JP2957312B2 - Method for measuring viable count of Staphylococcus aureus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the number of viable bacteria and for identifying the bacterial species, and a specific selective medium used for carrying out the method. More specifically, the present invention uses a specific selective medium to grow only Staphylococcus aureus in a specimen, and measures the viable cell count of the bacteria by capturing fluorescence emitted from a culture of the bacteria. Media for selection.

[0002]

2. Description of the Related Art Conventionally, identification of microorganism species has been carried out by using Berge (Ber
gey) by examining the morphological and physiological properties of the cultures. on the other hand,
The number of viable bacteria was 10 ^-1 , 10 ^-2 , ...,
After 10- ^n- fold dilution, a certain amount of the diluted solution was spread and inoculated on an agar plate medium, cultured for a certain period of time (for example, 24-48 hours), and then counted for the number of colonies that appeared on the agar plate. It was determined by multiplying by the dilution factor.

[0003] However, such a completely manual microbial testing method requires a testing period of 2 to 5 days, considerable skill, and may cause measurement differences between technicians or inspectors. Are known. In addition, the cost of testing is very high due to the use of large amounts of medium and dishes and the high labor costs of skilled technicians.

[0004] Microbial tests such as identification of microbial species and measurement of the number of viable bacteria are indispensable in departments such as clinical tests, food tests and pharmaceutical tests, and there is a high need for speeding up, labor saving and automation. In particular, the clinical testing department is developing various types of automation equipment. For example, a plater, which smears the sample solution from the center outward, is cultured on an agar plate medium rotating at 50 rpm, and the smeared agar medium is cultured, and the plate medium on which the colonies are formed is counted with a He-Ne laser. Spiral System Inc., a system that uses a cartridge containing various substrates to identify microbial species by a colorimetric method, and to automatically perform a drug susceptibility test by turbidimetric absorption method, such as the Auto Microbic System, MS-II, and Avantage Dynatech's MIC-2000 system using microplates instead of systems and cartridges has been developed mainly in the United States.

However, most of these instruments are used only for microbial tests such as urinary tract infection, and cannot be measured when samples are often turbid, such as in food tests. Yes, and sufficient accuracy could not be obtained. In addition, any method requires a culture time of 24 to 48 hours, so that rapid measurement is difficult. For this reason, it has not reached the stage where it can sufficiently respond to the urgent needs of the site. Research on these automatic devices and measuring methods has been actively conducted in the fields of clinical medicine and medical engineering, but very few have been put to practical use.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for measuring the number of viable bacteria which can be used in a wide range of fields, is highly accurate, is low in cost, and requires only a few hours for inspection.
An object of the present invention is to provide a method for identifying a bacterial species and a specific selective medium used for carrying out the method.

[0007]

In order to achieve the above-mentioned object, the present invention provides a method for preparing trypticase, phyton, N
Provided is a selective medium for Staphylococcus aureus, characterized by comprising aCl, Tween 80, and Azuleonam. The medium has the following composition, Trypticase
2.5 g / l, phyton 0.5 g / l, N
aCl 5 g / l, Tween 80
1.0 g / l, azuleonam 0.25 μg /
It is preferred to have ml. To estimate the viable cell count of Staphylococcus aureus from the fluorescence intensity of the culture, Try
pticase, phyton, NaCl, Tween 8
0 and azuleonam. After selectively cultivating Staphylococcus aureus in a medium having the above composition, AO-10 was added to the medium.
The present invention also provides a selective medium for measuring the viable cell count of Staphylococcus aureus, which is characterized by further adding dodecyl bromide.

[0008]

When the above-mentioned selective medium is used, only Staphylococcus aureus in a specimen can be selectively grown. The presence of Staphylococcus aureus among the causative pathogens such as food poisoning can be specifically confirmed by the selective medium of the present invention. Azreonam is a "monobactam" antibiotic developed by Squibb in the United States and has specific antibacterial activity against Escherichia coli and Salmonella, but has little activity against Staphylococcus aureus. Therefore, if a specimen is inoculated and cultured in this medium and a colony appears, it can be estimated that the colony is caused by Staphylococcus aureus.

