DISCRIMINATION OF Bacillus cereus BY FLUORESCENCE TITRATION USING
XANTHENE-BASED Zn(II) CHEMOSENSOR
Somruethai Khamsakhon1,*, Ippei Takashima2, Akio Ojida2, Itaru Hamachi3, Thunyarat
Pongtharangkul1, Jirarut Wongkongkatep1,#
1
Department of Biotechnology, Faculty of Science, Mahidol University, Thailand
2
Graduate School of Pharmaceutical Sciences, Kyushu University, Japan
3
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Japan
*e-mail: maprang.mu@gmail.com, #e-mail: jirarut.chu@mahidol.ac.th
Abstract
A xanthene-based Zn(II) fluorescent chemosensor is synthesized for binding with
polyphosphate such as ATP that can be found in every living organisms. The specificity of
this sensor to bacterial cells was demonstrated by the fluorescence titration between the
sensor and bacterial cells using a spectrofluorometer. Three strains of Bacillus spp. cells (B.
cereus ATCC 11778, B. subtilis 168 and B. megaterium ATCC 14581) in three different
stages which are vegetative, sporulation and late sporulation stages were determined. The
results reveal that this method is effective to discriminate the late sporulation state of B.
cereus ATCC 11778 from the vegetative state and from other two Bacillus spp. Moreover,
heat treatment of the bacterial cells at 80 °C for 15 min is capable of enhancement of the
fluorescent signal specific to B. cereus.
Keywords: Bacillus cereus, xanthene-based chemosensor, fluorescence titration, bacterial
discrimination, fluorescent sensor
Introduction
Bacteria are microorganisms which can be found in every environment. Some of them
are useful for food and pharmaceutical productions. However, many groups of bacteria can
cause serious effect on animals and human health if they contaminate in the products. One of
them is B. cereus which is the opportunistic pathogen and causes food poisoning in human
and food spoilage. (1) Therefore, bacterial detection is very important to help us controlling
and removing them from our products. One effective method is the use of fluorescent sensors
that have rapid response, high sensitivity and specificity, as well as easy performance.
1-2Zn(II), the binuclear Zn(II)-Dpa (2,2’-dipicolylamine) chemosensor, is the
xanthene-based Zn(II) fluorescent sensor that composed of two components. The first part is
two Zn2+ molecules as binding sites which is high selective and strong binding with
polyphosphates including nucleoside polyphosphates (ATP, ADP, GTP, CTP and UDP),
phosphorylated proteins (2), inorganic pyrophosphate (PPi), and inosital-1,3,4-trisphosphate
(IP3). Another part is a xanthene ring which is highly hydrophobic as a fluorescent sensing
unit or fluorophore. This sensor has excitation wavelength at 488 nm and fluorescence
emission at 522 nm. (3)
Our previous study indicated that the 1-2Zn(II) chemosensor presented a large
positive fluorescence response upon the addition of sporulating cells of some Bacillus spp.
when compared with other five strains of bacteria including spore forming and non-spore
forming bacteria. In addition, the fluorescence response was even higher when those bacterial
cells were heated at 80 °C for 15 min, while no change in fluorescence intensity was
observed in case of other bacteria after heated under the same condition. (4)
This study aimed to determine in quantitative analysis that whether the positive
response of the sensor to the Bacillus spp. can be observed in every stage and strain of
Bacillus spp. cells and the responses increase when those bacterial cells are heated using a
spectrofluorometer.
Methodology
B. cereus ATCC 11778, B. subtilis 168 and B. megaterium ATCC 14581 were used in
this study. The cultures were grown on nutrient agar (NA) and incubated at 37 °C (B. cereus
and B. subtilis) or 30 °C (B. megaterium) for 24 h. One single colony of each strain was
inoculated to 5 ml of nutrient broth (NB) and cultivated in a shaker incubator (200 rpm) at 37
°C for 6 h. while B. megaterium were inoculated in nutrient broth supplemented with 0.1%
yeast extract and 5 mg/l MnSO4 (NB+) and cultured at 30 °C under the same condition. B.
cereus and B. subtilis cells were transferred to a 1 ml-microtube, centrifuged at 9,660×g for 5
min and suspended in 0.85% sodium chloride solution to give an optical density at 660 nm
(OD660) of 0.5. The 100 µl of bacterial cell suspension was spread on a NA plate and cultured
at 37 °C. For B. megaterium, the cells were further transferred to a 250 ml-Erlenmeyer flask
containing 50 ml of NB+ to give an initial OD660 of 0.1.
The bacterial cells were harvested at desired growth stages (vegetative, sporulation
and late sporulation stages) by washing from the solid nutrient agar plates with 0.85% sodium
chloride solution. Those samples were centrifuged at 4,024×g at 4 °C for 10 min and washed
twice with 0.85% sodium chloride solution. After that, the washed cells were suspended in
HEPES buffer solution (pH 7.4) to give an OD660 of 2.0 for using as unheated samples,
whereas heated samples were prepared by heat treatment at 80 °C for 15 min prior to the
fluorescence measurement.
