The data were published several years ago in PNAS (Wilson et al. 2007 Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. PNAS 104:16299-16304). The paper reports differences in gene expression when Salmonella typhimurium cells growing on the space shuttle were compared to the same bacteria growing under identical conditions on the ground. I remember thinking this was a weird result at the time, but I didn't take the trouble to carefully investigate exactly what had been done. But now I have.
- The array results were inconsistent.
- The effects were due to suspension vs settling of the cells, not microgravity.
- The doses of space-grown and Earth-grown bacteria in the virulence experiment were not well controlled and differences could account for the apparent differences in virulence.
- The evidence for changes in biofilm-forming ability is very weak.
- The culture conditions used are not at all relevant for infections.
- The difference between changes in gene expression and genetically heritable changes were not considered.
The experiments: About 7x10^6 cells of a strain of S. typhimurium that readily kills mice were pre-packed into vessels with a separate chamber containing culture medium (rich broth). In space (and simultaneously on the ground), the cells and medium were mixed and let grow and divide for 25 hr (temperature not specified but the shuttle maintains 18-27°C). The amount of cell growth is not reported but was probably about 1000-fold (assuming doubling time of ~1 hr and maximum density of ~10^10 cells/ml). In some vessels the cells were then mixed with a fixative for later microscopy and RNA analysis; in others they were mixed with enough medium to allow one more cell doubling.
Once back on earth, the fixed bacteria were examined microscopically and their RNA and proteins were extracted and amounts of specific gene transcripts and proteins measured. Mice were infected with different doses of the not-killed bacteria from both space and ground cultures, and their survival monitored.
The microarray analysis of gene expression looks methodologically OK: Because Salmonella has about 4300 genes, microarray experiments of gene expression need to be carefully controlled and replicated to eliminate false-positive differences in gene expression. The authors did three biological replicates (three different cultures from space and from ground.
I. The array results were inconsistent. Some Hfq-regulated genes were found to be up-regulated in suspension (space) culture, and some were down-regulated. This isn't itself a problem, as Hfq is known to have different effects on different genes. But Table 1 doesn't tell us which genes are expected to be up-regulated and which down-regulated, so we can't easily compare the prediction with the result. I picked a few genes at random and compared the effect of space growth to the effect of a hfq deletion in E. coli (Guisbert et al 2007). Most of the genes listed as Hfq-regulated in Table 1 weren't listed in this paper. Of those that were, most had effects in the same direction, but some were in opposite directions (e.g. adhE was up 4.75-fold in space, but down 2.8-fold in a hfq deletion).
II. The differences were due to suspension culture, not microgravity. The paper and associated media coverage, and Ed's article, describe the experiment as testing the effects of spaceflight on bacterial growth and virulence, but as best I can tell it just compared bacteria that grew suspended in medium, more or less evenly distributed, with bacteria that were initially mixed but gradually settled to the bottom of the culture vessel, where their growth would have been limited by crowding. Although in principle the microgravity of space could have had specific effects, the effects of space shuttle growth were nicely reproduced by growth on the ground in a container that gently rotated so that cells never had time to settle but did not experience the shear forces created by typical shaken cultures (Figure S5 below). (However these 'validation' cultures were done at a different temperature (37°C), and the possible effects of this are not discussed.) The authors correctly interpret this as evidence that growth in space changes gene expression because it gently prevents cells from settling, but they ignore the implications.
III. The doses of space-grown and Earth-grown bacteria used in the virulence experiments were not well controlled. The inocula used are described simply as 10^4, 10^5...10^9, but no information is given about how cell numbers were measured. This is very important because the ground-grown cells would have settled to the bottom of their container and likely grown less than the space-grown cells, which would have remained in well-mixed suspension. Simple cell counts might have been used to determine the sizes of the inocula, but these would not control for any viability differences between the space and ground cultures. Using colony counts to retrospectively estimate innocula would have controlled for any differences in viability, but then the sizes on the innocula are unlikely to have been equivalent.
I think the investigators probably just immediately inoculated the mice with ten-fold serial dilutions of the cultures, and then, after the colony counts were obtained, rounded the inocula sizes up or down to powers of ten. This means that the numbers of viable space-grown and ground-grown cells given to the mice may differed by as much as 9-fold (e.g. ground-grown bacteria at 5.5x10^6 cells/ml and space-grown bacteria at 4.5x10^7 cells/ml would both have been rounded to 10^7 cells/ml). That could easily explain the virulence differences in Figure 1 B and C, as these are substantially less than ten-fold.
IV. The evidence for changes in biofilm-forming ability is very weak. The electron micrographs Figure 1E are claimed to show that space-grown bacteria are more aggregated and clumped due to presence of an extracellular matrix. But if anything, the ground-grown cells look more aggregated, and the chunks of 'unidentified extracellular matrix' in the space-grown cells are not associated with most of the cells.
V. The culture conditions used are not at all relevant for infections. The paper makes all sorts of claims that its results have big implications for infection control during space missions. They claim that the microgravity environment of space flight and the low-shear is like that experienced by pathogens during infection of the host. However the effects of growth in space were reproduced when the cells were grown in a gently mixed ground culture. which is totally unlike ANY natural infection condition, in space or on the ground. In infections, bacteria grow on the surface of or within tissues, or in bodily fluids such as blood (a very high-shear environment). Growth during gentle suspension in rich broth is also very different than environments likely to be experienced by free-living cells in space, where bacteria are typically either surface-associated or in aerosols.
VI. The differences between changes in gene expression and genetically heritable (mutational) change were not considered at all. This is a major oversight, confounding physiological and evolutionary changes. If the space-grown bacteria are just in an altered physiological state, they might be better able to initiate an infection but they would quickly alter their physiology to that determined by the host environment. If the differences were due to mutations favoured during growth in space, then we might have to worry about 'superbugs'. But the cells would have gone through only about 10 doublings, so it's very unlikely that any new mutations had accumulated to significant levels.
Overall I'm not convinced that this paper has any implications for space medicine at all. It certainly doesn't show that growth in space transforms bacteria into superbugs.