While the focus was on lunar contamination, analysis of crew specimens was performed to satisfy three additional objectives:
1) detection of potentially pathogenic microorganisms so that associated medical problems could be identified early and preventive measures could be established
2) identification of medically important microorganisms recovered from ill crewmembers to aid in diagnoses and treatment
3) evaluation of the space flight environment's effect on the crew's microbial autoflora and the resultant effect this potential change had on crewmembers. Studies conducted during the final missions of Apollo were designed to identify and prepare for possible microbial problems associated with the lengthy Skylab missions.
1) a 13 square centimeter area at the base of the neck below the hairline
2) the auditory canals of both ears
3) the internal area of the umbilicus and a surrounding 13 square centimeter area
4) a 6.5 square centimeter area below the hairline of each axilla
5) the area on the left and right of the groin, from front to rear
6) the area between the big and second toe of each foot
7) a 6.5 square centimeter area on each palm
8) both nostrils
9) the throat and mouth
10) a first-void midstream urine specimen
11) a fecal specimen
Body surface and nasal passage samples were obtained preflight and postflight (in duplicate) using sterile calcium alginate swabs moistened with phosphate buffer. One swab from each sample area was placed in a screw cap tube containing 10 ml of sterile trypticase soy broth (TSB) for aerobic analysis of microorganisms. The second swab was placed in a tube of sterile veal infusion broth (VIB) for anaerobic analysis. Throat and mouth samples were taken from a 60 ml sterile phosphate buffer gargle rinse. The gargle was rinsed three times orally to obtain a combination of throat-mouth sample, then emptied into a sterile wide-mouthed bottle. Urine and fecal specimens were collected on the mornings of preflight sampling days. Postflight, these samples were collected as they became available. Samples were maintained at 4 degrees Celsius during transport to the laboratory; approximately 12 hours elapsed from the time of sampling to the time of initial culture.
One milliliter aliquots of the throat-mouth gargle and urine sample were transferred to 9.0 ml each of TSB and VIB. Portions of fecal material, weighing 0.1 gram each, were transferred to TSB, VIB and tetrathionate broth. In addition, 0.1 gram of fecal material was heat shocked for five minutes at 80 degrees Celsius. Dilution series from each TSB and VIB sample tube were prepared by aseptically transferring 1.0 ml aliquots to 9.0 ml of TSB or VIB. Body and nasal samples were diluted to 10,000; throat-mouth gargle samples to 100,000; urine samples to 100; and fecal samples to 10 billion. All tubes were maintained at 4 degrees Celsius during the diluting process.
One-tenth milliliter aliquots from the initial TSB and VIB sample tubes and each dilution series tube were inoculated onto the surface of agar media for quantitation and isolation of aerobic and anaerobic microbial species. Aerobic media were incubated for 48 hours at 35 degrees Celsius. Anaerobic media were incubated for 96 hours at 35 degrees Celsius using an anaerobic Gas Pak for hydrogen gas generation. Colony counts were then performed on the aerobic and anaerobic quantitation media.
After 4.0 ml aliquots from each TSB sample tube (initial and dilution series) were transferred to labeled sterile screwtop tubes for mycological analysis, the TSB and VIB sample tubes were incubated for 24 hours at 35 degrees Celsius. Inocula from the sample tubes were then streaked onto chocolate agar isolation media and incubated at 35 degrees Celsius under an atmosphere of 8 to 10 percent carbon dioxide. These streaks were prepared to culture organisms too few in number to be isolated on quantitative media.
A portion of each colony type isolated on the quantitation and isolation media was transferred to a TSB or thioglycolate broth tube and incubated at 35 degrees Celsius until turbid. Pure cultures of each organism were used to prepare stained slides, inoculate biochemical media and perform biochemical tests for identification.
Swab samples were collected from the Apollo Command Module immediately before and after flight from the following sites:
1) the total surface area of the drink gun mouthpiece (the "drinking fountain" aboard Apollo)
2) a 13 square centimeter area of each pistol grip of the Command Module Pilot (CMP) maneuver controller
3) a 13 square centimeter area of each head strut
4) a 26 square centimeter area of the floor beneath the foot of the center couch
Two sterile calcium alginate swabs moistened with 0.85% saline containing 0.0003 molar phosphate buffer were used to swab each of the chosen sites in the spacecraft. After sampling, one swab from each site was placed in 5.0 ml TSB and the other in 5.0 ml VIB. The tubes were maintained at 4 degrees Celsius during transport to the laboratory. Each tube was vortexed and serially diluted with the appropriate medium. An aliquot of each TSB dilution was plated onto 5% sheep blood agar and incubated aerobically at 35 degrees Celsius for 48 hours. An aliquot of each VIB dilution was plated onto sheep blood agar containing 10 mg/liter vitamin K and 5 mg/liter hemin. The plates were incubated anaerobically at 35 degrees Celsius for 96 hours.
