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- MATERIALS AND METHODS
Bacillus cereus is a common environmental bacteria that can be found in soil as well as a common contaminant of milk (Griffiths 1992; Eneroth et al. 2001; Christiansson 2003). This endemic organism has been recognized as an agent of food spoilage and poisoning (Granum and Lund 1997). When the environmental conditions become unfavorable for the development of B. cereus vegetative form, those cells have the ability to enter in dormancy state, called spores (Setlow 1994). Spores can be produced in high numbers and survive for years, even decades (Driks 2002). The presence of B. cereus spores is a serious problem in the food industry, as spores are heat-resistant and very hydrophobic, and they can adhere to equipment surfaces (Koshikawa et al. 1989; Faille et al. 2002). Their remarkable resistance allows them to survive food processing and conservation methods. With the process of germination and outgrow, the bacterial spores return to the metabolically active vegetative state, which involves a rapid sequence of events that lead to the breakdown of the spores' structure and the simultaneous loss of the spores' resistance properties (Moir 2006).
The fast and specific detection of B. cereus spores in the environment is a challenging task. Culture on Petri plates is often used, but this technique is time-consuming. Polymerase chain reaction (PCR) assay can also be used, but may be affected by inhibition because of food components (Quarto and Chironna 2005). Thus, enrichment step and sample preparation is needed before PCR that increases the time to response (Fukushima et al. 2007; Park et al. 2007; Perry et al. 2007). Fluorescent in situ hybridization (FISH) is used to identify and characterize bacterial cells by using ribosomal RNA (rRNA) as hybridization targets for probes. FISH allows single cell detection of a specific taxon and is suitable for complex environments (Amann et al. 1990). Compared with PCR, this technique has the advantage of providing the visualization of the cells in their environment and evaluating their proportion. The technique is based upon a two-step sequence: cell permeabilization with proper fixative followed by hybridization under appropriate conditions with oligonucleotide probes. The FISH approach faces several constraints. Along with the cellular concentration of rRNA (probe target), the permeabilization step is another critical limitation (Oda et al. 2000; Wagner et al. 2003). In fact, FISH probes need to penetrate the cell to bind to their target.
A study published by the Kornberg group in the 1960s indicated that the rRNA content of dormant spores of Bacillus, in terms of relative amount and physicochemical property, are at the same level as log phase cells (Chambon et al. 1968). In order to be detectable by FISH, Bacillus spores would have to be (1) strongly permeabilized or (2) induced to germinate/growth (i.e. return to permeable vegetative phase) and permeabilized. Indeed, germination and outgrowth is facilitated by enzymes and energy reserves already present in Bacillus spores. Because the structure of the spore itself is the barrier, there is an advantage in using natural biophysical means like germination as a starting point for a permeabilization treatment. In fact, the germination of spores is accompanied by a loss of resistance properties. The inner membrane of the spore is supposed to be the major obstacle for the diffusion of small molecules into the spore's core (Setlow 2003). The permeability of this membrane increases by over 100-fold early in germination (Swerdlow et al. 1981). Furthermore and with respect to the first stages of spore germination, the large depot of dipicolinic acid (DPA) is excreted from the spore's core while water is absorbed to reduce dehydration of the spore (Setlow 2003; Moir 2006). The cortex hydrolysis that occurs later in germination but before outgrowth is needed for complete core rehydration. Eventually the spore's coat is degraded by an unresolved mechanism.
A report has demonstrated the feasibility of direct FISH on pure cultured spores (Fischer et al. 1995). The time required to obtain a FISH signal by using the protocol is 3 days and needs an extensive list of successive chemicals such as 4% parformaldehyde, 50% ethanol and lysozyme. The assay was performed on a microscope slide. A recent study showed that it was possible to obtain a FISH signal from germinating pure cultured spores after 6–8 h of treatment and handling using formaldehyde, lysozymes and different steps of drying and fixation of the samples on microscope slides. Additionally, a germination step of at least 2 h was necessary prior to fixation and permeabilization of the cells (Regamey et al. 2000). Therefore, there is a need to reduce the time and improve the conditions required to detect B. cereus spores by using FISH. Spore germination and growth induction followed by a nondestructive permeabilization treatment (like ethanol or aldehyde) is a promising strategy in terms of execution time, cost efficiency and simplicity for environmental samples.
