+1.41 ±1.69 ±1.06 ±1.52
mean
a Quantitation expressed in log10 colony-forming units per cm2 of internal nasal surface area sampled. b Significantly higher (P < 0.05 by comparison t test) than other entries in the column and the row.
30
14
Ia Q 0 2.10 5 5.33 3 4.39 5.19 5 4.92 Micrococcaceaee Staphylococcus au- 4.22 2 3.55
Qa 0 Bacillus sp. Corynebacterium sp. 4.72 Gram-negative rods +
Day of re-
covery
0 I
Q
2 4 4 5 2
+b 1
4.07 2.18 4.76 3.63
Q
4 6.09 3 1.40 5 5.51 1 6.60d
I 0 3 2 5 5
0 0
0
I 0
reus
0 1.40 1 0
Streptococcus sp.
0
Total
5.35 15 5.51 18 4.83 14 6.75
15
Q, Total quantitation in log,0 colony-forming units per cm2 of internal nasal surface area sampled; I, incidence, number of times isolated from the five nasal swabs. b +, Isolated but not quantitated. All members of Micrococcaceae except S. aureus. dSignificantly higher (P < 0.05 by comparison t test) than preflight recovery of S. aureus. a
662
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DECELLE AND TAYLOR
of aerobic bacteria from the gargle of the nasal inhabitants, the autoflora compo11 astronauts is outlined in Table 5. The family nents recovered from the healthy male adults Micrococcaceae and the genera Streptococcus, tested in this study generally agree with earlier Neisseria, and Haemophilus were generally re- reports (21, 22, 25, 26, 28). Aerobic corynebaccovered from each of the 44 gargle samples teria, Micrococcaceae, and anaerobic propioniobtained. Only with the genus Haemophilus bacteria predominated, with minor contribuwas the postflight recovery found to be signifitions from gram-negative rods and streptococci. cantly different from the preflight base line (P Although the nasal bioburden is less complex < 0.05). Members of the Enterobacteriaceae than that recovered from the mouth (21, 28, 37), and other gram-negative rods were infre- it is of major importance in evaluating autoquently observed, and then only in relatively flora responses to space flight. In fact, this low numbers. sparse population lends itself well to analysis because cross-contamination and microbial recovery
buildup are more easily observed against the low background. It has been proposed (Nefedov et al., 24th passages. Although few authors have critically evaluated Cong. Int. Astronaut. Fed., 1973) that the reTABLE 4. Recovery of anaerobic bacteria from gargle samples of 11 Apollo astronauts (Apollo 13, 14, 15, 16) DISCUSSION Bacterial population in the nasal
Recovery (%) frequencyG
Preflight quantitation
(log1O)
Group__
Postflight quantitation
(log1o)
___
Preflight Postflight Meanb SDC Mean SD Bacteroides 100 100 4.76 +0.71 3.85 ±+1.07 Fusobacterium 100 100 ±0.84 4.24 4.03 +0.92 Veillonella 90.9 100 4.47 ±1.08 4.28 ±0.84 Leptotrichia 66.7 45.5 ±0.87 3.03 2.57 ±0.50 Propionibacterium 51.5 54.6 +0.74 4.11 4.15 ±1.41 Lactobacillus 36.4 54.6 3.41 ±+1.21 4.35 ±1.46 Peptostreptococcus 33.3 27.3 4.70 +0.48 4.62 +0.28 Bifidobacterium 21.2 27.3 2.89 +0.99 1.92 ±0.79 Peptococcus 12.1 9.1 4.07 ±1.05 0.35 ±1.18 Eubacterium 9.1 18.2 4.20 3.77 ±0.12 +1.80 Actinomyces 3.0 0.0 0.07 +0.40 0 Total anaerobic load 5.37 +0.57 5.24 ±0.67 a Number of times isolated out of number of samples collected (x 100). Preflight, 33 samples (three per astronaut); postflight, 11 samples (one per astronaut). b Mean quantitation in log,0 colony-forming units per cm3 of gargle. Only samples positive for a group are used to calculate mean. c SD, Standard deviation. TABLE 5. Recovery of aerobic bacteria from gargle samples of 11 Apollo astronauts (Apollo 13, 14, 15, 16)
Preflight quantitation
Group
Streptococcus Neisseria
Recovery frequency" ( Preflight Postflight
100 97 97 96.3 90.9 81.5 36.4 26.3
100 100 81.8 88.9 90.9 66.7 18.2 45.5
Postflight quantitation
(loglo) Meanb
6.23 4.78 4.74 5.18 3.01 3.03 2.50 3.17
SDc ±0.88 ±0.99 ± 1.04 ±0.76 ±1.35 ±1.27 ±1.14 ±1.32
(log1o) Mean
SD
6.56 5.23 4.84
±0.64 ±0.99 ±0.90 +0.53 ±1.51 ±1.30 ±1.57 ±1.28
Corynebacterium Haemophilus 4.37d Micrococcaceae 3.59 Lactobacillus 3.06 Enterobacteriaceae 2.59 Other gram-negative 4.09 bacilli Total aerobic load 6.37 ±0.76 6.65 ±0.59 a Number of times isolated out of number of samples collected (x 100). Preflight, 33 samples (three per astronaut); postflight, 11 samples (one per astronaut). b Mean quantitation in log10 colony-forming units per cm3 of gargle. Only samples positive for a group are used to calculate mean. c SD, Standard deviation. d Significant at 5% level (comparison t test).
