JP2001275696A - Method for detecting physiological activity of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting the physiological activity of microorganisms, for example, the synthetic activity of cell walls in a short time in no need of installation, cost, labor, professional skills, and the like. SOLUTION: A microorganism is allowed to synthesize its cell wall, while it is cultured under the conditions where it causes no cell proliferation whereby the physiological activity of the microorganism is detected according to the degree of the increase of absorbancy with time of the cell suspension. This method is not only useful in the studies and inspections on the physiological activity of microorganisms relating to the cell wall synthesis but also for obtaining the information on the sensitivity of the cells to antimicrobial drugs that manifest their microbicidal activity through the suppression of cell wall synthesis. In addition, this inspection process is useful in simple and rapid detection of Staphylococcus aureus of low sensitivity to glycopeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting the biological activity of a microorganism.

[0002]

2. Description of the Related Art The synthesis of cell walls is indispensable for fungi and bacteria to survive and exhibit pathogenicity to humans and animals. Therefore, natural or synthetic antifungal and antibacterial agents that specifically inhibit the synthesis of cell walls are widely used as therapeutic agents for infection by these microorganisms.

[0003] In recent years, however, particularly in bacterial infections, the number of bacteria due to ineffective antimicrobial agents has increased, causing a serious medical problem. More than 80% of the antibacterial drugs used are penicillins, cephem and other β-lactams (beta-lact
am) Drugs and cell wall synthesis inhibitors such as glycopeptides such as vancomycin and teicoplanin. In 1997, the present inventors detected Staphylococcus aureus that acquired resistance to all of these cell wall synthesis inhibitors from clinical cases in which vancomycin treatment did not respond. This strain is either vancomycin-resistant S. aureus [VRSA] or V.
ISA [vancomycin-intermediate S. aureus] or GISA [g
lycopeptide-intermediate S. aureus]) (1,
2).

[0004] In 1997, the present inventors reported that VRSA
Not only, but also heterologous progenitor cell line
He clarified that VRSA (hetero-VRSA) exists (9). HeteroVRSA may also be resistant to vancomycin treatment by its own infection (5,6,7,
9). Of course, hospitals where there are many hetero VRSA,
In regions, VRSA is likely to appear with glycopeptide treatment, so to prevent the appearance of VRSA,
Detection of heterologous VRSA is also important. Glycopeptide antibacterial drugs such as vancomycin and teicoplanin are among the multi-drug resistant MRSA (methicillin-resi) among Staphylococcus aureus.
(stanat S. aureus) is one of the few treatments, so the development of a sensitive glycopeptide sensitivity test is crucial.

It has been found that VRSA is not easily detected by a conventional automated MIC measuring device or a glycopeptide sensitivity test using a disk diffusion method or the like (8). Furthermore, none of these commonly used conventional susceptibility tests can distinguish heterozygous VRSA from susceptible strains (10). Hetero-VRSA cannot be detected by MIC measurement under very strict quality control (10).

The present inventors have previously invented a method utilizing a Mu3 medium for detecting hetero VRSA (10). This method is a method that utilizes the antagonistic phenomenon when a combination of β-lactam drug and vancomycin is used, but it is difficult to distinguish between VRSA and hetero VRSA, and it is difficult to judge when it is in the middle between susceptible bacteria and hetero VRSA In this respect, the detection method is not yet complete. Also, in connection with this, the determination result is affected by an increase or decrease in the number of inoculated bacteria. Recently, it has become clear that the antagonistic phenomenon is a phenomenon observed in all common Staphylococcus aureus.
It turns out that it is not a phenomenon specific to RSA. Therefore, M
The method using three culture media cannot be said to be a detection method based on the resistance mechanism of these bacteria.

[0007] Hetero-VRSA, as a mechanism of its resistance, increases (activates) cell wall synthesis to form a thicker cell wall, traps vancomycin molecules in the cell wall, and causes vancomycin to undergo cell wall synthesis. It was found that the resistance was increased by making it impossible to easily reach the cytoplasmic membrane (3,4,5,6,7). This mechanism of tolerance is called affinity trapping (6, 7). Therefore, if the cell wall synthesis activity of an individual cell line of Staphylococcus aureus can be measured, the glycopeptide sensitivity of that line can be determined.

