Recovery of Bacillus Spore Contaminants from Rough Surfaces: a Challenge to Space Mission Cleanliness Control

Abstract

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load (“bioburden”) of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research.

The advent of space travel has raised concerns that biological cross-contaminations between solar bodies could occur due to a lack of cleanliness control of space missions. The concept of planetary protection includes the avoidance of so-called forward contaminations: spacecraft sent to foreign celestial bodies may inadvertently serve as a conveyer for biomolecules and/or life which originated on Earth. After landing or impact on the target, terrestrial contaminants may violate the scientific integrity of a mission by causing false-positive results in life detection—or even affect the extraterrestrial ecosystem.

For cleanliness and contamination control, spacecraft are assembled in clean rooms and must undergo a regular determination of bioload, as required by the United Nations Outer Space Treaty (2). Therefore, space agencies measure the aerobic, heterotrophic, and mesophilic spore loads of spacecraft as a proxy indicator to calculate the overall biomatter. Spore-forming bacilli were identified as frequent contaminants on spacecraft-related surfaces (19, 33, 39). Additionally, Bacillus spores are well known to withstand many stresses (28) and even to survive space conditions (17). Based on these observations, (Bacillus) spores are still considered the major contamination source that could affect the integrity of space missions, although the presence of vegetative, extremotolerant microorganisms in spacecraft assembly clean rooms has been reported (19, 31, 38).

Assays to sample spacecraft surfaces employ the wipe-rinse technique originally developed by Manheimer in the early 20th century (23). From that time on, many variants of this method have been established, but most previous studies of the method have been related to Bacillus anthracis spore detection due to terrorism incidents in 2001 (8-10, 13, 14, 16, 22, 35, 37). On that occasion, the Brentwood Mail Processing and Distribution Center in Washington, DC, was highly contaminated with spores of B. anthracis as a consequence of a terroristic attack (1).

Crucial factors for sampling success were attributed to the material of the sampling device (35), but the structure of the contaminated surface was also shown to influence the recovery of spores significantly (9, 11, 12, 30).

Most studies published so far have been based on analyzing the spore recovery of a single Bacillus strain, such as B. anthracis Sterne (6) or B. atrophaeus (26), serving as a surrogate for B. anthracis. Nevertheless, a significant difference in detection of the two spore types has been reported very recently, resulting in a 2-fold increase of B. atrophaeus spore recovery efficiency from stainless steel coupons over that of B. anthracis Sterne spores (30). In the aforementioned study, we also improved and simplified the current National Aeronautics and Space Administration (NASA) standard swab protocol for the detection of bacterial spores from various spacecraft surfaces but found an inefficient detection of spores on textiles. Moreover, swab protocols have been designed to sample small areas of spacecraft, but the agencies' polyester wipe assays are able to cover a sample area of 1 m² (3, 4). Little is known about the efficacy of these wipe assays, since their evaluation in 1979 was based on sampling exposed stainless steel and rocket booster materials (18).

The Viking Lander Capsule of the first Mars mission was subjected to extensive heat treatment, sterilizing the spacecraft comprehensively (33). To this day, spacecraft hardware is mostly manufactured under microbiologically uncontrolled conditions. Consequently, these novel components, which are very sensitive to dry-heat sterilization, must undergo alternative sterilization methods (e.g., H₂O₂) prior to assembly. Integration after sterilization poses a danger of recontamination, requiring a regular determination of bioburden during assembly, testing, and launching operations. Although Vectran fabric, a typical airbag material, can be sterilized via dry-heat treatment prior to clean room entry, the manual integration of, e.g., heat-prone pyrocutters poses a potential contamination risk arising from human activity, which is known to be a crucial factor in microbial contamination of clean room environments (25, 31, 33, 38).

Emerging improvements in space science hardware also present novel surface materials that are integrated into the spacecraft and must be monitored via microbiological sampling. Previous studies demonstrated negative effects of highly porous sample surfaces, resulting in a lower detection of contaminants (9, 11, 12, 30). In particular, the above-mentioned Vectran fabric was proven to retain the vast majority of contaminants when sampled with the present European Space Agency (ESA) swab assay (30). Hence, there is a potential threat to life detection missions arising from a biased detection of spores on rough surfaces. These issues, coupled with the ESA's interest in validating and improving the present standard methods in spore detection, led to setting up this comprehensive study. Its results will help researchers to understand the distribution of spores on and possible recovery from rough surfaces, which are also of great importance for the successful detection of, e.g., B. anthracis spores on textiles. The proposed improvements in sampling and spore recovery will be of high interest in the fields of planetary protection and public health.

MATERIALS AND METHODS

Experimental design and general background.

