WO2012043456A1 - Device for isolating spirillum and isolation method - Google Patents

Abstract

The present invention provides an improved filter method capable of more increasing the isolation rate of spirillum than the existing filter method and a device to be used for the same, and more specifically, a device for isolating spirillum, the device including a filter composed of polycarbonate, and an isolation method for spirillum using the device.

Description

Device and method for separating spiral bacteria

This patent application claims priority with respect to Japanese Patent Application No. 2010-214851, which is hereby incorporated by reference in its entirety.
The present invention relates to a novel device for separating spiral bacteria and a separation method that can be used for testing spiral bacteria such as Campylobacter in a specimen (clinical specimen, food, etc.).

In recent years, although food safety has been screamed and countermeasures have been taken by related parties, the incidence of food poisoning has not decreased at all. Among them, the number of food poisoning caused by Campylobacter spp. Is the highest in Japan, and is almost the same as that caused by norovirus. More than 19 species of genus Campylobacter have been reported.

In Japan, only Campylobacter jejuni and Campylobacter coli are designated as food poisoning bacteria, and both of these species account for 90% to 95% or more of Campylobacter infection-causing bacteria. Various problems in testing for Campylobacter spp .: (1) Special equipment is required because microaerobic conditions (85% N ₂ , 10% O ₂ , 5% C0 ₂ etc.) are required for culture, (2) It takes 2 to 3 days of culture, and it takes 8 to 10 days to determine the bacterial species. (3) The biochemical properties are very similar, making it difficult to distinguish bacterial species. Has been pointed out.

In addition, only C. jejuni and C. coli are tested in Japan, and the currently used medium was developed for C. jejuni and C. coli, so other Campylobacter spp. Infections caused by may be missed. Thus, the investigation of infectious diseases by Campylobacter in non-C. Jejuni / non-C. ク タ ー coli is not yet sufficient.

Since the identification of Campylobacter spp. As an important pathogen in humans and animals, various separation methods and media improvements and developments have been made. First used in 1972, Dekeyser et al. (Non-Patent Document 1) used a mixed cellulose filter with a pore size of 0.65 μm to physically separate the bacteria by motility.

Later, in 1977, Skirrow et al. Found that most Campylobacter bacteria suppressed the growth of antibacterial agents, trimethoprim (Trimethoprim; Gram-positive, negative Neisseria gonorrhoeae and cocci), and vancomycin (Vancomycin). ) And polymyxin B (Polymyxin B; suppresses the growth of Gram-negative gonococci), and developed a Skirrow medium utilizing the sensitivity to the normal bacterial flora in feces (Non-patent Document 2).

Further, developed by Lauwers, Butzler et al., Bacitracin (Bacitracin; suppresses the growth of Gram-positive and negative Neisseria gonorrhoeae and cocci), cycloheximide (Cycloheximide; suppresses the growth of fungi), colistin sulfate (Colistin sulfate; Gram-negative bacteria, Butzler medium (suppressing the growth of Pseudomonas aeruginosa), cephalothin (Cephalothin; suppressing the growth of Gram-positive and negative Neisseria gonorrhoeae and cocci), Novobiocin (Novobiocin; suppressing the growth of Gram-positive and Gram-negative bacteria) (non-patent literature) 3) Furthermore, Bolton et al. Developed charcoal, ferrous sulfate and sodium pyruvate, casein hydrolyzate, sodium deoxycholate instead of blood, and cefoperazone as an antibacterial agent. Modified 、 CCDA medium using amphotericin B (Amphotericin B; suppresses fungal growth) Developed (Non-Patent Document 4). In addition, a blood-free medium developed by Karmali et al., A Karmali medium containing charcoal, sodium pyruvate, cefoperazone, and cycloheximide (Non-patent Document 5) are known.

Thus, since the Skirrow medium was developed by Skirrow et al. In 1977, separation and selection media containing many antibacterial agents have been developed and compared. However, since the medium containing antibacterial agents and the current culture method are mainly intended for detection of C. jejuni and C. coli, it is highly possible that other than these two species are missed.

