JPH07308184A - Device for simply detecting resistant microorganism - Google Patents

Abstract

PURPOSE:To provide the subject device for readily detecting a microorganism resistant to many agents, comprising microorganism collecting ends to be mutually brought into contact with each other in an integrated way by action of external force, containing an antibiotic and a medium, arranging the antibiotic and the medium in a non-contact way in a container and having long-term shelf stability. CONSTITUTION:In a device for detecting a microorganism by culturing a microorganism in a medium and observing change of the medium, the device consists of a transparent lower tube 1 having an open top and a closed bottom and an upper tube 2 which has a closed top, a lower end closed with a porous plate 3, is used also as a cap to be hermetically engaged with the lower tube 1 and to seal the lower tube 1 and is provided with a glass ampule 4 having sealed the medium between the top and the porous plate 3 at the bottom. The porous plate 3 is provided with an applicator 6 for collecting a microorganism, having the other end extending in the lower tube 1 and a disc chip 5 impregnated with the antibiotic to suppress growth of microorganisms except the resistant microorganism. After a cell is collected by the applicator 6, the glass ampule 2 is broken, the medium is guided to the lower tube 1 and the microorganism is cultured to detect the microorganism by change of the medium.

Description

Detailed Description of the Invention

[0001]

The present invention has excellent long-term storage stability,
Easy-to-use and easy-to-carry resistant bacteria, especially methicillin-resistant Staphylococcus
cus aureus; hereinafter abbreviated as MRSA).

[0002]

2. Description of the Related Art With the recent advances in medical treatment and advances in medical treatment, antibacterial drugs, immunosuppressive drugs and the like have been widely used as therapeutic means, and have benefited from them. However, on the other hand, it is undeniable that many resistant bacteria have been produced as a secondary phenomenon, which is one of the causes of intractable infections.
The group is the representative one, and it is a big medical problem. In particular, infection in an intensive care unit that treats critically ill patients, or infection of people with weak resistance in nursing homes, etc. causes enteritis and pneumonia, and there is no effective drug treatment method, resulting in death from lung disease. Is increasing.

Not only these MRSAs but also multidrug-resistant coagulase negative Staph
ylococcus) (hereinafter abbreviated as multidrug-resistant CNS) is often detected by medical staff and tends to increase year by year.

On the other hand, in order not to increase MRSA and multidrug-resistant CNS infections, it is considered important to detect infections early and take appropriate preventive measures. In particular, in early detection of this infection, it has been strongly desired to be able to screen more samples quickly, easily, reliably and inexpensively.

In the conventional technique, a patient sample or a sample collected from a medical environment is cultured on an agar plate medium using a selective medium for resistant bacteria, and the colonies formed on the medium are visually observed, and other A method of confirming and detecting a resistant bacterium using a detection confirmation means has been adopted. For example, Yoshida Shigeru et al. "Prompt and simple methicillin-resistant Staphylococcus aureus (MR
SA) Development of agar plate for detection ”in Clinical Pathology (Vol. 41 No. 9)
p. 1037-1042 (1993)), and more recently,
MRSA using polymerase chain reaction (hereinafter abbreviated as PCR)
Has been proposed and applied.

[0006]

However, all of the above-mentioned conventional methods are complicated in operation; specialized techniques or skills are required for detection in order to determine colonies grown on an agar medium. In the raw medium to be used, a decrease in antibiotic activity is unavoidable and long-term storage is not possible; it has drawbacks such as high detection costs. Furthermore, in the method using PCR, for example, the MRSA resistance gene mecA Although it may be possible to amplify the gene, there is a problem of accuracy due to the presence of staphylococci containing the mecA gene in addition to MRSA, and at present it is only implemented in some hospitals such as university hospitals. Absent. Further, as a simple and particularly suitable device for detecting bacteria, JP-A-62-171671 proposes a device in which a liquid medium is contained in a capsule, and the capsule is also contained in a container together with a tampon for collecting bacteria. However, it cannot be denied that the added antibiotic loses its activity over time even in a liquid medium, and is not reliable enough to selectively detect resistant bacteria.

[0007]

[Means for Solving the Problems] In view of these circumstances, as a result of earnest research, a selective resistance bacterium detection device which is excellent in long-term storability and is easy to carry has been developed. And within 24 to 48 hours, resistant bacteria such as MR
The inventors have found that SA and multidrug resistant CNS can be detected, and completed the present invention.