Germination of Bacillus spores is induced by several nutrients called germinants (Paidhungat and Setlow 2002). These germinants are most often single amino acids, sugars and purine nucleosides (Hornstra et al. 2005). These components are recognized by spores as signals for appropriate germination conditions, but the mechanisms have not yet been elucidated (Paidhungat et al. 2001). Different combinations of germinants were used with B. cereus and among them, the addition of L-alanine and inosine was described as one of the most optimal mix for germination (Clements and Moir 1998; Barlass et al. 2002; Hornstra et al. 2005). Other combinations, such as the addition of calcium chloride (CaCl2) with L-alanine, produce synergistic effects (Kamat et al. 1985). Another way to improve germination is by adding DPA in the culture media (Ragkousi et al. 2003; Setlow 2003). This supplement is a natural constituent of the spore cortex, which is released when the spores germinate and then causes other spores in the environment to germinate. Furthermore, the inoculum concentration and heat activation at 70C is known to induce the germination (Keynan and Evenchik 1969; Caipo et al. 2002; Setlow 2003).
The main objective of this study was to evaluate the germination conditions leading to a rapid and detectable FISH signal when starting from B. cereus spores. The current study aimed to answer two questions: (1) By using the most favorable germinating and growth conditions found, could B. cereus cells be specifically detected by FISH within a milk sample? (2) How quickly can B. cereus spores be detected by FISH? Various germination conditions have been tested, and the proposed strategy leads to specific detection of B. cereus spores by FISH in less than 2 h. The procedure was tested on spiked milk samples with spores and allows for the specific detection of 103 colony-forming units (cfu) per milliliter in 2 h.
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- MATERIALS AND METHODS
In the present report, our optimized protocol leads to a FISH signal from B. cereus spores in less than 2 h including treatment and handling. The condition retained was: incubation at 37C for 1 h in TSB + L-alanine 100 mM and inosine 10 mM, 15 min permeabilization in 4% paraformaldehyde followed by 15 min of hybridization. A maximum of 30 min of manipulation between these steps is necessary: washings, transfers, addition of probes, preparations for flow cytometry or fluorescence microscopy.
This study shows that activation/germination and outgrowth of B. cereus spores facilitates fluorescence in situ hybridization protocols by reducing time to signal when compared with the direct spores' permeabilization. The FISH signal intensity depends on the number and accessibility of probe targets on the rRNA structure. Therefore, an enriched medium such as TSB reached this goal as vegetative cells reach a maximum of rRNA during the logarithmic growth phase. However, one study showed that an rRNA concentration for Bacillus megaterium spores are at the same level as log phase cells (Chambon et al. 1968). Considering this information, permeabilization of the spores and accessibility of the targets is probably more an issue here than the number of targets (rRNA) for FISH on Bacillus sp. spores. Previous authors have shown that it is possible to do FISH directly on spores without germination and outgrowth (Fischer et al. 1995). However, the proposed protocol needs a total of 3 days of incubation and the use of successive combinations of chemicals that can limit the application on environmental samples. Regamey et al. showed that 6–8 h of treatment and handling are required to obtain a FISH signal by using spore germination (Regamey et al. 2000). In the presence of germinants like L-alanine and inosine, those small molecules penetrate the outer layers, coat and cortex of the spore to bind to specific receptors located on the inner membrane (Setlow 2003; Moir 2006). This initiates the germination of spores that is an irreversible phenomenon. The presence of those germinant in the culture media improves the time to detection performed by Regamey et al.
A study shows that a mixture of L-alanine and inosine efficiently stimulates the germination of adhered spores, resulting in 3.2 decimal logarithms of germination (Hornstra et al. 2007). The result obtained in this study is in accordance with the observations of Hornstra et al. that the mixture of L-alanine and inosine is the strongest combination of germinants observed for B. cereus. We experimented minimal media composed of buffer (Tris, PBS) plus L-alanine and inosine (Table 1). After 2 h of incubation in those buffer-based media, no positive results were observed with FISH in flow cytometry and fluorescence microscopy. This observation demonstrated that germination/outgrowth of B. cereus spore is necessary to achieve a FISH signal by using the conditions of this study. This is not surprising as complete germination is necessary to remove the different layers that shield the spore. Those buffer-based media might be suitable for germination induction, but not for supporting growth. In fact, the germination induction of Bacillus spores is a rapid phenomenon, and amino acid synthesis occurs after 2–5 min after the spores are put in a buffer containing only the germinating agents (Garrick-Silversmith and Torriani 1973; Welkos et al. 2004). However, as the objective was to obtain a rapid FISH signal, those media were not suitable for our purpose.