For example, C. fetus subsp. Fetus, C. cinaedi (current Helicobacter cinaedi), C. fennelliae, C. hyointestinalis, etc. are sensitive to Cephalothin and do not grow in Butzler medium containing this. In addition, Goosens and Butzler oppose the use of a medium containing an antibacterial agent and recommend the use of a filtration culture method using a filter (Non-patent Document 6). In 1998, Roux and Lastvica performed filter filtration culture using non-selective medium at 37 ° C under hydrogenated microaerobic conditions (Cape Town Protocol), and C. 痢 concisus, C. upsaliensis, Various bacterial species such as C. fetus, C. hyointestinalis, and C. lari were isolated (Non-patent Document 7).

As described above, the advantages of the originally developed filter method that does not contain an antibacterial agent are widely known, and an improved filter method has also been developed (for example, Patent Document 1), but the operation is complicated. C. jejuni and C. coli are not widely used because of their disadvantages, such as slightly inferior to selective media containing other antibacterial agents. In Japan, the Skirrow medium is mainly used for patient specimens such as feces, and the mCCDA medium is often used for food inspection.

Special table 2009-532562

As described above, the separation medium containing an antibacterial agent is a medium mainly for the separation of C. jejuni and C. coli, and none can be applied to all Campylobacter species.
On the other hand, as a test method for Campylobacter spp., The filter method that can be separated regardless of the species of Campylobacter spp. Is mentioned.
The present invention is an improved filter method that can increase the separation rate of spiral bacteria than the existing filter method, a device (filter unit, etc.) that can be used in the method, and separation and detection of spiral bacteria that can be easily inspected. It is an object to provide a method.

As a result of examining the membrane material used for the filter method in detail for the purpose of solving the above-mentioned problems, the present inventors have found a filter showing a separation rate of spiral bacteria that is inferior to that of an existing selective medium. It came to be completed.
That is, the present invention includes the following:
[1] A device for separating spiral bacteria, comprising a filter comprising polycarbonate.
[2] The device according to [1] above, wherein the filter has a pore size in the range of 0.2 to 0.8 μm.
[3] The device according to [2] above, wherein the filter has a pore size in the range of 0.45 to 0.6 μm.
[4] The device according to [1] or [2] above, wherein the spiral bacterium is selected from bacteria belonging to the order of Campylobacter.
[5] The device according to [4] above, wherein the bacterium belonging to Campylobacter is selected from Campylobacter, Helicobacter and Arcobacter.
[6] Separation of spiral bacteria, comprising the step of bringing a specimen that may contain spiral bacteria into contact with the filter of the device according to any one of [1] to [3] above and allowing the spiral bacteria to pass through the filter Method.
[7] The method according to [6] above, wherein the step is performed under pressure, under reduced pressure by suction, or by centrifugal filtration.
[8] The method according to [6] or [7] above, wherein the spiral bacterium is selected from bacteria belonging to the order of Campylobacter.
[9] The method according to [8] above, wherein the bacterium belonging to Campylobacter is selected from Campylobacter, Helicobacter and Arcobacter.
[10] A method for detecting spiral bacteria comprising the following steps:
(I) a step of bringing a specimen capable of containing a spiral bacterium into contact with the filter of the device according to any one of the above [1] to [3], and allowing the spiral bacterium to pass through the filter;
(Ii) A step of detecting the filamentous fungus after passing through the filter.
[11] The method according to [10] above, further comprising a step of culturing the spiral fungus passing through the filter before detection.
[12] The method according to [10] or [11] above, wherein the step (i) is performed under pressure, reduced pressure by suction, or centrifugal filtration.
[13] The method according to any one of [10] to [12] above, wherein the spiral bacterium is selected from bacteria belonging to the order of Campylobacter.
[14] The method according to [13] above, wherein the bacterium belonging to Campylobacter is selected from Campylobacter, Helicobacter, and Arcobacter.