That is, in a device for detecting a bacterium by culturing the bacterium in a medium and observing a change in the medium, the bacterium is collected at the collecting end, antibiotics and medium, and before use (that is, before culturing). At least the non-contact of the antibiotic and the culture medium, when culturing subsequent to the bacteria collection, it is arranged in the container so that the bacteria collection end, the antibiotic and the culture medium are brought into contact with each other by applying an external force. The present invention has been accomplished by developing a selective resistant bacteria detection device characterized by:

Here, the bacteria collecting end is represented by a cotton swab structure, but when the object to be collected is liquid, a pipe-shaped dropper at least partially made of rubber or an elastic or flexible material. It may have a function or a spatula structure with various tips at the tip, in short, it can be stored aseptically in a sterile container with a shape suitable for collecting bacteria, and it can sufficiently contact with the medium after collecting bacteria. Any structure can be adopted as long as it is a structure. Further, it is preferable that the bacteria collection end is accommodated in a state in which it does not come into contact with an antibiotic or a medium described later before use, but there may be some contact with the antibiotic.

The type of the antibiotic is selected in relation to the resistant bacterium to be detected, and an appropriate amount is determined particularly in relation to the amount of medium. Although the device becomes more compact when antibiotics are added to the medium in advance, unfortunately, as mentioned above, the activity of antibiotics often decreases over time, making it a selective detection device for resistant bacteria that can be stored for a long period of time. In that case, it can be said that it is inevitable to accommodate these antibiotics and the medium in a non-contact manner. As a storage form, it can be stored directly in a container in powder form, but it is stored in the form of a disk chip by dissolving it in water or another suitable solvent, impregnating an appropriate amount into a carrier (for example, filter paper, etc.), freeze-drying. In addition, the solution is preferably added to a part of the container, which is to be a space for culture (hereinafter referred to as a culture part), and coated by a method such as vacuum drying to house the powder, or the powder thereof. Alternatively, the solution may be filled in a destructible ampoule or capsule to be stored. Regardless of where it is stored in the storage container, if it is stored in a state in which it can be contacted with the medium by shaking the container before culturing or rinsing with a liquid medium, it is in a non-contact state with the medium before culturing. Any storage method may be adopted as long as the above condition is maintained.

As the medium, a known medium for the resistant bacterium to be detected or a modified medium thereof can be used. The medium is preferably in a liquid state, and it is desirable to fill and store it in an ampoule or a capsule, but it is not limited to this, and for example, an agar medium stored so as not to come into contact with the antibiotic by a fragile partition wall. However, in this case, the partition that is fragile during the culture is destroyed through an external magnet with an iron piece coated with glass placed on the partition, or it is pierced by the stick part made of a hard material of a cotton swab, The bacteria at the collected end are brought into contact with the antibiotic and the agar medium by, for example, breaking the capsule of the antibiotic and water and pouring the solution onto the medium. By the way, it is also possible to store a dry medium instead of the agar medium and to restore the normal agar medium with water stored in various forms in the container. In addition, a pH indicator or a chemical substance that is decomposed by a bacterium and used as an indicator of the growth of the bacterium may be added to the medium so that the culture result can be accurately determined. Then, since some of them may inhibit the culture, it is natural that they are added in an appropriate amount.

[0012] Selection of various accommodation forms for the above-mentioned collection end and antibiotics, and various accommodation forms for the medium,
Regarding the combination, many combinations are allowed without departing from the spirit of the invention. In particular, when both the antibiotic and the medium (particularly the liquid medium) or one of them is contained in a synthetic resin or glass ampoule or capsule and destroyed to integrate the antibiotic and the medium, a container It is preferable that the capsule or ampoule housing section be flexible, because the capsule or ampoule can be easily broken by applying a force from the outside with a split tool or the like. In particular, ampules and capsules having various breakable mechanisms used in physical chemistry experiments are preferable for application. The more compact the storage container is, the more preferable it is, but it is indispensable to secure a volume in which the air necessary for culture can exist. In addition, the shape of the storage container is preferably cylindrical as a whole in terms of loading,
It is also possible to adopt a shape such as a protrusion on the upper surface of a dish-shaped disc, any form is acceptable without departing from the spirit of the invention, and the material is synthetic resin or natural rubber which is inert to various media and antibiotics. , Synthetic rubber, various types of glass, and various types of metals can be selected in whole or in part.

Further, a detailed description will be given with reference to FIG. 1 which is an example of a concrete apparatus according to the present invention which assumes MRSA detection. This detection device has a transparent lower tube 1 that is open at the upper end and closed at the lower end; closed at the upper end, closed at the lower end by a perforated plate 3, and is hermetically engaged with the open upper end of the transparent lower tube 1. A flexible synthetic resin that also serves as a cap for sealing the transparent lower tube 1 and that houses a glass ampoule 4 in which a culture solution described later is enclosed between the closed upper end and lower end perforated plate 3. An upper tube 2; a cotton swab 6 having one end held by the perforated plate 3 and the other end extending into the transparent lower tube 1; and a resistant bacterium placed in the transparent lower tube 1. It includes a dry disk chip 5 impregnated with the below-mentioned antibiotics that suppresses the growth of bacteria other than the above.