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AUTOFLORA IN APOLLO ASTRONAUTS
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duced gravitational attractions experienced flight. Likewise, there was a significant postduring space flight would increase the probabil- flight increase (P < 0.05) in the total number ity of respiratory infections by favoring the pro- of viable S. aureus cells recovered from the duction of larger particles, or droplets, expelled astronaut population. These data support the into the environment. The formation of these anticipated in-flight cross-contamination and larger particles, which would not settle out as buildup of pathogenic species, whereas nonthey do on earth, would effectively result in an pathogens remained essentially stable. increased infecting dose being drawn into the Bacterial population in the oral cavity. Innasal cavity. The increased particle size may vestigations of the oral autoflora have been also affect a change in the filtration ability of conducted with a variety of test subjects and the lungs and epithelial tissues (15; Nefedov et wide-ranging methods and have been repeatal., 24th Cong. Int. Astronaut. Fed., 1973). Fur- edly reviewed (5, 9, 11, 12, 14, 17, 28, 35). Rethermore, it has been suggested (2, 23, 38) that sults have most often been analyzed with rethis increased contamination potential would spect to whole saliva or specific oral surfaces not be the same for all microorganisms. In the such as the gingival crevice, surface of the absence of recontaminating sources, transient tongue, or coronal plaque. Additionally, such microorganisms would be removed by the investigations have usually been associated spacecraft air filtration system and thereby lost with studies of dental caries and peridontal from the autoflora. Therefore, the makeup of disease events and have emphasized the lactothe nasal microbial population could become bacilli and streptococci, which are of interest in simplified, with the remaining species being po- oral disease analyses. In general, previous tential pathogens and/or true members of the studies have shown the bacterial population nasal autoflora. Nefedov et al. (24), in summa- within the oral cavity of healthy persons to be rizing the results of several Soviet space quite stable from day to day, provided the influflights, have concluded that there sometimes is ences of eating, smoking, and mastication are a simplification of the nasal flora which in- controlled. Also, the composition of the oral cludes a disappearance of nonpathogenic micro- autoflora of the individual members of a reaorganisms and a predominance of pathogenic sonably homogeneous group is usually found to be quite similar. species. The study reported herein was conducted in Data obtained from the analysis of astronaut nasal samples were evaluated to determine if an effort to determine if the various pressures similar changes occurred during the subject of short-term (143 to 295 h) space flight exerted Apollo missions. The recovery incidence and any detectable alteration in the qualitative or quantitation of all anaerobic bacteria recovered quantitative composition of the oral autoflora of from the nasal passages after flight was not the crew members. Accordingly, it was necessignificantly different (P < 0.05) from values sary to collect a sample that was representative obtained before flight. Because of the normally of the oral cavity as a unit. For this reason, low incidence of anaerobes other than Propioni- loose microorganisms were washed from the bacterium, it is appropriate to evaluate this mouth and oral-pharyngeal vault of each subgenus alone. Even within this group, such vari- ject. Because larger numbers of bacteria accuability was demonstrated that a space flight- mulate in the oral cavity during sleep, when induced shift could not be shown. Although the salivary flow is greatly reduced (18, 29), collecpostflight recovery from one astronaut (Table 2) tion just after arising provided the best opporwas significantly high (P < 0.05) postflight, the tunity to recover oral inhabitants. In all cases, overall pattern mediates against alterations as- the subjects were healthy male adults without apparent oral disease symptomatology. The resociated with space flight. Compared with anaerobes, aerobic bacteria sulting data should relate most closely to inforwere more numerous both in terms of species mation obtained from studies with whole sarecovered and total quantitation. These data liva, because both reflect the sum of the contriare counter to those reported by Watson et al. butions from all oral surfaces (9, 10). Anaerobic bacteria. Analysis of the generic (37), in which anaerobes were generally present at a level 10 times higher than the aerobic recovery frequency data presented in Table 4 quantitation. These conflicting reports could demonstrates the ubiquitous appearance of the easily result from the different methods used. genera Bacteroides, Fusobacterium, and VeilThe data presented in Table 3 do not indicate lonella. The mean recovery of these common simplification of the aerobic autoflora. How- inhabitants of the healthy mouth during the ever, there is evidence of a significant increase preflight collection period was always in excess in number of individuals (to the maximum of of 10,000 viable cells per cm3 of wash. Gramfive) from which S. aureus was recovered post- positive rods were often recovered, especially
664
DECELLE AND TAYLOR
Leptotrichia and Propionibacterium. Veillonella was recovered at higher quantitation than expected, whereas actinomycetes were recovered less frequently than usually reported (13). Although the mean recovery of Bacteroides from the mouth of astronauts immediately after their return from space flight was somewhat lower than the preflight base line, no statistically significant quantitative changes in the three most common genera were observed (P < 0.05). The other genera as well, although less frequently recovered and therefore less stable, were not observed to change significantly postflight (P < 0.05). The dysbacteriosis, or flooding of the oral cavity with large numbers of bacterial cells, previously reported after certain space flights from the Soviet Union (1, 3, 38) was shown not to occur during the four Apollo missions analyzed in this report. The continued stability of the incidence of anaerobic genera contraindicated microbial simplification within the oral cavity. There was no discernible difference between the recovery of anaerobic bacteria from the oral cavity of the 11 subject astronauts before and after space flight. Aerobic bacteria. The data presented in Table 5 indicate that during the preflight period, members of the genera Streptococcus, Neisseria, Corynebacterium, and Haemophilus were virtually omnipresent at values ranging from 55,000 to 1,700,000 cells/cm3 of wash. As reported by others (27, 28), the streptococci were both the most frequently isolated and the most numerous of all aerobic bacteria recovered. Neisseria was isolated in larger numbers than typically reported (13). The genus Haemophilus, which has only recently been fully recognized as a major component of human oral autoflora (13, 34), was routinely isolated. Although there was no significant change in the total aerobic load or in any other reported microbial group, the preflight stability of the genus Haemophilus was adequate to detect a significant postflight decrease in quantitation (P < 0.05). Members of the family Micrococcaceae and the genus Lactobacillus were frequently isolated but at lower quantitative levels. This relatively low recovery of lactobacilli reflects the lack of carious lesions within the astronaut population (16, 32, 33). The absence of any increase in the quantitation of gram-negative rods indicates that flooding of the oral cavity with these microorganisms, reported by Zaloguev et al. (38), did not occur. It appears that this flooding, which has been reported only by investigators in the Soviet Union, was an isolated incident and was not related to space flight. These data, comparing pre- and postflight analyses from
APPL. ENVIRON. MICROBIOL.