One of our recent studies, evaluating a nylon-flocked-swab protocol, revealed high aberrations in collection efficiency depending on the porosity of the test surface (30). The material retaining most of its spores turned out to be Vectran fabric type A, which was consequently the reference test surface in this subsequent study. Ten different sampling devices and their corresponding sampling protocols were evaluated for B. atrophaeus spore recovery from Vectran fabric type A, the reference surface in this study. The nominal CFU concentration was set to 4 × 10⁴ per m² according to observations made in International Organization for Standardization standard 8 (ISO 8) clean rooms (20). This concentration corresponds to 100 CFU per 25 cm², 400 per 100 cm², and 1,600 per 400 cm². This standard concentration was also used for specificity tests using five different Bacillus strains. Spore solutions were spotted onto the test surface in a 50% (vol/vol) ethanol (EtOH) solution, if not given otherwise. In addition to efficiency measurements, the sampling devices were evaluated with regard to usability, handling, and possible disintegration during sampling. Different criteria thereof were documented and are listed in Table 1. For linearity studies, different amounts of B. atrophaeus spores were spotted on Vectran fabric type A in order to gain information about the efficiency of the foam-spatula method when varying the concentrations in inocula (1,600, 1,200, 800, 600, 400, 200, and 100 spore CFU per 400 cm²). Tests to determine the dependence of the recovery on different inoculation methods (spotting and aerosolization) were also performed at 1,600 CFU per 400 cm² (foam-spatula protocol).

TABLE 1.

Comparison of sampling methods and tools for rough surface sampling^f

Spore detection method	Sampling tool	Tip materiala	Supplier	Handling		Sampling area (cm2)	Inoculum CFU (nominal/actual)	No. of replicates	Recovered CFU		Recovery efficiency (%)	95% CIb
				Positive properties	Negative properties				Mean	Range
Swab assay	Foam-tipped applicator	Unknownc	Puritan Medical Products, Guilford, ME	Singly packed, PBP,d stable handle	High absorption of extraction solution	25	100/104.8	10	1.5	0.0-5.0	1.4	0.7-2.7
	Nylon-flocked swab, 552Ce	Nylon	Copan International, Brescia, Italy	Singly packed, PBP	Thin handle	25	100/104.8	20	6.1	2.5-10.0	5.9	4.7-7.2
	Puritan swab 3600	Polyester	Puritan Medical Products	No PBP necessary	Delivered unsterile, thin handle	25	100/104.8	10	2.5	0.0-5.0	2.4	1.5-3.7
	Puritan swab 3655	Polyester	Puritan Medical Products	Stable handle	Delivered unsterile, no PBP	25	100/104.8	11	5.6	1.3-11.3	5.3	3.9-7.1
	Whatman FTA card applicators	Unknownc	Fisher Scientific, Pittsburgh, PA	Singly packed, stable handle	No PBP	25	100/104.8	10	6.9	3.8-8.8	6.6	4.9-8.6
Foam-spatula protocol	Foam spatula 939CS01	Polyurethane	Copan International	Singly packed, PBP, stable handle	Some batches revealed residue problems	400	1,600/1,551.8	13	204.4	136.7-246.7	13.2	12.5-13.9
Nylon-flocked-spatula protocol	Nylon-flocked spatula, prototype	Nylon	Copan International	PBP, stable handle	Delivered unsterile, residue problems	400	1,600/1,551.8	10	64.8	50.0-87.6	4.2	3.8-4.6
SpongeSicle protocol	SpongeSicle	Cellulose	Biotrace International, Bridgend, United Kingdom	Easy handling	Disintegration of the cellulose material	400	1,600/1,551.8	10	53	40.0-80.0	3.4	3.1-3.8
Wipe assay	Spec wipe 7 wipers, 115-0043 (ESA standard)	Polyester	VWR, Darmstadt, Germany		Not delivered in proper size, requires the usage of gloves, direct contact	400	1,600/1,551.8	10	8.1	3.8-17.5	0.5	0.4-0.7
	Vectra Alphasorb TX 1050	Polyester	Texwipe, Ogdensburg, NY		Not delivered in proper size, foaming effect, requires the usage of gloves, direct contact	400	1,600/1,551.8	10	7.1	2.5-13.8	0.5	0.4-0.6
Direct extraction (bulk method)				Rapid	Not applicable to spacecraft	100	400/388.0	10	59.8	40.0-82.5	15.4	14.0-16.9

^aMaterial composition of the forefront of the sampling tool that is in direct contact with the sample surface and therefore responsible for recovery of microbes.

^bCI, confidence interval, in percentage of detected CFU.

^cClassified as confidential by the supplier.

^dPBP, predetermined breaking point.

^eResults from the work of Probst et al. (30).

^fThe surface was Vectran fabric type A; the organism was B. atrophaeus; the spore concentration (nominal) was 4 × 10⁴ CFU per m²; inoculation was performed via spotting.

Bacillus species.

Five different Bacillus species were used for specificity tests within this study: B. atrophaeus DSM 675^T (Deutsche Sammlung von Mikroorganismen und Zellkulturen [DSMZ]) served as the reference test strain. B. anthracis Sterne 34F2 (U. Reischl, Universitaetsklinik, Regensburg, Germany), B. megaterium 2c1 and B. thuringensis E24 (original clean room isolates; P. Rettberg, DLR, Cologne, Germany), and B. safensis DSM 19292^T (DSMZ) were the same strains as described in one of our previous publications (30).

