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LOCAD-PTS: Operation of a new system for microbial monitoring aboard the International Space Station (ISS)
Abstract
Microorganisms within the space stations Salyut, Mir and the International Space Station (ISS), have traditionally been monitored with culture-based techniques. These techniques involve growing environmental samples (cabin water, air or surfaces) on agar-type media for several days, followed by visualization of resulting colonies; and return of samples to Earth for ground-based analysis. This approach has provided a wealth of useful data and enhanced our understanding of the microbial ecology within space stations. However, the approach is also limited by the following: i) More than 95% microorganisms in the environment cannot grow on conventional growth media; ii) Significant time lags occur between onboard sampling and colony visualization (3-5 days) and ground-based analysis (as long as several months); iii) Colonies are often difficult to visualize due to condensation within contact slide media plates; and iv) Techniques involve growth of potentially harmful microorganisms, which must then be disposed of safely. This report describes the operation of a new culture-independent technique onboard the ISS for rapid analysis (within minutes) of endotoxin and β-1, 3-glucan, found in the cell walls of gram-negative bacteria and fungi, respectively. This technique involves analysis of environmental samples with the Limulus Amebocyte Lysate (LAL) assay in a handheld device. This handheld device and sampling system is known as the Lab-On-a-Chip Application Development Portable Test System (LOCAD-PTS). A poster will be presented that describes a comparative study between LOCAD-PTS analysis and existing culture-based methods onboard the ISS; together with an exploratory survey of surface endotoxin throughout the ISS. It is concluded that while a general correlation between LOCAD-PTS and traditional culture-based methods should not necessarily be expected, a combinatorial approach can be adopted where both sets of data are used together to generate a more complete story of the microbial ecology on the ISS.
... Surface sampling and culture: the main sampling methods are the medium contact method and sampling stick smear method. The Surface Sampler Kit [17] provided by the American cabin adopts the medium contact method, in which the medium conducive to the growth of microorganisms is made into a rectangular nutritional pad, which is then loaded onto a PVC plate. The collection area of the contact piece is 25cm 2 . ...
... "LOCAD-PTS" [6], a handheld microbial detection device, obtains microbial information by detecting characteristic biomolecules. The detecting system consists of a reader, a reaction box, a swabbing unit and swabbing kits, etc. (Figure 1) [17]. The sample solution reacts with a limulus reagent to produce color changes, and the number of bacteria is determined by the color changes after the reaction. ...
During a spaceflight, astronauts need to live in a spacecraft on orbit for a long time, and the relationship between humans and microorganisms in the closed environment of space is not the same as on the ground. The dynamic study of microorganisms in confined space shows that with the extension of the isolation time, harmful bacteria gradually accumulate. Monitoring and controlling microbial pollution in a confined environment system are very important for crew health and the sustainable operation of a space life support system. Culture-based assays have been used traditionally to assess the microbial loads in a spacecraft, and uncultured-based techniques are already under way according to the NASA global exploration roadmap. High-throughput sequencing technology has been used generally to study the communities of the environment and human on the ground and shows its broad prospects applied onboard. We here review the recent application of high-throughput sequencing on space microbiology and analyze its feasibility and potential as an on-orbit detection technology.
... In other words, why not benefit from the already optimized miniaturization of smartphones for the further development of sophisticated sensing devices. As previously mentioned, similar systems (in terms of compactness and simplicity), are already being developed by NASA such as the mentioned LOCAD system [14,21] and water monitoring systems for in-flight microbial contamination [22]. Such systems are being developed to enable more in-flight analysis instead of relying on analysis on the ground using bulky bench-top instruments, which is often still the case [22]. ...
A human mission to Mars can be viewed as the apex of human technological achievement. However, to make this dream a reality several obstacles need to be overcome. One is devising practical ways to safeguard the crew health during the mission through the development of easy operable and compact sensors. Lately, several smartphone-based sensing devices (SBDs) with the purpose to enable the immediate sensitive detection of chemicals, proteins or pathogens in remote settings have emerged. In this critical review, the potential to piggyback these systems for in situ analysis in space has been investigated on application of a systematic keyword search whereby the most relevant articles were examined comprehensively and existing SBDs were divided into 4 relevant groups for the monitoring of crew health during space missions. Recently developed recognition elements (REs), which could offer the enhanced ability to tolerate those harsh conditions in space, have been reviewed with recommendations offered. In addition, the potential use of cell free synthetic biology to obtain long-term shelf-stable reagents was reviewed. Finally, a synopsis of the possibilities of combining novel SBD, RE and nanomaterials to create a compact sensor-platform ensuring adequate crew health monitoring has been provided
... In other words, why not benefit from the already optimized miniaturization of smartphones for the further development of sophisticated sensing devices. As previously mentioned, similar systems (in terms of compactness and simplicity), are already being developed by NASA such as the mentioned LOCAD system [14,21] and water monitoring systems for in-flight microbial contamination [22]. Such systems are being developed to enable more in-flight analysis instead of relying on analysis on the ground using bulky bench-top instruments, which is often still the case [22]. ...