four different short-duration space flights, demonstrate that the aerobic bacteria in the oral cavity are quite stable both in terms of the genera and the total number of viable cells present. The quantitative recovery from these wash samples is understandably lower than those reported for whole saliva (where quantitation for aerobes and anaerobes may reach 8 x 1010 and 9 x 1010 cells per cm3 of saliva, respectively). However, an important difference is the ratio of aerobes to anaerobes. Whereas the anaerobes in whole saliva may outnumber the aerobes (27), the opposite was observed with whole mouth washes. Viable aerobic cells were consistently recovered at a level 10 times greater than that obtained for anaerobic cells, a condition which was unchanged postflight. Studies associated with early United States and Soviet Union space flights and groundbased simulations have indicated that the microflora inhabiting the upper respiratory tract of crew members may be altered during such missions. To evaluate this possibility, samples collected from the nasal passages of Apollo 13 and 14 crew members and from the oral cavity of Apollo 13 through 16 astronauts were quantitatively evaluated for the presence of aerobic and anaerobic bacteria. The anaerobic autoflora in the nasal passages, consisting almost exclusively of members of the genus Propionibacterium, remained unchanged postflight (with the exception of one subject). Representatives of Micrococcaceae and Corynebacterium were predominant in the nasal autoflora which demonstrated a postflight increase in the incidence and quantitation of S. aureus, concurrent with a decrease in the incidence of other bacterial isolations. Members of the genera Bacteroides, Fusobacterium, and Veillonella were ubiquitou-sly present in the mouth and remained unchanged after the space flights. The major aerobes present in the mouth belonged to Micrococcaceae, Streptococcus, Neisseria, and Haemophilus. There was a significant decrease in the number of viable Haemophilus cells recovered after flight. With the above exceptions, the bacterial flora inhabiting the upper respiratory tract of Apollo astronauts was found to remain stable after space flight of up to 295 h. ACKNOWLEDGMENTS This study was partially funded by National Aeronautics and Space Administration contract NAS 9-13000 to Northrop Services, Inc., Houston, Tex. 77034. We especially wish to thank Kathryn Kropp, Theron Groves, Carolyn Carmichael, Mary Arredondo, Nem Bryan, Walter Ellis, and Sara F. Wright for their technical assistance.
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LITERATURE CITED 1. Alekseyeva, 0. G. 1965. Some natural immunity factors and cosmnonaut autoflora during the training period and following the flights of "Vostok", "Vostok 2", "Vostok 3", "Vostok 4", p. 278-289. In N. M. Sisakyan (ed.), Problems of space biology, vol. 4. Gordon and Breach Publishers, New York. 2. Berry, C. A. 1970. Summary of medical experiences in the Apollo 7 through 11 manned spaceflight. Aerosp. Med. 41:500-519. 3. Bychlov, V. P., M. I. Kozar, S. N. Zaloguev, M. V. Markarian, and M. M. Shinkareva. 1970. Immunological reactivity of the human body during 120-day feeding on dehydrated diets. Kosm. Biol. Med. 4:4751. 4. Chukhlovin, B. A., P. B. Ostroumon, and S. P. Ivanova. 1971. Development of staphylococcal infection in human subjects under the influence of some spaceflight factors. Kosm. Biol. Med. 5:61-65. 5. Davis, R. M. 1972. General ecology of the commensal microflora of the mouth, p. 31-37. In T. MacPhee (ed.), Host resistance to commensal bacteria: the response to dental plaque. Churchill Livingstone, Edin-
burgh. 6. Drozdova, V. I., R. V. Petrov, and V. M. Shilov. 1970. Study of streptococcal flora of the human oral cavity during prolonged chamber confinement. Kosm. Biol. Med. 4:51-55. 7. Drozdova, V. I., R. V. Petrov, and V. M. Shilov. 1976. Study of streptococcal flora of the human pharynx in isolated human subjects. Kosm. Biol. Med. 5:26-29. 8. Ferguson, J. K., G. R. Taylor, and B. J. Mieszkuc. 1975. Microbiological investigations. Biomedical results of Apollo NASA special publication (NASA SP368), p. 83-103. National Aeronautic and Space Administration, Houston. 9. Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19-44. 10. Gibbons, R. J., B. Kapsimalis, and S. S. Socransky. 1964. The source of salivary bacteria. Arch. Oral Biol. 9:101-103. 11. Gordon, D. F., and B. B. Jong. 1968. Indigenous flora from human saliva. Appl. Microbiol. 16:428-429. 12. Handelman, S. L., and J. R. Mills. 1965. Enumeration of selected salivary bacterial groups. J. Dent. Res. 44:1343-1353. 13. Hardie, J. M., and G. H. Bowden. 1974. The normal microbial flora of the mouth, p. 47-84. In F. A. Skinner and J. E. Carr (ed.), The normal microbial flora of man. Academic Press Inc., London. 14. Hoffman, H. 1966. Oral microbiology. Adv. Appl. Microbiol. 8:195-251. 15. Kass, E. H. 1971. Resistance to infections in extended spaceflight. Life Sci. Space Res. 9:35-41. 16. Kesel, R. G., I. L. Shklair, G. H. Green, and H. R. Englander. 1958. Further studies on Lactobacilli counts after elimination of carious lesions. J. Dent. Res. 37:50-51. 17. Kraus, F. W., and C. Gaston. 1956. Individual constancy of numbers among the oral flora. J. Bacteriol. 71:703-707. 18. Lear, C. S. C., J. B. Flanagan, and C. F. A. Moorrees. 1965. The frequency of deglutition in man. Arch. Oral Biol. 10:83-99.