Spore stock solutions.

Pure Bacillus cultures were grown on plates until a sporulation of >95% was observed. Isolation and purification were performed as described previously with DNase and lysozyme (24). Afterwards, spores were stored in 50% (vol/vol) and absolute (abs.) EtOH, respectively. Dilution series were performed to create spore stock solutions with an appropriate number of CFU determined via positive controls (see below).

Sample surface materials were (i) Vectran fabric type A, (ii) Vectran fabric type B, (iii) carbon fiber-reinforced plastic (CFRP), and (iv) roughened carbon fiber-reinforced plastic (Fig. 1; Table 1). The sizes of the test surfaces varied depending on the assay: 5- by 5-cm² surfaces were used for swab assays, 10- by 10-cm² surfaces were used for direct extraction from the surface, and 20- by 20-cm² surfaces were used for other protocols. Detailed descriptions of the properties of the materials are provided elsewhere (30).

FIG. 1.

Scanning electron micrographs of different surfaces for recovery tests. White boxes indicate enlarged areas shown in the next picture of a series. Bars, 200 μm (row 1), 50 μm (row 2), and 20 μm (row 3). (A) Vectran fabric type A; (B) Vectran fabric type B; (C) carbon fiber-reinforced plastic (CFRP); (D) roughened CFRP.

Inoculation.

Prior to inoculation, surface materials were rinsed with 70% (vol/vol) EtOH, dried, and sterilized via UV irradiation (360 s; GS Gene Linker, UV Chamber; Bio-Rad) and autoclaving (121°C, 200 kPa, 1 h). Spore stock solutions were sonicated (3 min, 120 W, 35 kHz; Sonorex super DK 102P; Bandelin, Germany) prior to usage in order to ensure seeding of single spores. For wet inoculation, spores were spotted in 50% (vol/vol) EtOH solutions onto test surfaces and dried for 24 h in a laminar flow hood. For aerosolization, spore stock solutions (abs. EtOH) were sprayed into a settling chamber (dimensions, 28 by 28 by 25 cm; zinc sheet) as an alternate inoculation procedure. Two contrasting types of spraying methods were used: aerosolization via a spray gun (Sogolee HP-200 [0.2 mm]; Taiwan Airbrushes, Taoyuan, Taiwan) and via a spray diffuser (Roth GmbH & Co. KG, Karlsruhe, Germany).

In the case of the spray diffuser, the chamber, including the sample surface material, was preheated to 60°C for 15 min prior to each seeding in order to increase the aerosolization effect. Here, three pump strokes were necessary to meet CFU requirements. After the seeding process, spores were allowed to settle in the chamber for 15 min before the test surface was removed.

General handling and procedures.

A biosafety cabinet (Microflow biological safety cabinet; Nunc, Wiesbaden, Germany) ensured the cleanliness and sterility of the sampling assays described herein. Spacecraft bioburden assays usually focus on enumerating the number of germinable spores; vegetative cells are killed by implementing a heat step (15 min, 80°C) during the spore extraction procedure (3, 4). Therefore, this step was omitted for the recovery efficiency studies.

Sampling methods.

The procedures described herein were based on the wipe-rinse technique (23) if not stated otherwise. An overview of the methodologies, the sampling devices (including manufacturers), and the corresponding sample surface size is provided in Table 1. In general, sampling was performed by wiping the surface three times with a premoistened sampling device, rotating the direction of motion 0°, 90°, and 135°. For extraction of spores from the sampling material, phosphate-buffered saline including Tween 80 (0.02% [vol/vol]; PBST) was added to the removed head of the sampling tool; this suspension was incubated at room temperature (RT; 22°C ± 2°C) for 1 h prior to extraction in order to simulate transport from the sampling site to the laboratory. Cultivation was performed according to ESA standard assays (3): the cultivation medium for growing the extracted spores was R2A (BD, Heidelberg, Germany), and colony counts of the spread/pour plates were performed after 24, 48, and 72 h of incubation at 32°C.

The sampling protocols of the study were as follows.

(i) Swab assays.

Swab assays were carried out according to protocol A in the work of Probst et al. (3, 30). The sterile swabs were removed from their container, and the head of the swab was premoistened with water. Swabbing was performed by moving the swab in one direction while rotating the head and applying steady pressure. This was repeated twice by changing the linear direction of the swabbing process by 90° and 135°, as explained above. Afterwards, the swab head was broken into a tube containing 2.5 ml sterile PBST. For sample processing, the tube was vortexed at full speed for 5 to 6 s before four 0.5-ml samples of the spore suspensions were plated. Modifications of this protocol were accomplished when and where necessary, depending on the type of swab used: Whatman FTA card applicators were processed in a 50-ml Falcon tube (VWR, Darmstadt, Germany) for extraction due to their size; for Puritan foam-tipped applicators, only three instead of four spread plates were achieved due to Puritan foam's high absorption of the extraction solution.

