Recurrent Isolation of Hydrogen Peroxide-Resistant Spores ofBacillus pumilusfrom a Spacecraft Assembly Facility

Abstract

While the microbial diversity of a spacecraft assembly facility at the Jet Propulsion Laboratory (Pasadena, CA) was being monitored, H2O2-resistant bacterial strains were repeatedly isolated from various surface locations. H2O2 is a possible sterilant for spacecraft hardware because it is a low-temperature process and compatible with various modern-day spacecraft materials, electronics, and components. Both conventional biochemical testing and molecular analyses identified these strains as Bacillus pumilus. This Bacillus species was found in both unclassified (entrance floors, anteroom, and air-lock) and classified (floors, cabinet tops, and air) locations. Both vegetative cells and spores of several B. pumilus isolates were exposed to 5% liquid H2O2 for 60 min. Spores of each strain exhibited higher resistance than their respective vegetative cells to liquid H2O2. Results indicate that the H2O2 resistance observed in both vegetative cells and spores is strain-specific, as certain B. pumilus strains were two to three times more resistant than a standard Bacillus subtilis dosimetry strain. An example of this trend was observed when the type strain of B. pumilus, ATCC 7061, proved sensitive, whereas several environmental strains exhibited varying degrees of resistance, to H2O2. Repeated isolation of H2O2-resistant strains of B. pumilus in a clean-room is a concern because their persistence might potentially compromise life-detection missions, which have very strict cleanliness and sterility requirements for spacecraft hardware.

Glycosylation Genes Expressed in Seam Cells Determine Complex Surface Properties and Bacterial Adhesion to the Cuticle of Caenorhabditis elegans

“…(Darby et al 2007), which requires a bacterial exopolysaccharide (Tan and Darby 2004). The contaminant was isolated and identified as a strain of the ubiquitous and important Gram-positive species B. pumilus, which has attracted detailed study as a common contaminant of spacecraft, among other reasons (Kempf et al 2005). A sample of one of the well-studied and fully sequenced strains of B. pumilus, SAFR-032, was obtained and found to lack the nematode stickiness observed in CBX120.…”

Bacillus safensis sp. nov., isolated from spacecraft and assembly-facility surfaces

“…During monitoring of the microbial diversity of spacecraftassociated environments over a period of 5 years (1999)(2000)(2001)(2002)(2003)(2004), Bacillus pumilus was found to be the second most dominant species among the aerobic spore-forming bacteria (the predominant species being Bacillus licheniformis; La Duc et al, 2004a). Several of these B. pumilus isolates have exhibited elevated resistance to H 2 O 2 (Venkateswaran et al, 2001;Kempf et al, 2005) and are thus considered as 'problematic' micro-organisms, since H 2 O 2 is recommended for the bioreduction of spacecraft components.In previous studies using MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) Abbreviation: MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences referred to in this communication are given in The bacterial strains examined in this study are shown in Table 1. A total of 13 strains of this novel micro-organism were isolated from spacecraft and assembly-facility surfaces by standard swabbing procedures (Anonymous, 1980;La Duc et al, 2004b) at various times and locations.…”

“…During monitoring of the microbial diversity of spacecraftassociated environments over a period of 5 years (1999)(2000)(2001)(2002)(2003)(2004), Bacillus pumilus was found to be the second most dominant species among the aerobic spore-forming bacteria (the predominant species being Bacillus licheniformis; La Duc et al, 2004a). Several of these B. pumilus isolates have exhibited elevated resistance to H 2 O 2 (Venkateswaran et al, 2001;Kempf et al, 2005) and are thus considered as 'problematic' micro-organisms, since H 2 O 2 is recommended for the bioreduction of spacecraft components.…”

Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation

“…Recently, in several microbial diversity surveys performed over a period of 3 years, 125 aerobic microbial strains were isolated from spacecraft assembly facilities (20,24,25,52,53), and their phylogenetic affiliations were determined (23, 54). Eighty-five percent of these strains were identified as gram-positive bacteria.…”