Along with the identification of the bacterial species, it is also possible to estimate the number of viable bacteria. For example, a microorganism is cultured in the selective medium, the culture is irradiated with excitation light, and the measured value is plotted on a calibration curve of the number of viable bacteria prepared in advance for a known microorganism species and the fluorescence intensity corresponding to the number of bacteria. Is applied to obtain the estimated number of viable bacteria. Since the combination of the microorganism species and the medium is known in advance, the microorganism is cultured in this medium by a standard method, and the number of viable cells at each culture time is counted by a known and established method. Is measured, a calibration curve is prepared, and stored as a database. If an unknown sample is cultured in a selective medium and grows, the bacterial species can be easily identified from the type of the medium. At the same time, if the fluorescence intensity at the time of the predetermined culture time is compared with the database of the corresponding medium / microorganism, the viable cell count of the microorganism can be immediately obtained.

However, in the case of Staphylococcus aureus, the fluorescence from the culture alone is not sufficient for measurement, so a fluorescent label called A0-10 dodecyl bromide is added to the culture, and the fluorescence intensity is measured. The added A0-10 dodecyl bromide specifically binds to Staphylococcus aureus and shows a fluorescence intensity according to the number of bacteria. That is, A0-10 dodecyl bromide itself does not emit fluorescence, but emits fluorescence only when bound to S. aureus, so that it can be used as a fluorescent label in the method of the present invention. AO-10 dodecyl bromide has a chemical name of 3,6-bis (dimethylamino)-
It is a 10-dodecyl acridinium bromide, red-orange crystalline powder with a molecular weight of 514.59. This substance is non-fluorescent in aqueous solution, but 53% in organic solvent.
It is known that it shows strong fluorescence near 0 nm.

[0011]

The present invention will be described in more detail with reference to the following examples. The method for preparing the medium itself is not particularly limited. In general, it can be prepared by any method known to those skilled in the art. For example, the components are mixed in an appropriate order, the resulting mixture is sterilized in an autoclave or the like, and finally stored in an appropriate container under aseptic conditions.

The medium prepared as described above is dispensed under sterile conditions into three sterilized dishes, each of which is inoculated with Escherichia coli, Salmonella, and Staphylococcus aureus, respectively, at 37 ° C. in the atmosphere. After culturing for a predetermined time,
Colonies occurred only in petri dishes inoculated with Staphylococcus aureus. This confirmed that the medium of the present invention had specific selectivity for Staphylococcus aureus.

Next, specific examples of the use of the selective medium of the present invention will be described in more detail with reference to the drawings. FIG. 1 shows a microplate 1 for using the selection medium of the present invention.
FIG. 4 is a schematic plan view of an example. The material of the plate itself is not particularly limited, but any material can be used as long as it scatters excitation light as little as possible and does not hinder the generation and reception of fluorescence. For example, a transparent polycarbonate can be used. However, it is a matter of course that the present invention is not limited to this. The plate shown is of 96 holes. The mode of use of the holes is not particularly limited. As an example, as shown in the figure, of the 12 rows, the first and seventh rows are filled with a bacterial solution or a sample, and the second to sixth rows and the eighth to eighth rows are placed. Use up to column 12 for serial dilution. In this example, the first to sixth columns are used as a control (for example, for measuring the background value of fluorescence before culture), and the seventh and subsequent columns are used for fluorescence measurement after culture. Used for Lines A to H can contain the same bacteria or medium, or can be divided into lines A to D and lines E to H, each containing the above-mentioned medium. The specific manner of using such holes is well known to those skilled in the art and need not be further described.

Next, a series of procedures for carrying out the method of the present invention will be described with reference to FIG. First, FIG.
A microplate 1 as shown in (1) is prepared, and a predetermined selective medium is dispensed into a predetermined hole of the plate in a medium dispensing zone 2. It is well known to those skilled in the art that dispensing can be performed either manually or by an automatic machine. Next, in the bacterial solution dispensing zone 3, the specimen (solid or liquid) or the bacterial solution is adjusted using a commercially available blender or stomacher.
Emulsify and dispense into the first and seventh rows of the plate as described above. This dispensing can also be performed manually or by an automatic machine. However, although not particularly limited, the medium dispensing and the bacterial solution dispensing are performed by the automatic viable cell count measurement method of the present invention.
It is not incorporated into the strain identification system. Therefore, it should be performed by a person using the system of the present invention as preprocessing.