A spectrofluorometer (Jasco FP-6200, Tokyo, Japan) was used for the determination
of fluorescence response of the sensor toward those bacterial cells. The measurement was the
titration of 3 µl of 1 mM xanthene-based Zn(II) chemosensor in 3 ml of HEPES buffer
solution with bacterial cell suspensions from 0 - 150 µl in a quartz cell. (3) The responses
were expressed as the fluorescence intensity. Therefore, the fluorescence titration profiles of
the xanthene-based Zn(II) chemosensor for bacterial cell suspensions were constructed
between F/F0, i.e. fluorescent intensity of the titrated sample (F) divided by the fluorescent
intensity of the sensor (F0) at 522 nm, and the amount of bacterial cell suspensions.
Results
The colonial morphology of B. cereus ATCC 11778, B. subtilis 168 and B.
megaterium ATCC 14581 were quite similar resulting in the difficulty of the differentiation
by naked eyes (Table 1). Similarly, the examination of microscopic bacterial cell morphology
under a light microscope using Gram stain and spore stain techniques were uneasy leading to
the requirement of the expertise. Therefore, the simple method for discrimination of these
bacteria which is the fluorescence titration using the xanthene-based Zn(II) chemosensor
would be the another effective choice.
Table 1. Bacterial morphology profiles used in this study
No.
Bacterial strains
1.
B. cereus ATCC 11778
2.
B. subtilis 168
3.
B. megaterium ATCC 14581
Colonial morphology
on NA
Gram stain
Spore stain
The comparison of fluorescence response of 1-2Zn(II) toward bacterial cells in different
Bacillus species
In order to determine whether the difference species of Bacillus correlated with the
response of the sensor to the bacterial cells, the comparison of the responses in quantitative
analysis among three different strains of Bacillus spp. used in this study in the same stages
and conditions were studied. As a result, the fluorescence responses in vegetative and
sporulation stages of all bacterial strains under unheated condition were indifferent as the
similarity of the negative relationship between the addition of bacterial cell suspensions and
F/F0 (Fig 1A and 1B). On the other hand, the different responses from various species were
observed in late sporulation stage cells. Only B. cereus ATCC 11778 showed the positive
fluorescence responses at this stage (Fig 1C). Moreover, the positive response increased at
the beginning of the experiment to the highest point (F/F0 = 1.7) at 45 µl and continuously
decreased at after 45 µl. B. cereus ATCC 11778 was differentiated from B. subtilis 168 and
B. megaterium ATCC 14581 by grouping as positive fluorescence response species at late
sporulation stage.
A
F/F0 at 522 nm
2.0
1.5
1.0
0.5
0.0
0
20 40 60 80 100 120 140 160
amount of bacterial cell suspension (µl)
B
F/F0 at 522 nm
2.0
1.5
1.0
0.5
0.0
0
20 40 60 80 100 120 140 160
amount of bacterial cell suspension (µl)
C
F/F0 at 522 nm
2.0
1.5
1.0
0.5
0.0
0
20 40 60 80 100 120 140 160
amount of bacterial cell suspension (µl)
Figure 1. Fluorescence titration profile of 1-2Zn(II) at 522 nm for three strains of Bacillus cell suspension in
three different stages from 0 - 150 µl under unheated conditions: Vegetative stage (A), sporulation stage (B),
and late sporulation stages (C). B. cereus ATCC 11778 (), B. subtilis 168 (), and B. megaterium ATCC
14581 (); λex = 488 nm
The bacterial cells were determined at vegetative, sporulation and late sporulation
stage that were suggested by the bacterial cell morphology under a light microscope.
Vegetative stage was indicated by none of sporulating cells. Sporulation was the stage that
almost of the cells were sporulating cells. Finally, the spores were split out of the parent cells
that meant late sporulation stage. B. cereus ATCC 11778 exhibited the highest fluorescence
response at late sporulation stage that represented the spores of Bacillus. In contrast, B.
subtilis 168 and B. megaterium ATCC 14581 cells in all stages presented negative responses.
The effect of heat treatment on the fluorescence response of 1-2Zn(II) toward bacterial cells
Our previous study suggested that heat treatment had effects on the increase of
fluorescence response of the sensor toward some Bacillus spp. Cells as also indicated by
several reports. In order to clarify whether heat affected the fluorescence response toward
Bacillus spp. cells in every stage, unheated and heated samples of three different stages cells
(vegetative, sporulation and late sporulation stages) of Bacillus spp. were subjected to a
spectrofluorometer. The fluorescence titration showed that the heated Bacillus had an effect
on an increase in fluorescent changes when compared to unheated cells in every strains and
stages (Fig 2). However, the increases in fluorescent response were obviously observed at
sporulation and late sporulation stages especially in the case of B. cereus ATCC 11778. The
responses of this strain increased 2 and 5 times compared to unheated samples at sporulation
and late sporulation stage, respectively.
