The biomass, thickness, and surface area coverage of biofilms formed on SS316 were not statistically different from those of biofilms grown on pSS316 (α = 0.05) at any incubation day (Fig. S1). Similarly, no morphological differences were observed between biofilms formed on the two materials (Fig. S2) for either Earth-based or microgravity cultures. Similarly, there were no differentially expressed genes (DEG) between SS316 and pSS316 biofilms in any conditions tested (α = 0.05) except for microgravity day 2 where pSS316 had 44 DEG (Table S1). Based on this, SS316 and pSS316 were pooled into one “SS” data set which is hereon compared and contrasted to that of biofilms formed on LIS.
Biofilm Morphology and Observations from the Liquid Cultures
In 1 g, biofilms on SS grew throughout the surface with no marked boundaries, usually creating conglomerates that formed ‘mounts’ of biofilm with dispersed single cells in between (Fig. 2a). Conversely, the few biofilms grown on LIS were restricted to certain areas with sharp edges, had a more compact appearance, and were thicker than those grown on SS. Additionally, the LIS surface showed individual cells or small multiple cell groups attached in random isolated areas and a thin layer of nucleic acids in the areas where there was no biofilm. Bacterial cells attached on LIS were often found growing in the perimeter or inside of a circle (as seen for 1-day old biofilms on 1 g) as well as small, connected clusters. Overall, biofilms on LIS seemed to be restricted to grow in smaller areas than on SS. The morphology of biofilms formed on SS in space was similar to that of the Earth controls, in that the space samples also exhibited a lack of clear boundaries and biofilm ‘mounts’ were also observed, albeit with bigger gaps between them (Fig. 2b). Biofilms grown on LIS in microgravity did not form as many sharp, compact, and thick biofilms (only a few in smaller size where found) compared to those formed in 1 g. Instead, most of the surface of LIS was covered with a thin layer of nucleic acids.
The PA14 liquid cultures in which the coupons were submerged became more turbid for the microgravity set than for the 1 g controls. Specifically, for the samples cultured in space for three days, OD600 values were 0.400 and 0.422 for SS and LIS, respectively, compared to the 0.283 for SS and 0.342 for LIS measured on the 1 g set (Fig. S3). Additionally, it is worth noting that the color of the liquid culture changed as well. In microgravity the cultures were light green on day 1, light brown on day 2, and light pink on day 3, while the ground cultures remained clear for days 1 and 2 and then turned light yellow on day 3 (Fig. S4). The increased culture turbidity in microgravity could suggest more bacterial growth in general; however, microgravity biofilms exhibited the opposite trend.
The SS and LIS biofilms on 1 g appeared to grow in size with time, with the largest growth observed between day 1 and day 2 (Fig. 2a). This trend also held true for SS in microgravity; although with a lower growth rate (Fig. 2b). For biofilms on LIS in microgravity, there was little to no change in mass as a function of time.
Biofilm Biomass, Thickness, and Surface Coverage
PA14 biofilms formed in microgravity had significantly lower (p ≤ 0.05) biomass (Fig. 3), and thickness (Fig. 4) than those formed at 1 g. This is true for both SS and LIS, the only exception being for the biofilms formed on LIS at one day of incubation, which exhibited no difference between gravitational regimes. In the case of SS, by day 3 biofilms formed in microgravity had only 17.6% and 53.6% of the ground control biomass and thickness, respectively. The same phenomenon was observed on LIS, where biofilms formed in space had 8.7% and 21.3% of the ground control biomass and thickness. Furthermore, biofilms formed in space covered less surface than their 1 g counterparts (31.4% and 33.0% of surface area for SS and LIS, respectively) (Fig. 5). The values for the three parameters assessed are summarized in Table 1.
Biofilms grown on SS were significantly different than biofilms grown on LIS in terms of mass and surface area coverage. On both Earth and in microgravity, at day 3, the median of biomass and surface area coverage in LIS was significantly lower (p ≤ 0.001) than in SS biofilms (Table 1).