Four milliliters of undiluted TSB samples were used for mycological analysis. Each sample was centrifuged at 2500 rpm for 15 minutes. The supernatant was mixed with 10 ml of yeast-malt broth containing 33,000 units /liter penicillin G and 62 mg/liter streptomycin. The sediment was sampled with sterile calcium alginate swabs and streaked onto cornmeal-malt-yeast agar (containing antibiotics), Sabouraud dextrose agar (containing antibiotics) and Czapek Dox agar. The sediment swabs were then placed into 10 ml of yeast-malt broth (containing antibiotics). All mycological media was incubated at 25 degrees Celsius for 120 hours.
Final identification resulted in approximately 150 to 175 microorganisms per sampling period. The same microorganism was often isolated at more than one site. When the first lunar material was returned by Apollo 11 crewmen, the catalog contained identification data for approximately 4000 microbial isolations. Throughout the Apollo missions, no microorganisms were isolated from lunar soil. This confirmed the successful operation of the Lunar Receiving Laboratory and development of adequate aseptic techniques for handling and processing lunar soil.
Crew Medical Microbiology
Increased Incidence and Transfer of Microorganisms Between Crewmen: An increased incidence of medically important gram-positive cocci was found for Apollo 7. Immediately before flight, five Staphylococcus aureus isolations were made from two crewmen. Postflight, 16 isolations of S. aureus were made at various sampling sites on all three crewmembers. After this apparent transfer of microorganisms between crewmen, strain-specific bacteriophage typing was developed and performed on all S. aureus recovered from subsequent missions to better substantiate the suspected transfer.
Although not present preflight, beta-hemolytic streptococci were present postflight in the gargle sample of all three Apollo 7 crewmen. Each sample contained 1000 streptococcal cells/ml of gargle. The presence and abundance of beta-hemolytic streptococci and S. aureus on Apollo 7 undoubtedly contributed to the nasal congestion and discomfort experienced by crewmembers.
A third microorganism, Aspergillus fumigatus, increased in number and apparently spread over the body surfaces of Apollo 7 crewmembers. With a single exception, all preflight A. fumigatus isolations were from one crewmember. Postflight, isolations were made on all crewmembers, indicating one crewmember transferred the fungi to the others. No significant increase of A. fumigatus or any other fungus occurred on any mission through Apollo 12.
An increased incidence of S. aureus did not reoccur until the Apollo 12 flight. Although only two isolations of S. aureus (phage type 3A) were made from one crewmember at L-0 (preflight measurements on launch day), seven of the twelve postflight crewmember samples (taken from 3 crewmembers) were positive for S. aureus. The microorganism was apparently transferred from the initial crewman to a second crewmember, to the urine collection device (UCD) of the third crewmember and to the couch support struts of the Command Module. Although no inflight samples were obtained and pustules on the crewmen's skin had dried by the postflight examination, S. aureus may have caused skin infections aboard Apollo 12. On Apollo 13, two crewmembers carried S. aureus preflight, but of different strains. Transfer was again demonstrated when both strains were recovered postflight from the third crewman, who had not exhibited either strain preflight.
During Apollo missions 8, 9, 10 and 11, S. aureus was not exchanged between crewmembers and numbers did not increase, even though at least one crew member on two of the missions was carrying a nasal strain.