(ii) Foam-spatula protocol.

The foam spatula (22) had a 17-cm-long and 1.5-cm-wide handle. The bilateral broadening at the forefront was covered by a 4- by 4-cm large piece of polyurethane sponge on both sides. For premoistening, the sponge was slightly swirled in a wide-necked bottle containing 30 ml of sterile water. During sampling, the sampling device was held at a 30° angle toward the 400-cm² test surface and the side of the sponge was changed for each of the three sampling steps (0°, 90°, and 135°). Then the sponge was broken into a wide-necked bottle (VWR; 100-ml total volume, with screw cap) by using its front breaking point, and 40 ml of PBST was added. For extraction, the capped bottle was shaken for 20 s horizontally and sonicated for 120 s (120 W, 35 kHz). Twenty-four milliliters of the extracted solution was portioned into six petri dishes of 4 ml each, and pour plates were achieved by adding molten (50°C), sterile R2A agar.

(iii) Nylon-flocked-spatula protocol.

This sampling device had the same basic structure as that described for the foam spatula, but its forefront was flocked with nylon fibers as described previously (30). The sampling protocol was identical to the foam-spatula protocol, with the exception of using 10 ml of PBST for extraction to result in two pour plates only.

(iv) SpongeSicle protocol.

The SpongeSicle sampling device was 20 cm long and had a 2-cm-wide handle. The two-sided forefront was covered by a cellulose sponge having a size of 4 by 4 cm. For moistening the sponge, 10 ml of sterile water was added into the sterile bag of the sampling tool. The rest of the protocol was accomplished according to the foam-spatula protocol, except that 20 ml of PBST was used for extraction and three pour plates were achieved.

(v) Wipe assays.

Wipe assays were performed as described for the ESA standard assay for microbial examination of flight hardware (3), except that the sample surface area was 400 cm². In brief, a sterile 15- by 15-cm polyester wipe was premoistened with 3 ml sterile water and placed flat onto the test surface. After each step of wiping (0°, 90°, and 135°), the wipe was folded, so that the used surface was wrapped inside. Afterwards, it was placed into a 50-ml Falcon tube (VWR, Germany) and 40 ml PBST was added, as given in the ESA standard procedure (3). Spores were extracted from the wipe by shaking the tube for 15 s followed by sonication (120 s, 120 W, 35 kHz). Subsequently, 32 ml of the extraction solution was utilized for eight pour plates.

(vi) Direct extraction.

The direct-extraction bulk method was not based on the wipe-rinse technique; instead, spores were directly extracted from the surface material. Therefore, a 10- by 10-cm piece of (contaminated) Vectran fabric was rolled and placed into a 50-ml Falcon tube followed by addition of 40 ml of extraction solution. For extraction, the tube was shaken for 15 s and sonicated for 120 s (120 W, 35 kHz).

Controls.

For positive controls, spore stock solutions that were used for spotting spores onto test surfaces were directly plated on R2A agar plates. For aerosolization controls, sterile bioassay dishes (24 by 24 cm) with R2A medium were placed into the settling chamber and seeding was performed as described above. Colony counting was done after 24, 48, and 72 h of incubation (32°C) in order to determine the number of CFU.

For each test series, one negative control was carried along; that is, sterile 50% (vol/vol) EtOH and abs. EtOH were spotted or sprayed, respectively, onto the test surface as mentioned above. Then the corresponding sampling method was applied, and samples were processed as described above.

Direct inoculation.

For determination of the extraction efficiency of the foam-spatula protocol, 1,600 CFU of B. atrophaeus spores was directly spotted onto two-thirds of the sponge, 1 ml of the spore solution (50% EtOH [vol/vol]) for each side. Afterwards, the sponge was broken into a wide-necked bottle followed by extraction and CFU enumeration as already described above. For statistical analysis (see below), the volume was adjusted to 42 ml (40-ml storage and 2-ml spore stock solution).

Statistical analysis.

In adherence to the stipulation of USP (United States Pharmacopeia) 1223 (5), the statistical processing of the data makes proper use of the underlying probability distribution of the raw data, i.e., the Poisson distribution, instead of approximations to the normal distribution. In particular, two features of Poisson distributed data are exploited in the analysis (29, 32, 36). Given count data, x and y, from two Poisson populations with expectation values (means) ξ and η, i.e., X∼Poisson(ξ) and Y∼Poisson(η), then the sum, s = x + y, is again a Poisson variable, S∼Poisson(ζ), with mean ζ = ξ + η. This can be generalized to more than two samples. The test of equality of two means or the construction of confidence intervals for an observed ratio r = x/y is based on a second property of the Poisson distribution. Given the above observed sum, s = x + y, the distribution of Y or X conditional on S = s is a binomial distribution.

