SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

ABSTRACT

Methods for the specific detection of Bacillus spores are needed in many situations such as the recognition of food poisoning. This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples.

B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus-specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra heat-treated milk samples. FISH-labeled cells were detected by using flow cytometry, and the results were confirmed by fluorescence microscopy. The optimal condition allows the detection of B. cereus spores in less than 2 h. Overall, a ninefold reduction in total time for detection was achieved when comparing with previous works. Therefore, the permeabilization and hybridization optimizations mentioned in this study are major improvements for the detection time of B. cereus spores.

PRACTICAL APPLICATIONS

By using the optimized conditions of germination/outgrowth, permeabilization and hybridization, the detection of 103 cfu/mL of Bacillus cereus spores using fluorescent in situ hybridization is possible within 2 h in milk sample.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Bacterial Strains

B. cereus ATCC 14579 and B. cereus HER 1414 strain (Ahmed et al. 1995), encountered in food poisoning, were used. B. cereus HER 1414 was provided by the Felix d'Hérelle Reference Center for Bacterial Viruses.

Production and Purification of Spores

One aliquot (one loop) of each liquid culture, previously incubated in tryptic soy broth (TSB) overnight at 37C, was used to inoculate a solid sporulation medium (nutrient agar [Difco, Sparks, MD] with 0.5% yeast extract, 7 × 10−4 M CaCl2, 10−3 M MgCl2•6 H2O and 5 × 10−5 M MnCl2•4 H2O, final pH of 6.8), and it was incubated for a minimum 72 h at 37C (Holt and Krieg 1994). Colonies were harvested with a sterile cotton swab and transferred in 1 mL of 0.22 µm filtered phosphate buffer saline (PBS). After two washing steps by using PBS, the samples were gently deposited on top of a NaBr density gradient and centrifuged at 2,400 × g at 25C for 45 min as previously described (Laflamme et al. 2004).

Spore Count and Purity Evaluation

The purity of the spore preparations was evaluated by phase contrast microscopy: a wet mount was used to estimate the degree of purity of the spores isolated from the NaBr density gradient. In addition, to confirm the absence of debris and to quantify the spores, a fluorescent DNA marker, the 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), was used to stain the spores (Laflamme et al. 2005). According to the count obtained, spores were resuspended at a concentration of 109 spores/mL. By using this technique, the spore preparations were at least 95% pure, bright phase spores without debris or vegetative cells.

Growth Media and Experimental Design

A total of 20 different growth media were tested in this study (Table 1). An optimal concentration of L-alanine, inosine, CaCl2 and DPA for B. cereus germination was chosen following a literature review (Raso et al. 1998; Paidhungat et al. 2001; Ireland and Hanna 2002; Hornstra et al. 2006). Purified B. cereus spores of each strain were incubated at 37C under agitation at 150 rpm and at a concentration of 5 × 106 spores/mL in each growth media listed in Table 1 (n = 3). Samples (1 mL) were taken after 0, 30, 60 and 120 min of incubation. The collected samples were pelleted by centrifugation at 12,000 × g for 2 min and resuspended in 50% ethanol-PBS for 18 h in order to permeabilize the cells and obtain a maximum of positive cells for FISH. This method of permeabilization is frequently used in literature for gram-positive bacteria like Bacillus cells (Moter and Göbel 2000). The best growth media for each strain were selected according to the best FISH signal in those media at 37C after 120 min. The best FISH signal is defined as one that gave the highest percentage of positive cells and fluorescence intensity. Moreover, the FISH result must be visible under fluorescence microscopy. The selected media were used for the rest of the experiment to test the influence of three germination factors, permeabilization and hybridization conditions. Finally, the best conditions found were validated on a milk sample spiked with B. cereus HER 1414. Figure 1 summarizes the experimental design.

Table 1.  GROWTH MEDIA TESTED
MediaComposition
  1. CaCl2, calcium chloride; DPA, dipicolinic acid; PBS, phosphate buffer saline; TSB, tryptic soy broth.