Here, the culture solution is a mannitol salt medium,
It may be any common liquid medium such as Staphylococcus broth, contains phenol red as an indicator, and the initial pH of the culture solution is 7.4, which is red. The dried disc chip 5 also contains oxacillin which suppresses gram-positive cocci, which has no resistance, azothreonam which suppresses gram-negative bacilli, and polymyxin B antibiotics. The dry disk chip 5 may be of any kind as long as it is an aggregate of fibers such as synthetic resin or natural resin as an adsorption carrier, but a cellulose paper chip is most suitable.

Thus, since at least the antibiotic and the medium are stored in a state where they do not come into contact with each other, it is possible to provide a simple detection device for resistant bacteria which can be stored for a long period of time.

[0016]

In the use of the detection device of the present invention, the sterilized or sterilized bacterium sampling end is taken out from the container, the sample is quickly brought into contact with the subject, and the bacterium is quickly hermetically sealed again. Then, an external force is applied from the outside of the container to integrally mix and contact the bacteria collection end, which has kept the non-contact state, the antibiotic and the medium, and culture them at a constant temperature for a certain period of time by a conventional method. , Turbidity of the medium, growth of colonies, changes in physicochemical properties based on the production substances derived from the proliferated bacteria are detected as "changes in the medium", or the proliferation of compounds previously added to the medium It means that the presence of the bacterium is determined by observing the change in the physicochemical properties of the medium due to the decomposition by the bacterium as "change of the medium". Of course, after culturing, it is possible to take out the medium from the container and make the above observations,
It is preferable that the cells can be observed as they are, and it is preferable that at least a portion of the container for observing the culture condition is transparent. On the contrary, if the contained antibiotics or culture medium may change its properties due to light because it is transparent, a light-shielding coating such as a black tape may be wrapped around the transparent part. It is preferable to keep it in advance, and it is preferable that it is easy to remove when observing the change of the medium.

When an agar medium is used as the medium, the partition wall above the agar medium is made thin and the partition wall is crushed with a cotton swab, followed by applying an external force to the capsule containing the antibiotic and the capsule containing water. A method of destroying and uniformly supplying on the medium is preferable.

Although various explanations have been given on the destruction of the capsule, the ampoule or the partition wall by applying an external force, in short, when applying a force from the outside of the container,
Even if the container is deformed, it does not interfere with culturing, or as long as the culture system is isolated from the outside, in other words, the inside of the container is not contaminated by outside air, and As long as the culture medium and the antibiotic can form a culture system, any action of force can be applied, and any action of force such as dent or light impact can be adopted.

Again referring to FIG. 1, a concrete example of MRSA
Describe the detection method. In "using the detection device" of the present invention, the upper tube 2 is replaced with the lower tube 1.
Then, the upper tube 2 is held and the tip of the swab 6 is rubbed on the nasal mucosa of the patient, the pharynx, or the floor, wall, or the surface of furniture or the like in the area to be inspected. Then, the upper tube 2 from which the sample has been collected is inserted into the lower tube 1 as before, and is engaged and hermetically sealed. The glass ampoule 4 containing the culture solution held in the upper tube 2 is split from the outside using, for example, a commercially available splitting tool, and the enclosed culture solution passes through the perforated plate 3 and moves downward along the cotton swab 6. The tip of the cotton swab is dropped in the tube 1 so that the tip of the cotton swab is immersed in the culture solution. At this time, the broken glass remains on the porous plate 3 and does not hinder the observation after the culture. The culture solution contained in the lower tube 1 comes into contact with the above-mentioned dried disk tip 5 impregnated with the antibiotic, and the antibiotic held by the dried disk tip 5 is dissolved in the culture solution. Then, the lower tube 1
Incubate at 35 ° C. for 24 to 48 hours with the upper tube 2 engaged with. After culturing, when MRSA is present, these decompose mannitol in the culture broth, so that the pH of the culture broth becomes acidic, and the color of the indicator phenol red changes from red to yellow. Therefore, in the determination, the presence of resistant bacteria can be detected by the yellowing of the culture solution.

