JP2014183802A - Culture medium for detecting group b streptococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture medium enabling detection of group B Streptococcus in a simple process regardless of pigment production.SOLUTION: The culture medium contains a β-glucuronidase substrate having a group that emits detectable fluorescence when released. To this culture medium, a swab sample is stabbed and group B Streptococcus is cultured to detect fluorescence. Further, when the culture medium contains glucuronic acid salt that induces β-glucuronidase, the sensitivity is improved to a large extent. Use of the culture medium of the invention enables detection of non red pigment-producing group B Streptococcus not detectable by Granada medium.

Description

  The present invention relates to a medium used for detection of group B streptococci.

  Group B Streptococcus is a Gram-positive gonococci and is a permanent bacterium in the vagina and intestinal tract. When a pregnant woman holds the bacterium in the vagina, it may cause death or meningitis to the newborn, and management of the bacterium is important in terms of countermeasures against infection of the newborn. (Non-Patent Document 1). In the United States, CDC (American Center for Disease Control and Control) has prepared guidelines for screening tests for this bacterium as a countermeasure against perinatal infection. If the presence of this bacterium is detected during the perinatal period, measures will be taken to prevent mother-to-child transmission.

  The screening method for group B streptococci recommended by the CDC guidelines is to perform selective enrichment culture of a vaginal or rectal swab specimen in a medium called Granada medium (Non-patent Document 2) at 35 ° C. for 24 hours. Many group B streptococci produce a red carotenoid pigment in this medium. A specimen in which pigment production is observed in the medium is determined to be positive for this bacterium. On the other hand, if the growth of bacteria is observed in the medium but no pigment production is observed, antigens are extracted from the grown bacteria in the medium and confirmed by immunological techniques such as latex agglutination. It is supposed to do. (Non-Patent Document 3)

Various media for selectively growing group B streptococci in swab specimens have been devised (Patent Document 1), all of which use red pigment productivity in the presence of starch in Granada medium as an indicator of group B streptococci Although detected, about 10% of group B streptococci have been reported as non-pigmenting strains (Non-patent Document 4). Therefore, when the test is performed according to the CDC guidelines, the growth of bacteria is observed in many cases, but the pigment production is not observed, because the selectivity of the medium using many Granada medium is not perfect. Results are frequent.
This means that a screening test using Granada medium requires a deterministic test using an immunological technique for many specimens, and this is a method that requires a lot of cost and labor.

Patent Publication No. Hei 9-313171

Guidelines for Obstetrics and Gynecology Practice-Obstetrics 2011 Japan Gynecology Association, Japan Gynecology Association pp239-241 Manuel de la Rosa, Ricardo Villareal, Dolores Vega, Consuelo Miranda, and Antonio Martinezbrocal: Granada medium for detection and identification of group BStreptococci. Journal of Clinical Microbiology, vol.18 (1983) pp779-785. U.S. Center for Disease Control and Prevention, Prevention of Perinatal Group B Streptococcal Disease, Revised Guidelines from CDC, 2010, Morbidity and Mortality Weekly Report, vol.59 (2010) SilvieNickmans, ElineVerhoye, An Boel, Kristien Van Vaerenbergh, Hans De Beenhouwer: Possible solution to the problem of nonhemolytic group B Streptococcus on granada medium.Journal of Clinical Microbiology, vol.50 (2012) pp1132-1133.

  An object of the present invention is to provide a medium capable of detecting all group B streptococci including non-pigmented group B streptococci by simple operations.

  In view of such circumstances, the present inventor has conducted intensive research. First, many group B streptococci produce red carotenoid pigments, but they are chromogenic enzyme substrates with chromogenic free radicals commonly used in culture media, such as the β-glucuronidase possessed by group B streptococci. For coloring with 5-bromo-4-chloro-3-indoxyl-β-glucuronide, 5-bromo-6-chloro-3-indoxyl-β-glucuronide, which are chromogenic β-glucuronidase substrates that can be used as substrates It is difficult to visually confirm the formation of a clear chromogen due to interference with carotenoid pigments by Granada Medium. Thus, when a substrate having a free group capable of detecting fluorescence by β-glucuronidase possessed by group B streptococci was added alone, fluorescence excited by fluorescence excitation means was observed. However, since the formation was weak, it was similarly difficult to judge visually. Therefore, various studies were conducted, and surprisingly, by using a β-glucuronidase substrate having a detectable fluorescent free group and glucuronate in combination, regardless of the ability to produce a red carotenoid pigment, The present invention was completed by finding that the presence of group B streptococci can be detected by fluorescence of the medium.