One-sided type I error probabilities, α⁽¹⁾, for the null hypothesis H₀: ξ = η are obtained by summing the probabilities of this binomial distribution pertaining to those events which belong to the alternative hypothesis H₁ for an envisaged deviation from H₀ (H₁: less than and/or H₁: greater than).

Confidence intervals for ratios, R, estimating ρ = ξ/η from observations of counts u and v, U∼Poisson(aξ) and V∼Poisson(bη) with u + v = w and known a and b, again are derived from the binomial distribution.

Raw data were processed by a program script for the statistical programming environment R (http://www.R-project.org). Further details are given in the work of Probst et al. (30). Confidence levels were chosen as 0.95 (95%). Nonoverlapping confidence intervals of two assortments of sampling methods were evidence for a significant difference of their recovery efficiencies.

For different recovery methods, average recoveries across the test species or across the test inocula were determined as weighted averages (for the procedure, see reference 30).

SEM and spore preparation.

Scanning electron microscopy (SEM) (30) was carried out with a digital scanning microscope (DSM 950; Zeiss, Oberkochen, Germany). Prior to the analysis, preparations were coated with a gold-palladium target, creating a layer of 1.4 nm (Polaron SC 515, SEM coating system). To analyze the polyurethane material of the foam spatula, a 1- by 1-cm-large piece was cut out of its sponge and glued onto a stab by using nonconductive adhesion tabs (Plano GmbH, Wetzlar, Germany). For analysis of the sample surface material, pieces of 1 cm in diameter were prepared and placed on SEM stabs also by using nonconductive adhesion tabs. For further purification of spores, ultracentrifugation was performed using a CsCl gradient (40% [wt/wt], 16 h; 50,000 rpm [336,239 × g]) and a swing-out rotor (SW 60 Ti; Beckman Optima LE-80K; Beckman Coulter Inc.) (15, 30). In order to gain insights into the distribution of spores on the sample surface material, highly purified spore solutions (50% [vol/vol] EtOH) were sonicated (3 min, 120 W, 35 kHz) and spotted onto prepared test surfaces. For aerosolization, the sample surface was preheated to 60°C and highly purified spores were sprayed (abs. EtOH) onto the test surface by using a spray diffuser (Roth GmbH & Co. KG, Karlsruhe, Germany).

RESULTS

Comparison of sampling methods.

In general, means of the CFU recovery efficiencies from Vectran fabric type A ranged from 0.5% (wipe protocol) to 15.4% (direct extraction) depending on the sampling method applied (Table 1). Considering the swab protocol, Whatman FTA card applicators collected the highest number of CFU from the textile (6.7% recovery efficiency) but showed no significant difference from Puritan 3655 swabs (5.3%). Fewer CFU were recovered by foam-tipped applicators and Puritan 3600 swabs (1.4% and 2.4% recovery efficiencies, respectively). The wipe methods based on Spec wipe 7 wipers (ESA standard method [3]) and Vectra Alphasorb turned out to be insignificantly different in detection, whereas both resulted in a very poor recovery efficiency of 0.5%. The nylon-flocked spatula and SpongeSicle protocols revealed efficiencies of 4.2% and 3.4% recovery, respectively. Moreover, no significant differences were detected between nylon-flocked spatulas and Puritan 3655 swabs or SpongeSicles and Puritan 3600 swabs. Direct extraction (bulk method; 15.4%) and the foam-spatula protocol (13.2%) revealed the highest recovery efficiencies, both being significantly different from all other methods. For the bulk method, however, the surface sample material itself was used for extraction; hence, this procedure was studied for comparison but not as a method applicable to spacecraft surfaces. The foam-spatula protocol consequently out-performed all other methods in spore detection on Vectran fabric type A.

Sampling surfaces of different degrees of roughness.

The present ESA standard procedure for large surface sampling was compared to the foam-spatula protocol in CFU recovery of B. atrophaeus spores from four different spacecraft surfaces (Fig. 1). An overview of the results of this section is provided in Table 2, whereas Fig. 2 additionally shows results from the nylon-flocked-swab protocol of a previous study for comparison (30). The wipe method was most efficient when sampling roughened CFRP (19.6% recovery efficiency) and detected fewer CFU on smooth CFRP (9.0%). Sampling of Vectran fabric type B resulted in a significantly higher recovery of CFU (6.5%) than that of Vectran fabric type A (0.5%). Considering the foam-spatula protocol, it revealed the highest recovery efficiencies when sampling smooth CFRP (57.1%) and roughened CFRP (38.1%), with significant differences. In the case of Vectran fabric, more CFU were detected on type B than on type A (24.4% and 13.2% recovery efficiencies, respectively), which correlates with results from the wipe assay. In summary, at a concentration of 1,600 CFU per 400 cm², the foam-spatula protocol was superior to the present ESA standard wipe assay regardless of the porosity of the surface analyzed. The average recovery efficiency for B. atrophaeus spores (CFU) for the foam-spatula protocol was 41.1%, approximately 3 times higher than that for the standard wipe assay (13.9%).

TABLE 2.