In determining whether the bacterium whose presence was detected was MRSA or another resistant bacterium, a portion of the culture broth of a sample showing a positive reaction was taken, and 4 -Methylumbelliferyl phosphate is added, and after further culturing for 1 to 2 hours, it can be confirmed by detecting the fluorescence of 4-methylumbelliferone produced by MRSA-produced phosphatase at a wavelength of 365 nm.
At the same time, another part of the sample showing a positive reaction was added to a solution containing rabbit plasma in the same manner as usual, and after culturing, the gelling reaction caused by the coagulase produced by MRSA was confirmed and It can be determined that the drug-resistant bacterium is MRSA.

In this case, the 4-methylumbelliferyl phosphate was impregnated into the disk chip and freeze-dried in the same manner as the above-mentioned antibiotics, while the rabbit plasma was dissolved in an aqueous gelatin solution and molded into a columnar shape. Then, by freeze-drying it into chips, long-term storability is secured.

Hereinafter, specific examples of use of the present invention will be described with reference to examples, but the present invention is not limited thereto.

[0023]

[Example 1] Tryptone 1.0 (w / v)% (hereinafter simply expressed as%), lactose 0.20%, yeast extract
0.25%, D-mannite 1.0%, lithium chloride 0.50
%, Sodium chloride 0.40%, phenol red 0.17%
1.5 ml of liquid medium consisting of purified water and purified water was sealed in a glass ampoule and sterilized at 121 ° C. for 120 minutes. For antibiotics, oxacillin (6 μg / m
l), aztreonam (5 μg / ml) and polymyxin B (10 μg / ml) were impregnated into the disk chips and freeze-dried. The following tests were conducted using the apparatus of Figure 1 containing a glass ampoule and a dry disc chip.

The biopsy specimens were collected from 20 MRSA-affected patients and 20 healthcare workers admitted to Hospital A.
Regarding the positive standard sample (n = 10) as a control, MRSA was used as the tested multidrug resistant standard sample 1 and multidrug resistant CNS was used as the positive standard sample 2 in 104 / tube, respectively. As a negative standard sample, Staphylococcus epidermidis 104 /
Used in tubes. Specimens are collected from each subject's pharynx, nasal cavity, and fingers with a cotton swab, and the cotton swab is placed in a lower tube made of resin (polyethylene: simply referred to as resin below), and the cap is hermetically engaged, and then made of resin. The glass ampoule enclosing the liquid medium in the upper tube of No. 1 was split from outside the upper tube with a splitting tool, and the liquid medium was allowed to flow into the lower resin tube. After culturing at a culturing temperature of 35 ° C. for 24 to 48 hours, the medium change was judged. The judgment was made by observing the change in the color tone of the medium, and those that remained red were judged to be negative, and those that changed to yellow were judged to be positive.

It should be noted that the presence of production of phosphatase and coagulase in the positive sample in which the medium turned yellow was further checked for 4
-Methylumbelliferone was investigated by fluorescence and rabbit plasma gelation.

Table 1 shows the test results by the discoloration of the medium for the presence of multidrug-resistant bacteria, and Table 2 shows the results of the phosphatase and coagulase tests conducted on the positive samples.

[0027]

[Table 1] Test results Species Target site MRSA sufferers Medical staff (Change in color tone of medium) ( Change in color tone of medium ) Pharynx 20/20 3/20 Nasal cavity 20/20 2/20 Fingers 12/20 4/20 Positive standard sample 1 10/10 Positive standard sample 2 10/10 Negative standard sample 0/10

[0028]

[Table 2] Test results Species Target site MRSA Affected patient Medical patient Fosufatase Bu test Koaku Bu Bu Rase test Fosufatase Bu test Koaku Bu Bu Rase test throat head 20/20 20/20 2/20 2/20 nose cavity 20/20 20/20 1/20 1/20 fingers 8/20 8/20 2/20 2/20 Positive standard sample 1 10/10 10/10 Positive standard sample 2 10/10 10/10 Negative standard sample 0/10 0/10

In Tables 1 and 2 above, the test results are displayed as follows. Change in color tone of liquid medium: Number of cases where color tone of medium changed from red to yellow / total number of samples Phosphatase test: Number of positive cases that fluoresce in blue /
Total number of samples Coagulase test: Number of positive cases of gelation of rabbit plasma solution / Total number of samples

As is clear from Table 1, the detection result is MRSA.
All cases were positive in the pharynx and nasal cavity of affected patients. The finger had a detection rate of 60%. The pharynx, nasal cavity,
In the fingers, the detection rates were 15%, 10%, and 20%, respectively.