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19. Lebedev, K. A., and R. V. Petrov. 1971. Immunological problems of closed environments and gnotobiology. Usp. Sovrem. Biol. 71:235-252. 20. Luckey, T. D. 1966. Potential microbic shock in manned aerospace systems. Aerosp. Med. 37:1223-1228. 21. Marples, M. J., R. R. Marples, and B. Muir. 1960. Microbiological studies in Western Samoa - I. Effects of a Yaws control programme on the incidence of superficial skin lesions and their aerobic bacterial flora, with an appendix on the normal flora of the nose. Trans. R. Soc. Trop. Med. Hyg. 54:155-165. 22. Marples, R. R., J. E. Fulton, J. Leyden, and K. J. McGinley. 1969. Effect of antibiotics on the nasal flora in acne patients. Arch. Dermatol. 99:647-651. 23. Moyer, J. E., D. G. Farrell, W. L. Lamb, and J. L. Mitchell. 1966. Study of man during a 56-day exposure to an oxygen-helium atmosphere at 28 mm Hg total pressure. Aerosp. Med. 37:597-600. 24. Nefedov, Yu. G., V. M. Shilov, I. V. Koustantinova, and S. N. Zaloguev. 1971. Microbiological and immunological aspects of extended manned space flights. Life Sci. Space Res., Comm. Space Res. 9:11-16. 25. Nobel, W. C. 1969. Distribution of the Micrococcaceae. Br. J. Dermatol. 81:27-32. 26. Nobel, W. C. 1969. Skin carriage of the Micrococcaceae. J. Clin. Pathol. 22:249-253. 27. Richardson, R. L., and M. Jones. 1958. A bacteriologic census of human saliva. J. Dent. Res. 37:697-709. 28. Rosebury, T. 1962. Microorganisns indigenous to man. McGraw-Hill, New York. 29. Schneyer, L. H., W. Pigman, L. Hanaban, and R. W. Gilmore. 1956. Rate of flow of human parotid, sublingual, and submaxillary secretions during sleep. J. Dent. Res. 35:109-114. 30. Shilov, V. M., N. N. Lizko, 0. K. Borisova, and V. Ya. Prokhorov. 1971. Changes in the microflora of man during long-term confinement. Life Sci. Space Res. 9:4349. 31. Shilov, V. M., N. N. Lizko, and 0. K. Borisova. 1972. Changes in intestinal microflora during man's longterm confinement in an isolation chamber. Kosm. Biol. Med. 6:78-81. 32. Shklair, I. L., H. R. Englander, L. M. Stein, and R. G. Kesel. 1956. Preliminary report of the effect of complete mouth rehabilitation on oral Lactobacilli counts. J. Am. Dent. Assoc. 53:155-158. 33. Shklair, I. L., and M. A. Mazzarella. 1961. Effects of full-mouth extractions on oral microbiota. Dent. Prog. 1:275-280. 34. Sims, W. 1970. Oral Haemophili. J. Med. Microbiol. 3:615-625. 35. Socransky, S. S., and S. D. Manganiella. 1971. The oral microbiota of man from birth to senility. J. Periodont. 42:485. 36. Spizizen, J. 1971. Microbiological problems of manned spaceflight. Life Sci. Space Res., Comm. Space Res. 9:65-68. 37. Watson, E. D., N. J. Hoffman, R. W. Simmers, and T. Rosebury. 1962. Aerobic and anaerobic bacterial counts of nasal washings. Presence of organisms resembling Corynebacterium acnes. J. Bacteriol. 83:144-148. 38. Zaloguev, S. N., T. G. Utkina, and M. M. Skinkareva. 1971. The microflora of the human integument during prolonged confinement. Life Sci. Space Res. 9:55-59.
1976,
p. 659-665
Copyright © 1976 American Society for Microbiology
Vol. 32, No. 5 Printed in U.S.A.
Autoflora in the Upper Respiratory Tract of Apollo Astronauts J. GLEE DECELLE AND GERALD R. TAYLOR* Northrop Services, Incorporated, Houston, Texas 77034, and National Aeronautics Administration, L. B. Johnson Space Center, Houston, Texas 77058*
and Space
Received for publication 23 April 1976
The typical microbial inhabitants of the oral and nasal cavities of Apollo astronauts were identified before space flight and generally found to be similar to those previously reported for healthy male adults. Additional analyses of samples collected immediately after return of the Apollo 13, 14, 15, and 16 crew members to earth were performed to evaluate the effects of space travel on the microbial bioburden of the upper respiratory tract. In-flight cross-contamination and buildup of pathogens such as Staphylococcus aureus were noted, although significant increases in nonpathogenic species were absent. Other proposed alterations, such as dysbacteriosis (flooding of the mouth with a single species) and simplification of the autoflora, did not occur. Generally, the incidence and
quantitation of each species after flight was within the preflight range, although the number of viable Haemophilus cells recovered from the mouth decreased significantly after space flight. Except for those minor alterations listed above, the aerobic and anaerobic bacterial components of the upper respiratory autoflora of Apollo astronauts was found to be stable after space flight of up to 295 h.
The behavior of infectious, or potentially in- C. A. Berry, Bacteriol. Proc., p. 16, 1967). This fectious, agents in the upper respiratory tract of could presumably result from the efficiency of astronauts is of importance when considering the spacecraft cabin filtration system and the the habitability of the space flight environ- lack of contaminating sources other than acment. Space cabins are by necessity very small, companying crew members. It has further been completely closed chambers, generally with a suggested that this "simplification," typified as recirculating ventilation system. During space a decrease in the number of different types of flight, crews must live close together for ex- microorganisms composing the microbial poputended periods, with frequent intervals of high lation, could be accompanied by a significant activity. This contributes to opportunities for quantitative increase in a single species (1, 4, 6, cross-infections of the type often observed in the 38). This dysbacteriosis (1) has been theorized confined environments of space flight simula- by Zaloguev and his co-workers to result when microorganisms better fitted for life in the tion chambers (6, 7, 23, 30, 31). In addition, certain aspects of space flight space cabin environment crowd out certain othcreate an unusual situation which also must be ers commonly found in the autoflora of the crew evaluated. For example, it has been proposed members (1, 3, 38). Although some of the works referenced above that because of the reduced gravity, droplets expelled from the upper respiratory tract would involve actual space flight analyses, the fragbe larger and would remain in the space cabin mentary data have as yet been inconclusive. At atmosphere longer, thus increasing the oppor- any rate, these studies are not generally applitunity for exchange between astronauts (Yu cable to the Apollo space flight series because of Nefedov, S. N. Zaloguev, and V. P. Savina, important environmental differences. Such dif24th Cong. Int. Astronaut. Fed., 1973). The ferences include, among other things, atmosresults of studies with space simulation cham- pheric pressure and composition (including parbers, and the rudimentary results from the tial pressures of 02 and C02), environmental space flights of Vostok 1 through 4, have control systems, and crew hygiene regimens. prompted investigators in the United States However, these studies should be accepted as and the Soviet Union to suggest that the auto- indicators that the activity of the upper respiflora of the upper respiratory tract could be- ratory autoflora during space flight are potencome simplified (1, 19, 20, 24, 35, 38; H. 0. tially dangerous to the health of the astronaut Wheeler, W. W. Kemmerer, L. F. Dietlein, and (2). Accordingly, the activity of the upper respi659
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DECELLE AND TAYLOR
APPL. ENVIRON. MICROBIOL.
