1. Introduction

A human mission to Mars is one of the most important steps in the exploration of the Solar System in the decades to come (Mitchell and Staretz 2010; Zubrin 2010). While the benefits of a human mission to Mars are obvious from a scientific to technological point, one has to insure that such a mission is undertaken with the appropriate safety measures (Straume et al., 2010; Stuster 2010). If the history of space exploration can be used as a lesson, the highest risk for such a mission will be catastrophic vehicle failure. However, visiting an alien planet will also come with a certain degree of biological risk, in terms of infection or contamination of either the astronauts, the technical crew on the ground, or even Earth ecosystems upon return of the mission. In this article we will review the current knowledge concerning the biohazard of potential Martian microbes.

2. The Probability of Life and Pathogenic Microorganisms on Mars

The probability of life on Mars is one of the most exciting questions to the scientific community (Houtkooper and Schulze-Makuch, 2010; Levin 2010; Leuko et al. 2010; Sephton 2010), but no firm answers have been given during the decades of space exploration. Many believed that life on Earth began in water (Russell and Kanik, 2010), and it is also currently thought that liquid water is one of the prerequisites of life on any planet (Brack, 1999). Mars mapping by Mariner 9 and by Viking 1 and 2 revealed channels resembling riverbeds, and information collected by the Mars Global Surveyor (MGS) strengthened the case for early surface water on Mars (Malin and Edgett, 2000a). Another investigation based on photographic images provided by MGS shows relatively young geological features compatible with the presence of liquid water possible as recent as a few million years ago (Malin and Edgett, 2000b). Even more striking, data from Mars Exploration Rovers discovered round pebbles scattered on the surface of Meridiani Planum, suggesting that this region has once been submerged (Squyres, et al., 2004).

Current water on Mars appears to be concentrated in the Northern as well as Southern Polar Ice Cap (Titus, et al., 2003), and as subsurface ice in other areas of the planet (Picardi, et al., 2005). In addition to water, one would consider the presence of organic substances as an additional argument for possible biological processes (Levin 2010). In several Martian meteorites organic matter of extraterrestrial origin has been identified (Jull, et al., 2000; Wright, et al., 1989). However, the in-situ experiments searching for Martian life carried out by Viking 1 and 2 in 1976 could not provide unequivocal signs of life or metabolic activity (Klein, et al., 1976), though some investigators believe otherwise (Levin, 2010). Specifially, Levin (2010), argues that since the Viking Mission Labeled Release experiment, which exploits the sensitivity of ¹⁴C respirometry, provided positive responses at both the Viking 1 and 2 sites on Mars, that this evidence favors the presence of life. Even so, the consensus favored chemical or physical agents in the Martian surface material, not life.

Studies on the Martian meteorite ALH84001 that reported the discovery of carbonate granules resembling microfossils, have been hailed as the first probable direct evidence of life outside Earth (McKay, et al., 1996). Within the same meteorite, magnetite crystals with properties compatible with biogenic terrestrial magnetite have also been found (Thomas-Keprta, et al., 2002). However, these reports remain controversial, as non-biological processes have also been proposed to explain the features found in ALH84001 meteorite (Scott, 1999).

The detection of methane and formaldehyde in Mars’s atmosphere could be another indication that microbes exist on Mars (Formisano, et al., 2004; Yung et al., 2010). Although the presence of methane could be a mere sign of geographical processes (e.g. serpentinization), it is well known that most of the methane in Earth’s atmosphere is produced by microbes. Furthermore, methane concentrations have been found to vary in space and time, suggesting that it is produced or released in a dynamic process and this could support the idea of present life on Mars (Sephton 2010).

All in all, the direct arguments for microbial life on Mars remain equivocal. While proof of liquid water, plate tectonics and a dense atmosphere on early Mars is convincing, the current habitats on Mars are currently extremely hostile. The absence of a magnetic field results in intense ionizing radiation on the Martian surface. The associated damage to DNA or RNA based life forms would presumably wipe out any forms of life we know down to 7.5 m in the sediment (in the absence of active DNA repair, Dartnell et al 2007). The absence of plate tectonics (the major potential chemotrophic energy source for growth) and the low water activity at the surface are the other major challenges to life as we know it. If there is any analogue for Martian life on Earth, it is the deep biosphere, with bacteria in low cell numbers, estimated doubling times of thousands of years in substrate depleted, anaerobic environments. It is interesting to note that the deep biosphere may contain up to 30% of the living biomass on Earth (Whitman et al 1998) and from that perspective the two planets may not be so different than their completely different planetary surfaces suggest. Therefore, an analysis of the potential biohazard imposed by Martian microbes is warranted.

