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At least 91 records · Page 5

Bacillus cereus strain MCN as a debriding agent

Biologically active means are effective for rapidly removing scar tissue caused by burns or corrosive agents. Specially selected strain of bacteria applied to injury site releases enzymes which are active against eschar. These bacteria tend to locate between eschar and unburned tissue, thus providing optimal cell surface area arrangement for enzyme dispersal. Procedure may prove especially useful in treatment of disaster casualties under relatively primitive conditions.

Dalton, H. P.↗

Evaluation of Various Cleaning Methods to Remove Bacillus Spores from Spacecraft Hardware Materials

A detailed study was made of the biological cleaning effectiveness, defined in terms of the ability to remove bacterial spores, of a number of methods used to clean hardware surfaces. Aluminum (Al 6061) and titanium (Ti 6Al-4V) were chosen for the study as they were deemed the two materials most likely to be used in spacecraft extraterrestrial sampler construction. None of the cleaning protocols tested completely removed viable spores from the surface of the aluminum. In contrast, titanium was capable of being cleaned to sterility by two methods, the JPL standard and the commercial SAMS cleaning process. Further investigation showed that the passivation step employed in the JPL standard method is an effective surface sterilant on both metals but not compatible with aluminum. It is recommended that titanium (Ti 6Al-4V) be considered superior to aluminum (Al 6061) for use in spacecraft sampling hardware, both for its potential to be cleaned to sterilization and for its ability to withstand the most effective cleaning protocols.

planetary protection↗

Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach

The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

Non-NASA Center↗

Congruence of Clusters Defined By Whole Genome Sequencing and MALDI-TOF for Bacteria Isolated From Cleanrooms

Introduction: Oligotrophic conditions can render cleanrooms inhospitable to microbes. Despite these constraints, fungi and bacteria are frequently isolated from surfaces in astromaterials cleanrooms at the Johnson Space Center. Bacillus species are of particular concern because endospores belonging to this genus are resilient and can affect astromaterials. Current monitoring programs rely on 16S rRNA sequencing and the VITEK2 Compact system. These methods have limited power to resolve Bacillus species. Matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF MS), provides a rapid, low cost, method of identifying bacterial isolates and has a higher resolution than 16S rRNA sequencing, particularly for Bacillus species; however, few studies have compared this method to the industry gold standard, whole genome sequencing (WGS). Methods: Based on 16S rRNA classification, we selected 14 isolates for analysis with MALDI-TOF and WGS. Mass spectra were generated with MALDI-TOF MS and processed with custom scripts to identify clusters of closely related isolates and calculate a matrix of pairwise cosine similarity scores. Hybrid Illumina and Nanopore sequencing were used to generate draft genomes. Pairwise similarity scores were calculated from these genomes based on the average amino acid identity (AAI) predicted from single copy core genes. Congruence of clustering between these methods, was assessed by calculating adjusted Rand and Wallace coefficients. Results: Clusters of species generated from MALDI-TOF MS showed good agreement of phylotypes generated with WGS. Pairs of strains that were > 94% similar to each other in terms of predicted amino acid sequences consistently showed cosine similarities of mass spectra > 0.65 and, of the 9 clusters identified with WGS, 8 were identical with MALDI-TOF. This corresponds to an adjusted Rand index of 0.95 and a 95% confidence interval of 0.80 – 1.00 for adjusted Wallace coefficients. The only discordance was for a pair of isolates that were classified as Paenibacillus species. This pair showed relatively high similarity (0.84) in terms of MALDI-TOF MS but only 85% similarity in terms of AAI. Conclusion: This study shows that MALDI-TOF and WGS exhibit a similar ability to delineate Bacillus species isolated from cleanrooms and taxonomic units described by these two methods are consistent with one another. Since MALDI-TOF MS is low in cost and high in throughput, this approach appears to be an ideal option for routine microbial monitoring and identifying Bacillus species.

Farnaz Mazhari↗

Survival of spacecraft-associated microorganisms under simulated Martian UV irradiation

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA_B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m_2 of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA_B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the anitation capabilities of UV irradiation are determined.

Venkateswaran, Kasthuri↗

Services provided in support of the planetary quarantine requirements of the National Aeronautics and Space Administration

The project to evaluate thermal sterilization for unmanned landers is reported. A temperature controlled oven with a nitrogen gas supply containing a known concentration of water is discussed. The studies show that bacillus lentus, bacillus brevis, bacillus coagulans, atypical bacillus spp., and actinomycete are isolated heat survivors. The thermal resistance is given for naturally occurring airborne bacterial spores collected on exposed teflon ribbons.

