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- MATERIALS AND METHODS
Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H2O2, desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate).
Bacterial spores (endospores) produced by the genera Bacillus and Clostridium are a dormant form of cells that can persist for a long time in harsh conditions without dividing and display resistance towards chemical disinfectants, UV- and γ-radiation, and extreme pH, temperature, pressure and dryness (1, 2). These dormant spores are capable of monitoring the surrounding environmental conditions, and germinate to physiologically active vegetative cells in favorable situations (3). Several species of spore-forming bacteria are reported as pathogenic to humans and terrestrial and aquatic life, and can survive hospital disinfection procedures (2).
These highly resistant spores are a major concern to public and environmental health (2), as well as to the extraterrestrial environment (Mars, Europa or Enceladus) during search for life via spacecraft (reviewed in 4). To avoid biocontamination, the fabrication, assembly and processing of many industrial products, including medical devices, pharmaceuticals, spacecraft components, and bulk and canned foods are carried out inside the clean room facility. Despite the application of stringent sterilization steps in the clean room to minimize microbial contamination, reports have documented the isolation of spore-forming bacteria from surfaces of spacecraft that have been assembled inside the clean room facility (4–6), and from pharmaceutical manufacturing clean rooms (7). While procedures to analyze the bacterial spore burden on surfaces of polymeric materials are reasonably well developed, methods to examine the presence of embedded/encapsulated bacterial spores within polymeric materials are rather scarce. As a consequence, there is considerable interest in the development of a method for rapid monitoring and visualization of spores embedded/encapsulated in the spacecraft-associated polymeric materials to assess the effectiveness of clean room sterilization procedures (8, 9).
When discussing life forms trapped within a solid matrix, it is pertinent to address the extent of the effect of the ambient environment on such organisms. For the purposes of the present study, microbes enveloped by materials having a high (>15 g/m2 per day) WVTR, and thus able to interact with ambient gases are said to be embedded. Alternatively, microorganisms encased in materials having a much lower WVTR are said to be encapsulated. In this study, PMMA (otherwise known as Lucite or Plexiglas) with a WVTR value approaching zero was selected as a model polymeric material to encapsulate bacterial spores because of the extensive utilization of PMMA polymer not only in various aerospace applications, but also in medical, pharmaceutical and food-processing industries.
The objectives of the present study centered on developing a cultivation-independent, molecular means of efficiently assessing the Bacillus pumilus SAFR-032 spore burden artificially encapsulated in, and released from, PMMA. B. pumilus SAFR-032 is a common contaminant of the Jet Propulsion Laboratory spacecraft assembly facility clean room (6, 10). The spores of B. pumilus SAFR-032 have been reported as extremely resistant to UV radiation, H2O2 treatment, desiccation and starvation (6, 10–13).
One of the major obstacles in gauging the embedded/encapsulated bacterial spore burden in polymeric materials is the lack of reported protocols for releasing spores from their encapsulating solid materials that are amenable to downstream molecular biological detection and quantification techniques. In addition to the loss of substantial amounts of biomatter upon physical and/or chemical extraction from polymeric materials, embedded/encapsulated spores are often accompanied by elevated levels of particulate debris. These particulates shed from the polymeric materials confound many downstream molecular analyses by non-specifically binding fluorescent dyes used in microscopy (e.g. DAPI, acridine orange), and by either totally inhibiting, and/or introducing considerable bias in DNA-based PCR analyses.
FISH microscopy facilitates the simultaneous identification and enumeration of microorganisms without isolation and cultivation (14, 15). In FISH, the fluorescently labeled oligonucleotide probes seep through the permeabilized microbial cells and hybridize to complementary rRNA sequences. This allows for the screening of a particular subset of cell lineages present in a heterogeneous population of microorganisms. Typically, oligonucleotide probes used for FISH analysis are designed to target the 16S rRNA sequences of a particular taxonomic group of microbes, as these sequences are highly conserved compared to other protein-encoding genes (16). One of the major demerits of FISH is photobleaching of the fluorescent oligonucleotide probes. Recently, the coupling of Alexa-fluor dyes, popularly known as Alexa-FISH, has substantially improved the performance and robustness of FISH techniques. In the present study, Alexa-FISH techniques were optimized and used to quantitatively assess the recovery of B. pumilus SAFR-032 spores artificially encapsulated within PMMA. The results obtained from this research endeavor are of immense relevance to current and future planetary protection policy-making and implementation, as well as to medical, pharmaceutical, and food-processing industries for the improved assessment of the embedded/encapsulated bacterial endospores burden.