According to the device and method of the present invention, by using a specific filter that selectively allows spiral bacteria to pass through, the spiral bacteria contained in the sample can be easily separated at a separation rate comparable to that of an existing selective medium, Can be detected. Therefore, the device and method of the present invention can be suitably applied to the inspection and identification of causative agents of food poisoning.
In addition, according to the present invention, spiral bacteria such as Campylobacter spp., Helicobacter spp., Arcobacter spp., Etc. that have been overlooked other than C. jejuni and C. coli, and C. jejuni sensitive to antibacterial agents C. coli can be separated and examined efficiently.
Furthermore, by applying the present invention to the inspection, it is possible to easily identify and inspect colonies of spiral bacteria including Campylobacter spp.

It is a figure which shows the filter passage experiment using Campylobacter genus bacteria and Alcobacter genus bacteria.

In the present invention, the term “spiral bacterium” means a bacterium having a spiral shape, for example, a bacterium belonging to the order of Campylobacterales, including, for example, the genus Campylobacter (for example, C. jejuni, C. coli, C. fetus, C. hyointestinalis, C. concisus, C. upsaliensis, C. lari, C. helveticus, C. hominis, C. lanienae, C. canadensis, C. curvus, C. insulaenigrae, etc.), Helicobacter genus Examples include fungi (for example, Helicobacter pylori, Helicobacter cinaedi, etc.), Alcobacter bacteria (for example, A. butzleri, A. Cryaerophilus, A. skirrowii, A. nitrofigilis, etc.).

The filter according to the present invention comprises polycarbonate as a material and can selectively pass spiral bacteria.
The filter may contain a material other than polycarbonate as long as the effects of the present invention are not impaired. For example, the filter contains other materials up to 50% by weight, preferably up to 60% by weight. May be. Such a material is not particularly limited as long as it can be used for a filter.
Further, the polycarbonate may be chemically modified, for example, may be subjected to treatment with vinyl chloride, polyester, epoxy, urethane, silicon, polyethylene, fluorine, or the like.

The pore size of the filter is preferably in the range of 0.2 to 0.8 μm, more preferably in the range of 0.45 to 0.6 μm, from the viewpoint of selectively separating spiral bacteria.
In addition, the hole diameter as used in this specification is the maximum hole diameter determined by the bubble point test method.

Further, the filter preferably has a thickness of about 7 to 22 μm, is translucent, and has a smooth surface such as glass. Moreover, what is produced so that a hole diameter is exact and a hole diameter distribution becomes constant is preferable.

The device for separating spiral bacteria of the present invention is not particularly limited as long as it contains the filter as an essential component. For example, the filter itself may be used, or a filter unit (one integrated with the filter) or a kit (one not integrated with the filter) may be configured together with other components.
Examples of components other than the filter in the device include, for example, a member for supporting the filter (such as a frame member), a specimen receiver, and a member for introducing the specimen into the filter by applying pressure (for syringes and centrifuges). Cups, adapters such as suction units, etc.), various members of conventional filter units such as filtration filters, medium for cultivating fungi when passing through the filter, members for collecting fungi when passing through the filter, etc. Can be mentioned.

The medium for culturing the above spiral bacteria is not particularly limited, for example, blood agar medium, Brucella medium, Mueller Hinton medium, Nutrient broth medium, skilow medium, mCCDA medium, Kalmari medium, Butler medium, Bolton medium, Examples include media usually used for culturing spiral bacteria such as Preston's medium and CAT medium.
The medium may contain various antibacterial agents, and may contain, for example, trimethoprim, vancomycin, polymyxin B, bacitracin, cycloheximide, colistin sulfate, cephalothin, novobiocin, cefoperazone, amphotericin B, and the like.

The present invention also provides a method for separating spiral bacteria using the above device. The method includes the step of bringing a specimen that may contain a spiral bacterium into contact with the filter of the device and allowing the spiral bacterium to pass through the filter.