Thereafter, the plate 1 is loaded on a serial dilution / fluorescence probe addition device 4. Here, for example, 10 μl of the bacterial liquids in the first and seventh rows are automatically collected, respectively, and injected into the holes in the second and eighth rows, respectively. Next, 10 μm from the holes in the second and eighth rows, respectively.
1 volume is automatically collected and injected into the holes in the third and ninth rows, respectively. Thereafter, this is sequentially repeated up to the sixth and twelfth columns. Then, 10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 ,
Seven dilution series of 10 ^-4 , 10 ^-5 and 10 ^-6 are obtained.
According to the experiments performed by the present inventors, it was confirmed that a dilution of 10 ⁻⁶ or more did not produce colonies and was meaningless.

When the serial dilution is completed, the AO-10 dodecyl bromide probe solution is dispensed only to all the wells on the control side of the plate. As mentioned above, the AO-10 dodecyl bromide probe is specific for S. aureus only.

After dispensing the probe solution into the control side hole of the plate, the plate is transferred to the fluorescence measuring device 5. Here, the fluorescence intensity before culturing is measured and stored in the external storage device of the data processing / control device 6 as a background value. Thereafter, the plate is transferred to the incubator 7 and cultured under predetermined conditions for a predetermined time. After the culturing, the plate is returned to the fluorescent probe adding device again, and the AO-10 dodecyl bromide probe solution is dispensed into all the holes on the culture side of the plate. To ensure the binding reaction between the bacteria and the probe, the plate is left for several minutes to 10 minutes after dispensing the probe. Thereafter, the fluorescence intensity on the culture side of the plate is measured by a fluorescence measurement device, and the measurement result is transmitted to an external storage device of the data processing / control device 6. The true fluorescence intensity is obtained by subtracting the background value before the culture from the fluorescence intensity after the culture. By comparing the result of the subtraction with a database in the external storage device, the bacterial species and the viable cell count are determined.

FIG. 3 is a flowchart showing the above processing procedure. After the pretreatment 10 of the medium dispensing and the bacterial solution dispensing, it enters the serial dilution / probe dispensing 11. Next, an initial fluorescence intensity measurement 12 is performed. The measured values are processed in line 13
It is transmitted to the control device 14. After the initial fluorescence intensity measurement, culture 14 is performed. After the culture, the process returns to step 11, and probe dispensing is performed again. After that, fluorescence intensity measurement 15 is performed after the culture. The measured values are transmitted on line 16 to the data processing and control unit 14. The data processing / control device 14 has a database 17 as an external storage device. The transmitted measured fluorescence value is collated with the database, the number of bacteria and the type of bacteria are determined, and output at 18.

FIG. 4 shows a schematic configuration of a viable cell count measurement / species identification system 20 that can use the selective medium of the present invention. The illustrated system basically includes the serial dilution / fluorescence probe addition device 4, the fluorescence measurement device 5, and the data processing / control device 6, as described above. Although not specifically excluded, incubator 7 is generally not included in this system 20. That is, this system 20
Is to be prepared by the person who uses. In the illustrated embodiment, the serial dilution / fluorescence probe addition device 4 and the fluorescence measurement device 5 are configured as separate devices, but it can be understood by those skilled in the art that the two can be combined into a single system. It is obvious. In the figure, the white thick arrow line
I to IV indicate the transport direction of the plate, and I and IV can be performed by an automatic handling mechanism (not shown) in the system. II and III are manual.