However, the cell wall synthesizing activity requires timely measurement of the uptake of isotope-labeled N-acetyl-glucosamine or the like, or measurement of the cell wall thickness using a transmission electron microscope. Requires facilities, equipment, skilled technicians or researchers. In addition, this method does not meet the conditions required to test many strains within a short period of time, such as in daily clinical sample tests.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for detecting the biological activity of a microorganism in a short time without requiring equipment, cost, labor, and specialized procedures.

[0010]

Means for Solving the Problems The present inventor uses a medium containing a large amount of precursor nutrients for cell wall components, and further restricts the growth of microorganisms in the medium, thereby preventing nutrient depletion due to growth. We found a method for synthesizing a cell wall that reflects the cell wall synthesis ability of the microorganism and detecting it. Further, the present inventors have found a method for measuring the sensitivity of a bacterium to an antimicrobial drug by synthesizing or synthesizing a cell wall using this medium and then growing the bacterium in a culture solution containing an antimicrobial drug.

That is, a first aspect of the present invention is a method for detecting the physiological activity of a microorganism by synthesizing a cell wall under culture conditions that do not cause cell growth of the microorganism and measuring the degree of the cell wall synthesis ability of the microorganism. is there. The second present invention provides an antimicrobial drug susceptibility test method comprising inoculating a medium containing an antimicrobial agent, which particularly contains a large amount of nutrients that assist the synthesis of cell walls, with Staphylococcus aureus, and observing the cell growth. It is.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism in the present invention includes bacteria and fungi having a cell wall. For example, Gram-positive bacteria can be exemplified, and Staphylococcus aureus can be typically exemplified.

[0013] Culture conditions that do not cause cell growth include the use of a medium capable of causing cell growth with the addition of an antibacterial agent or a chemical substance that hinders cell growth, or the substantial inclusion of essential amino acids required for cell division. Using no media.

Although essential amino acids vary depending on the strain of Staphylococcus aureus, arginine and valine are almost always essential amino acids. Furthermore, leucine, cystein
e, proline, etc. are also often required. If a medium not containing one or more of these amino acids is used, Staphylococcus aureus does not grow. However, in the case of other strains or other strains, deletion of other amino acids may serve the purpose. Those amino acids are, for example, alan
ine, aspratic acid, histidine, isoleucine, methio
nine, phenylalanine, serine, threonine, tryptopha
n, tyrosine, etc. Whether or not the amino acid is an essential amino acid can be easily determined by, for example, culturing the target strain or strain in a medium containing the amino acid and a medium lacking the amino acid, and examining the degree of proliferation.

However, even such essential amino acids required for cell wall synthesis need to be contained in the medium. The amino acids required for cell wall synthesis include Glycine, D-glutamic acid, DL-alanyl-DL-alanine, and Glycine included in those exemplified as components of the culture medium that supports cell wall synthesis described below.
DL-lysine and the like can be exemplified. Whether or not an amino acid is necessary for cell wall synthesis can be easily determined by culturing the target strain and strain in a medium containing the amino acid and a medium lacking the amino acid, and examining the degree of cell wall thickening. be able to.

As a medium lacking essential amino acids, not causing cell proliferation and assisting the synthesis of cell walls, for example, a synthetic medium having the following composition can be mentioned. Glycine, 0.5-5 mM; D-glutamic acid, 0.5-5 mM; DL-alanyl-DL-alanine, 0.5-5 mM DL-lysine, 0.3-5 mM; MgCl ₂ , 0.5-2 mM; MnCl ₂ , 0.05 -0.2 mM; Uracil, 0.1-0.4 mM; Nicotinamide, 0.005-0.02 mM; K ₂ HPO ₄ , 20-100 mM; Thiamine, 0.001-0.005 mM; Glucose 10-80 mM;

Commercially available and commonly used culture media include:
Both contain all essential amino acids and allow cell growth. Examples of such a medium include commercially available Luria medium, Mueller-Hinton medium, and Heart Infusion medium.
Examples include a medium, a Brain Heart Infusion medium, and a tryptic soy medium.