 1Tris–HCl 10 mM (pH 8)
 2Tris–HCl 10 mM (pH 8) + L-alanine 100 mM
 3Tris–HCl 10 mM (pH 8) + inosine 10 mM
 4Tris–HCl 10 mM (pH 8) + L-alanine 100 mM + inosine 10 mM
 5Tris–HCl 10 mM (pH 8) + L-alanine 100 mM + CaCl2 10 mM
 6PBS
 7PBS + L-alanine 100 mM
 8PBS + inosine 10 mM
 9PBS + L-alanine 100 mM + inosine 10 mM
10PBS + L-alanine 100 mM + CaCl2 10 mM
11TSB
12TSB + L-alanine 100 mM
13TSB + inosine 10 mM
14TSB + L-alanine 100 mM + inosine 10 mM
15TSB + CaCl2 10 mM
16TSB + L-alanine 100 mM + CaCl2 10 mM
17TSB + DPA 60 mM
18TSB + L-alanine 100 mM + DPA 60 mM
19TSB + L-alanine 100 mM + CaCl2 10 mM + DPA 60 mM
20TSB + CaCl2 10 mM + DPA 60 mM
image

Figure 1. EXPERIMENTAL DESIGN OF THE STUDY

Download figure to PowerPoint

Germination Factors

Three factors known to increase the germination efficiency were tested: (1) incubation temperature (30C versus 37C); (2) inoculum concentration (from 5 × 106 to 5 × 108 spores/mL); and (3) a heat shock treatment. The spores (5 × 106 spores/mL) were inoculated in the optimal growth media and incubated for 30 min at 70C, under agitation in a hybridization incubator (Robbins Scientific, model 310 Robbins Scientific Corp., Sunnyvale, CA) prior to incubation at 37C under agitation at 150 rpm. To test the germination factors, 1 mL samples were taken after 0, 30, 60 and 120 min. The cells were pelleted by centrifugation at 12,000 × g for 2 min and resuspended in 50% ethanol-PBS for 18 h before the FISH experiment.

Permeabilization

By using the best growth medium and germination conditions determined in steps 1 and 2, three standard fixation/permeabilization treatments for FISH were tested: 1% formaldehyde, 4% paraformaldehyde and 50% ethanol-PBS (Amann et al. 1990; Moter and Göbel 2000). Fixation time intervals of 15, 30 min, 1, 2, 4, 8 and 18 h were tested for each of them.

FISH

Universal FISH probe designed for eubacteria 16S rRNA, EUB338 (5′-GCT GCC TCC CGT AGG AGT-3′) and the antisense sequence notEUB338 (Amann et al. 1990), both labeled with FITC, were used to test steps 1–4 of the experimental design (Fig. 1) (Invitrogen Life Technologies, Frederick, MD). These probes were chosen to set up the rapid conditions as their target is well characterized and their use in FISH protocols was thoroughly described and validated (Amann et al. 1990; Fischer et al. 1995; Fuchs et al. 1998; Wagner et al. 2003). In addition, a B. cereus-specific probe, pB394 (5′-ATG CGG TTC AAA ATG TTA TCC GG-3′) and its corresponding antisensenotpB394 were used to validate the best conditions obtained at steps 5 and 6 of the experimental design (Fig. 1). The probes were labeled with Alexa 488 (Integrated DNA Technology, Coralville, IA). The pB394 probe targeted a unique sequence present in the 16S rDNA of B. cereus (Liu et al. 2001). Prior to each FISH reaction, the hybridization buffer was preheated at 46C. The reaction tubes contained 95 µL of hybridization buffer (0.9 M, NaCl, 0.01% SDS, 20 mM Tris–HCl pH 7.6 with 6.5 ng/mL of probe) and 5 µL of germinating spore suspension. Each sample was hybridized for 2 h at 46C. FISH reactions were performed by using a thermal cycler (DNA engine, DYAD, MJ Research, BIO-RAD, Waltham, MA) for temperature accuracy.