Favero, M. S.↗

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

The experiments conducted to determine the heat resistance of Bacillus megaterium ATCC 6458 at 90 and 100 C were completed. Estimates from replicate experiments at eight percent relative humidities (less than 0.001 to 100% RH) for each temperature were computed. A Bacillus cereus strain with high heat resistance was cultured and the resistance determined in phosphate buffer (D sub 121.1 = 2.16 min and z = 8.7 C). The profile of the dry heat resistance of B. megaterium is summarized and the most resistant condition to the three spores (Bacillus subtilis var. niger, ATCC 29669, and Bacillus stearothermophilus, strain 1518) is compared.

Reyes, A. L.↗

Response of selected microorganisms to experimental planetary environments

Anaerobic and aerobic sporeformers and non-sporeformers were cultivated anaerobically in nutrient media under various pressures (up to 1800 psi) of pure H2, CH4, NH3, and H2S. Viability assays were performed periodically to determine growth, survival, or spore survival. Hydrogen up to 1800 psi demonstrated little or no suppression of growth with the possible exception of Bacillus coagulans at 1800 psi. The obligate anaerobes grew very well. Under CH4 the obligate anaerobes again exhibited the most prolific growth, whereas the facultative anaerobes grew well except under higher pressures. Ammonia at low pressure was extremely toxic to all test organisms. At 100 psi all populations were killed within 24 hours except Staphylococcus aureus which survived for 72 hours and the Bacillus spp. which produced a surviving population of approximately 10,000 spores/ml. All populations in H2S were killed within 24 to 48 hours except Proteus mirabilis which decreased to 100 cells/ml and the Bacillus spp. Spore survival studies of two months duration demonstrated that B. coagulans and B. pumilus survived under all experimental conditions. Clostridium novyi type B and C. sporogenes were killed rapidly in NH3 and H2S and demonstrated no sporulation.

Foster, T. L.↗

Isolation of the Paenibacillus phoenicis, a Spore-Forming Bacterium

A microorganism was isolated from the surfaces of the cleanroom facility in which the Phoenix lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Paenibacillus and represents a novel species. Bacillus spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Spores of Bacillus species are of particular concern to planetary protection due to the extreme resistance of some members of the genus to space environmental conditions such as UV and gamma radiation, vacuum, oxidation, and temperature fluctuation. These resistive spore phenotypes have enhanced potential for transfer, and subsequent proliferation, of terrestrial microbes on another solar body. Due to decreased nutrient conditions within spacecraft assembly facility clean rooms, the vegetative cells of Bacillus species and other spore-forming Paenibacillus species are induced to sporulate, thereby enhancing their survivability of bioreduction

Benardini, James N.↗

Heat Melt Compaction as an Effective Treatment for Eliminating Microorganisms from Solid Waste

One of the technologies being tested at Ames Research Center as part of the logistics and repurposing project is heat melt compaction (HMC) of solid waste to reduce volume, remove water and render a biologically stable and safe product. Studies at Kennedy Space Center have focused on the efficacy of the heat melt compaction process for killing microorganisms in waste and specific compacter operation protocols, i.e., time and temperature, required to achieve a sterile, stable product. The work reported here includes a controlled study to examine the survival and potential re-growth of specific microorganisms over a 6-month period of storage after heating and compaction. Before heating and compaction, ersatz solid wastes were inoculated with Bacillus amyloliquefaciens and Rhodotorula mucilaginosa, previously isolated from recovered space shuttle mission food and packaging waste. Compacted HMC tiles were sampled for microbiological analysis at time points between 0 and 180 days of storage in a controlled environment chamber. In addition, biological indicator strips containing spores of Bacillus atrophaeus and Ceo bacillus stearothermophilus were imbedded in trash to assess the efficacy of the HMC process to achieve sterilization. Analysis of several tiles compacted at 180 C for times of 40 minutes to over 2 hours detected organisms in all tile samples with the exception of one exposed to 180 C for approximately 2 hours. Neither of the inoculated organisms was recovered, and the biological indicator strips were negative for growth in all tiles indicating at least local sterilization of tile areas. The findings suggest that minimum time/temperature combination is required for complete sterilization. Microbial analysis of tiles processed at lower temperatures from 130 C-150 C at varying times will be discussed, as well as analysis of the bacteria and fungi present on the compactor hardware as a result of exposure to the waste and the surrounding environment. The two organisms inoculated into the waste were among those isolated and identified from the HMC surfaces indicating the possibility of cross contamination.

Hummerick, Mary P.↗