Antibacterial drugs and chemicals that hinder cell growth
For example, as antibacterial agents and chemical substances, those having a protein synthesis inhibitory action such as aminoglycoside, macrolide and tetracycline, and those having a DNA synthesis inhibitory action such as quinolone antibacterial drugs. The amount of these additives is the minimum concentration MIC (minimal inhibitory conc
(entration).

Further, in the present invention, a medium containing a particularly large amount of nutrients for assisting the synthesis of cell walls and containing an antimicrobial agent is defined as a medium containing Luria medium, Mueller-Hinton
A medium containing more antibacterial agents typified by glycopeptides than the medium, Heart Infusion medium, Brain Heart Infusion medium, tryptic soy medium and the like. The concentration of nutrients in the synthetic medium shown in paragraph [0016] in a commonly used medium is not exactly known. Therefore, by further adding this synthetic medium to these mediums, a medium containing a particularly large amount of nutrients that assist the synthesis of the cell wall can be prepared.

The biological activity of the microorganism to be detected by the detection method of the present invention is, for example, cell wall synthesis activity and glycopeptide antimicrobial susceptibility. The glycopeptide antibacterial agent here means D-alanyl-D-alanine binding antibiotics with
a heptapeptidic structure (dalbaheptide)
Vancomycin, teicoplanin (teico)
planin), including avoparcin.

The method of the present invention is carried out as follows, for example, in the case of detecting vancomycin-resistant bacteria. A medium in which the above-described cell growth can be performed, a medium to which an antibacterial agent or a chemical substance that hinders cell growth has been added, or a medium that does not contain some of the nutrients necessary for the growth and division of microorganisms and contains a large amount of nutrients that assist in the synthesis of cell walls The sample is cultured using the medium for detecting the physiological activity of the contained microorganism, and the cell wall is synthesized. At this time, it is preferable to keep the temperature substantially constant. The absorbance of the culture during this culture is measured over time. For example, in the case of vancomycin-resistant bacteria, the absorbance sharply increases despite little change in the number of bacteria. In contrast, the absorbance of vancomycin-sensitive bacteria increases slowly. Vancomycin-sensitive bacteria, hetero-resistant bacteria, and resistant bacteria can be distinguished from the change in absorbance with time.

When culturing a sample in the medium and synthesizing a cell wall, vancomycin is added within a range of 0.5-30 mg / l, and the presence or absence of an increase in OD and the speed of the increase are observed.
It is also possible to compare with vancomycin resistant, hetero resistant and susceptible bacteria. In our experiments, 10 mg
When vancomycin / l was added, the increase in the OD value after 2 hours was the mean and SD value of the four experiments, with a Mu50 of 0.72 ± 0.13,
Mu3 is 0.41 ± 0.22, H1 and FDA209P are both 0.05 or less (range
0.01-0.05). Therefore, also by this method, resistant, heteroresistant and susceptible bacteria can be distinguished.

The bacteria cultured as described above are inoculated into a medium containing vancomycin, and the time until the start of bacterial growth, the presence or absence of bacterial growth after a certain period of time, or the consumption of vancomycin are measured. Thereby, bacteria can be detected. For example, vancomycin resistant and hetero resistant bacteria grow after 8-18 hours, but susceptible bacteria do not. In addition, in the case of vancomycin-resistant bacteria and hetero-resistant bacteria, the time until the start of growth is short,
Long for susceptible bacteria. In the case of vancomycin resistant bacteria, consumption of vancomycin is fast, and in the case of susceptible bacteria, consumption is slow.

[0024]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Example 1 [Correlation between synthesis of cell wall and increase of absorbance] Cui-Hiramatsu (CH) broth, which contains a large amount of nutrients necessary for cell wall synthesis and lacks amino acids essential for growth, and C
CHglu- further lacking glucose from H, CHGn with glutamine (glutamine) added to CH to enhance cell wall synthesis,
N-acetyl-gluc, a precursor of cell wall amino sugars in CHglu-
Using CHNAG with osamine, Staphylococcus aureus strain Mu
50 was kept at 37 ° C. for 2 hours, and the thickness of the cell wall was observed with an electron microscope. The concentrations of the components of each medium are as follows.