Flow Cytometry

Flow cytometry analyses were performed by using an EPICS XL-MCL flow cytometer (Beckman-Coulter, Miami, FL) with the acquisition software EXPO 32 (version 1.1c). The flow cytometer was equipped with an air-cooled 15 mw argon laser as a light source. Fluorescence signals were collected through a 525 nm bandpass filter (FL1). The acquisition of fluorescence data were gated by forward angle light scatter and side scatter while the data rate was set at less than 500 events/s. Samples were allowed to run approximately 1 min before the acquisition of a minimum of 5,000 events. The percentage of positive cells as well as the mean fluorescence intensity were calculated and normalized between samples by using a standard method by subtracting the corresponding antisense probe results. The flow cytometer performance and stability over time was periodically controlled by using flow set beads (Beckman-Coulter) in order to evaluate the possible fluorescence drift because of the instrument. No variation was observed during the period of experimentations (data not shown). Flow cytometry was the instrument used to determine the percentage of FISH positive cells and the mean fluorescence that is an indication of the FISH signal intensity.

Fluorescence Microscopy

The fluorescence microscopy analyses were performed by using a Nikon Eclipse 6600 with three sets of spectral filters (UV-2A, FITC and B-2A). The microscope was connected to a system of picture capture (Retiga 1300, QImaging). The software Simple PCI version 5.1 (Campix, Inc., Cranberry Town, PA) was used for picture capture and image analysis. Fluorescence microscopy was used at each step of the experimental design in order to confirm the signal obtained with flow cytometry and for the environmental sample analysis.

Specific Detection of B. cereus HER 1414 in a Milk Sample

The number of colony-forming units (cfu) of freshly isolated B. cereus HER 1414 spores was determined by plate counts using tryptic soy agar. Various quantities of spores (106–101 cfu) were inoculated directly in 1 mL of 1% ultra heat-treated milk. In order to remove the major part of fat present in milk, 60 µL of 25% sodium citrate (Sigma-Aldrich, Oakville, ON, Canada) was put into the 1 mL of spiked milk and shaken for 5 min at 200 rpm (Lucore et al. 2000). The tubes were centrifuged at 15,000 × g for 5 min. The parts of the cream that adhered to the wall of the tube were removed and withdrawn by using a sterile cotton swab. The tubes were then emptied by inversion while the pellets remained at the bottom. The residual cream was scrapped and removed from the tube, and the pellet was resuspended by using 1 mL of TSB +  L-alanine + inosine (medium 14, Table 1). The tubes were incubated for 1 h at 37C under agitation at 150 rpm. The tubes were centrifuged at 12,000 rpm for 2 min, and the pellets were resuspended in 1 mL of 4% paraformaldehyde for 15 min. Then, they were centrifuged at 12,000 rpm for 2 min, and the FISH protocol was performed for 15 min by using pB394 and notpB394 probes.

Statistical Analyses

The statistical analyses were completed by using the Statistical Analytical Software. The results were expressed as mean values ± standard error of the mean. The data were analyzed by using a three-way ANOVA. All reported P values were declared significant at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Step 1: Selection of the Best Growth Media

Purified spores of B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media (Table 1). Samples of cells were taken after 0, 30, 60 and 120 min, fixed and FISH was completed for each sample by using an EUB338 probe and a notEUB338 probe (n = 3). Each sample was analyzed by using flow cytometry and fluorescence microscopy. For B. cereus ATCC 14579 and HER 1414, the growth media 1–10 and 19 yielded negative results after 120 min of incubation. In order to simplify Fig. 2, negative results were omitted. In addition, the results obtained at time intervals of 0 and 30 min were omitted in Fig. 2 as the results were consistently too low to be detected. At 60 min, only medium 14 provided a statistical difference with that obtained from medium 12 (P = 0.048). At 120 min, media 11, 12, 14, 15 and 16 represented the best growth media for the percentage of positive results (Fig. 2A). The mean fluorescence is an indication of the averaged intensity of fluorescence emitted by the entire cell population analyzed. At 60 min, there was no significant difference between each medium. However, at 120 min, media 14, 15 and 18 led to similar results and were better than the other media (Fig. 2B).