ratory tract autoflora was studied during the throat-mouth gargle were maintained at + 277°K series of Apollo space flights, with special em- (+4°C) during transportation to the L. B. Johnson phasis on the Apollo 13 through 16 missions. Space Center. Storage time was standardized at 10 h The results are evaluated in terms of the effects for all samples collected. The nasal samples in TSB were diluted 10' to of space flight on the quantitative bioburden 10-4 TSB for quantitation of aerobic microorgaand on the activity of microorganisms fre- nisms.in Those samples in VIB were diluted to 10-4 in quently isolated from the nasal and oral cavi- VIB for anaerobic quantitation. A 0.1-cm3 sample ties of the crew members. from each TSB sample and dilution tube was placed on blood agar (Trypticase soy agar with 5% sheep MATERIALS AND METHODS added) and Staphylococcus 110 agar (S110Sample collection schedule. The time periods blood and spread across the surface of the medium during which the upper respiratory tract areas of BBL) with a sterile glass rod. The VIB samples and diluApollo astronauts were sampled are illustrated in tion tubes were plated on anaerobic blood agar (conTable 1. Data from four sampling periods are reTrypticase soy agar, 0.5% yeast extract ported for each Apollo flight. The Apollo 13 and 14 taining crew members were sampled 30 days before, 2 weeks [Difco], 5% sheep blood, and 1.0% vitamin K-hemin solution). The undiluted samples and the 10-1 dilubefore, and immediately preceding flight, as well tions were also plated on paromomycin-vancomycinas immediately after return of the Apollo from space menadione agar. Rogosa agar (Difco) was plated flight. For Apollo 15 and 16, a sampling period 5 with 0.1-cm3 portions from each of the undiluted days before launch was substituted for the 2-week tubes, 10-1, and 10-2 dilution tubes. preflight sampling period of Apollo 13 and 14. Be- sample The gargle samples were diluted 10-1 to 10-5 in cause one crew position was substituted 3 days before launch (8), only the two permanent subjects TSB for aerobic quantitation and in VIB for anaerowere evaluated for the Apollo 13 mission. Three bic quantitation. A 0.1-cm3 sample from each sample and TSB dilution tube was plated on blood agar, astronauts were studied with all of the other re- S110 agar, and mitis-salivarius agar (Difco). Samported missions. from the Apollo 15 and 16 astronauts were also Sample collection procedure. Each crew member ples inoculated onto bacitracin chocolate agar for quantiwas cautioned not to brush the teeth, use a mouth- tation of Haemophilus species. The gargle samples wash, eat, drink, or smoke before sample collection. A pair of calcium alginate swabs (Wilson Diagnos- and VIB dilution tubes were plated on anaerobic tics, Inc., Glenwood, Ill.) moistened with sterile 0.3 blood agar, paromomycin-vancomycin-menadione, mM phosphate buffer was used to sample a 2-cm2 and Rogosa agar. The aerobic plates were incubated at +308°K surface area in each of the two nasal chambers of the Apollo 13 and 14 astronauts. The nasal passages of (+35°C) for 48 h, after which each morphologically colony was quantitated and picked to a the Apollo 15 and 16 crew members were not sam- different pled. To obtain a throat-mouth gargle, all 11 of the tube of TSB, incubated at +308°K, and allowed to until turbid. The resulting broth suspension subject astronauts gargled with 60 cm3 of a 0.3 mM grow phosphate buffer solution which was rinsed through was used to make a Gram stain and to streak onto a the oral cavity three times. All preflight samples blood agar plate to check the purity of the culture. If were taken upon arising, whereas postflight sam- the specimen was shown to be a pure culture, bioples were taken within 1 h after the astronauts were chemical media were inoculated for identification of aboard the recovery ship and before any hygiene the microorganism. The anaerobic plates were incuregimen was initiated. One of each pair of swabs bated at +308°K (+35°C) in an atmosphere of hydrowas placed in a tube containing 5 cm3 of Trypticase gen and carbon dioxide generated by the GasPak soy broth (TSB; BBL), and the remaining swab was (BBL) method. After incubation for 96 h, each morplaced in a tube containing 5 cm3 of veal infusion phologically different colony was quantitated and broth (VIB; BBL). These tubes and containers of picked to an anaerobic broth. An aerobic blood agar plate was also streaked to verify that the picked colony was not capable of growth under aerobic conTABLE 1. Time periods for collection of Apollo ditions. Each broth culture was incubated at +3080K astronaut upper respiratory tract samples (+35°C) until turbid. Gram stains were made and biochemical identification media were subsequently Days before Apollo Crew launch Recov- inoculated from the anaerobic broth. mis- member Sample type ain 5~~Ofl no.o ~~30 15 5 0 ery day RESULTS Recovery of anaerobic bacteria from nasal 13 and 1, 2, 3, 4, Nasal swab +b + -c + + 14 passages of Apollo 13 and 14 crew members. + + _ + + 5 Gargle An average of 1.2 species of anaerobic bacteria . 15 and 6, 7, 8, 9, Nasal swab with a mean quantitation of 2,520 cells/cm2 of 16 10, 11 Gargle + _ + + surface area was recovered from the nasal pasa sages of each astronaut at each sample time Crew member numbers: Apollo 13 = 1, 2; Apollo before flight. The average recovery was 1.0 spe14 = 3, 4, 5; Apollo 15 = 6, 7, 8; Apollo 16 = 9, 10, 11. cies, with a mean quantitation of 41,700 cells/ +, Sample collected. c -, Sample not collected. cm2 the day of return from space flight. Al-