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- MATERIALS AND METHODS
When the commonly used nucleic acid-specific fluorescent DAPI dye was used to estimate the recovery of once-encapsulated spores released via PolyGone-500 or acetone, a strong autofluorescence originating from simultaneous staining of PMMA- and solvent-associated organic debris precluded accurate enumeration. Additionally, the negative control (PMMA without spores) samples dissolved in PolyGone-500 or acetone also produced fluorescent signals due to uptake of DAPI dye by PMMA- and solvent-associated organic debris. To circumvent this problem, an Alexa-FISH microscopy approach using novel BP15 (oligonucleotide specifically designed for B. pumilus SAFR-032 16S rRNA sequences) and EUB338 (oligonucleotide targeting all eubacterial 16S rRNA sequences) probes was evaluated for the enumeration of once-encapsulated recovered spores. FISH signals were not detected when spores (both non-treated and once-encapsulated) were pre-incubated for 24 hrs at 4°C in a gradient of standard fixative reagents, including paraformaldehyde (1–10%, in 2% increments) and ethanol (20–70%, in 5% increments). The complex and recalcitrant structure of bacterial endospores restricts the accessibility of fluorescent oligonucleotide probes for hybridization with nucleic acids at the spore core. To this end, various combinations of protease (proteinase-K), glycosidases (lysozyme and mutanolysin) and non-ionic surfactants (Tween 80 and Triton X-100) were tested to improve the permeabilization of control and test spores and thus achieve efficient FISH signaling for accurate enumeration. Table 1 summarizes the effects of treatment with each enzyme alone, and in combination with the surfactants, upon enumerating spores (untreated and those recovered from PMMA encapsulation and PolyGone-500 release) via Alexa-FISH microscopy with BP15 probes. The results demonstrated a synergistic action among the three enzymes and Triton X-100 that enabled the effective permeabilization of both positive-control and test spores for subsequent detection and enumeration by Alexa-FISH microscopy.
Table 1. No. spores detected after permeabilization using different enzymes (1.5 U proteinase-K, 7 mg/mL lysozyme and 7 U mutanolysin) and non-ionic surfactants (0.5% Tween 80 and 1% Triton X-100)†
|Enzyme(s)||Control spores||Encapsulated spores|
|and surfactant||(×105 spores/mL)||(×105 spores/g)|
|Proteinase-K||0.260 ± 0.024||0.177 ± 0.026|
|Lysozyme||0.590 ± 0.017||0.281 ± 0.038|
|Mutanolysin||0.200 ± 0.022||0.101 ± 0.013|
|Proteinase-K + lysozyme + mutanolysin||1.821 ± 0.253||0.778 ± 0.028|
|Proteinase-K + lysozyme + mutanolysin + Tween 80||1.841 ± 0.261||0.780 ± 0.027|
|Proteinase-K + lysozyme + mutanolysin + Triton X-100||6.590 ± 1.421||2.951 ± 0.383|
The novel BP15 probe used in concert with the novel permeabilization method referred to above was able to detect a total of 6.761 ± 1.303 × 105 non-treated control spores/mL (Fig. 1a), and 3.051 ± 0.450 × 105 spores/g (Fig. 1b) and 6.890 ± 0.450 × 104 spores/g (Fig. 1c) of PMMA-encapsulated spores extracted with PolyGone-500 or acetone, respectively. The addition of 5% formamide to the hybridization reactions decreased (by approximately 95%) the detectable counts for both non-treated spores and PMMA-encapsulated spores, which indicated that formamide was not required for hybridization reactions with the novel BP15 probe.
Figure 1. Alexa-FISH with Bacillus pumilus-specific BP15 probe at 1000× magnification. (a) Non-treated control spores, (b) PMMA-encapsulated spores released via PolyGone-500, (c) PMMA-encapsulated spores released via acetone.