The specific configuration of the serial dilution / fluorescence probe addition device 4 is not particularly limited. Any device configuration that can achieve the intended purpose of serial dilution and addition of a fluorescent probe may be used. As an example, as shown, X-
The YZ-Z 'axis moving mechanism 22 is used, the quad tip dispensing mechanism 24 is arranged on the Z' axis, and the probe liquid tank 26 can be provided. Although not shown, since the chips are disposable, a chip supply / discard mechanism can be provided. It is preferable that the serial dilution / fluorescence probe addition device is housed in a closed box, and the inside of the box is maintained in a sterile state by a UV (ultraviolet) sterilizing lamp 28.

The specific configuration of the fluorescence measuring device 5 is not particularly limited. However, in the illustrated system 20, it is preferable to use a measuring device having an optical system as shown in FIG. 5 below. The illustrated fluorescence measuring device 5 has, for example, an optical system unit 30 and an XY table 32 for moving a plate. The feature of the illustrated optical system unit 30 is that fluorescence is received by the optical fiber 36 held in the holder 34, which will be described in detail below. It is preferable that the fluorescence measurement device is also housed in a closed box, and the inside of the box is maintained in a sterile state by a UV (ultraviolet) germicidal lamp 28. Further, as described above, the serial dilution / fluorescence probe addition device 4 and the fluorescence measurement device 5 can be accommodated in one closed box.

In the illustrated system 20, a serial dilution
The fluorescent probe adding device 4 and the fluorescence measuring device 5 are connected to a data processing / control device 6. If necessary, they can be connected via the expansion slot box 40. The data processing and control are performed so that the devices 4 and 5 operate in accordance with a procedure programmed in the data processing and control device 6 in advance.
The control device 6 also fulfills the function of a controller for the devices 4 and 5. The data processing and control device 6 is a CPU in the main body.
In addition, an external storage device 42 such as an HDD for storing a database can be provided. Further, a CRT 44 and a printer 46 for displaying control contents and outputting measurement results can be provided.

FIG. 5 shows a conceptual configuration diagram of an optical system in the fluorescence measuring device of the viable cell count measuring / species identification system of FIG.
As a light source, for example, a xenon lamp 50 is used,
A light source system is configured by arranging an auxiliary mirror 52 behind, a condenser lens 54 in front, and a heat ray absorption filter 56 in front of the lens. The light beam collimated by the condenser lens 54 is passed through an excitation light bandpass filter 58 in order to extract only excitation light having a specific wavelength. This filter is rotated by a motor so that excitation light of four wavelengths 0 to 3 can be obtained as shown in the figure. However, the number of filters is not limited. Although the wavelength of the excitation light itself is not an essential requirement of the present invention, it is preferable to use excitation light having a wavelength of 490 nm as a general index in the case of Staphylococcus aureus.

The excitation light passing through the band-pass filter 58 passes through the relay lens 62 and the variable stop 64. The collimated excitation light is stopped down by the variable stop 64,
° Refracted. The refraction excitation light is focused by the imaging lens 68 and is applied to the sample in the well (hole) 70 of the microplate. Alternatively, the pump light exiting the pump light bandpass filter 58 may be coupled to an optical fiber (not shown).
To irradiate the specimen. The use of an optical fiber eliminates the need for a relay lens, a variable aperture, a reflecting mirror, and an imaging lens, and can suppress energy attenuation of excitation light.

The fluorescence generated from the specimen is received by an optical fiber. The number of optical fibers for fluorescence reception may be one or more (for example, six). The optical fiber is preferably arranged so as to surround the specimen in a circular shape.
For this reason, it is preferable to hold by the holder 34 as shown simply in FIG. The holding angle of the optical fiber with respect to the vertically incident excitation light is not particularly limited. Any angle that can achieve the maximum efficiency for receiving the fluorescent light may be used. Such an angle can be easily determined by those skilled in the art.
Generally, 45 ° is preferred.

The fluorescence received by the optical fiber 72 is further guided to a fluorescence-side bandpass filter 74 and an excitation light-side bandpass filter 76. Filters 74 and 76
Must use the same numbered filter as filter 58. Although not shown, the filters 74 and 76 are simultaneously driven and controlled by the motor 60 at the same time as the filter 58. In the case of Staphylococcus aureus, the fluorescence-side band-pass filter 74 is preferably a filter that transmits fluorescence at 535 and 540 nm. The filter 76 can be used to remove stray light excitation data from the data so as not to be a factor in disturbing the fluorescence intensity.
However, this filter 76 does not have to be used. Immediately after each filter, photomultipliers 78 and 80
Is arranged. The electric signal from the photomultiplier is transmitted to the data processing / control device as shown in FIGS. 2 and 4 through the amplifier circuits 82 and 84.