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though there was a slight reduction in the number of different isolates and a slight increase in the total load of viable anaerobic cells, these changes were not statistically significant (P < 0.05). Although representatives of the genera Peptococcus and Peptostreptococcus were infrequently recovered from the nasal passages, only the genus Propionibacterium was isolated frequently enough to be considered a component of the nasal autoflora. Accordingly, only that genus is evaluated here. The preflight recovery pattern of this genus, outlined in Table 2, demonstrates no statistically significant (P < 0.05) difference between the five crew members, indicating that propionibacteria regularly inhabited each crew member at about the same quantitative level. Such regularity provided an opportunity to evaluate the effect of short-duration (Apollo 13, 143 h; Apollo 14, 217 h) space flight on the stable portion of the nasal autoflora. The data shown in Table 2 indicate that the recovery of viable cells, belonging to the genus Propionibacterium, from crew members immediately upon their return from space flight was not significantly (P < 0.05) different than for any preflight sample collection period, except in the case of astronaut 4. This value was higher than the values obtained from the other astronauts postflight and was also higher than the preflight values for this crew member. Because this postflight elevation was not duplicated by the other crew members, it cannot be considered a typical response to space flight. Recovery of aerobic bacteria from nasal passages of Apollo 13 and 14 crew members. The qualitative and quantitative recoveries of
aerobic bacteria from the nasal passages of the five subject crew members at various times before and after space flight are shown in Table 3. When compared to preflight recovery, there is a marked decrease in the number of times the bacilli, corynebacteria, gram-negative rods, and streptococci were isolated immediately after flight. Simultaneous with this decrease, the incidence of the potentially pathogenic Staphylococcus aureus increased from a maximum of two isolates during any preflight period to a maximum of five postflight, being transferred to all of the crew members. Quantitatively, the total nasal population increased significantly (P < 0.05) postflight (Table 3), due to the accession of S. aureus mentioned above as well as an increase in quantitation of corynebacteria and Micrococcaceae. Other microorganisms were isolated too infrequently to establish any pattern. Recovery of anaerobic bacteria from the oral cavity of crew members from Apollo 13 through 16. The quantitative and qualitative recovery of anaerobic bacterial genera from the oral cavity of the 11 subject astronauts is presented in Table 4 in decreasing frequency of isolation. Analysis of the generic recovery frequency data shows no major difference between the occurrence of any particular genus before and after flight. Similarly, quantitative analysis of the mean number of viable cells present belonging to any particular genus indicates that, in general, the postflight load was within the preflight range. Recovery of aerobic bacteria from the oral cavity of crew members from Apollo 13 through 16 crew members. A summary of the
TABLE 2. Quantitationa of the genus Propionibacterium cultured from nasal passages of five Apollo astronauts (Apollo 13 and 14)
TABLE 3. Recovery of aerobic bacteria from the nasal passages offive Apollo astronauts (Apollo 13 and 14)
Days before
Crew member Apollo launch
Day
1
2 3 4 5
14
3.48 1.88 0 2.80 4.00 2.40 3.55 3.58 3.40 0
0
2.58 2.40 2.88 2.80 2.40
ryc
mean
viation
recovery
2.00 3.10 0
5.31b 2.96
2.49 2.08 2.32 3.81 2.19
2.89 2.13 2.61
2.67
2.58
1.63 1.34 0.22 Standard deviation
1.92
1.35
Geometric
Group
Apollo Geomet dard-
no.
30
Days before launch
Stan-
of
+0.73 ±+1.41 ±1.69 ±1.06 ±1.52
mean
a Quantitation expressed in log10 colony-forming units per cm2 of internal nasal surface area sampled. b Significantly higher (P < 0.05 by comparison t test) than other entries in the column and the row.
30
14
Ia Q 0 2.10 5 5.33 3 4.39 5.19 5 4.92 Micrococcaceaee Staphylococcus au- 4.22 2 3.55
Qa 0 Bacillus sp. Corynebacterium sp. 4.72 Gram-negative rods +
Day of re-
covery
0 I
Q
2 4 4 5 2
+b 1
4.07 2.18 4.76 3.63
Q
4 6.09 3 1.40 5 5.51 1 6.60d
I 0 3 2 5 5
0 0
0
I 0
reus
0 1.40 1 0
Streptococcus sp.