image

Figure 2. FLOW CYTOMETRY RESULT FOR BACILLUS CEREUS ATCC 14579 (A, B) AND HER 1414 (C, D) FOR FLUORESCENT IN SITU HYBRIDIZATION (FISH) REACTION BY USING AN EUB338 PROBE IN SELECTED MEDIA (M) (A, C) The percentage of positive cells at 60 and 120 min. (B, D) Mean fluorescence intensity obtained at 60 and 120 min.

Download figure to PowerPoint

At 60 min, the media 11, 13, 14, 15 and 16 constituted the best growth media in terms of percentage of positive cells. At 120 min, media 11, 12, 14, 17 and 20 were the five best media (Fig. 2C).

At 60 min, media 11, 14, 15, 16 and 20 gave the best results. At 120 min, the media 11, 12, 14 16 and 17 gave the best result (Fig. 2D). The FISH results were validated by fluorescence microscopy (data not shown). After 60 min of incubation, positive cells can be observed. Control experiments by using the antisense probe notEUB338 were still negative under fluorescence microscopy.

Following the experimental design in Fig. 1, step 1 was performed to select the medium that gave the best results in terms of percentage of positive cells and mean fluorescence intensity in flow cytometry. Moreover, the FISH results had to be validated in fluorescence microscopy. For the percentage of positive cells and mean fluorescence obtained with B. cereus ATCC 14579 (Fig. 2A,B), at 60 and 120 min, the media 14 and 15 were the best according to ANOVA statistics. For HER 1414, by using the same criteria, (Fig. 2C,D), media 11 and 14 were the best. Therefore and in accordance with these results, the best growth medium for the rest of the experiment was medium 14. In fact and based upon the two strains studied, only this medium was significantly equal or better in terms of the percentage of positive cells and mean fluorescence intensity.

Step 2: Influence of Germination Factors

None of the germination factors tested gave a significant increase in the signal obtained after 120 min of incubation (data not shown). Therefore, the further experiments were performed in medium 14 incubated at 37C, by using a starting inoculum of 5 × 10spores/mL and without heat shock treatment as used in step 1.

Step 3: Optimal and Minimal Time of Permeabilization Treatments

Permeabilization with 50% ethanol, 4% paraformaldehyde and 1% formaldehyde were compared. The two B. cereus strains were incubated in growth medium 14 for 60 and 120 min as positive results can be observed using flow cytometry and fluorescence microscopy at those incubation periods. Figure 3A presents the percentage of positive cells for the different permeabilization treatments after 60 and 120 min of incubation in growth medium 14 obtained for B. cereus ATCC 14579. Treatment with 50% ethanol required longer incubation time to be optimal (percentage of positive cells and mean fluorescence) compared with 4% paraformaldehyde and 1% formaldehyde that had almost reached their maximum after 15 min of incubation. The results obtained for the B. cereus HER 1414 were similar to those obtained for B. cereus ATCC 14579 (data not shown).

image

Figure 3. PERMEABILIZATION TREATMENT AND MINIMAL TIME OF PERMEABILIZATION FROM THE BACILLUS CEREUS ATCC 14579 SPORE The spores were incubated in culture medium 14 for 60 and 120 min before permeabilization with 50% ethanol (EtOH 50%), 4% paraformaldehyde (Para 4%) or 1% formaldehyde (Form 1%) for 15, 30, 60, 120 min, 4, 8 and 18 h. (A) The percentage of positive cells obtained for fluorescent in situ hybridization (FISH) reaction by using EUB338 probe. (B) Mean fluorescence intensity obtained for FISH reaction by using an EUB338 probe.

