Introduction

Because the search for life on other planets will involve ultrasensitive technologies for the detection of cells and biomarkers, contamination of extraterrestrial samples with cells or biomarkers originating from Earth (forward contamination) would seriously compromise the interpretation that life signatures, if found, were indeed of extraterrestrial origin. This concern has been heightened by recent data indicating routine meteorite exchange between Earth and Mars [3]; the probability that living microbes, particularly bacterial spores, could survive interplanetary transfer [6, 10]; and the resulting possibility that martian life could resemble life on Earth [14]. Consequently, current planetary protection protocols require that spacecraft be constructed and assembled under conditions as nearly as possible approaching sterility. To achieve disinfection, robotic spacecraft are assembled in clean rooms where air circulation is controlled and strict hygienic practices are implemented to minimize microbial contamination. In addition to physical containment methods, a number of sterilants including hydrogen peroxide (H2O2) vapor and ultraviolet radiation (UV) are considered to be viable options for sterilization of spacecraft hardware because they are low-heat sterilization processes compatible with various modern-day spacecraft electronics and components [1, 2].

As part of the NASA planetary protection program, recent monitoring of microbial diversity in the NASA Jet Propulsion Laboratories Spacecraft Assembly Facility (JPL-SAF) resulted in the isolation of a number of microbial species inhabiting various parts of the facility [16]. Whereas a highly diverse collection of microbes was isolated from entrance areas, microbial diversity decreased as samples were obtained from successively cleaner interior areas of the JPL-SAF. Interestingly, predominant isolates found to have penetrated deepest within the cleanest parts of the JPL-SAF were various strains of spore forming bacteria identified by biochemical testing and 16S rDNA analysis as being most closely related to Bacillus pumilus [16]; Kempf, MJ, Quigley MS, Chen F, Satomi M, Kern R, and Venkateswaran K, unpublished data). Not only were B. pumilus spores found to survive and persist in the extreme environment of the JPL-SAF, characterized by very low nutrient levels, strictly controlled humidity, and periodic disinfection (extensive details of JPL-SAF clean room disinfection procedures can be viewed at the Web site http://dnp- lib.jpl.nasa.gov/dnp-lib/dscgi/ds.py/Get/File-1671/Toc495979317), but B. pumilus isolates have also been recently recovered from hardware surfaces and air particles aboard the International Space Station (ISS) [15]. Taken together, the data strongly suggest that spores of B. pumilus are capable of escaping current spacecraft disinfection regimens and being inadvertently transported into space.

In this communication we tested spores of 10 JPL-SAF B. pumilus isolates for their resistance to germicidal (254-nm) UV and report that a subset of these strains exhibit elevated levels of UV resistance. The results are discussed in light of their important implications for spacecraft sterilization, planetary protection, and our understanding of spore UV resistance mechanisms.

Materials and Methods

Bacterial Strains and Culture Conditions

The bacterial strains used are listed in Table 1. All JPL-SAF isolates are described in detail elsewhere [16]; Kempf MJ, Quigley MS, Chen F, Satomi M, Kern R, and Venkateswaran K, unpublished data). All bacteria were sporulated by incubation in liquid Schaeffer Sporulation Medium (SSM) [13] at 37°C for 48 h in a rotary shaker with vigorous aeration. Cultures were harvested by centrifugation (10,000 g, 10 min, 25°C); spores were purified using the lysozyme and buffer washing method described by Nicholson and Setlow [11], heat-shocked (80°C, 10 min) and stored in deionized water at 4°C. All spore preparations were determined to be essentially free of vegetative cells and to consist of >99.9% phase-bright spores by phase-contrast microscopy. The viable titer of all spore preparations was determined by serial 10-fold dilution in phosphate-buffered saline (PBS) (10 mM potassium phosphate, 150 mM NaCl, pH 7.4) and plating on SSM solidified with 1.7% agar.

Table 1 Bacterial strains used in this study and their resistances to 254-nm UV in the preliminary screen
Full size table

Spore UV Resistance Assay

UV irradiation was performed using a commercial low-pressure mercury vapor lamp (model UVGL-25, UV Products, Upland, CA) with the filter removed, producing predominantly 254-nm UV-C radiation. Throughout the experiments the lamp was placed at a constant height of 42 cm above the target. Lamp UV output was measured using a UVX radiometer fitted with the UVX-25 filter (UV Products), recently calibrated and traceable to the National Institute of Standards and Technology standard. Purified spores were diluted to a final concentration of 106 colony-forming units (cfu)/mL in 10 mL of PBS; the spore suspension was pipetted into the bottom of an uncovered 6-cm diameter Petri dish with a sterile magnetic stir bar added, and the dish was placed on a magnetic stir plate beneath the UV lamp. Spores were exposed to UV with constant mixing and samples removed from the suspension at the desired UV dose, calculated from the fluence rate and the time of exposure. Spores were diluted serially 10-fold in PBS, plated in duplicate on solid SSM medium, and incubated at 37°C for 48 h, after which colonies were counted.