0
Total
5.35 15 5.51 18 4.83 14 6.75
15
Q, Total quantitation in log,0 colony-forming units per cm2 of internal nasal surface area sampled; I, incidence, number of times isolated from the five nasal swabs. b +, Isolated but not quantitated. All members of Micrococcaceae except S. aureus. dSignificantly higher (P < 0.05 by comparison t test) than preflight recovery of S. aureus. a
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DECELLE AND TAYLOR
of aerobic bacteria from the gargle of the nasal inhabitants, the autoflora compo11 astronauts is outlined in Table 5. The family nents recovered from the healthy male adults Micrococcaceae and the genera Streptococcus, tested in this study generally agree with earlier Neisseria, and Haemophilus were generally re- reports (21, 22, 25, 26, 28). Aerobic corynebaccovered from each of the 44 gargle samples teria, Micrococcaceae, and anaerobic propioniobtained. Only with the genus Haemophilus bacteria predominated, with minor contribuwas the postflight recovery found to be signifitions from gram-negative rods and streptococci. cantly different from the preflight base line (P Although the nasal bioburden is less complex < 0.05). Members of the Enterobacteriaceae than that recovered from the mouth (21, 28, 37), and other gram-negative rods were infre- it is of major importance in evaluating autoquently observed, and then only in relatively flora responses to space flight. In fact, this low numbers. sparse population lends itself well to analysis because cross-contamination and microbial recovery
buildup are more easily observed against the low background. It has been proposed (Nefedov et al., 24th passages. Although few authors have critically evaluated Cong. Int. Astronaut. Fed., 1973) that the reTABLE 4. Recovery of anaerobic bacteria from gargle samples of 11 Apollo astronauts (Apollo 13, 14, 15, 16) DISCUSSION Bacterial population in the nasal
Recovery (%) frequencyG
Preflight quantitation
(log1O)
Group__
Postflight quantitation
(log1o)
___
Preflight Postflight Meanb SDC Mean SD Bacteroides 100 100 4.76 +0.71 3.85 ±+1.07 Fusobacterium 100 100 ±0.84 4.24 4.03 +0.92 Veillonella 90.9 100 4.47 ±1.08 4.28 ±0.84 Leptotrichia 66.7 45.5 ±0.87 3.03 2.57 ±0.50 Propionibacterium 51.5 54.6 +0.74 4.11 4.15 ±1.41 Lactobacillus 36.4 54.6 3.41 ±+1.21 4.35 ±1.46 Peptostreptococcus 33.3 27.3 4.70 +0.48 4.62 +0.28 Bifidobacterium 21.2 27.3 2.89 +0.99 1.92 ±0.79 Peptococcus 12.1 9.1 4.07 ±1.05 0.35 ±1.18 Eubacterium 9.1 18.2 4.20 3.77 ±0.12 +1.80 Actinomyces 3.0 0.0 0.07 +0.40 0 Total anaerobic load 5.37 +0.57 5.24 ±0.67 a Number of times isolated out of number of samples collected (x 100). Preflight, 33 samples (three per astronaut); postflight, 11 samples (one per astronaut). b Mean quantitation in log,0 colony-forming units per cm3 of gargle. Only samples positive for a group are used to calculate mean. c SD, Standard deviation. TABLE 5. Recovery of aerobic bacteria from gargle samples of 11 Apollo astronauts (Apollo 13, 14, 15, 16)
Preflight quantitation
Group
Streptococcus Neisseria
Recovery frequency" ( Preflight Postflight
100 97 97 96.3 90.9 81.5 36.4 26.3
100 100 81.8 88.9 90.9 66.7 18.2 45.5
Postflight quantitation
(loglo) Meanb
6.23 4.78 4.74 5.18 3.01 3.03 2.50 3.17
SDc ±0.88 ±0.99 ± 1.04 ±0.76 ±1.35 ±1.27 ±1.14 ±1.32
(log1o) Mean
SD
6.56 5.23 4.84
±0.64 ±0.99 ±0.90 +0.53 ±1.51 ±1.30 ±1.57 ±1.28
Corynebacterium Haemophilus 4.37d Micrococcaceae 3.59 Lactobacillus 3.06 Enterobacteriaceae 2.59 Other gram-negative 4.09 bacilli Total aerobic load 6.37 ±0.76 6.65 ±0.59 a Number of times isolated out of number of samples collected (x 100). Preflight, 33 samples (three per astronaut); postflight, 11 samples (one per astronaut). b Mean quantitation in log10 colony-forming units per cm3 of gargle. Only samples positive for a group are used to calculate mean. c SD, Standard deviation. d Significant at 5% level (comparison t test).
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duced gravitational attractions experienced flight. Likewise, there was a significant postduring space flight would increase the probabil- flight increase (P < 0.05) in the total number ity of respiratory infections by favoring the pro- of viable S. aureus cells recovered from the duction of larger particles, or droplets, expelled astronaut population. These data support the into the environment. The formation of these anticipated in-flight cross-contamination and larger particles, which would not settle out as buildup of pathogenic species, whereas nonthey do on earth, would effectively result in an pathogens remained essentially stable. increased infecting dose being drawn into the Bacterial population in the oral cavity. Innasal cavity. The increased particle size may vestigations of the oral autoflora have been also affect a change in the filtration ability of conducted with a variety of test subjects and the lungs and epithelial tissues (15; Nefedov et wide-ranging methods and have been repeatal., 24th Cong. Int. Astronaut. Fed., 1973). Fur- edly reviewed (5, 9, 11, 12, 14, 17, 28, 35). Rethermore, it has been suggested (2, 23, 38) that sults have most often been analyzed with rethis increased contamination potential would spect to whole saliva or specific oral surfaces not be the same for all microorganisms. In the such as the gingival crevice, surface of the absence of recontaminating sources, transient tongue, or coronal plaque. Additionally, such microorganisms would be removed by the investigations have usually been associated spacecraft air filtration system and thereby lost with studies of dental caries and peridontal from the autoflora. Therefore, the makeup of disease events and have emphasized the lactothe nasal microbial population could become bacilli and streptococci, which are of interest in simplified, with the remaining species being po- oral disease analyses. In general, previous tential pathogens and/or true members of the studies have shown the bacterial population nasal autoflora. Nefedov et al. (24), in summa- within the oral cavity of healthy persons to be rizing the results of several Soviet space quite stable from day to day, provided the influflights, have concluded that there sometimes is ences of eating, smoking, and mastication are a simplification of the nasal flora which in- controlled. Also, the composition of the oral cludes a disappearance of nonpathogenic micro- autoflora of the individual members of a reaorganisms and a predominance of pathogenic sonably homogeneous group is usually found to be quite similar. species. The study reported herein was conducted in Data obtained from the analysis of astronaut nasal samples were evaluated to determine if an effort to determine if the various pressures similar changes occurred during the subject of short-term (143 to 295 h) space flight exerted Apollo missions. The recovery incidence and any detectable alteration in the qualitative or quantitation of all anaerobic bacteria recovered quantitative composition of the oral autoflora of from the nasal passages after flight was not the crew members. Accordingly, it was necessignificantly different (P < 0.05) from values sary to collect a sample that was representative obtained before flight. Because of the normally of the oral cavity as a unit. For this reason, low incidence