3. What is the Pathogenic Potential of Putative Martian Life Forms?

The first aspect when one asks of the potential of existence of life on Mars regards the life forms that would be most likely to be encountered. The most obvious conclusion based on the observations made until now is that Martian life forms will be necessarily microscopic. Microscopic life on Earth includes viruses, archaea, bacteria, fungi, unicellular plants, and protozoa. The more complex forms of unicellular life are unlikely to be present in the hostile environment of Mars (such as fungi, unicellular plants and protozoa).

Viruses are obligate intracellular organisms, needing a host to replicate and transmit genetic information, which make them additionally vulnerable to extreme environments. However, the example of bacteriophage viruses of extremophile bacteria provides the example of virus presence in extreme environments (Prigent, et al., 2005; Sawstrom, et al., 2008). Thus the possibility of Martian viruses (in case of the presence of bacteria) cannot be completely ruled out.

Therefore, the most likely life form on Mars would be bacterial life. Bacteria are not only the most resilient and adaptable of known life forms but they are also the only class of organisms that can survive "stand alone" and do not rely on other life forms. In addition, the microfossils present in the ALH84001 meteorite are most plausible bacterial (nanobacteria), if they are indeed of biologic origin (McKay, et al., 1996).

How likely is that a Martian bacteria would be pathogenic for humans, or disruptive for an Earth ecosystem? An excellent analysis of these aspects have been provided by Schuerger, based on a plant infection model of pathogenicity (Schuerger, 1998). Terrestrial plant-microbe interaction can be classified by non-interactive, saprotrophism, necrotrophism, biotrophism, symbiosis and commensalism (Schuerger, 1998). The last four categories imply a certain degree of adaptation and co-evolution between the microbial life and the multicellular organism, and this is absent between microbial Martian life forms and humans (or other Earth organisms). This would imply that the most likely interaction between microbes on Mars and astronauts would be non-interactive or saprotrophic, and hence most likely nonpathogenic. The chance of a Martian microbe, adapted to extremely slow, cold and anaerobic conditions having the ability to attach to cells of a terrestrial host and invade its cells or tissues, and hence produce infection, in full competition with terrestrial microbes, is very small. Less likely is even transmission to a second 'vulnerable' host.

However, a pathogenic potential of Martian microbes cannot be excluded either. Even if they were not capable of directly invading the host and causing infection, Martian microbes could still have pathogenic potential by secreting toxins that could indirectly harm the astronauts (e.g. through wounds, contaminated food). Examples of powerful microbial toxins secreted by terrestrial bacteria indeed abound, e.g. clostridial toxins (Lebrun, et al., 2009). Still, one has to recognize that the majority of such toxins of terrestrial bacteria are proteins, which in turn are recognized by specific cellular receptors, again requiring a history of previous interaction between the pathogenic agent and the host. Would such putative toxins of Martian microbes also be proteins, would they have similar biochemistry, would they even be made of the same aminoacids? Although it is possible that through mechanisms know as panspermia (Joseph and Schild 2010a,b) that microbes from Earth could be transported to Mars (and vice versa) thereby providing opportunities for horizontal gene transfer and thus giving Martian microbes human-infective properties (Joseph and Wickramasinghe 2010), at present there is no hard evidence to substantiate these theories. Thus, these are all questions that cannot be answered at present. Still, how minimal the chance that there may be pathogenic microorganisms on Mars, one cannot completely rule it out (Rummel et al. 2010).

A different aspect of the biohazard potential of Martian microbes is the capacity of such microorganisms to disrupt Earth ecosystems, should contaminated material from a Mars mission reach the environment upon return (Rummel et al. 2010). This risk is most likely also small, as environmental conditions such as temperature, humidity, chemistry, atmospheric pressure, and nutrients fundamentally differ between Earth and Mars. From an evolutionary point of view, it is highly unlikely that a Martian microbe that in Earth terms would be characterized as an extremophile would be able to compete successfully with terrestrial microorganisms, which are optimally adapted to the environment through millions years of evolution. However, long-term subtle influences on terrestrial ecosystems might be induced by introduction of Martian microorganisms and thereby represent a potential hazard. As a conclusion, the National Research Council Space Studies Board who assessed the biohazard posed by Martian microorganisms considered the risk of "back-contamination" as small, but not inexistent, and recommended that spacecraft and samples returning from Mars to be treated as potentially hazardous (National Research Council Space Studies Board, 1997; see also Rummel et al. 2010).

The reverse should also be considered, i.e., the potential contamination of Mars with Earth microorganisms. Although it is not likely that hitchhiking microbes can survive on the harsh surface of Mars, - likewise they should behave as extremophiles - it could be that during drilling missions in the search for water or during accidents such as a vehicle crash during landing, terrestrial microbes gain access to spots where they could potentially survive and multiply. Although this will not immediately result in a risk for human explorers, time and adaptation may result in a biohazard during returning missions to Mars.