Comparison of B. atrophaeus spore recoveries from surfaces with different degrees of roughness^d

Sampling method/tool	Sample surface material	Sampling area (cm2)	No. of replicates	Recovered CFU		Recovery efficiency (%)	95% CIa	Avg recovery (%)b
				Mean	Range
Foam-spatula protocol	CFRPc	400	10	886.4	680.0-1,111.7	57.1	55.4-58.6	41.1 ± 9.7 (19.5)
	Roughened CFRP	400	10	591.0	461.7-711.7	38.1	36.7-39.5
	Vectran fabric type A	400	13	204.4	136.7-246.7	13.2	12.5-13.9
	Vectran fabric type B	400	10	378.0	280.0-458.3	24.4	23.3-25.5
Wipe assay	CFRP	400	10	138.9	61.3-235.0	9.0	8.4-9.5	13.9 ± 3.8 (7.68)
	Roughened CFRP	400	10	304.5	236.3-426.3	19.6	18.8-20.5
	Vectran fabric type A	400	10	8.1	3.8-17.5	0.5	0.4-0.7
	Vectran fabric type B	400	10	101.5	48.8-143.8	6.5	6.1-7.0

^aCI, confidence interval of recovery efficiency.

^bMean ± standard error of the mean (standard deviation) of the recovery efficiencies of the different test series of a single method.

^cCFRP, carbon fiber-reinforced plastic.

^dThe spore concentration (nominal) was 4 × 10⁴ CFU per m². Inoculation was performed via spotting. The spore inocula were 1,600 (nominal) and 1,551.8 (actual) CFU.

FIG. 2.

Histogram of recovery efficiencies of B. atrophaeus spores (1,600 CFU per 400 cm² or 100 CFU per 25 cm²) from different surfaces using the foam-spatula protocol, the ESA standard wipe assay, and the nylon-flocked-swab protocol. Error bars indicate the confidence interval (95%). CFRP, carbon fiber-reinforced plastic. The asterisk indicates that data are from the work of Probst et al. (30).

Operational range of the foam-spatula protocol.

A summary of the results of these test series is provided in Table 3. The method's recovery efficiency declined from 13.2% to 7.0% for inocula of 1,600 and 1,200 CFU per 400 cm², respectively. This was also observed in additionally performed experiments. At and below 1,200 CFU per sample area, the recovery efficiency stayed constant with an average of 6.7%.

TABLE 3.

Recovery efficiency of the foam-spatula protocol at different spore concentrations^b

Nominal inoculum (CFU)/m2	Nominal inoculum (CFU)/400 cm2	Actual inoculum (CFU)/400 cm2	No. of replicates	Recovered CFU		Recovery efficiency (%)	95% CIa
				Mean	Range
2,500	100	104.8	11	6.8	1.7-15.0	6.5	4.7-8.7
5,000	200	209.6	10	12.5	8.3-18.3	6.0	4.7-7.5
10,000	400	387.5	11	26.8	15.0-50.0	6.9	5.9-8.0
15,000	600	632.5	10	47.5	36.7-60.0	7.5	6.7-8.5
20,000	800	775.9	10	52.3	50.0-76.7	6.7	6.0-7.5
30,000	1,200	1,265.0	13	87.9	41.7-135.0	7.0	6.4-7.5
40,000	1,600	1,551.8	13	204.4	136.7-246.7	13.2	12.5-13.9

^aCI, confidence interval, in percentage of recovered spores.

^bThe sample surface was Vectran fabric type A, 400 cm². The organism was B. atrophaeus spores; inoculation was performed via spotting.

The specificity of the foam-spatula protocol was tested by varying the Bacillus species used for the recovery test; numerical values of those recovery efficiency experiments are provided in Table 4. Spores of B. atrophaeus were recovered with the highest efficiency (13.2%), and those of B. anthracis were recovered with the lowest efficiency (0.3%); the two were significantly different from one another. Spores of clean room isolates, B. megaterium 2c1 and B. thuringiensis E24, were detected with an insignificant difference (5.1% and 5.4% recovery efficiencies, respectively). B. safensis spores were recovered with an efficiency of 0.5%. The average recovery efficiency based on all species was determined to be 4.9%.

TABLE 4.

Specificity of the foam-spatula protocol^b

Organism	Actual CFU in inoculum (nominal, 1,600)	No. of replicates	Recovered CFU		Recovery efficiency (%)	95% CIa
			Mean	Range
Bacillus anthracis Sterne	1,566.6	10	5.3	1.7-15.0	0.3	0.2-0.5
Bacillus atrophaeus	1,551.8	13	204.4	136.7-246.7	13.2	12.5-13.9
Bacillus megaterium 2c1	1,509.4	10	76.8	40.0-123.3	5.1	4.6-5.6
Bacillus safensis	1,640.2	11	8.5	5.0-15.0	0.5	0.4-0.7
Bacillus thuringiensis E24	1,519.6	10	81.7	51.7-108.3	5.4	4.9-5.9

^aCI, confidence interval, in percentage of recovered spores.

