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Effects of Microgravity and Clinorotation on the Virulence of Klebsiella, Streptococcus, Proteus, and Pseudomonas

Timothy G. Hammond
Timothy G. Hammond
, 
Louis Stodieck
Louis Stodieck
, 
Paul Koenig
Paul Koenig
, 
Jeffrey S. Hammond
Jeffrey S. Hammond
, 
Margaret A. Gunter
Margaret A. Gunter
, 
Patricia L. Allen
Patricia L. Allen
 e 
Holly H. Birdsall
Holly H. Birdsall
   | 17 lug 2020
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Article Category: Research Article
Pubblicato online: 17 lug 2020
Pagine: 39 - 50
DOI: https://doi.org/10.2478/gsr-2016-0004
Parole chiave
© 2016 Timothy G. Hammond et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Flight Processing Apparatus (FPA). The left end of the FPA is plugged with a breathable Gortex membrane. To activate the experiment, the plunger is advanced to mix C. elegans in chamber 1 with Luria or Tryptic Soy broth in chamber 2, and microorganisms in saline in chamber 3. At the end of 48 hours, the plunger is further advanced to empty the paraformaldehyde from chamber 4 into the mixture and terminate the experiment.
Flight Processing Apparatus (FPA). The left end of the FPA is plugged with a breathable Gortex membrane. To activate the experiment, the plunger is advanced to mix C. elegans in chamber 1 with Luria or Tryptic Soy broth in chamber 2, and microorganisms in saline in chamber 3. At the end of 48 hours, the plunger is further advanced to empty the paraformaldehyde from chamber 4 into the mixture and terminate the experiment.

Figure 2

Design and timeline of the experiments. Growth controls were set up with live microorganisms grown in the absence of worms. Worm feeding controls were conducted with L2 larvae and heat-killed E. coli. Virulence in adult and larval worms was evaluated by preparing C. elegans eggs just before launch. All Group Activation Packs (GAPs) were loaded one day before launch and passed off. Eggs hatching in basal medium are growth-arrested at the L2 stage, whereas larvae hatching in the presence of heat-killed E. coli as a food source can mature into L3/4 larvae. Virulence assays were activated approximately 46 hours after launch and terminated by fixation after an additional 48 hours. The timeline reflects hours prior to and after launch, which is defined as zero.
Design and timeline of the experiments. Growth controls were set up with live microorganisms grown in the absence of worms. Worm feeding controls were conducted with L2 larvae and heat-killed E. coli. Virulence in adult and larval worms was evaluated by preparing C. elegans eggs just before launch. All Group Activation Packs (GAPs) were loaded one day before launch and passed off. Eggs hatching in basal medium are growth-arrested at the L2 stage, whereas larvae hatching in the presence of heat-killed E. coli as a food source can mature into L3/4 larvae. Virulence assays were activated approximately 46 hours after launch and terminated by fixation after an additional 48 hours. The timeline reflects hours prior to and after launch, which is defined as zero.

Figure 3

Virulence of four microorganisms towards adult and larval worms in ground/static, spaceflight, and clinorotation. Microorganisms were cultured with nematodes in identical hardware under static conditions, clinorotation, and space-flight and fixed with formaldehyde after 48 hours of co-incubation. Delta (Δ) OD620 values are the OD620 for microorganism in the presence of larval or adult worms minus the OD620 for microorganism cultured alone. Error bars indicate ± 1 SEM of quadruplicates. Two-tailed unpaired t-tests were used to estimate the significance between the ΔOD620 for spaceflight or clinorotation, versus static conditions and clinorotation versus spaceflight. * indicates p<0.05 relative to static controls, # indicates p<0.05 for clinorotation versus flight. The positive ΔOD620 with the static control suggests that there is some debris generated when microorganisms are incubated with larvae or adult worms (or that the microorganisms grew better in the presence of C. elegans). Smaller ΔOD620 indicates more consumption of microbes and/or less generation of debris under spaceflight. Either explanation is consistent with decreased virulence. Assays of virulence were done concurrently in spaceflight, static ground, and clinorotation.
Virulence of four microorganisms towards adult and larval worms in ground/static, spaceflight, and clinorotation. Microorganisms were cultured with nematodes in identical hardware under static conditions, clinorotation, and space-flight and fixed with formaldehyde after 48 hours of co-incubation. Delta (Δ) OD620 values are the OD620 for microorganism in the presence of larval or adult worms minus the OD620 for microorganism cultured alone. Error bars indicate ± 1 SEM of quadruplicates. Two-tailed unpaired t-tests were used to estimate the significance between the ΔOD620 for spaceflight or clinorotation, versus static conditions and clinorotation versus spaceflight. * indicates p<0.05 relative to static controls, # indicates p<0.05 for clinorotation versus flight. The positive ΔOD620 with the static control suggests that there is some debris generated when microorganisms are incubated with larvae or adult worms (or that the microorganisms grew better in the presence of C. elegans). Smaller ΔOD620 indicates more consumption of microbes and/or less generation of debris under spaceflight. Either explanation is consistent with decreased virulence. Assays of virulence were done concurrently in spaceflight, static ground, and clinorotation.

