Antibiotic resistance of pathogenic bacteria

Who it is for

• Antibiotic developers looking for stress-response targets (sigma-s / rpoS, Hfq) that change drug tolerance without genetic selection
• Space agency crew health offices needing dosing evidence for long missions
• Academic microbiology and infectious disease labs with modeled-microgravity results that need a real flight test
• Antimicrobial susceptibility testing and diagnostics companies checking how their assays behave under low shear
• Biofilm and medical device companies studying aggregate formation and matrix production
• National AMR research programmes funding mechanism work on tolerance and persistence

What earlier flights found

How LELP-1 runs it

Sample. Uropathogenic E. coli, BSL-2 clinical isolate, flown as a matched pair: wild type and an isogenic deletion mutant of the stress-response sigma factor gene rpoS. 4 gentamicin levels (0, 25, 55, 100 micrograms/mL) x 2 strains x 3 wells = 24 wells. Container. One 24-well sealed microfluidic cassette in a large bay, about 400 microlitres per well, each well with its own inlet, outlet and LED/photodiode pair. BSL-2 triple containment with an absorbent secondary tray and a seal pressure sensor. Launch. Live cells, no cryopreservation: loaded at the launch site within 24 h of liftoff and held dormant at 4 C in the sealed wells, with growth medium kept in a separate reservoir and not in contact with the cells. No thaw step and no -80 C cassette. Load-to-dose time is kept to days, because EcAMSat reported that multi-week storage in payload hardware stressed the cells. Light and gas. No light needed for growth; the only light is the 570 nm and 600 nm LED pairs used for the dye readout, plus the microscope illuminator. No CO2 supply needed: the medium is HEPES-buffered and the sealed air headspace is sized for the well volume. Headspace oxygen is monitored, not replenished.

1. day 0

   Launch. Bay held at 4 C, cells dormant, seal and pressure logged every minute. Dosimeter running.

2. day 1

   Orbit reached and the lab is quiet. Bay ramps to 37 C over 1 h. Pump fills all 24 wells with HEPES-buffered growth medium. Optical density read every 15 min.

3. day 2

   Cultures reach stationary phase (expected 18 to 24 h), then are held without fresh medium so they starve, as EcAMSat did for over 24 h before dosing. Microscope takes a brightfield image of every well to check for aggregates before dosing.

4. day 3

   Dosing. Gentamicin injected at 0, 25, 55 and 100 micrograms/mL, three wells per strain per level, then alamarBlue redox dye into all 24 wells. Optical readout at 570 and 600 nm every 30 min starts here and runs 72 h. Live/dead fluorescence images every 6 h.

5. day 4

   Dose-response window continues. Downlink the optical series once per orbit so the team can confirm the assay is working while there is still time to extend the window.

6. day 5

   Robotic arm carries the bay to the central lab for one 40x fluorescence look at the highest-dose wells (aggregates, matrix, vesicle-scale structures). Arm motion window logged, since quasi-steady microgravity is lost to 1e-4 to 1e-3 g while it moves.

7. day 6

   End of exposure. 12 wells get RNA stabiliser (transcriptome snapshot taken in orbit), 6 wells get glutaraldehyde fixative for electron microscopy, 6 wells are left live for ground plating. Bay drops to 4 C and holds. Waste line chemically inactivated.

8. day 7

   Deorbit, re-entry under the inflatable heat shield, splashdown. Bay stays at 4 C through recovery; samples handed to the customer courier on the recovery ship about 20 min after splashdown.

The payload module, in CAD

Every item in the module sheet, drawn and packed into the large bay deck by deck. The back face mates with the bay: blind-mate connector, and a cold wall against the bay's thermoelectric block. The front face carries Dexter-L's grapple fixture, and the fluid lines run down a channel just behind it. What you load before launch is marked blue in the configurator's payload guide.

CAD generated by cad/build_modules.py (CadQuery, OpenCascade kernel) from the catalog's module items: concept geometry, not a released design. Standard parts keep their real formats; the rest are sized from their catalog mass. The front panel and grapple fixture are left off in the 3-D view and are in the STEP.