of anaerobes other than Propioni- loose microorganisms were washed from the bacterium, it is appropriate to evaluate this mouth and oral-pharyngeal vault of each subgenus alone. Even within this group, such vari- ject. Because larger numbers of bacteria accuability was demonstrated that a space flight- mulate in the oral cavity during sleep, when induced shift could not be shown. Although the salivary flow is greatly reduced (18, 29), collecpostflight recovery from one astronaut (Table 2) tion just after arising provided the best opporwas significantly high (P < 0.05) postflight, the tunity to recover oral inhabitants. In all cases, overall pattern mediates against alterations as- the subjects were healthy male adults without apparent oral disease symptomatology. The resociated with space flight. Compared with anaerobes, aerobic bacteria sulting data should relate most closely to inforwere more numerous both in terms of species mation obtained from studies with whole sarecovered and total quantitation. These data liva, because both reflect the sum of the contriare counter to those reported by Watson et al. butions from all oral surfaces (9, 10). Anaerobic bacteria. Analysis of the generic (37), in which anaerobes were generally present at a level 10 times higher than the aerobic recovery frequency data presented in Table 4 quantitation. These conflicting reports could demonstrates the ubiquitous appearance of the easily result from the different methods used. genera Bacteroides, Fusobacterium, and VeilThe data presented in Table 3 do not indicate lonella. The mean recovery of these common simplification of the aerobic autoflora. How- inhabitants of the healthy mouth during the ever, there is evidence of a significant increase preflight collection period was always in excess in number of individuals (to the maximum of of 10,000 viable cells per cm3 of wash. Gramfive) from which S. aureus was recovered post- positive rods were often recovered, especially
664
DECELLE AND TAYLOR
Leptotrichia and Propionibacterium. Veillonella was recovered at higher quantitation than expected, whereas actinomycetes were recovered less frequently than usually reported (13). Although the mean recovery of Bacteroides from the mouth of astronauts immediately after their return from space flight was somewhat lower than the preflight base line, no statistically significant quantitative changes in the three most common genera were observed (P < 0.05). The other genera as well, although less frequently recovered and therefore less stable, were not observed to change significantly postflight (P < 0.05). The dysbacteriosis, or flooding of the oral cavity with large numbers of bacterial cells, previously reported after certain space flights from the Soviet Union (1, 3, 38) was shown not to occur during the four Apollo missions analyzed in this report. The continued stability of the incidence of anaerobic genera contraindicated microbial simplification within the oral cavity. There was no discernible difference between the recovery of anaerobic bacteria from the oral cavity of the 11 subject astronauts before and after space flight. Aerobic bacteria. The data presented in Table 5 indicate that during the preflight period, members of the genera Streptococcus, Neisseria, Corynebacterium, and Haemophilus were virtually omnipresent at values ranging from 55,000 to 1,700,000 cells/cm3 of wash. As reported by others (27, 28), the streptococci were both the most frequently isolated and the most numerous of all aerobic bacteria recovered. Neisseria was isolated in larger numbers than typically reported (13). The genus Haemophilus, which has only recently been fully recognized as a major component of human oral autoflora (13, 34), was routinely isolated. Although there was no significant change in the total aerobic load or in any other reported microbial group, the preflight stability of the genus Haemophilus was adequate to detect a significant postflight decrease in quantitation (P < 0.05). Members of the family Micrococcaceae and the genus Lactobacillus were frequently isolated but at lower quantitative levels. This relatively low recovery of lactobacilli reflects the lack of carious lesions within the astronaut population (16, 32, 33). The absence of any increase in the quantitation of gram-negative rods indicates that flooding of the oral cavity with these microorganisms, reported by Zaloguev et al. (38), did not occur. It appears that this flooding, which has been reported only by investigators in the Soviet Union, was an isolated incident and was not related to space flight. These data, comparing pre- and postflight analyses from
APPL. ENVIRON. MICROBIOL.
four different short-duration space flights, demonstrate that the aerobic bacteria in the oral cavity are quite stable both in terms of the genera and the total number of viable cells present. The quantitative recovery from these wash samples is understandably lower than those reported for whole saliva (where quantitation for aerobes and anaerobes may reach 8 x 1010 and 9 x 1010 cells per cm3 of saliva, respectively). However, an important difference is the ratio of aerobes to anaerobes. Whereas the anaerobes in whole saliva may outnumber the aerobes (27), the opposite was observed with whole mouth washes. Viable aerobic cells were consistently recovered at a level 10 times greater than that obtained for anaerobic cells, a condition which was unchanged postflight. Studies associated with early United States and Soviet Union space flights and groundbased simulations have indicated that the microflora inhabiting the upper respiratory tract of crew members may be altered during such missions. To evaluate this possibility, samples collected from the nasal passages of Apollo 13 and 14 crew members and from the oral cavity of Apollo 13 through 16 astronauts were quantitatively evaluated for the presence of aerobic and anaerobic bacteria. The anaerobic autoflora in the nasal passages, consisting almost exclusively of members of the genus Propionibacterium, remained unchanged postflight (with the exception of one subject). Representatives of Micrococcaceae and Corynebacterium were predominant in the nasal autoflora which demonstrated a postflight increase in the incidence and quantitation of S. aureus, concurrent with a decrease in the incidence of other bacterial isolations. Members of the genera Bacteroides, Fusobacterium, and Veillonella were ubiquitou-sly present in the mouth and remained unchanged after the space flights. The major aerobes present in the mouth belonged to Micrococcaceae, Streptococcus, Neisseria, and Haemophilus. There was a significant decrease in the number of viable Haemophilus cells recovered after flight. With the above exceptions, the bacterial flora inhabiting the upper respiratory tract of Apollo astronauts was found to remain stable after space flight of up to 295 h. ACKNOWLEDGMENTS This study was partially funded by National Aeronautics and Space Administration contract NAS 9-13000 to Northrop Services, Inc., Houston, Tex. 77034. We especially wish to thank Kathryn Kropp, Theron Groves, Carolyn Carmichael, Mary Arredondo, Nem Bryan, Walter Ellis, and Sara F. Wright for their technical assistance.
VOL. 32, 1976
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