The specimen that can contain the above spiral fungus is not particularly limited as long as it is a specimen that may contain a spiral fungus, for example, feces, food, milk, pet food, excrement, rivers, ponds, Environmental water such as aquarium, well water, and drinking water.

When a specimen that can contain the above spiral bacteria is brought into contact with the filter of the above device, if spiral bacteria are present in the specimen, the spiral bacteria are likely to pass through the filter, and other bacterial species are difficult to pass through the filter. It is possible to selectively isolate spiral bacteria.
The method for bringing the specimen into contact with the filter of the device is not particularly limited. For example, the specimen may be dropped or poured on the filter, or the specimen may be applied on the filter.

The above step may be performed without applying pressure, or may be performed under pressure (under pressure). When performing the said process under pressure, it can carry out, for example, pressing a test substance on a filter using a syringe etc. Alternatively, suction filtration may be performed using a suction unit. Alternatively, centrifugation can be performed after the sample is introduced onto the filter.
When carried out under pressure, the rate of the spiral bacteria passing through the filter is increased, so that the spiral bacteria can be separated from other bacterial species more rapidly.

In addition, in the above-mentioned method for separating spiral bacteria, a selective medium such as a skilow medium, mCCDA medium, butler medium, and karmari medium can be used in combination.

The present invention also provides a method for separating spiral bacteria using the above device and then detecting the spiral bacteria. The method includes the step of (i) bringing the specimen that may contain a spiral bacterium into contact with the filter of the device and allowing the spiral bacterium to pass through the filter, and (ii) detecting the bacterium after passing through the filter.
The said process (i) can be performed similarly to the isolation | separation method of the said spiral bacterium.

The detection of spiral bacteria in the above step (ii) was performed using Gram staining, catalase test and oxidase test, Multiplex PCR, PCR of hippuric acid hydrolase gene specifically possessed by C. jejuni, and live using API Campy (BIOMERIEUX). It can be carried out by using conventional methods for identifying spiral bacteria such as chemical property tests and modified methods based thereon.

The method for detecting a spiral bacterium according to the present invention may further include a step of culturing the bacterium after passing through the filter before detecting the spiral bacterium in the step (ii). Thereby, even when the number of spiral bacteria contained in the sample is small, the spiral bacteria can be detected.
As a culture medium used for the said culture | cultivation, the culture medium etc. which were illustrated in description of the said device are mentioned, for example. In addition, the culture medium may contain an antibacterial agent such as the antibacterial agent exemplified in the description of the above device. In that case, without growing other bacterial species sensitive to the antibacterial agent to be used, a spiral bacterium Can be selectively grown.
Moreover, the culture conditions such as temperature and carbon dioxide (or oxygen) concentration are not particularly limited, and conditions usually used for culture of spiral bacteria can be used. For example, a microaerobic condition at 37 ° C. on a blood agar medium (O ₂ concentration: about 3 to 10%, CO ₂ concentration: 5 to 15%, N ₂ concentration: about 75% to 92%, for bacterial species that require hydrogen gas replaces with H ₂ concentration of about 0-60% in place of nitrogen gas as required) under about 12 to 72 hours, with respect to the slow strain of growth can be cultured under the conditions of about 10 days.

In the above method for detecting spiral bacteria, colonies characteristic of clear or grayish light pink spiral bacteria formed on an agar medium are formed, and gram-staining and spiraling are performed under the microscope by Gram staining. This can be done by observing the shape. It can also be observed directly under a microscope to observe spirally-shaped cells with high mobility and linear movement. Further, if necessary, genetic tests such as PCR, colony hybridization, and analysis of 16S rRNA gene targeting genes capable of detecting spiral bacteria can be used in combination.
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

In the following examples, C. jejuni 81-176 (hereinafter referred to as Cj 81-176) was used as Campylobacter, and Citrobacter amalonaticus (patient specimen isolate P3211f + 4), Escherichia coli ( Three strains of patient specimen isolate P3210f + 6) and Pseudomonas aeruginosa (patient specimen isolate P2497f + 4) were used.
As filters, mixed cellulose filters with pore sizes of 0.45 μm, 0.65 μm, and 0.8 μm (hereinafter referred to as MC filters) (Nihon Millipore Corporation) and polycarbonate filters with pore sizes of 0.4 μm, 0.6 μm, and 0.8 μm (hereinafter referred to as PC filters) ( Japan Millipore Corporation) was used.