Table 1
Median values of biofilm biomass, thickness, and surface area coverage per day and material
| Biomass (µm3/µm2) | Thickness (µm) | Surface area coverage (%) |
| 1g | µg | 1g | µg | 1g | µg |
SS day 1 | 0.69 (0.38) | 0.22 (0.13) | 3.70 (2.19) | 3.44 (1.52) | 38.76 (8.57) | 15.48 (10.24) |
SS day 2 | 2.06 (1.90) | 0.27 (0.18) | 6.30 (4.53) | 2.77 (1.38) | 65.38 (25.77) | 21.19 (9.48) |
SS day 3 | 1.87 (1.64) | 0.33 (0.38) | 5.86 (2.82) | 3.14 (1.76) | 74.79 (28.97) | 23.49 (15.93) |
LIS day 1 | 0.07 (0.06) | 0.05 (0.05) | 4.41 (3.41) | 5.06 (4.32) | 3.40 (2.55) | 4.20 (4.30) |
LIS day 2 | 0.49 (0.64) | 0.02 (0.04) | 7.09 (5.29) | 4.21 (3.66) | 11.17 (18.79) | 2.00 (2.22) |
LIS day 3 | 0.46 (0.28) | 0.04 (0.05) | 8.04 (10.63) | 1.71 (1.18) | 10.87 (10.21) | 3.60 (4.30) |
SS = Stainless Steel, LIS = Lubricant Impregnated Surface. Results are presented as median and the interquartile range Q3-Q1 is indicated in parenthesis. For SS, n = 8 biological replicates each imaged in 4 fields of view. For LIS, n = 4 biological replicates each imaged in 4 fields of view.
Biofilm Gene Expression
Bacteria adapt to their environment through gene expression modulation. The mechanisms used by bacteria to adapt to changes can be observed in their transcriptomic profile. Comparing biofilms formed on SS for three days in microgravity against Earth controls showed nine differentially expressed genes (DEG) with a fold change of two or more (Fig. 6a). Seven had increased expression in space (rsmY, rmf, pchB, PA14_08570, PA14_62090, PA14_55637, PA14_70910), and the remaining two had decreased expression in space (PA14_63220, PA14_21970). Genes PA14_08570, PA14_62090, PA14_55637, and PA14_70910 code for rRNA. Thus, the increased expression of these genes suggests a limitation during rRNA depletion. Because all samples were prepared the same, comparisons of other genes should not be affected.
To understand how the P. aeruginosa biofilm metabolic pathways changed between microgravity and 1 g, a KEGG pathways enrichment analysis was performed. This analysis revealed three pathways that were enriched in the space samples (Fig. 6b). The most enriched pathway was that of “nitrogen metabolism”. Additionally, analyses of the differential gene expression of genes involved in P. aeruginosa virulence and antimicrobial resistance were performed to assess the effects of microgravity on these processes. For the virulome analysis, six genes involved in pyochelin production had significant increased expression in microgravity compared to earth controls (Fig. 6c); pchB (2.2-fold), pchI (1.4-fold), pchE (1.9-fold), pchD (1.5-fold), pchC (1.9-fold), and fptA (1.8-fold). For the resistome, there was no significant effect > 2-fold.
Gene expression analyses were also performed to compare 3-day-old biofilms grown on LIS to those grown on SS (baseline). On Earth, biofilms grown on LIS had 313 DEG with a fold change of 2 or more (Fig. 7a) when compared to SS. The top five DEG were PA14_09380, pchC, PA14_06170, PA14_52500, and pchE; all with increased expression on LIS. Gene PA14_52500 is annotated as coding for a hypothetical protein and had the highest fold change (3.9) of all. Some of the genes that code for alginate production had significantly increased expression (below 2-fold) on LIS: mucB (1.1-fold), mucD (1.1-fold), algR (1.5-fold), mucE (1.9-fold), mucP (1.4-fold). The pathways enrichment analysis (Fig. 7b) showed the “carbon metabolism” pathway as the most enriched in LIS compared to SS. For the virulome analysis (Fig. 7c), several genes of Hcp secretion island I (HSI-I), type three secretion systems (TTSS), lipopolysaccharide (LPS), and pyocyanin had decreased expression (see bold genes in Fig. 6c) in LIS. Conversely, almost all the genes involved in synthesis of pyochelin had increased expression in LIS more than 2.5-fold: pchI (3.1-fold), pchH (2.6-fold), pchG (2.7-fold), pchF (3.1-fold), pchE (3.1-fold), pchD (2.5-fold), pchC (3.1-fold), pchB (3.2-fold), pchA (3.0-fold), fptA (2.6-fold). For the resistome analysis, gene nouJ had 2.2-fold increased expression on LIS with respect to SS. Additionally, genes involved in apoptosis were differentially expressed, rpoS had increased expression (2.4-fold) and the antitoxins higA and PA14_21720 had decreased expression (-1.4 and − 2.4-fold, respectively) on LIS.