The permeabilization treatments tested were standard in FISH studies. The envelope of B. cereus cells differs greatly depending on the growth stage of the cell. The challenge of the permeabilization procedure is to enable the FISH probe to enter the cell in order to reach its target without disrupting the cell's morphology or lose its cytoplasmic content. There are no universal cell permeabilization protocols for FISH. This study shows that 50% ethanol needs more incubation time (minimum 8 h) to be fully efficient compared with 1% formaldehyde and 4% paraformaldehyde. The latter two are similar in terms of efficiency (15 min of treatment is sufficient) and mechanisms of action. In fact, ethanol is a precipitating agent, and formaldehyde and paraformaldehyde are cross-linking agents (Moter and Göbel 2000). Thus, a cross-linking agent is more suitable with vegetative B. cereus cells for fast and efficient permeabilization. This result is crucial for a rapid in situ detection protocol.
A reduction of the time of hybridization was also evaluated. The results showed that a hybridization of 1 h versus 2 h gave the same FISH result. Reducing the hybridization to 30 min and 15 min slightly decreases the FISH signal in flow cytometry. This result was confirmed by fluorescence microscopy. However, a good and acceptable detection was reached after 15 min of hybridization by using both flow cytometry and fluorescence microscopy. It is difficult to draw a line when a signal is considered too weak, but after 15 min of hybridization by using the optimized conditions, the FISH signal is clearly detectable.
The B. cereus-specific probe pB394 in this study was originally validated in a microchip system of detection (Liu et al. 2001). This probe binds a variable region in the 16S rRNA. When used with the optimized conditions determined in this study, the pB394 shows comparable results for the detection with flow cytometry in terms of the percentage of positive cells compared with the universal probe for eubacteria EUB338, but presents a reduction in the fluorescence intensity. When the results of pB394 are observed by fluorescence microscopy, the percentage of positive cells and the fluorescence intensity are weaker when compared with those of the universal probe EUB338. The difference in the percentage of positive cells obtained between flow cytometry (pB394 similar to EUB338) and the fluorescence microscopy (pB394 result in less positive cells number than EUB338) can be explained by the fact that flow cytometry is equipped with a photomultiplier that renders this instrument more sensitive to low signal compared with fluorescence microscopy. Because the fluorescence intensity is weaker with pB394 than with EUB338, the fluorescence microscopy does not allow the observation of positive cells with the lowest fluorescence intensity. This difference in the intensity of fluorescence between the universal and specific probes can be explained, in part, by the accessibility of the probe to the target. In a previous study conducted by Fuchs et al., the accessibility of FISH probes that targeted the 16S rRNA was performed in flow cytometry by using the mean fluorescence intensity (Fuchs et al. 1998). The three-dimensional structure of the ribosome compromises the access of some targets for the probes, thus reducing the signal. The pB394 probe targeted the position 162–185 of the 16S rRNA according to Escherichia coli numbering (Brosius et al. 1981). At this position, the study of Fuchs et al. has shown that this target gave low–moderate fluorescence signal (category IV of VI) whereas a category I gave the strongest signal. In comparison, EUB338 probe targeted a position overlapping categories II and III.
The germination of spores results in an immediate loss of the spores' resistance that makes the permeabilization more efficient. Our findings open the possibility of including sporulating bacteria in overall taxonomic distribution assessment studies and in detecting specific agents with the FISH approach. This study shows that a brief exposure of Bacillus spores to TSB plus L-alanine and inosine makes them more permeable to be fixed with 4% paraformaldehyde and enables probe binding for standard FISH protocols. With this new rapid germination method and optimized FISH protocol, B. cereus spores present in a milk sample can be detected in 2 h.