Download figure to PowerPoint

Using fluorescence microscopy, results obtained after 15 min of incubation in 1% formaldehyde and 4% paraformaldehyde were still the same, but slightly better in terms of the quality of images in fluorescence microscopy for 4% paraformaldehyde (data not shown). A permeabilization treatment of 15 min in 4% paraformaldehyde was chosen for the rest of the experiments.

Step 4: Minimal Hybridization Time

The hybridization time used thus far was 2 h with the universal probe. Figure 4 presents the results for B. cereus ATCC 14579 and B. cereus HER 1414 for the optimization of hybridization time. Comparable results were obtained for the two strains for the percentage of positive cells (Fig. 4A) and fluorescence intensity (Fig. 4B). Reducing hybridization to 15 min and 30 min gave fewer positive cells and less intensity of fluorescence than with 1 and 2 h of hybridization. Equivalent results were obtained for a hybridization of 1 or 2 h. A FISH signal was obtained in fluorescence microscopy with all the incubation periods (data not shown). Even if the results were slightly better with 1 h of incubation, hybridization time of 15 min was kept for the rest of the experiment to reduce the overall time of the protocol to achieve a fast detection.

image

Figure 4. INFLUENCE OF HYBRIDIZATION TIME ON FLUORESCENT IN SITU HYBRIDIZATION (FISH) SIGNAL FOR BACILLUS CEREUS HER 1414 (BLACK BAR) AND B. CEREUS ATCC 14579 (GRAY BAR) The spores were incubated for 60 min in culture medium 14, permeabilized in 4% paraformaldehyde for 15 min and hybridized for 15, 30 min, 1 and 2 h. (A) The percentage of positive cells obtained for FISH reaction by using an EUB338 probe. (B) Mean fluorescence intensity obtained for FISH reaction by using an EUB338 probe.

Download figure to PowerPoint

Step 5: Validating the Optimal Condition Using Specific Probes

The next step was to validate the optimal conditions determined by using the specific probes for B. cereus pB394. Figure 5 shows the results for B. cereus ATCC 14579 and HER 1414 obtained with the pB394 probe (gray bar) compared with the EUB38 probe (black bar) for the percentage of positive cells and mean fluorescence intensity. Comparable results were obtained, by flow cytometry, for the percentage of positive cells (Fig. 5A), but differences were observed for the mean fluorescence intensity between the two stains of B. cereus (Fig. 5B).

image

Figure 5. VALIDATION OF THE OPTIMAL CONDITION BY USING THE BACILLUS CEREUS-SPECIFIC PROBE PB394 (GRAY BAR) COMPARED WITH EUB338 PROBE (BLACK BAR) FROM B. CEREUS ATCC 14579 AND B. CEREUS HER 1414 SPORE The spores were incubated for 60 min in culture medium 14, permeabilized in 4% paraformaldehyde for 15 min and hybridized for 15 min. (A) The percentage of positive cells obtained for fluorescent in situ hybridization (FISH) reaction by using a pB394 and EUB338 probe. (B) Mean fluorescence intensity obtained for FISH reaction by using a pB394 and EUB338 probe.

Download figure to PowerPoint

Figure 6 shows representative results with B. cereus ATCC 14579 obtained in fluorescence microscopy comparing the optimal conditions. Visible difference is obtained in the number of positive cells when hybridization time was decreased from 2 h (Fig. 6A) to 15 min (Fig. 6B) by using EUB338 probes. Also, the percentage of positive cells observed by fluorescence microscopy with the pB394 probe was lower compared with the EUB338 probe at 15 min of hybridization (Fig. 6C). Control experiments using antisense probe notEUB338 and notpB394 were still negative under fluorescence microscopy.

image

Figure 6. PHOTOMICROGRAPH OF THE BACILLUS CEREUS ATCC 14579 CELLS INCUBATED FOR 60 MIN IN CULTURE MEDIUM 14 (TRYPTIC SOY BROTH + L-ALANINE + INOSINE) (A) The cells were permeabilized with 4% paraformaldehyde for 15 min followed by fluorescent in situ hybridization (FISH) reaction for 2 h by using the EUB338 probe. (B) The cells were permeabilized with 4% paraformaldehyde for 15 min followed by FISH reaction for 15 min by using the EUB338 probes. (C) The cells were permeabilized for 15 min by using 4% paraformaldehyde, and FISH protocol was performed for 15 min by using the pB394 probe.