CH: Glycine, 1 mM; D-glutamic acid, 1 mM; DL-alanyl-DL-alanine, 0.5 mM; DL-lysine, 0.3 mM; MgCl ₂ , 1 mM; MnCl ₂ , 0.1 mM; Uracil, 0.17 mM; Nicotinamide, 0.0082 mM, K ₂ HPO ₄ , 80 mM; Thiamin, 0.003 mM; glucose 28.8 mM GHglu-: Removes glucose from CH. CHGn: 30 mM glutamine added to CH. CHNAG: Add 30 mM N-acetyl-glucosamine to CHglu-.

FIG. 1 (A, CHglu-; B, CH; C, CHNAG; D,
As shown in CHGn), the cell wall thickness of CHglu- was 31.03n
m ± 2.30 nm is the thinnest, 38.13 nm ± 3.49 nm for CHNAG, and 53.29 nm ± 3.01 nm, 57.1 for CH and CHGn, respectively.
The thickness was 8 nm ± 3.18 nm. The viable cell count at 2 hours of incubation was A, 3.95 ± 0.87 × 10 ⁷ CFU; B, 3.65 ±
0.92 × 10 ⁷ CFU; C, 3.87 ± 0.34 × 10 ⁷ CFU; D, 3.26 ± 0.4
6 × 10 ⁷ CFU, no statistical difference. That is,
It was found that there was no difference in the change in the number of bacteria during the incubation time between the AD conditions under the incubation conditions that did not support this cell growth.

FIG. 2 shows the turbidity of the bacterial suspension measured using an absorbance meter during the incubation. OD600nm (optical density at 600nm; below
OD) was observed to increase in media other than CHglu- (A). The degree of this rise (OD in the second hour in Figure 2)
1) and the thickness of the cell wall in FIG. 1 correlated very well (correlation coefficient: 0.928).

Example 2 [Correlation with Increase in Cell Wall Thickness, Absorbance, and Low Sensitivity to Vancomycin] In Example 1, bacteria (AD) incubated for 2 hours
And a fixed number (8.5 × 10 ⁸ CFU) of brain heart infusion (BHI) broth containing 10 ml of vancomycin 30 mg / l.
After inoculation of h, and culturing at 37 ° C., as shown in FIG. 3, after a certain period of time, each bacterium started to grow (the solid line of AD indicates the growth curve of the bacterium). The length of time was inversely correlated with the cell wall thickness of each bacterial cell. That is, the thicker the cell wall, the higher the degree of resistance (low sensitivity) to vancomycin (that is, the earlier the growth curve rises). Furthermore, as shown in FIG. 3, a decrease in the concentration of vancomycin (dashed line graph of ad) was observed in one hour after inoculation of each bacterium, in correlation with the cell wall thickness. That is, the thicker the cell wall, the more vancomycin was consumed.

Example 3 [Glycopeptide-sensitive bacteria, heteroresistant bacteria, resistant bacteria
An experiment to determine the increase in OD in CH medium] glycopeptide-sensitive Staphylococcus aureus strains FDA09P (methicillin-sensitive) and H1
(Methicillin resistance), heteroglycopeptide resistant Staphylococcus aureus strain Mu3, glycopeptide resistant Staphylococcus aureus strain M
When u50 (VRSA) is cultured for 2 hours in CH medium, commercially available B
Compared to those cultured in the HI medium, as shown in FIG.
3.97nm ± 3.61nm (FDA209P), 25.99nm ± 2.29nm to 2
9.09nm ± 3.26nm (H1), 26.17nm ± 2.17nm to 38.44nm
± 1.65nm (Mu3), 31.88nm ± 1.56nm to 53.29nm ± 3.01
The cell wall thickness increased to nm (Mu50). However, the rate of increase in cell wall thickening was in the order of Mu50>Mu3>H1> FDA209,
The percentage of this increase for each strain was statistically significant (P <0.001).

FIG. 5 shows the increase in the OD value of these strains while keeping them in a CH medium. The rise is apparently Mu50>Mu3> H
1> In order of FDA209P, the increase in cell wall thickness and the OD value at 2 hours were well correlated (correlation coefficient: 0.998).