That is, the present invention is as follows.
1. A medium for detecting group B streptococci, comprising a β-glucuronidase substrate having a detectable fluorescent free group.
2. 2. The culture medium according to 1 above, containing glucuronate for inducing a β-glucuronidase substrate.
3. 3. The medium according to 1 or 2 above, which contains xanthan gum in order to enhance the carotenoid pigment-producing ability of the pigment-producing group B streptococci.
4). A method for detecting group B streptococcus, comprising inoculating and culturing a sample in the medium according to any one of 1 to 3, and observing the presence or absence of a red pigment in the medium.
5. A method for detecting group B streptococcus, comprising inoculating and culturing a sample in the medium according to any one of 1 to 4 and observing the generation of fluorescence in the medium excited by fluorescence excitation means. .

  By using the medium of the present invention, non-red pigment-producing group B streptococci that cannot be detected on the Granada medium can be detected by fluorescence with a β-glucuronidase substrate having a detectable fluorescent free group, and glucuronic acid By using a salt in combination, it can be detected more reliably and easily.

  The medium of the present invention is obtained by adding a β-glucuronidase substrate having a fluorescent free group described below and other components to a basic medium. The basic medium is preferably a Granada medium.

Examples of the β-glucuronidase substrate having a detectable fluorescent free group for use in the present invention include β-glucuronide or a salt thereof capable of releasing a fluorescent chromogenic compound. Examples include 4-methylumbelliferyl-β-D-glucuronide, 4-trifluoro-methylumbelliferyl-β-D-glucuronide, 6-bromo-2-naphthyl-β-D-glucuronide, and the like.
Of these, 4-methylumbelliferyl-β-D-glucuronide is preferable from the viewpoint of versatility.
The concentration of β-glucuronidase substrate having a detectable fluorescent free group is preferably 0.001 g / L to 2 g / L, more preferably 0.01 g / L to 0.25 g / L, and 0.05 g / L to 0.15 g. / L is particularly preferred.

  The β-glucuronidase-inducing substrate used in combination with the fluorescent β-glucuronidase substrate is preferably sodium glucuronate, preferably 0.001 g / L to 2 g / L, more preferably 0.01 g / L to 0.25 g / L, 0.05 g Particularly preferred is / L to 0.15 g / L.

  The medium of the present invention preferably further contains starch from the viewpoint of producing a red carotenoid pigment of group B streptococci. The starch concentration is preferably 1 g / L to 50 g / L, more preferably 5 g / L to 50 g / L, and particularly preferably 10 g / L to 30 g / L.

  The medium of the present invention preferably further contains xanthan gum from the viewpoint of enhancing the color development of red carotenoid pigment production of group B streptococci. The concentration of xanthan gum is preferably 0.01 g / L to 10 g / L, more preferably 0.05 g / L to 5 g / L, and particularly preferably 0.5 g / L to 2 g / L.

  From the viewpoint of stabilizing the fluorescence intensity of the group B streptococci, the medium of the present invention preferably further contains Tricine and the pH is adjusted to 7.8 ± 0.1. The concentration of Tricine is preferably 5 g / L to 50 g / L, more preferably 10 g / L to 50 g / L, and particularly preferably 15 g / L to 20 g / L.

Furthermore, the medium of the present invention preferably contains an antibacterial substance from the viewpoint of suppression of bacteria other than group B streptococci. Examples of such antibacterial substances include methotrexate hydrate, crystal violet, colistin sulfate, polymyxin B, metronidazole, amphotericin B, and the like. The concentration of these antibacterial substances is preferably 0.01 mg / L to 50 mg / L, more preferably 0.05 mg / L to 15 mg / L, and particularly preferably 0.1 mg / L to 10 mg / L.
The medium of the present invention is further added with components usually used in the medium, for example, inorganic salts such as disodium hydrogen phosphate, organic acid salts such as sodium pyruvate, nutrients such as peptone and meat extract, vitamins and the like. It is preferable.

  The form of the medium of the present invention is not particularly limited, and a high-layer semi-fluid agar medium, a liquid medium, an agar plate medium, or the like can be used.

A specific example of the method for detecting group B streptococci according to the method of the present invention is as follows.
The medium of the present invention containing a β-glucuronidase substrate is punctured and cultured, and the presence or absence of red pigment production in the medium is observed to detect group B streptococci. Since the basic medium is Granada medium, most group B streptococci can be detected at this stage, but non-pigmented group B streptococci, which are said to be present at about 10%, cannot be detected. However, when it contains a β-glucuronidase substrate, non-pigmented group B streptococci can be detected by fluorescence of the medium excited by fluorescence excitation means such as a UV lamp. However, since the fluorescence is extremely weak, detection may be difficult. However, when glucuronate that induces a β-glucuronidase substrate is used in combination with the medium, the fluorescence increases, so that the bacteria can be detected more clearly and easily.