(Examples 1 to 3 and Comparative Examples 1 to 3)
Cj 81-176 was cultured on a blood agar medium for 18 hours, colonies on the medium were scraped off, suspended in PBS, and a bacterial solution with an OD ₆₀₀ = 0.1 was prepared. 10 ³ , 10 ² , 10 ¹ cfu / 100 μL of bacterial solution prepared by serially diluting bacterial solution with turbidity of 0.1 on a blood agar medium 0.45 μm, 0.65 μm, 0.8 μm Three 100 μL each were inoculated on a filter and a PC filter having a pore size of 0.4 μm, 0.6 μm, and 0.8 μm. After standing at 37 ° C. for 30 minutes under microaerobic conditions, the filter was removed, and the cells were cultured for 2 days under 37 ° C microaerobic conditions, and the average number of colonies that passed through (hereinafter referred to as the number of bacteria passing through) was calculated. The colony count in the added bacterial solution was similarly performed using a blood agar medium, and the ratio of the number of passaged bacteria to the number of bacteria in the added bacterial solution was calculated as the passage rate.
In the filter passage experiment using a pure culture of Cj 81-176, the passage rates were 0.6%, 0.7%, and 1.7% for the MC filter pore sizes of 0.45 μm, 0.65 μm, and 0.8 μm, respectively. Moreover, when the pore sizes of the PC filter were 0.4 μm, 0.6 μm, and 0.8 μm, the passing rates were 1.9%, 10.9%, and 21.8%, respectively (Table 1). Both filters showed a tendency for the passage rate to increase as the pore size increased. However, with the pore sizes of 0.6 μm and 0.8 μm, the MC filter and the PC filter showed a PC filter passage rate 10 times higher. .

(Example 4 and Comparative Example 4)
The methods for culturing Cj 81-176 and non-Campylobacter and preparing a diluted bacterial solution were the same as described above. Cj 81-176 of ^{10 2 cfu / 100 μL, C} . Amalonaticus, E. Coli, and were prepared to P. aeruginosa of ^{10 4, 10 3, 10 2} , 10 1, 10 0 cfu / 100 μL. C. amalonaticus, E. coli, and P. aeruginosa are mixed with Cj 81-176, and the number of non-Campylobacter bacteria against Cj 81-176 is 1: 100, 1:10, 1: 1. , 10: 1, 100: 1 mixed bacterial solution was prepared. 100 μL each of the mixed bacterial solution was inoculated on two types of filters, a MC filter having a pore size of 0.65 μm and a PC filter having a pore size of 0.6 μm, placed on a blood agar medium. After leaving still at 37 ° C under microaerobic condition for 30 minutes, the filter was removed, and further cultured under microaerobic condition for 2 days. After culturing, the numbers of bacteria passing through Cj 81-176 and C. amalonaticus, E. coli and P. aeruginosa at each concentration were calculated according to the above method. Each colony was identified by colony shape and Gram staining.