Specific Medical Microbiological Problems Associated with the Apollo 13, 14 and 17 Flights:
A Service Module malfunction, which caused early termination of the Apollo 13 mission, created a suboptimal environment and stressful situation for its crew. Crew examination immediately after flight revealed that one crewmember had a severe urinary tract infection caused by Pseudomonas aeruginosa. Antibiotic therapy was administered and closely monitored for 48 hours following recovery. Viable microbes had disappeared from midstream urine samples within nine days following splashdown, but P. aeruginosa could still be recovered following prostatic massage after 16 days. Also noted was an increased incidence of pathogenic microorganisms on the body surface. Whereas only three pathogenic species (S. aureus, H. vaginicola and Escherichia coli) were recovered on the morning of launch, seven medically important species were recovered immediately after splashdown (S. aureus, Pseudomonas sp., Streptococcus sp., Klebsiella sp., H. vaginicola, E. coli and Mima polymorpha). In addition, the number of isolates of each species was higher after flight. Although there was a slight postflight increase of pathogens in other crews, the occurrence on Apollo 13 was significantly elevated. An average of 175% more medically important species were recovered from the seven Apollo 13 postflight skin swabs as compared with an average increase of only 33% for the same samples from Apollo 14. This illustrates infectious problems that can occur when the life support system operates suboptimally for even a short period.
Urinalyses were performed on one Apollo 14 crewmember several times during the 26 months preceding liftoff in response to a recurrent urethritis of possible microbial origin. However, no microorganisms were recovered until seven months before launch. Urine samples were evaluated periodically through the day of launch, and seven medically important microorganisms were isolated. Of the microorganisms recovered, Haemophilus sp. was most likely to cause bacteria-mediated recurrent urethritis. No symptoms occurred during the Apollo 14 space flight, although Haemophilus sp. was again isolated two weeks following landing. As was usually the case, the presence of potentially pathogenic microorganisms Klebsiella pneumoniae, Proteus mirabilis and Herellea vaginicola in the postflight urine reflected the similar buildup observed in the UCD.
It was not unusual to find at least one crewmember from each Apollo team harboring Candida albicans, a pathogenic yeast in the mouth. The presence of this species does not pose a significant threat to healthy adults. However, fungi that control C. albicans populations through microbial competition decreased dramatically during space flight. This population shift creates a situation in which the natural resistance to infection may be depressed at a time when clinical diagnosis and treatment are most difficult. The presence of C. albicans, as well as other Candida sp. implicated in a variety of pathogenic situations, was monitored during each Apollo flight. No anomalies traceable to yeast infections were noted among any Apollo crewmembers. However, Apollo data demonstrated that a lack of microbial competition could mediate a disease state during missions of longer duration.
A crewmember of Apollo 17 exhibited chronic dermatitis of the groin and feet, preflight and postflight. The pathogenic fungus Trichophyton rubrum was isolated as the causative agent at each sampling period. A similar dermatitis was present on the toes of another crewmember, although the causative agent could not be cultured. The presence of active dermatophyte infections on two Apollo 17 crewmembers afforded an opportunity to study the response of this disease to short-term space flight. Postflight lesion analysis revealed no discernible change from the preflight condition. Likewise, there was no evidence of transfer of T. rubrum to other parts of the body. The opportunistic pathogen, Pseudomonas aeruginosa, present on the toes of one Apollo 17 crewmember, created a possible avenue for a secondary infection. P. aeruginosa was present preflight and spread to the toes of other crewmembers during flight. However, presence of this species near the dermatophytic lesions did not result in a secondary P. aeruginosa mediated infection.
Spacecraft and Clothing Microbiology
Spacecraft microbial samples from Apollo missions 7 through 12 were evaluated and grouped by morphological type (gram-positive cocci, Bacillus sp., Diphtheroids, gram-negative rods, filamentous fungi and yeasts). Although the sample population was small, increased numbers of potential pathogens were observed. S. aureus was isolated during preflight sampling of only the Apollo 10 spacecraft, but could not be recovered postflight. However, several medically important organisms were isolated postflight from many Apollo missions.
Of the 79 morphological types isolated preflight, only ten species were reisolated postflight. The reisolated types were primarily Staphylococcus epidermidis and Micrococcus sp. These isolates are predominant in the human microflora and their reisolation from spacecraft samples is probably caused by recontamination of sample sites by crew members rather than survival in the Command Module.
The transfer of microorganisms between crewmen and Command Module or extravehicular activity clothing became more obvious in subsequent mission analysis. Apollo 14 data showed various sites harbored a variety of microbial species. Command Module habitation did not affect a significant change in the number of contaminating species. However, there was an obvious loss of original contaminants on each site with a concurrent invasion of different microbe species. Apollo 14 data also illustrate the general buildup phenomena of medically important species during the space flight. Only one pathogenic species (Pseudomonas maltophilia) was recovered from the Command Module sites on L-0 (launch day), although four pathogens (H. vaginicola, Klebsiella pneumoniae, Proteus mirabilis and Streptococcus faecalis) were recovered postflight. This same pattern was noted for each flight.