Download figure to PowerPoint

Step 6: Validating the Optimal Conditions in Spiked Milk Samples

Various concentrations of viable B. cereus HER 1414 spores were inoculated in sterile milk samples. Specific detection of 103 cfu/mL B. cereus spores by using pB394 probes was possible within 2 h using the optimized protocol. The milk clarification protocol using sodium citrate did not affect FISH detection. The antisense probe notpB394 gave no signal. Figure 7 shows a representative result. B. cereus ATCC 14579 gave the same result as HER 1414 (data not shown).

image

Figure 7. PHOTOMICROGRAPH OF BACILLUS CEREUS HER 1414 DETECTED FROM A MILK SAMPLE BY USING A PB394 PROBE The milk sample was inoculated at a concentration of 103 cfu/mL, clarified by using sodium citrate and growth for 1 h in tryptic soy broth + L-alanine + inosine. The cells were permeabilized for 15 min by using 4% paraformaldehyde, and fluorescent in situ hybridization protocol was performed for 15 min by using a pB394 probe (A) and a notpB394 probe (B).

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

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.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

These studies were undertaken under a Department of National Defense (DND) contract W7702-00R802/001/EDM: Dr. J. Ho was the scientific authority acting for DND. The authors are thankful to Mr. Luc Trudel and Dr. Benjamin Nehmé for their insightful review of the manuscript and thank Serge Simard for statistical analysis. Christian Laflamme received a Natural Sciences and Engineering Research Council of Canada (NSERC)/Institut de recherche Robert-Sauvé en santé et sécurité du travail (IRSST) studentship. Dr. C. Duchaine acknowledges IRSST/Canadian Institutes of Health Research (CIHR) and Fonds de la Recherche en Santé du Québec (FRSQ) Junior 2 Scholarships and a NSERC Discovery Grant.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  • AHMED, R., SANKAR-MISTRY, P., JACKSON, S., ACKERMANN, H.-W. and KASATIYA, S.S. 1995. Bacillus cereus phage typing as an epidemiological tool in outbreaks of food poisoning. J. Clin. Microbiol. 33, 636640.
  • AMANN, R.I., BINDER, B.J., OLSON, R.J., CHISHOLM, S.W., DEVEREUX, R. and STAHL, D.A. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 19191925.
  • BARLASS, P.J., HOUSTON, C.W., CLEMENT, M.O. and MOIR, A. 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: The roles of gerL and gerQ operons. Microbiology 148, 20892095.
  • BROSIUS, J., DULL, T.J., SLEETER, D.D. and NOLLER, H.F. 1981. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148, 107127.
  • CAIPO, M.L., DUFFY, S., ZHAO, L. and SCHAFFNER, D.W. 2002. Bacillus megaterium spore germination is influenced by inoculum size. J. Appl. Microbiol. 92, 879884.
  • CHAMBON, P., DEUTSCHER, M.P. and KORNBERG, A. 1968. Biochemical studies of bacterial sporulation and germination. X. Ribosomes and nucleic acid of vegetative cells and spore of Bacillus megaterium. J. Biol. Chem. 243, 51105116.
  • CHRISTIANSSON, A. 2003. Bacillus cereus. In Encyclopedia of Dairy Sciences (H.Roginsky, J.W.Fuquay and P.F.Fox, eds.) pp. 123128, Academic Press, Amsterdam, The Netherlands.
  • CLEMENTS, M.O. and MOIR, A. 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. J. Bacteriol 180, 67296735.