OD620 of Microorganisms after Growth and Virulence Assays. OD620 of microorganisms grown in medium alone (growth) or after addition into cultures of larval or adult worms. Cultures in identical hardware were maintained under static/ground, spaceflight, or clinorotation conditions. After 48 hours of growth, samples were fixed with paraformaldehyde. Values are the mean + 1 SEM of quadruplicates.

PseudomonasKlebsiellaProteusStreptococcus
Growth Static/Ground0.263 ± 0.1060.307 ± 0.0220.386 ± 0.0550.256 ± 0.013
Larval Worms Static/Ground0.511 ± 0.01030.532 ± 0.0120.539 ± 0.0040.423 ± 0.017
Adult Worms Static/Ground0.508 ± 0.0340.601 ± 0.0010.686 ± 0.0230.557 ± 0.024
Growth Spaceflight0.227 ± 0.0170.302 ± 0.0130.307 ± 0.0030.207 ± 0.050
Larval Worms Spaceflight0.501 ± 0.0160.458 ± 0.0060.475 ± 0.0130.344 ± 0.005
Adult Worms Spaceflight0.558 ± 0.0200.565 ± 0.0240.580 ± 0.0380.381 ± 0.050
Growth Clinorotation0.553 ± 0.0120.597 + 0.0100.617 ± 0.0110.440 ± 0.080
Larval Worms Clinorotation0.589 ± 0.0210.485 + 0.0250.602 ± 0.0120.523 ± 0.008
Adult Worms Clinorotation0.722 ± 0.0100.685 + 0.0180.708 ± 0.0220.676 ± 0.011

Effect of Spaceflight and Clinorotation on Virulence of Microorganisms towards Adult and Larval C. elegans. The effect of spaceflight and clinorotation on virulence of four microorganisms for adult and larval worms is summarized along with the morphology of the microorganisms. Statistical significance of spaceflight or clinorotation versus static conditions was estimated by two-tailed unpaired Student's t-test.

MicroorganismShapeAdult Worms SpaceflightLarval Worms SpaceflightAdult Worms ClinorotationLarval Worms Clinorotation
PseudomonasRodMotile⇔p = 0.07⇔p = 0.2⇔p = 0.08⇓⇓⇓p <0.001
KlebsiellaRodNon-motile⇔p = 0.2⇓⇓p <0.01⇓⇓⇓p <0.001⇓⇓⇓p <0.001
ProteusRodMotile⇔p = 0.6⇔p = 0.3⇓⇓⇓p <0.001⇓⇓⇓p=0.001
StreptococcusCocciChains⇓p = 0.06⇔p =0.14⇓⇓p = 0.05⇓⇓p <0.01

Effect of Spaceflight and Clinorotation on the Virulence of Nine Different Microorganisms. Compilation of our laboratory's studies using the C. elegans model for assaying virulence in spaceflight and clinorotation. Results for Listeria monocytogenes, Enterococcus faecalis, Candida albicans, and Methicillin-resistant Staphylococcus aureus (MRSA) are taken from Hammond et al. (Hammond et al., 2013b). Results for Salmonella are from Hammond et al. (Hammond et al., 2013c).

Increased VirulenceUnchanged VirulenceDecreased Virulence
Adult Worms SpaceflightKlebsiella Proteus Pseudomonas SalmonellaCandida Enterococcus Listeria MRSA Streptococcus
Larval Worms SpaceflightProteus Pseudomonas Salmonella StreptococcusKlebsiella Candida Enterococcus Listeria MRSA
Adult Worms ClinorotationEnterococcus Listeria MRSA Pseudomonas SalmonellaKlebsiella Proteus Streptococcus
Larval Worms ClinorotationListeria MRSA SalmonellaCandida Enterococcus Klebsiella Proteus Pseudomonas Streptococcus
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