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When the EUB338 universal probes were used in the absence of formamide the number of detectable untreated spores was 9.231 ± 0.614 × 103 spores/mL, while counts of PMMA-encapsulated spores subsequently released via PolyGone-500 or acetone were 3.212 ± 0.432 × 103 spores/g and 9.540 ± 1.061 ×102 spores/g, respectively. A gradient of formamide (5–50%, in 5% increments) was tested to determine the stringency of and optimize the EUB338 hybridization reactions (Table 2). The detectable abundance of non-treated and encapsulated spores extracted with either PolyGone-500 or acetone increased by approximately 96% following the addition of 30% formamide, yielding counts of 6.262 ± 1.405 × 105 spores/mL (Fig. 2a), 2.150 ± 0.370 × 105 spores/g (Fig. 2b) and 6.603 ± 0.316 × 104 spores/g (Fig. 2c), respectively.
Table 2. Effect of formamide on the stringency of hybridization of the EUB338 probe
|Formamide (%)||Control (×105 spores/mL)||Encapsulated PolyGone-500 (×105 spores/g)||Encapsulated- acetone (×105 spores/g)|
|0||0.092 ± 0.006||0.032 ± 0.004||0.009 ± 0.001|
|5||0.251 ± 0.032||0.108 ± 0.012||0.031 ± 0.003|
|10||0.622 ± 0.067||0.258 ± 0.026||0.085 ± 0.009|
|15||2.814 ± 0.173||0.281 ± 0.038||0.343 ± 0.033|
|20||5.751 ± 0.396||1.097 ± 0.112||0.594 ± 0.058|
|25||5.942 ± 0.638||2.064 ± 0.138||0.640 ± 0.065|
|30||6.262 ± 1.405||2.150 ± 0.370||0.660 ± 0.032|
|35||6.134 ± 0.823||2.085 ± 0.173||0.639 ± 0.064|
|40||4.382 ± 0.671||1.763 ± 0.176||0.533 ± 0.052|
|45||3.441 ± 0.413||1.354 ± 0.135||0.423 ± 0.042|
|50||2.810 ± 0.232||1.011 ± 0.106||0.318 ± 0.030|
Figure 2. Alexa-FISH with universal EUB338 probe using 30% formamide at 1000× magnification. (a) Non-treated control spores, (b) PMMA-encapsulated spores released via PolyGone-500, (c) PMMA-encapsulated spores released via acetone.
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Unlike with DAPI staining, the negative control (PMMA without spores) samples dissolved in PolyGone-500 or acetone did not stain to yield considerable levels of background fluorescent signal with neither the BP15 nor EUB338 probes. Furthermore, there were no fluorescent signals resulting from any of the permeabilized non-treated control spores, encapsulated spores released via PolyGone-500 or with acetone, and negative control (PMMA without spores) samples dissolved in PolyGone-500 or acetone hybridization reactions upon using the antisense non-BP15 and non-EUB338 probes. This confirmed the absence of non-specific binding of the Alexa Fluor® 488 dye.
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- MATERIALS AND METHODS
Standard culture-based plate-counting (on TSA medium) methods are typically used for routine surveys of the presence and abundance of bacterial spores, a physiologically dormant form capable of withstanding extended periods of time in harsh conditions (e.g. extreme temperature, UV and γ radiation) (1, 6, 12). In recent years, however, more attractive methods, such as microfluidic chips (21), terbium dipicolinate fluorescence spectroscopy and microscopy (22), most probable number (MPN)-PCR (23), q-PCR (24), ATP bioluminescence (25) and FISH (26) have been developed for more rapid and sensitive detection and identification of bacterial endospores. The majority of these approaches have focused primarily on enumerating spores by way of a coupling germinant, such as alanine and/or glucose, which initiates and drives the outgrowth and germination into vegetative cells (3). It is well documented that such germination-dependent methodologies can underestimate spore numbers due to the transition to viable but not-cultivable (VBNC) physiological states (27).
Direct epifluorescence microscopy techniques using DAPI dye have proven successful in enumerating spores originating from ice cores (22), pure bacterial cultures (28) and soil (29). In the present study, DAPI staining procedures failed to prove effective due to the constant emission of background autofluorescence upon visualization of encapsulated spores released with either PolyGone-500 or acetone. Such autofluorescence could arise from the simultaneous staining of the composition of PMMA, and/or chemical deposits indigenous to the solvents used. The seemingly inescapable autofluorescence associated with DAPI-based microscopy necessitated the application of more sensitive FISH methodologies.