^bThe sample surface was Vectran fabric type A, 400 cm². The nominal spore concentration was 4 × 10⁴ CFU per m²; inoculation was performed via spotting.

Extraction efficiency of the foam-spatula protocol.

The mean of the extraction efficiency was determined to be 84.2% with a confidence interval (95%) ranging from 81.9% to 86.6%. Moreover, the mean of recovered CFU was 1,306.4 per test with a maximum of 1,442.0 and a minimum of 1,209.3 spores. CFU that were consequently lost during sample processing or retained in the sponge were calculated to add up to 15.8% of the inoculum.

SEM analysis of the foam-spatula sponge.

The tip of the sampling device was a spongy network of polyurethane with bubble-like cavities (Fig. 3). The sponge comprised several layers of this network, whereas only the uppermost layer was in direct contact with the sample surface material. The cavities of the top surface of the sponge had a diameter of approximately 60 μm, and the polyurethane junctions varied from 10 to 30 μm in width. Moreover, these connections had gaps and holes of different sizes ranging from approximately 2 to 10 μm.

FIG. 3.

SEM images of the sponge material of the foam spatula (polyurethane, macrofoam). (A) Top view; bar, 500 μm. (B) Enlargement of the white box in panel A; bar, 50 μm. (C) Oblique view (85°); bar, 100 μm. (D) Enlargement of the white box in panel C; bar, 10 μm.

SEM analysis of the sample surface material.

Vectran fabric type A was made of bundles of single fibers, which were interwoven into a network in a 90° angle. These cylindrical fibers had a diameter of approximately 20 μm. An oblique view of the bundles (Fig. 1A) showed that there were also gaps between the fibers of a single bundle (Fig. 1A3). Additionally, there were free thin fibers found to be detached from the actual fibers and protruding in a disorderly way from the surface. When these free, thin fibers, which were variable in width (2 to 15 μm) and length, came apart, furrows were left on the main fiber, thus increasing the porosity of the surface.

The surface structure of Vectran fabric type B was almost identical to that of type A (Fig. 1B). However, the fibers had a diameter of 25 μm and were flattened cylinders in shape. Consequently no gaps occurred between the fibers within a bundle.

CFRP was shown to be a very smooth surface with sporadic, low rises (Fig. 1C). In comparison, roughened CFRP consisted of blocks of 20 grooves that alternated at 90° angles (Fig. 1D). These grooves were approximately 20 μm wide and 20 μm deep. Although the structure of the surface was very regular, it revealed many particulates of different sizes and diffuse structures protruding from its surface.

Distribution analysis of spores on sample surfaces.

When spotted onto the test surfaces, B. atrophaeus spores (1.3 × 10⁴ spores per mm²) turned out to be well distributed on CFRP and roughened CFRP, occurring individually or in pairs (not shown). Only about 24% of 150 spores enumerated were attached to diffuse structures or to large particles in the case of roughened CFRP (not shown). On Vectran fabric type B, a highly accumulative effect, resulting in single layers of clustering spores, was observed (not shown). Analysis of spore distribution on Vectran fabric type A was carried out by enumerating more than 1,700 spores via SEM (Fig. 4). Spores of B. atrophaeus and other species (B. megaterium 2c1, B. thuringiensis E24, and B. safensis), which were spotted on Vectran fabric type A, were observed to cluster between the single fibers of the textile (56%) (Fig. 5). Others attached to free, thin fibers protruding from the surface or were located in furrows, but only 13% of the spores were lying on the fibers. In contrast, when aerosolized/sprayed onto Vectran fabric type A, spores of B. atrophaeus were observed to be mainly free on the fibers (62%), and only 7% were located in between.

FIG. 4.

Scanning electron micrograph of Vectran fabric type A showing four fibers and B. atrophaeus spores spotted onto the surface (concentration, 1.3 × 10⁴ per mm²). Bar, 10 μm. A, spores free on fibers; B, spores between fibers; C, spores attached to free, thin fibers; D, spores in furrows of the fibers.

FIG. 5.

Histogram showing the statistical distribution of Bacillus spores on Vectran fabric type A. Defined localizations are according to Fig. 4. Spores were spotted onto the test surface if not stated otherwise. Numbers in parentheses give the numbers of preparations analyzed. Sp, spores.

Variation of inoculation method.

Based on the different distributions of B. atrophaeus spores when sprayed and spotted onto the test surface (see above), different inoculation methods were applied to seed B. atrophaeus spores onto Vectran fabric type A; the results are summarized in Table 5. Using the spray diffuser and a preheated settling chamber for inoculation, the foam-spatula protocol resulted in low recovery efficiency (3.0%) compared to previous results from spotting spores (50% EtOH; 13.2% recovery efficiency). In addition, the spray gun method for inoculation allowed a recovery of spores from the surface of 18.7%. The comparison of all three inoculation methods revealed significantly different results for the foam-spatula protocol based on confidence interval analysis (95%).

TABLE 5.