Data Analysis

The surviving fraction of spores was calculated as log10(N/N 0), in which N is the viable spore titer at any given UV dose and N 0 is the spore titer obtained from the untreated suspension. For establishing dose-response curves, each experiment was repeated at least three times; averages and standard deviations of each set of data points were calculated and compared by analysis of variance (ANOVA) using Minitab version 10.5 software for Macintosh.

Results

In order to screen for extreme UV resistance, 10 JPL-SAF B. pumilus isolates were sporulated in SSM medium and their purified spores were exposed to a final UV dose of 1000 J/m2. From this preliminary screen it was determined that spores of four isolates (SAFN-001, FO-033, SAFN-027, and KO-052) exhibited approximately the same level of UV resistance as did spores of the B. pumilus type strain ATCC 7061 (Table 1). Spores of five JPL-SAF isolates (SAFN-029, SAFN-037, SAFN-036, FO-036b, and FO-038) exhibited between 10-fold and 100-fold greater survival at 1000 J/m2 than did the B. pumilus type strain (Table 1), and spores of one JPL-SAF isolate (SAFR-032) proved to produce the most UV-resistant spores tested, exhibiting ~340-fold greater survival to UV at 1000 J/m2 than did spores of the B. pumilus type strain ATCC 7061 (Table 1).

In order to confirm and extend the above observations, UV dose-response curves were generated for the six JPL-SAF isolates which exhibited elevated levels of spore UV resistance in the preliminary screen, and the resulting UV inactivation curves were compared with that of the standard laboratory UV biodosimetry strain, B. subtilis WN624 [8] (Fig. 1). From the data of Fig. 1 it appeared that spores of all JPL-SAF isolates tested were more UV-resistant than spores of the B. subtilis biodosimetry standard strain WN624. Moreover, it appeared that strains SAFN-029, SAFN-037, SAFN-036, FO-036b, and FO-038 formed a loose cluster exhibiting rather similar UV resistances, but that strain SAFR-032 was distinct in producing by far the most UV-resistant spores of all of the SAF-JPL isolates (Fig. 1). This notion was further tested quantitatively as follows. Spore UV inactivation curves characteristically consist of a “shoulder” at low UV doses followed by an exponential decline in viability at higher UV doses (see Fig. 1). To quantify spore resistance properties two parameters, the LD90 value and the D value, are often used [7, 8]. The LD90 value is defined as the lethal dose for 90% of the population and reflects mostly the length of the “shoulder”; the D (“decimal reduction”) value, defined as the dose producing 1 log10 of inactivation, is derived from the exponential portion of the inactivation curve and reflects its slope [5]. The LD90 and D values were derived from the inactivation curves of each strain (n = 3 for SAF-JPL isolates; n = 4 for B. subtilis WN624), the averages and standard deviations for each data set were calculated, and the data set from each strain was compared to all other strains in pairwise combination by ANOVA. Differences among the resulting LD90 and D values of P ≤ 0.05 were considered significant, and isolates were grouped accordingly (Fig. 2). When compared by either LD90 (Fig. 2A) or D-value (Fig. 2B), spores of the standard B. subtilis biodosimtery strain WN624 were significantly less UV resistant than spores of all JPL-SAF isolates tested; thus WN624 formed its own group (“a” in Fig. 2). Spores of JPL-SAF isolates SAFN-029, SAFN-036, SAFN-037, and FO-038 were found to form a second cluster (“b”) of similar UV resistance when compared using either LD90 (Fig. 2A) or D-value (Fig. 2B) as the criterion. Spores of strain FO-036b also belonged to cluster “b” when D-value was used as the comparison criterion (Fig. 2B), but formed its own, slightly more UV-resistant, group (“d”) when evaluated using LD90 as the criterion (Fig. 2A). Spores of strain SAFR-032 were found to be significantly more UV resistant than spores of all other strains tested, thus forming their own group (“c” in Fig. 2). It is noteworthy that spores of strain SAFR-032, exhibiting LD90 and D values of 1689 ± 279 and 686 ±123 J/m2, respectively, are the most highly UV-resistant spores yet to be discovered and are at least five-fold more UV-resistant than spores of B. subtilis reference strains commonly used for UV biodosimetry [8].

Figure 1
figure 1

UV inactivation curves of spores of JPL-SAF isolates SAFR-032 (filled circles), SAFN-029 (filled squares), SAFN-037 (open squares), SAFN-036 (open triangles), FO-036B (filled triangles), and FO-038 (open circles). The dashed line represents the spore UV inactivation curve of B. subtilis biodosimetry strain WN624 [8] for comparison. All curves representaverages of three independent determinations (four independent determinations for WN624). Error bars are omitted for clarity, as the data points differed by less than 10%.