  • DRIKS, A. 2002. Maximum shields: The assembly and function of the bacterial coat. Trends Microbiol. 10, 251254.
  • ENEROTH, A., SVENSSON, B., MOLLIN, G. and CHRISTIANSSON, A. 2001. Contamination of pasteurized milk by Bacillus cereus in the filling machine. J. Dairy Res. 68, 186196.
  • FAILLE, C., JULLIEN, C., FONTAINE, F., BELLON-FONTAINE, M.N., SLOMIANNY, C. and BENEZECH, T. 2002. Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: Role of surface hydrophobicity. Can. J. Microbiol. 48, 728738.
  • FISCHER, K., HAHN, D., HÖNERLAGE, W., SCHÖNHOLZER, F. and ZEYER, J. 1995. In situ detection of spore and vegetative cells of Bacillus megaterium in soil by whole cell hybridization. System Appl. Microbiol. 18, 265273.
  • FUCHS, B.M., WALLNER, G., BEISKER, W., SCHWIPPL, I., LUDWIG, W. and AMANN, R. 1998. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labelled oligonucleotide probes. Appl. Environ. Microbiol. 64, 49734982.
  • FUKUSHIMA, H., KATSUBE, K., HATA, Y., KISHI, R. and FUJIWARA, S. 2007. Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR. Appl. Environ. Microbiol. 73, 92100.
  • GARRICK-SILVERSMITH, L. and TORRIANI, A. 1973. Macromolecular syntheses during germination and outgrowth of Bacillus subtilis spores. J. Bacteriol 114, 507516.
  • GRANUM, P.E. and LUND, T. 1997. Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett. 157, 223228.
  • GRIFFITHS, M.W. 1992. Bacillus cereus in liquid milk and other milk products. Bull. Int. Dairy Fed. 275, 3639.
  • HOLT, J.G. and KRIEG, N.R. 1994. Growth, enrichment and isolation. In Methods for General and Molecular Bacteriology (P.Gerhardt, R.G.E.Murray, W.Wood and N.R.Krieg, eds.) pp. 179215, American Society for Microbiology, Washington, DC.
  • HORNSTRA, L.M., DE VRIE, Y.P., DE VOS, W.M., ABEE, T. and WELL-BENNIK, M.H. 2005. gerR, a novel ger operon involved in L-alanine and iosine-initiated germination of Bacillus cereus ATCC 14579. Appl. Environ. Microbiol. 71, 774781.
  • HORNSTRA, L.M., DE VRIES, Y.P., WELLS-BENNIK, M.H.J., DE VOS, W.M. and ABEE, T. 2006. Characterization of germination receptors of Bacillus cereus ATCC 14579. Appl. Environ. Microbiol. 72, 4453.
  • HORNSTRA, L.M., DE LEEUW, P.L., MOEZELAAR, R., WOLBERT, E.J., DE VRIES, Y.P. and ABEE, T. 2007. Germination of Bacillus cereus spores adhered to stainless steel. Int. J. Food Microbiol. 116, 367371.
  • IRELAND, J.A.W. and HANNA, P.C. 2002. Amino acid- and purine ribonucleoside-induced germination of Bacillus anthracis Δsterne endospore: gerS mediates responses to aromatic ring structures. J. Bacteriol 184, 12961303.
  • KAMAT, A.S., LEWIS, N.F. and PRADHAN, D.S. 1985. Mechanism of Ca2+ and dipicolinic acid requirement for L-alanine induced germination Bacillus cereus BIS-59 spores. Microbios 44, 3344.
  • KEYNAN, A. and EVENCHIK, Z. 1969. Activation. In The Bacterial Spore (G.W.Gould and A.Hurst, eds.) pp. 359396, Academic Press, New York.
  • KOSHIKAWA, T., YAMAZAKI, M., YOSHIMI, M., OGAWA, S., YAMADA, A., WATABE, K. and TORII, M. 1989. Surface hydrophobicity of spores of Bacillus spp. J. Gen. Microbiol. 135, 27172722.
  • LAFLAMME, C., LAVIGNE, S., HO, J. and DUCHAINE, C. 2004. Assessment of bacterial endospore viability with fluorescent dyes. J. Appl. Microbiol. 96, 684692.