Recovery efficiency of the foam-spatula protocol when sampling Vectran fabric type A inoculated with B. atrophaeus spores by different methods^b

Inoculation method	Actual CFU in inoculum (nominal, 1,600)	No. of replicates	Recovered CFU		Recovery efficiency (%)	95% CIa
			Mean	Range
Spray diffuser	1,600.5	7	48.1	20.0-85.0	3.0	2.6-3.5
Spray gun	1,601.9	10	298.8	200.0-328.3	18.7	17.8-19.6
Spotting	1,551.8	13	204.4	136.7-246.7	13.2	12.5-13.9

^aCI, confidence interval, in percentage of recovered spores.

^bThe nominal spore concentration was 4 × 10⁴ CFU per m².

DISCUSSION

In a recent study evaluating a nylon-flocked-swab protocol, we reported an unsatisfactory recovery of B. atrophaeus spores from Vectran fabric, an airbag material used for space travel (30). As a matter of fact, the present study was subsequently undertaken to further investigate this problematic surface, elucidate the recovery efficiency of ESA's current wipe assay, and find a possible alternative.

Many of the current spore detection protocols have been evaluated on stainless steel (16, 18, 35), some studies have included alternative test surfaces like bare concrete coupons (8-10, 12, 22), and only a few investigations have been performed on rough surfaces, wherein carpeting has been of primary interest (11, 14). Direct extraction of spores from the Vectran fabric type A (bulk method) showed low recovery efficiency compared to previous data based on carpeting (11), indicating that this fabric has a higher potential to retain spores. Interestingly, the wipe-rinse protocol that out-performed other sampling strategies—including ESA's wipe assay—was based on a foam spatula. This sampling device is made of polyurethane (macrofoam [16]) and has already been identified as a sampling material with high recovery efficiencies (12, 13, 16, 22, 35). The evaluation of this protocol according to international standards (5) showed a decrease of the spore recovery at lower concentrations of B. atrophaeus spores (13.2% to 6.7%). This is in contrast to a previous report in the literature (22) in which the same sampling device was used. Hence, the rough surface (Vectran fabric) rather than the foam spatula may be responsible for the nonlinearity, which, however, has been observed for other protocols before (7-10, 14, 16).

Based on the constant recovery of ∼6.7% at low spore concentrations, the limit of detection was calculated to be approximately 15 CFU per 400 cm² and 368 CFU per m² for the foam-spatula protocol. In contrast, planetary protection requirements demand a spore load equal to or lower than 300 spores (bioburden) per m² of spacecraft surface (2)—a value that can hardly be proven with present sampling methods. Similar to ESA's nylon-flocked-swab protocol (30), sampling methods applied in this study are unable to meet this requirement for Vectran fabric type A. These methods also included the agencies' wipe protocol, which has been used in several microbial community analyses and screening surveys in the last decade (19-21, 25, 27, 31). Furthermore, Vectran fabric has been used for previous Mars landing missions (Mars Exploration Rover, http://marsrover.nasa.gov/mission/spacecraft_edl_airvbags.html; Mars Pathfinder, http://marsprogram.jpl.nasa.gov/MPF/mpf/mpfairbags.html), yet data about the performance of space agencies' spore detection methods on this surface have not been reported in the literature up to now. As a conclusive statement, positive CFU detection on Vectran fabric is indicative of a high contamination level, whereas negative detections on the fabric do not constitute fulfillment of planetary protection requirements. Moreover, we assert that rough surfaces should be reduced to a minimum on spacecraft in order to ensure proper cleanliness control and meet planetary protection requirements.

For the purpose of evaluating the specificity of the foam-spatula protocol, spores of different Bacillus species were recovered from Vectran fabric type A. The causes of aberrations in the recovery of different types of Bacillus spores, based on, e.g., hydrophobicity of the spore sheath (34), have already been discussed previously (30). In addition, data from SEM analysis in this study clearly support this assumption, as all spores were distributed similarly on Vectran fabric, although they were recovered with different efficiencies. However, the more-than-40-fold-higher recovery efficiencies of B. atrophaeus than of B. anthracis Sterne spores reemphasize our previous statement that B. atrophaeus cannot be used as a surrogate for B. anthracis contaminants (30), and investigations based on this model organism (8-13, 22) should be interpreted with high skepticism concerning their transferability in the case of a B. anthracis terrorism incident.

Although this communication clearly showed the limitations of present spore detection methods with regard to fabrics, our foam-spatula protocol represents a strong advantage in spore load detection on large (spacecraft-related) surfaces: this method was superior in that it detected an up-to-26-fold increase of CFU over that in the present ESA and NASA wipe assay, whereas the applicability of the foam-spatula method in terms of particle loss and usability still has to be approved. Clearly, the results presented here have shed more light onto the distribution of spore contaminants on spacecraft surfaces and have a striking significance for anthrax spore-related studies: those should no longer be based on B. atrophaeus as a surrogate and should also consider textiles and cloths as contaminated materials.