Preliminary testing with the BP15 and EUB338 probes on paraformaldehyde and ethanol treated control and encapsulated B. pumilus SAFR-032 spores demonstrated that Alexa-FISH tailored methods commonly used for vegetative cells were not effective for bacterial spores. Standard reagents (e.g. paraformaldehyde and ethanol) routinely used to treat vegetative cells for FISH analysis target relatively malleable phospholipid and lipopolysaccharide membranes (30) whereas the structure of bacterial endospores is highly complex and rigid (3, 31). Spores are surrounded by an exosporium composed mostly of proteinaceous compounds, followed by coat and cortex layers primarily composed of peptidoglycan and lipids (3, 32).
Filion et al. (33) have carried out FISH analysis using spores of three Bacillus species (B. megaterium, B. atrophaeus and B. cereus) and developed a universal permeabilization protocol for detection of Bacillus spores in the environment. Application of the universal permeabilization protocol using BP15 and EUB338 probes did not produce efficient fluorescent signals for enumeration of untreated and encapsulated B. pumilus SAFR-032 spores released via PolyGone-500 or acetone. This might be attributed to the complexity of the B. pumilus SAFR-032 spore structure. Generally, the spore structure differs with the species of bacteria and/or with the type of environmental stress exposure (31, 32). B. pumilus SAFR-032 is found to survive the standard decontamination methods of the Jet Propulsion Laboratory spacecraft assembly facility clean room (6, 10). It has also been documented that spores of B. pumilus SAFR-032 are 10-fold more resistant to simulated Martian UV radiation than B. subtilis spores (34). This finding prompted the design and development of a novel permeabilization protocol for B. pumilus SAFR-032 spores.
The key to the novel permeabilization protocol for B. pumilus SAFR-032 described in the present study was the mixing and matching of various chemicals (EDTA, DTT, urea and SDS) and enzymes (protease and glycosidases) reported previously (33, 35), and inclusion of additional Triton X-100 treatment steps. Triton X-100 is one of the most widely used non-ionic surfactants for lysing or permeabilizing prokaryotic and eukaryotic cell membranes (36, 37). The efficacy of proteinase-K, lysozyme, mutanolysin and Triton X-100 were synergistic once thoroughly optimized with the BP15 probe, resulting in the detection of approximately 72% more spores than any protocol based on the combination of three enzymes only.
Neither of the Alexa-FISH probes was capable of detecting the true, 100% spore density (approximately 106 spores/g) initially encapsulated in the PMMA. This might be attributed to the disintegration of spore-borne genetic materials during the chemically abrasive encapsulation procedures, and/or the deleterious action of the organic solvents (PolyGone-500 or acetone). The novel BP15 and universal EUB338 Alexa-FISH probes used in this study did not yield detectable autofluorescence upon epifluorescent microscopic enumeration of encapsulated spores released with either PolyGone-500 or acetone. However, the signals emanating from hybridization events between Alexa-FISH probes and encapsulated spores varied dramatically between the two types of solvent tested to dissolve the model polymeric material (PMMA). When PolyGone-500 was used to dissolve the spore-laden PMMA solid, the percent recovery of detectable spores was 31% and 21% with BP15 and EUB338 probes, respectively. By comparison, these detectable recovery rates were only 6.9% with the BP15 probe and 6.6% with the EUB338 probe upon degrading the spore-laden PMMA solid with acetone. The exact mechanism causing this sharp decrease in Alexa-FISH signals in the presence of acetone was not examined. However, it is not unreasonable to hypothesize that acetone might impose a more destructive impact on the spore.
In conclusion, the results of the present study demonstrate that Alexa-FISH microscopy can be considered an effective molecular diagnostic tool for the direct enumeration of encapsulated B. pumilus SAFR-032 spores released from PMMA polymer, which is commonly used in various aerospace applications, and in medical, pharmaceutical and food-processing industries. The ability to detect and enumerate the presence of embedded/encapsulated bacterial spores directly by a FISH-based approach will lead to effective strategies for monitoring and mitigating the spore-specific bioburden in a given environment. Bacterial spores originating from an industrial clean room environment, including spacecraft assembly and encapsulation facilities, are often referred to as the hardiest of all forms of microbial life. Therefore, future research efforts should be focused on determining the sensitivity and versatility of the Alexa-FISH method with spores of different bacterial species isolated from clean room facilities.