Vegetative stage
Sporulation stage
Late sporulation stage
5.0
4.0
3.0
2.0
1.0
0.0
0
40
80
120
160
F/F0 at 522 nm
C 6.0
5.0
4.0
3.0
2.0
1.0
0.0
F/F0 at 522 nm
B 6.0
5.0
4.0
3.0
2.0
1.0
0.0
F/F0 at 522 nm
A 6.0
0
amount of bacterial cell suspension (µl)
40
80
120
0
160
amount of bacterial cell suspension (µl)
5.0
4.0
3.0
2.0
1.0
0.0
40
80
120
F/F0 at 522 nm
F 6.0
5.0
4.0
3.0
2.0
1.0
0.0
F/F0 at 522 nm
E 6.0
5.0
4.0
3.0
2.0
1.0
0.0
F/F0 at 522 nm
D 6.0
0
0
160
40
80
120
0
160
amount of bacterial cell suspension (µl)
amount of bacterial cell suspension (µl)
80
120
160
amount of bacterial cell suspension (µl)
F/F0 at 522 nm
5.0
4.0
3.0
2.0
1.0
0.0
F/F0 at 522 nm
I 6.0
5.0
4.0
3.0
2.0
1.0
0.0
F/F0 at 522 nm
H 6.0
5.0
4.0
3.0
2.0
1.0
0.0
40
0
40
80
120
160
amount of bacterial cell suspension (µl)
80
120
160
40
80
120
160
amount of bacterial cell suspension (µl)
G 6.0
0
40
amount of bacterial cell suspension (µl)
0
40
80
120
160
amount of bacterial cell suspension (µl)
Figure 2. Fluorescence titration profile of 1-2Zn(II) at 522 nm for unheated () and heated () Bacillus spp.
cell suspensions from 0 - 150 µl in three different stages: B. cereus ATCC 11778 (A, B and C), B. subtilis
168 (D, E and F), and B. megaterium ATCC 14581 (G, H and I).
Discussion and Conclusion
Nowadays, various methods for bacterial detection have been developed to increase
their specificity, sensitivity and easy performance. Our study confirmed that the fluorescence
titration using xanthene-based Zn(II) fluorescent chemosensor could be one of the effective
methods which is fast and simple without the requirement of any complicated instruments
and tedious procedures. Moreover, the calibration curve between fluorescent change and the
amount of bacteria can be constructed which applicable as quantitative analysis that is
reliable and the data are easy to observe and compare. Our newly developed method presents
high efficiency to discriminate B. cereus ATCC 11778 at late sporulation stage from other
two Bacillus spp. at the same stage as the notably positive fluorescence response of the sensor
was observed (1.7 times compare to the initial fluorescence intensity). According to the
highly hydrophobic property of the xanthene ring of the sensor, the positive responses might
be correlated with the bacterial cell-surface hydrophobicity because there are several reports
that the spores of Bacillus were more hydrophobic than vegetative cells as determined by
bacterial adhesion to hydrocarbon or BATH assays. (5-6) Furthermore, the fluorescence
responses were increased several times when B. cereus ATCC 11778 cells were heated while
no obvious change in fluorescence intensity was observed in case of other two Bacillus spp.
after heated under the same condition. This finding indicated that heat treatment enhanced the
sensitivity of the responses by the increase of cell permeability as same as the use of heat in
spore stain technique using malachite green. Heat was able to destroy the spore coat that was
a permeability barrier. (7)
In conclusion, the fluorescence titration using the xanthenes-based Zn(II)
chemosensor might be the fast and simple method to discriminate the spore of B. cereus from
other Bacillus spp. and the sensitivity was increased by the treatment of heat with the
bacterial cells. For the further study, more various strains and species of Bacillus should be
determined to clarify the specificity of the sensor. Moreover, the detection limit and the
probable mechanisms of the fluorescence responses are under investigation.
References
1.
2.
3.
4.
5.
6.
7.
Rasko DA, Altherr MR, Han CS, Ravel J. Genomics of the Bacillus cereus group of organisms. FEMS
Microbiology Reviews. 2005;29(2):303-29.
Ishida Y, Inoue M-a, Inoue T, Ojida A, Hamachi I. Sequence selective dual-emission detection of (i, i + 1)
bis-phosphorylated peptide using diazastilbene-type Zn(II)-Dpa chemosensor. Chem Commun. 2009:284850.
Ojida A, Takashima I, Kohira T, Nonaka H, Hamachi I. Turn-on fluorescence sensing of nucleoside
polyphosphates using a xanthene-based Zn(II) complex chemosensor. J Am Chem Soc.
2008;130(36):12095-101.
Ketsub N, Phupancharoensuk R. Detection of bacteria using fluorescent molecular sensor. Bangkok:
Mahidol University; 2011.
Thwaite JE, Laws TR, Atkins TP, Atkins HS. Differential cell surface properties of vegetative Bacillus. Lett
Appl Microbiol. 2009;48(3):373-8.
Wiencek KM, Klapes NA, Foegeding PM. Hydrophobicity of Bacillus and Clostridium spores. Appl
Environ Microbiol. 1990;56(9):2600-5.
Kozuka S, Tochikubo K. Permeability of dormant spores of Bacillus subtilis to malachite green and crystal
violet. J Gen Microbiol. 1991;137:607-13.
Acknowledgement:
This research was supported by Thailand Research Fund and Mahidol University (Grant No.
RSA 5580001).