Next, a method of creating a database will be described. Data on the viable cell count is obtained by the conventional agar plate method. For example, from a sample with a dilution ratio of 10 ^-6 times, 0.1
An agar plate is inoculated on the agar plate, and cultured at 37 ° C. in an air atmosphere for a predetermined time. Further, 0.1 ml is collected from a sample having a dilution factor of 10 ^-7 times, inoculated on an agar plate, and similarly cultured at 37 ° C. in an atmosphere in the atmosphere for a predetermined time. After culture
The number of colonies appearing on the agar plate is counted. For example,
In the case of 10 ^-6 samples, 100 colonies appeared and 10
^-7 sample, 10 colonies appeared, 10
^The number of bacteria in the sample of ^-6 was 100 × 10 ⁶ × 10 = 10
It becomes ⁹ / ml, in 10 ^-7 specimens, 10 × 10 ⁷ ×
10 = 10 ⁹ cells / ml. From this result, the number of viable bacteria in the sample is estimated to be 10 ⁹ . If this measurement is performed, for example, at 3, 4.5, 6, and 7.5 hours after the start of the culture, a characteristic curve showing the change in the number of bacteria over time can be obtained.

At the same time, the fluorescence measurement system of the present invention was used for 3 hours, 4.5 hours,
The fluorescence intensity at time 7.5 hours is measured. When a characteristic curve is created from this data, it can be confirmed that the fluorescence intensity increases over time. Therefore, by combining the data of the number of viable bacteria at each culture time with the data of the fluorescence intensity at each culture time, a calibration curve of the fluorescence intensity with respect to the number of viable bacteria can be created. This is stored and stored as a database. Therefore, if the species of the unknown sample is identified,
The number of viable cells can be obtained by applying the measured fluorescence intensity to the viable cell count / fluorescence intensity calibration curve database of the species.

The basic concept of creating a database for use in the method and system of the present invention is as described above. Therefore, the reliability of the method and system of the present invention is not remedied, but depends on the reliability of the database. For this reason, it is preferable to repeat as many culture experiments as possible, statistically process the obtained results, minimize variations, and construct a universal database as much as possible. For example, there are many types of Staphylococcus aureus and mutants, so even if the culture conditions and fluorescence measurement conditions are the same, the fluorescence intensity slightly changes depending on each strain or strain even with the same number of viable bacteria. Occurs. In the method of the present invention, since it is not possible to identify up to a specific strain in the Staphylococcus aureus group, even if various Staphylococcus aureus strains are mixed in the sample,
It is estimated as a viable bacterial count of generic generic Staphylococcus aureus.

Due to factors such as the difference between strains as described above, noise can greatly affect the predicted value of the initial bacterial concentration. Can be processed by fuzzy logic. A function that associates a specific variable value (such as a measured fluorescence intensity) with a fuzzy variable expressed by a language (such as a large fluorescence intensity) is called a membership function. In addition, a relation between events, such as "If the amount of change in fluorescence intensity is large, the initial bacterial concentration is extremely large", is called a rule base. According to the fuzzy theory, to estimate the initial bacterial concentration from the change in fluorescence intensity, a database of rules (that is, a rule base) for estimating the initial bacterial concentration from the change in fluorescence intensity for each identified bacterial species and dilution concentration is used. A membership function for each fuzzy variable is created and stored in the data processing device. According to this method, even if there is an error in the measured value of the fluorescence intensity, the deviation from the expected value is smoothed / corrected to some extent by the processing of “ambiguity” which is a characteristic of fuzzy, and the noise is canceled. Thus, the initial bacterial concentration can be measured with high reliability. With the accumulation of the rule base, the membership function, and the experimental data, it is possible to further improve the measurement accuracy by sequentially performing correction and tuning. In addition, by adding a learning function, these tunings can be automatically performed, and the function can be improved.