Example 4 As in Example 3, bacteria incubated in a CH medium were inoculated into BHI broth containing 30 mg / l vancomycin, and the degree of vancomycin resistance was compared as shown in FIG. In addition, the reciprocal of the time until the start of the growth of each bacterium and the thickness of the cell wall, and the reciprocal of the time until the start of the growth of each bacterium and the increase in the OD value after 2 hours in FIG. 5 were well correlated. (Correlation coefficients: 0.923 and 0.956, respectively).

Example 5 Each of the constituent nutrients of the CH medium was added to the BHI medium so that the final concentration was the same as that of the CH medium, and vancomycin was added to the medium for 30 minutes.
A medium BHI-CH containing a large amount of nutrients for cell wall synthesis in addition to mg / L was prepared. BHI-CH and BHI medium, respectively, a strain cultured overnight in BHI medium, Mu50,
Mu3, H1, and FDA09P were each inoculated at 1 × 10 ⁶ CFU. FIG.
As shown in the figure, in the case of BHI-CH (B), all strains started to grow earlier than in the case of BHI (A). However, the time until the start of the growth of Mu3 was much shorter in BHI-CH than in BHI-CH compared to other strains. Therefore, it was easy to distinguish the strain from the vancomycin-sensitive strain H1. That is, hetero-resistant bacteria and susceptible bacteria can be distinguished by determining the presence or absence of bacterial growth during 18 to 30 hours.

[0034]

The method of the present invention is useful not only for studying and examining the physiological activities of all microorganisms involved in the strength of cell wall synthesis, but also as an antimicrobial drug whose action mechanism is inhibition of cell wall synthesis. Can obtain information on the sensitivity of the cells. Furthermore, this test method is a useful test method for simple and rapid detection of S. aureus with low glycopeptide sensitivity. In particular, these methods have made it possible to detect vancomycin-insensitive bacteria containing hetero VRSA.

[0035]

[Literature] 1. Hiramatsu, K., H. Hanaki, T. Ino, K. Ya
buta, T. Oguri, and FCTenover. 1997. Methicilli
n-resistant Staphylococcus aureus clinical strainw
ith reduced vancomycin susceptibility. Journal of
Antimicrobial Chemotherapy. 40: 135-136. 2. Hiramatsu, K. 1998. The emergence of Staphyloco
ccus aureus with reduced susceptibility to vancomy
cin in Japan.American Journal of Medicine.104 (5
A): 7S-10S. 3. Hanaki, H., K. Kuwahara-Arai, S. Boyle-Vavra,
RS Daum, H. Labischinski, and K. Hiramatsu. 199
8. Activated cell-wall synthesis is associatedwith
vancomycin resistance in methicillin-resistant St
aphylococcus aureus clinical strains Mu3 and Mu50.
Journal of Antimicrobial Chemotherapy. 42: 199-209. 4. Hanaki, H., H. Labischinski, Y. Inaba, N. Kond.
o, H. Murakami, and K. Hiramatsu. 1998. Increase in
glutamine-non-amidated muropeptides in the peptid
oglycan of vancomycin-resistant Staphylococcus aur
eus strain Mu50.Journal of Antimicrobial Cheomothe
rapy. 42: 315-320. 5. Hiramatsu, K. 1998. Vancomycin resistance in st
aphylococci.Drug Resistance Updates.1: 135-150.

6. Hiramatsu, K., and H. Hanaki. 1998.
Glycopeptide resistance in staphylococci.Current
Opinion in Infectious Diseases. 11: 653-658. 7. Hiramatsu, K., T. Ito, and H. Hanaki. 1999. Ev
olution of methicillinand glycopeptide resistance
in Staphylococcus aureus., p. 221-242. InR. G. Fi
nch, and Williams, RJ, (ed.), Bailliere Yus Cli
nical Infectious Disease, vol. 5.Bailliere Tindal
l, London. 8. Tenover, FC, MV Lancaster, BC Hill, C.
D. Steward, SAStocker, GA Hancock, CM O '
Hara, NC Clark, and K. Hiramatsu. 1998.Charact
erization of staphylococci with reduced susceptibi
lity to vancomycin and other glycopeptides. Journa
l of Clinical Microbiology. 36: 1020-1027. 9. Hiramatsu, K., N. Aritaka, H. Hanaki, S. Kawasa
ki, Y. Hosoda, S. Hori, Y. Fukuchi, and I. Kobayas
hi. 1997. Dissemination in Japanese hospitals of s
trains of Staphylococcus aureus heterogeneously re
sistant to vancomycin. Lancet. 350: 1668-1671. 10. Hanaki, H., and K. Hiramatsu. 1998. A novel me
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[Brief description of the drawings]