A human mission to Mars can be viewed as the apex of human technological achievement. However, to make this dream a reality several obstacles need to be overcome. One is devising practical ways to safeguard the crew health during the mission through the development of easy operable and compact sensors. Lately, several smartphone-based sensing devices (SBDs) with the purpose to enable the immediate sensitive detection of chemicals, proteins or pathogens in remote settings have emerged. In this critical review, the potential to piggyback these systems for in situ analysis in space has been investigated on application of a systematic keyword search whereby the most relevant articles were examined comprehensively and existing SBDs were divided into 4 relevant groups for the monitoring of crew health during space missions. Recently developed recognition elements (REs), which could offer the enhanced ability to tolerate those harsh conditions in space, have been reviewed with recommendations offered. In addition, the potential use of cell free synthetic biology to obtain long-term shelf-stable reagents was reviewed. Finally, a synopsis of the possibilities of combining novel SBD, RE and nanomaterials to create a compact sensor-platform ensuring adequate crew health monitoring has been provided.
Planetary instrumentation has enabled scientists and citizen-scientists to learn more about the universe and beyond. Scientific payloads have been deployed over decades of exploration to answer questions of how the universe works, how our solar system evolved, what characteristics lead to life, and whether we are alone. Significant technology advances and the early identification of instrument requirements, such as environmental and sampling constraints, have facilitated a long history of successful missions. Simultaneously, engineering advances have enabled the development of complex mission concepts that aim to land on and explore challenging uncharted bodies. Therefore, it is critical to identify risks and dependencies across instrument technology trades and develop baseline system requirements in the mission design phase. This chapter discusses general instrument specifications and considerations, including size, mass, robustness, and cleanliness. In addition, the flight history, basic principles, and future developments of selected instrument classes are examined. The technologies reviewed in this chapter span remote and in-situ instruments including synthetic aperture radar, spectrometers, seismic instrumentation, and nano- and microtechnologies. The goal of the chapter is to provide an overview of the highlighted instrument classes, as well as general considerations, so that the reader may understand the operations, observations, and requirements of specific technologies.
A rapid method of performing the Limulus amoebocyte lysate (LAL) test in milk is proposed using the Toxinometer ET-201. This instrument measured the increase in turbidity due to the interaction between the endotoxins of the Gram-negative bacteria and the LAL reagent, monitored the ratio Rt of the sequential to the initial transmission at 12 s intervais and quantified endotoxins by determination of the reaction time Tr required to obtain a 5% decrease in Rt. There was a good correlation between the toxinometrically determined endotoxin concentrations and the number of Gram-negative bacteria (SD, 0·18 log(plate count units)), and the repeatability (CV, 6·10%) was high. The assay may be useful for screening raw materials for UHT milk production, as the endotoxin content of the raw material is related to the rest proteinase activity in the UHT milk.
Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.
Microbiological profiles were determined for surfaces of the command module, lunar module (ascent and descent stages), instrument unit, Saturn S-4B stage, and the spacecraft lunar module adapter of the Apollo 10 and 11 spacecraft. Average levels of contamination of the command module were 2.1 × 10⁴ and 2.7 × 10⁴ microorganisms per ft² for Apollo 10 and 11, respectively. With the exception of the exterior surfaces of the ascent stage of the lunar module and the interior surfaces of the command module, average levels of microbial contamination on all components of the Apollo 11 were found to be lower than those observed on Apollo 10. For each Apollo mission, approximately 2,000 colonies were picked from a variety of media and identified. The results showed that approximately 95% of all isolates were those considered indigenous to humans; the remaining were associated with soil and dust in the environment. However, the ratio of these two general groups varied depending on the degrees of personnel density and environmental control associated with each module.
Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (10⁴ per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch². A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft.
In the past years an assortment of samples of plasma proteins, enzymes, vaccines and blood substitutes were tested comparatively in rabbits (pyrogen test, European Pharmacopoeia) and with the LAL test (Pyrogent, Byk-Mallinckrodt, Inc.). Specificity and sensitivity were tested with endotoxins and lipid A of gram-negative bacteria. The limulus amebocyte lysate (LAL) test gave similar results or was tenfold more sensitive than the assay in rabbits. More than 300 samples of drugs were examined by both tests. All preparations positive in the rabbit test were positive in the LAL test too. In the testing of plasma proteins the LAL test was more sensitive. The examination of 45 samples of vaccines for pyrogens gave the same result in both assays. Streptokinase does not inhibit the LAL test unspecifically. The LAL test is not an alternative but an additional method in the detection of lipopolysaccharides in drugs.