FIG. 6 shows an example of a calibration curve created based on the basic concept as described above. FIG. 6 is a graph showing the relationship between the fluorescence intensity and the viable cell count in the culture of Staphylococcus aureus (JCM2413 strain). The white hexagonal spot is a characteristic curve showing the time-dependent change in the number of viable bacteria with respect to the culture time. On the other hand, ● is a characteristic curve showing the change over time of the fluorescence intensity with respect to the culture time.

[0032]

As described above, according to the selective medium of the present invention, the species of Staphylococcus aureus can be identified within a short time of about 8 hours, and the number of these bacteria can be measured. Can be.

[Brief description of the drawings]

FIG. 1 is a schematic plan view of an example of a microplate in which a selective medium of the present invention is actually used.

FIG. 2 is a schematic conceptual diagram showing the progress of the process of the method for identifying a bacterial species and estimating the number of viable cells using a selective medium according to the present invention.

FIG. 3 is a flowchart showing the flow of a processing procedure of a method for identifying a bacterial species and estimating the number of viable cells using a selective medium according to the present invention.

FIG. 4 is a schematic configuration diagram of an example of a system used for carrying out a method for identifying a bacterial species and estimating the number of viable cells using a selective medium according to the present invention.

FIG. 5 is a schematic configuration diagram of an optical system for measuring fluorescence used in the system of FIG. 4;

FIG. 6 is a characteristic curve showing the relationship between the fluorescence intensity and the viable cell count in the culture of Staphylococcus aureus.

[Explanation of symbols]

 Reference Signs List 1 Microplate 2 Medium dispensing zone 3 Bacterial solution dispensing zone 4 Serial dilution device 5 Fluorescence measuring device 6 Data processing / control device 7 Incubator 20 Viable cell count measurement / species identification system

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takao Kutsuno 2-6-1 Otemachi, Chiyoda-ku, Tokyo Within Hitachi Electronics Engineering Co., Ltd. (72) Rika Kasuga 2-6-Otemachi, Chiyoda-ku, Tokyo 2 Hitachi Electronics Engineering Co., Ltd. (72) Inventor Shinpei Suzuki 2-6-2 Otemachi, Chiyoda-ku, Tokyo Hitachi Electronics Engineering Co., Ltd. (72) Fukuo Iwatani 2-6-Otemachi, Chiyoda-ku, Tokyo 2 Hitachi Electronics Engineering Co., Ltd. (72) Inventor Yoshimi Benno 2-1 Hirosawa, Wako-shi, Saitama Pref., RIKEN (72) Inventor Teruyuki Nagato 2-1 Hirosawa, Wako-shi, Saitama Pref. Inventor Kazusa Asama 2-1 Hirosawa, Wako-shi, Saitama Pref. RIKEN (72) Inventor Far Isao Fuji 2-1, Hirosawa, Wako-shi, Saitama Pref., RIKEN (56) References JP-A-57-179073 (JP, A) US Patent 3278393 (US, A) Journal of Food Protection, Vol. 50, No 8, 669-672 (1987) Journal of Hygiene, Vol. 80, 57-67 (1978) Progress in Medicine, Vol. 8, No. 1,189-194 (1988) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12Q 1/00-3/00 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (1)

(57) [Claims] (1) Trypticase: 2.5 g /
1, phyton: 0.5 g / l, NaCl: 5 g / l, T
ween 80: 1.0 g / l and azleonam: 0.2
Separate with 5 μg / ml selective medium for Staphylococcus aureus
Culturing the analysis sample; (2) adding AO-10 dodecyl bromide to the obtained culture.
A step of an appropriate amount, (3) culture AO-10 dodecyl bromide was added
Measuring the fluorescence intensity of (4) viable bacteria prepared in advance for known Staphylococcus aureus
The measured value is plotted on a calibration curve of the number and the fluorescence intensity corresponding to the number of bacteria.
Is applied to the yellow globules in the analyte
Determining the viable cell count of the bacterium, the viable cell count of Staphylococcus aureus, comprising:
Method.