When Cj 81-176 and C. amalonaticus, E. coli and P. aeruginosa were mixed and the number of bacteria passed through each filter was compared, the MC filter with a pore size of 0.65 μm mixed Cj 81-176 and C. amalonaticus. In the bacterial solution, the average number of bacteria passing through Cj 81-176 was 0.1 cfu, while that of C. amalonaticus was 10 ⁴ , 10 ³ , 10 ² , 10 ¹ , 10 ⁰ cfu / 100 μL. And 9.7 cfu, 0.67 cfu, 0.3 cfu, 1.3 cfu, and 0 cfu, respectively. In the mixed bacterial solution of Cj 81-176 and E. coli, the average number of bacteria passing through Cj 81-176 was 0.3 cfu, whereas in E. coli, 2.0 cfu, 0.3 cfu, 0 cfu, 0.7 cfu, and 1.7 cfu. Similarly, in the mixed bacterial solution of Cj 81-176 and P. aeruginosa, the average passage number of Cj 81-176 was 0.3 cfu, whereas P. aeruginosa was 43 cfu and 5.7 at the above five types of bacterial concentrations, respectively. cfu, 0 cfu, 0.7 cfu, 1.0 cfu.

In the PC filter with a pore size of 0.6 μm, the average number of cells passing through Cj 81-176 was 5.9 cfu in the mixed bacterial solution of Cj 81-176 and C. amalonaticus, whereas C. amalonaticus passed at any bacterial concentration. Was not seen. In the mixed solution of Cj 81-176 and E. coli, the average passage number of Cj 81-176 was 10 cfu, whereas in E. coli, no passage was seen at any concentration. Similarly, in the mixed bacterial solution of Cj 81-176 and P. aeruginosa, the average number of bacteria passing through Cj 81-176 was 3.4 cfu, whereas P. aeruginosa had the highest concentration of 0.7 cfu, and the other 4 No passage was seen at different bacterial concentrations. That is, it was found that the PC filter not only has a high passing rate of Campylobacter but also eliminates other bacteria more efficiently than the MC filter.
The results are shown in Table 2.

From the results in Table 2, a sample prepared by mixing spiral bacteria (Campylobacter jejuni: Cj 81-176) and C.lonamalonaticus at a ratio of 1: 100 was applied to the filter method using mixed cellulose (MC) and polycarbonate (PC) filters. When isolated and cultured, the mixed cellulose (MC) filter showed no passage of spiral bacteria (Cj), and an average of 9.7 colonies were observed in the mixed C. amalonaticus. However, when a polycarbonate (PC) filter was used, mixed C. amalonaticus colonies were not observed, and an average of 3.7 colonies of spiral bacteria (Cj) were observed. Even if the mixing ratio is changed or other bacterial species are mixed, the same results are seen in the passage, so the filter method using a polycarbonate (PC) filter clearly eliminates bacteria other than spiral bacteria. I found out.

(Example 5 and Comparative Example 5)
44 rectal swabs from 44 Campylobacter-positive diarrhea patients were suspended in 500 μL of sterile physiological saline, and separated according to the above method using a filter method using a PC filter with a pore size of 0.6 μm and mCCDA medium. Comparison of methods was performed.
Using 44 specimens of Campylobacter positive diarrhea patients, we compared the mCCDA medium, which had the highest positive rate in the examination of patient specimens, with the filter method using a PC filter with a pore size of 0.6 μm.
As a result, Campylobacter was separated from 36 of 44 positive samples in mCCDA medium, and the separation rate was 81.8% (Table 3). This was almost the same result as when the patient specimens were first examined (Table 3). On the other hand, when a PC filter having a pore size of 0.6 μm was used, Campylobacter was separated from 37 samples out of 44 positive samples, and the separation rate was 84% (Table 3).
From these facts, the new Campylobacter separation method using a PC filter was considered to be an excellent separation method that facilitates visual discrimination of bacteria.