In microgravity, biofilms grown on LIS for three days had 423 DEG with fold change > 2 (Fig. 8a) compared to their spaceflight SS counterparts. The top five DEG were PA14_27520 (-5.5-fold), PA14_44920 (-3.1-fold), PA14_20060 (3.7-fold), PA14_51580 (-4.0-fold), and estA (3.1-fold). Three of these top five DEG code for hypothetical proteins that have not been characterized. In microgravity, no pathways were significantly enriched or depleted on LIS with respect to SS. Interestingly, the observations made on the virulome when comparing LIS and SS on Earth remain true in microgravity: HSI-I, TTSS, LPS, and pyocyanin-synthesis associated genes had reduced expression while genes associated with pyochelin and rhamnolipids had increased expression (Fig. 8b). Additionally, there was no increased expression of alginate-associated genes as seen on Earth. For the resistome analysis, there was increased expression on LIS of mexT (2.3-fold) and galU (2.1-fold), and decreased expression of ampD (-3.1). Finally, no significant differences in gene expression of rpoS or higA were observed in LIS with respect to SS in microgravity, but one putative toxin (PA14_28120) presented a 1.4-fold increased expression.
Changes in the transcriptome between 3- and 1-day-old biofilms provide insight into the biofilm formation process. A comparison between biofilms grown on Earth on SS for three versus one days exhibited 119 DEG with fold change > 2 (Fig. S5a) with respect to 1-day-old biofilms. The top five DEG had decreased expression (PA14_40310, trxB2, PA14_39700, PA14_10380, nirN) at 3-days with respect to 1-day. PA14_10380, the most differentially expressed (-5.2-fold), codes for a hypothetical protein. Day three had increased expression of several genes involved in production of Psl extracellular matrix polysaccharide, in particular pslO (3.1-fold). The pathways enrichment analysis (Fig. S5b) showed several depleted pathways; “sulfur relay system” was the most depleted, followed by “nitrogen metabolism”. For the virulome analysis (Fig. S5c) at three days and with respect to the 1-day old biofilms, there was a reduction in expression of genes in the Las (lasA − 2.2-fold, lasB − 2.3-fold) and Rhl systems (rhlA − 2.3-fold, rhlB − 2.2-fold), and of gene algP/algR3 (-2.1-fold) involved in alginate synthesis. Regarding the resistome analysis, 3-day old biofilms had decreased expression of genes that code for RND efflux pumps and outer membrane proteins: mexH (-2.1-fold), ompA (-2.6-fold), ompD (-2.3-fold).
A comparison between biofilms grown in microgravity on SS for three versus one days showed 70 DEG with fold change ≥ 2 (Fig. S6a). The top five DEG correspond to: PA14_40310 (-2.6-fold), malQ (-3.1-fold), PA14_36650 (-3.7-fold), PA14_72370 (-5.1-fold) and pchF (3.2-fold). Again, the most differentially (under)expressed gene (PA14_72370) codes for a hypothetical protein. In microgravity, there was no systemic increased expression on day 3 of extracellular matrix polysaccharides, but on the ground, the gene trxB2 (-2.3-fold) and the cytochrome c gene cluster (including nirN − 2.1-fold) had decreased expression. The pathways enrichment analysis (Fig. S6b) only showed three depleted pathways. “Porphyrin and chlorophyll metabolism” is the most depleted pathway. For the virulome analysis (Fig. S6c), rlhA (-2.2-fold) was less expressed on day three (as seen on Earth) while an increased expression of all pyochelin-associated genes was observed (pchI 2.1-fold, pchH 2.4-fold, pchG 3.5-fold, pchF 3.2-fold, pchE 3.2-fold, pchR 1.3-fold, pchD 2.6-fold, pchC 2.8-fold, pchB 3.3-fold, pchA 3.5-fold, fptA 2.5-fold). For the resistome analysis, ompA (-2.4-fold) showed a reduced expression at day 3 with respect to day 1; as with the ground samples.