4. Preventive Measures Against Biohazards During a Mars Mission

Despite the low probability of pathogenic microorganisms as indicated above, it cannot be excluded that Mars harbors microscopic life, and the possibility that astronauts would come in contact with it necessitate precautionary measures to insure safety of the crew and Earth habitats upon return of the mission. Based on the chances of an encounter with pathogenic life forms on Mars, a set of recommendations to insure the biosafety of a Mars mission has been recently proposed by Warmflash and colleagues (Warmflash, et al., 2007) (Table 1). Firstly, crew extravehicular activity (EVA) suits should be decontaminated upon habitat return of the astronauts from field activities. Secondly, laboratory facilities on Mars working with Martian samples should be equipped with a minimal biosafely level (BSL) 2 equipment. Thirdly, sterilization methods for the spacecraft upon return should be considered (Trofimov, et al., 1996). Finally, a quarantine program for crews and material brought from Mars also seems to be prudent. Protocols based on the Apollo Quarantine Program should be developed and followed strictly. The return from Mars to Earth has a long duration, which may allow for a disease to manifest itself in the astronauts during the flight back, but the certainty of such a scenario cannot be assumed. While the Apollo astronauts were quarantined for 21 days, breaches in the protocol occurred (National Research Council Space Studies Board, 1997), which should be strictly avoided upon the return of a Mars mission.

5. Measures in Case of Suspicion of Infection or Contamination

The facts and arguments assembled above present a situation in which the infection of humans with a pathogenic Martian microorganism is highly unlikely. This chance should be even further diminished by strict implementation of preventive biosafety measures. However, the possibility of infection can never be completely excluded. What measures should be taken in case of suspicion of infection with Martian microorganisms?

As with any other infection, one has to think at diagnostic and therapeutic measures, neither of which will be trivial in such a case. In order to diagnose an infection with a putative Mars pathogen, one will initially rely on microbiological culture techniques or direct detection of components or structures of extraterrestrial microbes (Table 1). Very little will be known about the microbiological culture techniques to be used, but one has to consider growth conditions close to those of the original Martian habitat from which the microbe originated.

Direct detection of chemical signatures of Martian microbes could be both challenging, as well as providing a possible window of opportunity. On the one hand, PCR-based diagnostics as used for detection of novel pathogens on Earth will be challenging, as indeed one has to assume the presence of nucleic acids as prerequisites of Martian life. These amplification techniques are based on common sequences within the 16s ribosomal RNA of terrestrial bacteria, and such sequences may be totally absent in Martian microbes. An additional challenge would be to exclude any contamination with terrestrial microbial sequences as the source of a potentially positive PCR-based test (Poole and Willerslev, 2007). We should be aware that the putative Martian microorganisms are likely to use as building blocks at least some aberrant materials such as aminoacids or sugars of varieties inexistent or rare on Earth. For example, the abundance of the aminoacids α–aminoisobutiric acid and isovaline in carbonaceous meteorites (Engel, et al., 1990) may indicate the possibility that such aminoacids are used as building blocks by extraterrestrial microbes. These aminoacids have a structure clearly different from terrestrial aminoacids are practically not found on Earth, and their presence in biologic material may indicate infection with extraterrestrial microbes.

However, Martian organisms may not have a DNA-based genome, or they may use other sugars or even of very different structure (such as PNA’s – peptide nucleic acids). If life did not arise on Earth or Mars by mechanisms of panspermia (Joseph and Schild 2010a,b), and unless life has been transferred back and forth between the planets, thereby exchanging DNA, then it must be recognized that if there is Martian life, it may not have arisen in the same way or completed all the same steps as life on Earth. Martian life could have stopped at the pre-RNA of RNA world levels, with replicating macromolecules different from the current one in terrestrial life, in which case, PCR will not work at all.

Treatment of such of infection would be a tremendous challenge. Extensive testing capabilities of substances with antibiotic properties should be in place once a Mars mission returns. Such testing of antibiotics should start as soon as a Martian microorganism is identified and cultured, and even if thought non-pathogenic. In addition, the possibility exists that the human (or other animal) immune system does not recognize a pathogen with which it has not shared billions of years of evolution. In that case, it is not possible to raise antibodies naturally and thus biotechnical methods to manufacture synthetic therapeutic tools (e.g., synthetic antibodies against Martian bacteria) should be considered (Table 1).

6. Conclusions

The chance of a human mission to Mars to encounter pathogenic microorganisms is small, but not zero. A set of safety measures to prevent, diagnose and eventually treat infections with Martian microorganisms should be considered (Table 1), and these may further diminish potential biohazards. Therefore, it may be concluded that the benefits of a mission to Mars, with all the scientific, technological and economical progress that is envisaged, heavily outweigh the low probability of an encounter with a pathogenic microbe, and therefore this should not be an impediment for pursuing human exploration of Mars.

Acknowledgements: M.G.N. was supported by a Vici Grant of the Netherlands Organization for Scientific Research. M. S. is supported by ERC grant "MASEM" (242635).