The increased incidence of medically important microorganisms is more obvious from UCD analysis. UCDs were first sampled in the clean room at NASA's John F. Kennedy Space Center on the morning of launch, and were generally free of microbes. However, samples collected in the Command Module immediately upon recovery contained a variety of contaminants. All but one species (Bacillus) recovered postflight from Apollo 14 devices were potential pathogens. The buildup of P. mirabilis on the UCD reoccurred throughout most Apollo missions.
Statistical Analysis of Crew Microflora:
Statistical analysis, using paired t-tests, was performed on the bacterial flora data from Apollo missions 7 through 11 to identify significant changes in the number or occurrence of microorganisms in the postflight period as compared with the preflight period. Comparisons were made by testing both the sum of bacterial counts within a genus and the sum of occurrence of a genus at each sampling site. Selected times for comparison were L-30 and L-0 (30 days before launch and launch day); L-30 and R+0 (30 days before launch and landing day); and L-0 and R+0 (launch day and landing day). The tests were performed on microflora of the stool, urine, throat-mouth gargle and body surface samples. The microflora from each sample were further divided into groups of aerobic gram-positives, anaerobic gram-positives, aerobic gram-negatives and anaerobic gram-negatives. A high degree of variation was observed in the microflora between sampling periods, between crewmembers and between missions. No other consistent microflora alteration was observed by this test method.
Apollo Crew Virology
Serological titers were determined preflight on crewmembers, crew contacts and key mission personnel to ascertain immune status to mumps, rubella and rubeola. The astronauts' immune status to poliomyelitis virus types 1, 2 and 3 was also determined. In addition, complement-fixation antibody titers to influenza A, influenza B, enteric cytopathogenic human orphan (ECHO) virus (group), adenovirus (group), parainfluenza, herpes simplex, Mycoplasma pneumoniae, cytomegalovirus and respiratory syncytial virus (RSV) were determined for the crew members.
Poliomyelitis virus was isolated from the preflight stool samples of the Apollo 11 crewmembers after receiving poliomyelitis boosters. Herpes simplex virus was isolated from the throat-mouth gargle collected on L-0 from one Apollo 15 crewmember. This virus was not isolated from postflight specimens. A2 Hong Kong influenza was established as the cause of the postflight illness of Apollo 7 crewmen. Postflight illnesses in two Apollo 9 crewmembers were confirmed as influenza B by virus isolation and identification.
Mycoplasma species were routinely isolated from preflight and postflight specimens from all Apollo crewmen. Throat specimens frequently yielded M. salivarium and M. orale I, and M. hominis was isolated from the urine. M. laidlawii A was isolated from throat and urine specimens of Apollo 12 crewmen. Some evidence of cross infection was noted. Usually, Mycoplasma sp. were isolated from one or two crewmembers, and the same species were isolated before and after flight.
In conclusion, significant changes were noted in the microfloral response during the Apollo missions. Host susceptibility, external environmental factors and ecological relationships among competing species of microorganisms were undoubtedly responsible for the observed response. Analysis of Command Module samples exhibited a loss of preflight microorganisms, which were replaced by crew microorganisms during spaceflight. An increased incidence and spread of potentially pathogenic microorganisms between crewmembers was demonstrated on several missions. However, observations did not suggest the spacecraft environment predisposed crewmembers to viral or mycoplasma induced illness.
|Mission||Launch/Start Date||Landing/End Date||Duration|
|Apollo 10||05/18/1969||05/26/1969||8 days|
|Apollo 11||07/16/1969||07/24/1969||8 days|
|Apollo 12||11/14/1969||11/24/1969||10 days|
|Apollo 13||04/11/1970||04/17/1970||6 days|
|Apollo 14||01/31/1971||02/09/1971||9 days|
|Apollo 15||07/26/1971||08/07/1971||12 days|
|Apollo 16||04/16/1972||04/27/1972||11 days|
|Apollo 17||12/07/1972||12/19/1972||12 days|
|Apollo 7||10/11/1968||10/22/1968||11 days|
|Apollo 8||12/21/1968||12/27/1968||6 days|
|Apollo 9||03/03/1969||03/13/1969||10 days|