Full size image
Figure 2
figure 2

Comparison of LD90 values (A) and D values (B) of JPL-SAF isolates by ANOVA. Bars and error bars represent averages and standard deviations, respectively. Strains with 13 P ≤ 0.05 were judged as belonging to separate groups (lowercase letters).

Full size image

Is there a relationship between the high UV resistance of B. pumilus SAF isolates and their resistance to H2O2? To assess H2O2 resistance, both vegetative cells and purified spores of B. pumilus SAF isolates were exposed to 5% liquid H2O2 for 60 min. As expected, spores were more highly resistant than were their vegetative counterparts. Although purified spores of the B. pumilus type strain ATCC 7061 did not show any survivors after exposure to liquid H2O2, spores of all B. pumilus SAF isolates exhibited survivors to the H2O2 treatment, as did spores of the standard laboratory strain B. subtilis 168. Spores of B. pumilus SAF isolates SAFN-036 and FO-038 were inactivated by three to five orders of magnitude, as was the B. subtilis 168 laboratory strain. In contrast, spores of B. pumilus SAF isolates SAFR-032, SAFN-001, FO-036b, and FO-033 were observed to be more resistant to 5% H2O2, exhibiting only zero to two orders of magnitude of reduction in viability. However, although spores of B. pumilus strains FO-33 and SAFN-001 exhibited the highest degree of resistance to liquid H2O2 (Kempf MJ, Quigley MS, Chen F, Satomi M, Kern R, and Venkateswaran K, unpublished data), spores of these two strains were essentially no more resistant to UV than the type strain of B. pumilus, ATCC 7061 (Table 1). Therefore, there appeared to be no strict correlation between high spore H2O2 resistance and high spore UV resistance among the B. pumilus SAF isolates.

Discussion

In this article we report that a number of B. pumilus strains isolated from the interior of the JPL-SAF produce spores which demonstrated an elevated level of resistance to inactivation by germicidal (254-nm) UV. Spores of one strain in particular, SAFR-032, exhibited the highest degree of UV resistance yet encountered. These observations have profound implications for efforts at prevention of forward bacterial contamination during planetary missions, and for UV disinfection of recycled drinking water and wastewater during long-duration space flight. For example, current standards for UV disinfection of municipal drinking water require a biologically effective dose of 400 J/m2 [4], which reduces the viability of B. subtilis spore UV biodosimetry strains, including ATCC 6633 and WN624, by approximately two orders of magnitude [4, 9]. For spores of JPL-SAF isolate SAFR-032 treated under our laboratory conditions, it would require a UV dose of ~2000–2500 J/m2 to achieve the same reduction in viability. In addition, it should be stressed that spore resistance properties can be affected by the sporulation environment. In the current study, SAFR-032 spores were produced in laboratory medium, but in earlier work, we have found that Bacillus spp. spores isolated and purified directly from the soil environment demonstrated an additional two- to three-fold higher UV resistance than when the same isolates were sporulated in laboratory medium [9]. Thus it is possible that our measurements underestimate the intrinsic spore UV resistance of SAFR-032 in its natural setting, which raises an interesting ecological issue. It is reasonable to assume that all of the B. pumilus isolates originally inhabited the soil environment outside the ultraclean JPL-SAF and were inadvertently brought inside the JPL-SAF along with myriad other bacteria and fungi. It appears that the rigorous maintenance of clean-room conditions within the JPL-SAF has resulted not in an absolute barrier to contamination, but the establishment of a selective “bottleneck” which is sufficient to prevent penetration or survival of all but the hardiest microbes, which not surprisingly are bacterial spores.

In conclusion, recent environmental sampling of the JPL-SAF resulted in the isolation of a number of B. pumilus strains whose spore resistance properties are being actively investigated ([16]; Kempf MJ, Quigley MS, Chen F, Satomi M, Kern R, and Venkateswaran K, unpublished data). In this communication we report that a subset of the JPL-SAF B. pumilus isolates exhibit elevated spore resistance to UV. Repeated isolation of highly resistant strains of B. pumilus in clean rooms is problematic because their persistence in SAFs and on spacecraft might compromise planetary protection efforts and extraterrestrial life-detection experiments. Elimination of such forward contamination will require novel cleaning and sterilization technologies which are not only compatible with modern spacecraft, but which can successfully remove or inactivate the most resistant of the microbes found in the SAFs. An understanding of the microbial diversity of spacecraft assembly areas, and any extreme characteristics these microbes might possess, is necessary to develop these technologies.