  That is, when a red pigment can be confirmed in this medium, the presence of group B streptococci is observed. On the other hand, if growth is observed but pigment production is not observed, or if the color development is weak and difficult to distinguish, the whole medium emits fluorescence by applying light that induces fluorescence, such as a UV lamp. Even in the presence of group B streptococci, it can be easily detected with a high probability. Therefore, all group B streptococci including non-pigmented group B streptococci can be detected.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
Example 1
Preparation of Medium Each medium having the composition shown in Table 1 was prepared as follows.

  As shown in Table 1 basal medium components, each component was added to 950 milliliters of purified water, autoclaved at 121 ° C. for 15 minutes, cooled to about 50 ° C., and then pre-dissolved and over-sterilized. Add antibacterial solution, mix well, dispense 5 ml each into a small tester with screw cap (φ10mm × 100mm) and let stand until the medium is solidified to prepare Group B Streptococcus Detection Medium A of the present invention did. Furthermore, 4-methylumbelliferyl-β-D-glucuronide which is a fluorescent β-glucuronidase substrate or sodium glucuronate which is a β-glucuronidase inducer, and both were prepared from this medium. As a control, a GBS medium manufactured by Fuji Pharmaceutical Co., Ltd. described in Patent Document 1 based on Granada medium was used.

Strain test The test strain was pre-cultured on sheep blood agar for 24 hours, and suspended in sterile physiological saline to give McFarland # 0.5 was used as a test bacterial stock solution. Each stock solution of test bacteria was appropriately diluted 10-fold with sterile physiological saline, and 30 μL of 10 ^{−2 of} each diluted solution was infiltrated into a sterile cotton swab, and the cotton swab was punctured into the medium to inoculate the medium of the present invention.

  Table 2 shows the results of culturing each strain for 24 hours at 35 ° C., and confirming the visual growth, production of red carotenoid pigment, and the presence or absence of fluorescence when irradiated with a 365 nm UV lamp.

In the medium of the present invention, good growth of chromogenic S. agalactiae which is a group B streptococcus, production of red pigment and generation of fluorescence were observed. At the same time, in the non-pigmenting S. agalactiae, fluorescence was observed in the medium A of the present invention. That is, by confirming the production of red pigment and the generation of fluorescence, it was confirmed that group B streptococci could be detected with high probability regardless of the production of red carotenoid pigment. Furthermore, even in the medium A ′ that does not contain the inducer sodium glucuronate, more than half of the chromogenic S. agalactiae was observed to generate fluorescence, and even in the non-pigmented S. agalactiae, Occurrence was observed.
Moreover, although the fluorescent β-glucuronidase substrate 4-methylumbelliferyl-β-D-glucuronide was not included, the production of red pigment was observed in medium B and medium C, but no fluorescence was observed. It was.

Example 2
Preparation of medium The medium of the present invention was prepared in exactly the same manner as in Example 1. At the same time, the medium D of the present invention containing no xanthan gum was prepared. As a control, a GBS medium manufactured by Fuji Pharmaceutical Co., Ltd. described in Patent Document 1 based on Granada medium was used.
The strain was tested in the same manner as in Example 1.
Table 3 shows the results of culturing each strain for 24 hours at 35 ° C. and confirming the growth by visual observation and the strength of the red carotenoid pigment.

  In the present invention medium A, xanthan gum was contained, and it was confirmed that the addition of xanthan gum enhanced the production of red carotenoid pigment of group B streptococci. In medium D containing no xanthan gum and the control medium, the production of red pigment was similar, but it was observed that the addition of xanthan gum enhanced the production of red carotenoid pigment.

  By using the culture medium of the present invention, it is possible to reliably and easily detect the red pigment non-producing group B streptococci that cannot be detected with the Granada medium with fluorescence.

Claims (5)

  A medium for detecting group B streptococci, comprising a β-glucuronidase substrate having a detectable fluorescent free group.   The medium according to claim 1, comprising glucuronate for inducing a β-glucuronidase substrate.   The medium according to claim 1 or 2, which contains xanthan gum in order to enhance the carotenoid pigment-producing ability of the pigment-producing group B streptococci.   A method for detecting group B streptococcus, comprising inoculating and culturing a specimen in the medium according to any one of claims 1 to 3 and observing the presence or absence of a red pigment in the medium.   Detection of group B streptococcus, characterized by observing the occurrence of fluorescence in the culture medium inoculated with the sample in any one of claims 1 to 4 and excited by fluorescence excitation means Law.