• Each well sits between its own LED and photodiode, as in EcAMSat's optical card: no microscope, no moving parts.
• Reagent volumes the catalog does not state are split evenly from 75 % of the reservoir item's mass. The waste bag holds the spent medium and washes; fixatives and stabilisers stay in the wells and come home.
• Fluid pack: 8 reagent bags in 4 layers (118 x 113 x 69 mm), every port on a front edge.
• The waste bag lies under the reagent bags and fills the room they free: they hold 275 mL, it needs 170 mL.
• The catalog gives 0.64 kg for structure and containment; as an aluminium box that is a 0.32 mm skin on 0.19 kg of posts and shelves. Thin: the bay rails have to carry the launch loads, or the module needs more structure mass.
• The grapple fixture stands 26 mm proud of the front panel, into the bay opening; everything else is inside the bay envelope.

Ground control

An identical cassette in an identical bay inside the flight-spare satellite on the ground, run on a 24 h delayed timeline that replays the actual flight telemetry: measured bay temperature, pressure, humidity and the real fluidics command log, same cell lot, same cassette fill batch, same reagent lots. EcAMSat ran a parallel ground-based control experiment against its flight data, and a second ground-control experiment that showed multi-week storage in the payload hardware stressed the cells - so our ground arm also carries a storage-matched control, and the flight plan keeps load-to-dose time short. Because launch loads, re-entry loads and the 550 km radiation dose are not reproduced on the ground, we also fly a 1 g reference: three wells of each strain in a bay on the onboard centrifuge, so microgravity can be separated from the rest of the flight environment. Without that 1 g arm, any flight-versus-ground difference stays confounded.

Coming home

Fixed and RNA-stabilised wells are robust: 4 C and up to 48 h from day-6 preservation to the lab is fine. The live wells are the tight ones - plate them within 24 h of splashdown, because cells sitting at 4 C in spent medium keep changing expression and losing viability. That is why the transcriptome and electron microscopy samples are preserved in orbit on day 6 rather than on the ship: the only clean snapshot of the flight state is one taken in flight.

Limits of this flight

• The flight evidence does not point one way. ISS E. coli grew to a 13-fold higher final cell count in space, while EcAMSat found uropathogenic E. coli more susceptible to gentamicin in microgravity and its growth rate unchanged. One 7-day flight will not settle that; it adds one well-controlled point.
• A 2 to 14 day flight gives one dose-response, not evolution. Resistance that arises by selection over weeks of serial passage cannot be studied here.
• EcAMSat incubated its cells with the dye for 144 h after dosing. Our 7-day baseline gives a 72 h window; matching EcAMSat's readout length needs a 10-day mission, which is inside the 2 to 14 day envelope but costs more.
• No crew. There is no plating, no colony picking, no re-dosing decision in the moment and no fixing a stuck valve. Every move must be in the fluidic plan before launch, and a single pump or valve failure can cost the whole bay.
• 24 wells means 3 replicates per condition. That resolves a large shift in tolerance, not a small one. EcAMSat's own report notes that cell stress in the hardware could obscure small differential effects, so customers wanting statistical power should buy more than one bay.
• The in-flight readout is dye reduction, that is metabolic activity, not colony-forming units. A clinical MIC number must be confirmed on the ground, and returned live cells spend 1 to 2 days at 4 C before plating.
• Only wells preserved in orbit give a clean flight-state transcriptome or ultrastructure. Anything judged after the courier hand-off has a day or two of cold storage written into it.
• Launch loads, re-entry loads and the 550 km radiation dose are confounders. Only the onboard 1 g control separates microgravity from them, and that control shares the same radiation and vibration history, not the same gravity history as a laboratory on Earth.
• Quasi-steady microgravity is about 1e-6 g near the centre of mass, but rises to 1e-4 to 1e-3 g while the robotic arm moves. Data taken in those windows is not a clean microgravity measurement and is flagged as such.
• No animal virulence test on board. The STS-115 result that flight Salmonella killed mice faster needed returned live cells and a ground animal facility; LELP-1 can return the cells, nothing more.
• No host. There is no infection model, no human pharmacokinetics and no immune system in the bay, so nothing here translates directly into a dosing recommendation for a crew member.
• No CO2 supply. Fine for E. coli, but a bacteria-plus-host-cell co-culture would need HEPES-buffered media and gas-permeable lids and is a harder sell in a sealed bay.
• LELP-1 is a design in a digital twin. No bay, cassette or thermoelectric block described here has flown, and every mass, power and timing number above is our estimate awaiting test.