  • LAFLAMME, C., HO, J., VEILLETTE, M., DE LATRÉMOILLE, M.C., VERREAULT, D., MÉRIAUX, A. and DUCHAINE, C. 2005. Flow cytometry analysis of germinating Bacillus spore, using membrane potential dye. Arch. Microbiol. 183, 107112.
  • LIU, W.T., MIRZABEKOV, A.D. and STAHL, D.A. 2001. Optimization of an oligonucleotide microchip for microbial identification studies: A non-equilibrium dissociation approach. Environ. Microbiol. 3, 619629.
  • LUCORE, L.A., CULLISON, M.A. and JAYKUS, L.A. 2000. Immobilization with metal hydroxides as a mean to concentrate food-borne bacteria for detection by cultural and molecular methods. Appl. Environ. Microbiol. 66, 17691776.
  • MOIR, A. 2006. How do spore germinate? J. Appl. Microbiol. 101, 526530.
  • MOTER, A. and GÖBEL, U.B. 2000. Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J. Microbiol. Methods 41, 85112.
  • ODA, Y., SLAGMAN, S.J., MEIJER, W.G., FORNEY, L.J. and GOTTSCHAL, J.C. 2000. Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris. FEMS Microbiol. Ecol. 32, 205213.
  • PAIDHUNGAT, M. and SETLOW, P. 2002. Spore germination and outgrowth. In Bacillus subtilis and Its Relatives : From Genes to Cells (J.A.Hosh, R.Losick and A.L.Sonenshein, eds.) pp. 537548, American Society for Microbiology, Washington, DC.
  • PAIDHUNGAT, M., RAGKOUSKI, K. and SETLOW, P. 2001. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2+-dipicolinate. J. Bacteriol 183, 48864893.
  • PARK, S.H., KIM, H.J., KIM, J.H., KIM, T.W. and KIM, H.Y. 2007. Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR. J. Microbiol. Biotechnol. 17, 11771182.
  • PERRY, L., HEARD, P., KANE, M., KIM, H., SAVIKHIN, S., DOMINGUEZ, W. and APPLEGATE, B. 2007. Application of multiplex polymerase chain reaction to the detection of pathogens in food. J Rapid Methods Autom. Microbiol. 15, 176198.
  • QUARTO, M. and CHIRONNA, M. 2005. Hepatitis A: Sources in food and risk for health. In Review in Food and Nutrition Toxicity. Vol. 2 (V.R.Preedy and R.R.Watson, eds.) pp. 91126, CRC Press, London, U.K.
  • RAGKOUSI, K., EICHENBERGER, P., VAN OOJI, C. and SETLOW, P. 2003. Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate. J. Bacteriol. 185, 23152329.
  • RASO, J., MARCELA GONGORA-NIETO, M., BARBOSA-CANOVAS, G.V. and SWANSON, B.G. 1998. Influence of several environmental factors of germination and inactivation of Bacillus cereus by high hydrostatic pressure. Int. J. Food Microbiol. 44, 125132.
  • REGAMEY, A., HARRY, E.J. and WAKE, R.G. 2000. Mid-cell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: Co-ordinating chromosome replication with cell division. Mol. Microbiol. 38, 423434.
  • SETLOW, P. 1994. Mechanisms which contribute to the long-term survival of spores of Bacillus species. J. Appl. Bacteriol. Suppl. 76, 49S60S.
  • SETLOW, P. 2003. Spore germination. Curr. Opin. Microbiol. 6, 550556.
  • SWERDLOW, B.M., SETLOW, B. and SETLOW, P. 1981. Level of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium. J. Bacteriol. 148, 2029.
  • WAGNER, M., HORN, M. and DAIMS, H. 2003. Fluorescence in situ hybridization for the identification and characterization of prokaryotes. Curr. Opin. Microbiol. 6, 302309.
  • WELKOS, S.L., COTE, C.K., REA, K.M. and GIBBS, P.H. 2004. A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies. J. Microbiol. Methods 56, 253265.