Furthermore, in the phosphatase test and the coagulase test performed on the positive cases in which the color tone of the liquid medium changed from red to yellow, it is clear from Table 2 that MRSA
All cases were positive in the pharynx and nasal cavity of affected patients. The finger had a detection rate of 60%. The pharynx, nasal cavity,
In the fingers, the detection rates were 10%, 5%, and 10%, respectively. This means that the difference between the positive result in the simple detection device of the present invention in the medical staff and the positive result in the phosphatase test or the coagulase test, other than MRSA,
This means that CNS with the same multidrug resistance was also detected.

[0032]

[Example 2] Using the simple detection device of the present invention prepared in Example 1, the storage stability of the conventional method was compared and examined.
In the conventional method, a commercially available agar plate medium for MRSA screening (Food Stamp "Nissui" MSO agar; Nissui Pharmaceutical Co., Ltd.) was used, and the agar plate medium was stored after refrigeration for 3 months before use. On the other hand, the simple detection device of the present invention was used after being stored at 15 ° C for 1 year and at 40 ° C for 2 months. The test was performed 3 times for each test strain.

In the assay, MRSA as Gram-positive bacteria,
CNS, Staphylococcus epidermidis, Staphylococcus aureus, Enterococus faecalis, Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Serratia marcesces, Serassia marcesces 104 / Proteus vulgaris, Pseudomonas aeruginosa
Tested using in tubes.

The results obtained are shown in Table 3.

[0035]

[Table 3] Test results Tested bacteria Detection device of the present invention Commercial agar medium for MRSA screening A B C D MRSA 3/3 3/3 3/3 3/3 CNS 3/3 3/3 3/3 3/3 S. Extended duty 0/3 0/3 1/3 3/3 S.E. Aureus 0/3 0/3 0/3 3/3 E. Faecalis 0/3 0/3 1/3 3/3 E. Kori 0/3 0/3 0/3 2/3 K. Pneumonier 0/3 0/3 0/3 3/3 S. Marcescence 0/3 0/3 0/3 3/3 P. Burgeris 0/3 0/3 0/3 2/3 P.I. Aeruginosa 0/3 0/3 0/3 3/3 A: 15 ° C, 1 year storage B: 40 ° C, 2 months storage C:
Store under refrigeration for 1 week D: Store under refrigeration for 3 months

From the above results, in the simple detection device of the present invention, both the stored product for 1 year at 15 ° C. and the storage for 2 months at 40 ° C., and the yellowing of the color tone of the liquid medium was observed in all MRSA and CNS cases. (Positive) was observed, and all other bacteria were negative. When the commercially available agar medium, which is a conventional method, was stored in a refrigerator for 3 months, bacteria other than MRSA and CNS also grew and MRSA and CNS could not be detected correctly. It is considered that this is because the antibiotic contained in the agar medium was decomposed and the growth inhibition of bacteria other than MRSA and CNS was weakened. As described above, the commercially available agar medium has a problem in storage stability. In the simple detection device of the present invention, since the disk chip in which the antibiotic is freeze-dried and the liquid medium enclosed in the glass ampoule are separated and not in contact with each other in the simple detection device until just before the culture, the antibiotic is activated. Stable and long-term storage can be achieved.

[0037]

EFFECTS OF THE INVENTION The simple detection device according to the present invention is suitable for detecting resistant bacteria capable of long-term storage because the bacteria collection end, the culture medium and the antibiotics are stored in a container in a contact manner and collectively. A device is provided, and as a result, the operations from bacterial collection to culture and the determination operation after culture can be performed in a very short time, and a large number of samples can be processed reliably without requiring skill and expertise. It provides a way to do it. Therefore, it is possible to detect a resistant bacterial infection in a large number of collected samples at an early stage, and it can be useful for preventing nosocomial infections such as MRSA, which is a problem. In addition, it can be applied and applied to a culture device in which not only antibiotics but also storage-unstable media are divided and stored to provide stability.

[Brief description of drawings]

FIG. 1 is a schematic view of a simple detection device for multidrug-resistant bacteria according to the present invention.

[Explanation of symbols]

 1 Lower tube 2 Upper tube 3 Perforated plate 4 Glass ampoule 5 Dry disc tip 6 Cotton swab

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Atsushi Sanwa 1-6-4 Onaridai, Wakaba-ku, Chiba-shi, Chiba

Claims (1)

[Claims] 1. An apparatus for detecting a bacterium by culturing the bacterium in a medium and observing a change in the medium, which comprises a bacterium sampling end, an antibiotic and a medium which are integrally brought into contact with each other by the action of an external force. At least the antibiotic and the medium are arranged in a non-contact manner in a container, and the selective resistant bacteria detecting device is characterized.