(Example 6 and Comparative Example 6)
<Purpose>
The performance of the separation device of the present invention is evaluated for bacteria belonging to the order of Campylobacterales.
<Method>
C. jejuni (81-176), C. coli (ATCC33559), C. fetus (ATCC27374), C. lari (ATCC43675), C. upsaliensis (ATCC43954), C. hyointestinalis (ATCC35217), A. butzleri (ATCC49616) Was cultured overnight at 37 ° C. under microaerobic conditions (5% O ₂ , 10% CO ₂ , 85% N ₂ ), and the resulting cells were diluted with sterile PBS (−). Dilute the diluted bacterial solution serially and inoculate 100 µl each on a mixed cellulose (MC) filter with a pore size (φ) of 0.65 µm and a polycarbonate (PC) filter with φ0.6 µm on a blood agar medium. did. After leaving still at 37 ° C under microaerobic condition for 30 minutes, the filter was removed, and the cells were cultured at 37 ° C under microaerobic condition for 2 to 3 days, and the number of colonies obtained was calculated. The prepared bacterial solution was similarly cultured on a blood agar medium, and the number of added bacteria was calculated. The number of bacteria passing through each filter was divided by the number of added bacteria, and the passage rate was calculated.
<Result>
The recovery rate of C. jejuni (81-176) with the MC filter was 1.74 ± 0.32%, while that of the PC filter was 12.63 ± 2.1%, about 7 times higher than that of the MC filter. In addition, C. fetus (ATCC27374), C. lari (ATCC43675), and A. butzleri (ATCC49616) also showed higher values of the PC filter, about 5 times, about 9 times, and about 4 times, respectively. C. upsaliensis (ATCC43954) and C. hyointestinalis (ATCC35217) could not be recovered at all with the MC filter, but the PC filter showed a high recovery rate of 41 ± 18% and 6.22 ± 1.68%, respectively. On the other hand, C. coli (ATCC33559) did not show a significant recovery improvement like other bacteria, but the recovery rate with the MC filter was 1.57 ± 0.71%, while the PC filter was 1.90 ± 1.24%, About 1.2 times the PC filter showed a higher value (Table 4, FIG. 1).
<Discussion>
Separation with PC filters was effective for C. jejuni and C. coli, which are important as food poisoning bacteria, as well as other Campylobacter bacteria, and Alcobacter bacteria that have recently attracted attention as food poisoning bacteria.

Claims (14)

1. A device for separating spiral bacteria, including a filter comprising polycarbonate.
2. 2. The device according to claim 1, wherein the filter has a pore diameter in the range of 0.2 to 0.8 μm.
3. 3. The device according to claim 2, wherein the filter has a pore diameter in the range of 0.45 to 0.6 μm.
4. The device according to claim 1, wherein the spiral bacterium is selected from bacteria belonging to Campylobacter.
5. The device according to claim 4, wherein the bacteria belonging to the order of Campylobacter are selected from Campylobacter, Helicobacter and Arcobacter.
6. A method for separating a spiral bacterium, comprising a step of bringing a specimen that may contain a spiral bacterium into contact with the filter of the device according to any one of claims 1 to 3, and allowing the spiral bacterium to pass through the filter.
7. The method according to claim 6, wherein the step is performed under pressure, under reduced pressure by suction, or by centrifugal filtration.
8. The method according to claim 6 or 7, wherein the spiral bacterium is selected from bacteria belonging to Campylobacter.
9. The method according to claim 8, wherein the bacterium belonging to the order Campylobacter is selected from Campylobacter, Helicobacter and Arcobacter.
10. A method for detecting spiral bacteria comprising the following steps:
    (I) contacting the sample that may contain spiral bacteria with the filter of the device according to any one of claims 1 to 3, and passing the spiral bacteria through the filter;
    (Ii) A step of detecting the filamentous fungus after passing through the filter.
11. The method according to claim 10, further comprising a step of culturing the filamentous fungus that passes through the filter before detection.
12. The method according to claim 10 or 11, wherein the step (i) is carried out under pressure, under reduced pressure by suction, or by centrifugal filtration.
13. The method according to any one of claims 10 to 12, wherein the spiral fungus is selected from bacteria belonging to the order of Campylobacter.
14. The method according to claim 13, wherein the bacterium belonging to the order Campylobacter is selected from Campylobacter, Helicobacter and Arcobacter.

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