The treatment of peritonitis in continuous ambulatory peritoneal dialysis patients is empiric until the bacteriological results are available. The Lymulus amebocyte lysate assay (LAL) is a very sensitive method for the detection of endotoxin, a structural component of gram-negative bacteria. We performed the LAL assay in a prospective study in 36 consecutive episodes of peritonitis. The LAL assay was positive in all 10 episodes of gram-negative peritonitis (100% specificity). Treatment directed specifically against gram-negative or -positive infection was started based on the LAL assay result. In 26 episodes with LAL-negative test, a gram-positive bacterium was cultured in 23 episodes, in 1 there was fungal infection and 2 were sterile. In summary: the LAL assay is a rapid (1 h) and sensitive method for the differentiation of gram-positive or -negative peritonitis and enables starting an immediate and more appropriate antibiotic therapy.
Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (10(4) per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch(2). A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft.
Microbiological profiles were determined for surfaces of the command module, lunar module (ascent and descent stages), instrument unit, Saturn S-4B stage, and the spacecraft lunar module adapter of the Apollo 10 and 11 spacecraft. Average levels of contamination of the command module were 2.1 x 10(4) and 2.7 x 10(4) microorganisms per ft(2) for Apollo 10 and 11, respectively. With the exception of the exterior surfaces of the ascent stage of the lunar module and the interior surfaces of the command module, average levels of microbial contamination on all components of the Apollo 11 were found to be lower than those observed on Apollo 10. For each Apollo mission, approximately 2,000 colonies were picked from a variety of media and identified. The results showed that approximately 95% of all isolates were those considered indigenous to humans; the remaining were associated with soil and dust in the environment. However, the ratio of these two general groups varied depending on the degrees of personnel density and environmental control associated with each module.
Cardiopulmonary bypass (CPB) is associated with blood heparin level fluctuations and a reduction in haematocrit due to crystalloid haemodilution. The effect of these changes on the reliability of the Limulus amoebocyte lysate (LAL) chromogenic microassay for the measurement of plasma endotoxin was assessed in vitro. It was shown that the assay could be significantly compromised by twofold haemodilution which can occur during CPB. The interference effect on the assay caused by CPB-associated heparin was not significant if a comparatively large amount of heparin (25 IU/ml) was added to the blood at the time of sampling. The effect of haemodilution was counteracted by prediluting plasma samples with crystalloid by a factor dependent on the sample haematocrit (to ensure that the proportion of plasma was similar in all samples). A correction was then required to determine the endotoxin level in the original sample. The modified assay was used to determine sequential plasma endotoxin levels in 14 patients undergoing hypothermic nonpulsatile CPB. Endotoxaemia occurred at the time of aortic cross-clamp release and reached a peak of 48.9 +/- 12.9 ng/l shortly before the end of CPB, which was significantly higher than baseline values pre-CPB (p < 0.05). Thereafter, there was a decline in endotoxin levels to 28.9 +/- 13.6 ng/l 24 hours later which was still significantly higher than baseline levels (p < 0.05). Peak endotoxaemia was a predictor of protracted hospital stay when compared with haemodynamic and tissue perfusion parameters.
The hypothesis being tested in this paper is that endotoxin levels in donors and in recipients during liver transplantation influences postoperative outcome.
Endotoxin levels in systemic venous and portal venous blood were measured using in 46 adult donors and 44 adult recipients (47 liver transplants) during the period 1992-95. Endotoxin was measured using a modification of the Limulus amoebocyte lysate (LAL) assay.
In the donor, systemic endotoxin levels were above normal levels at 10.0 +/- 1.3 pg/mL from the start and rose to 15.8 +/- 2.9 pg/mL after dissection of the hilar structures, but fell to 10.6 +/- 0.8 pg/mL just prior to the removal of the liver (control = 7.8 pg/mL). The mean portal venous endotoxin levels were 18.2 +/- 3.4 pg/mL after dissection of the hilar structures and 12.6 +/- 0.9 pg/mL after cannulation of the portal vein. In the recipients, the highest level in the portal venous blood occurred at the end of the anhepatic phase (46.5 +/- 6.7 pg/mL). The systemic venous samples in the recipients were elevated to start with, but fell rapidly to 19.3 +/- 1.5 pg/mL 24 h postoperatively, and to 13.2 +/- 1.0 pg/mL by day 7. The endotoxin concentrations were higher in recipients who developed complications.
Endotoxin is elevated throughout the recipient transplantation procedure and up to 7 days postoperatively. High levels of endotoxin at induction, the anhepatic phase and at certain time points correlated with patients who developed postoperative complications.