FIG. 1 In Example 1, cell wall synthesis was enhanced by Cui-Hiramatsu (CH) broth (B), a medium lacking amino acids essential for growth, and CHglu- (A), CH further lacking glucose from CH. CHGn with glutamine
(D), N-acetyl, a precursor of cell wall amino sugars on CHglu-
5 is a photograph obtained by observing, with an electron microscope, the thickness of cell walls after keeping S. aureus strain Mu50 at 37 ° C. for 2 hours using CHNAG (C) to which -glucosamine has been added.

FIG. 2 shows the turbidity of the bacterial suspension during the incubation in Example 1.
It is the figure measured using the absorptiometer.

FIG. 3 is a graph showing bacterial growth and changes in vancomycin concentration when bacteria (AD) incubated for 2 hours were inoculated and cultured in BHIbroth containing vancomycin 30 mg / l in Example 1.

FIG. 4 shows glycopeptide-sensitive Staphylococcus aureus strains FDA09P (methicillin-sensitive) and H1 (methicillin-resistant), heteroglycopeptide-resistant Staphylococcus aureus strain Mu3, and glycopeptide-resistant Staphylococcus aureus strain Mu50 (V) in Example 3.
7 is a photograph obtained by observing, under an electron microscope, a change in cell wall thickness when (RSA) was cultured in a commercially available BHI medium and CH medium.

FIG. 5: Incubation of the culture strain in Example 3 with CH medium
It is a figure showing rise of OD value.

FIG. 6 is a diagram comparing each degree of vancomycin resistance by inoculating each of the bacteria kept in a CH medium into BHI broth containing 30 mg / L vancomycin.

FIG. 7: BHI-CH (B) containing 30 mg / L vancomycin and BHI
FIG. 3 is a diagram comparing cultures of cells by culturing Mu50, Mu3, H1, and FDA09 in a medium (A).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/20 (C12N 1/20 C12R 1: 445) C12R 1: 445)

Claims (19)

[Claims] 1. A method for detecting a biological activity of a microorganism by synthesizing a cell wall under culture conditions that do not cause cell growth of the microorganism, and measuring the degree of cell wall synthesis ability of the microorganism. 2. The detection method according to claim 1, wherein the method for measuring the cell wall synthesizing ability of the microorganism is an absorbance measurement of the microorganism suspension. 3. The method according to claim 1, wherein the physiological activity is sensitivity to a cell wall synthesis inhibitor. 4. The method according to claim 1, wherein the physiological activity is sensitivity to a glycopeptide antibacterial drug. 5. The method according to claim 4, wherein the microorganism is Staphylococcus aureus.
the method of. 6. The method according to claim 5, wherein a culture medium used does not contain a part of nutrients necessary for the growth and division of the microorganism, and uses a medium rich in nutrients for assisting cell wall synthesis. 7. The method according to claim 6, wherein the glycopeptide antibacterial agent is vancomycin. 8. A cell wall is synthesized using a medium that does not contain some of the nutrients necessary for the growth and division of microorganisms and contains a large amount of nutrients that assist in cell wall synthesis, and then an antimicrobial drug susceptibility test is performed. An antimicrobial drug susceptibility test method, characterized in that: 9. The method according to claim 8, wherein the microorganism is Staphylococcus aureus and the antimicrobial agent is an antibacterial agent inhibiting cell wall synthesis. 10. The method according to claim 9, wherein the cell wall synthesis inhibitor is a glycopeptide antibacterial agent. 11. The method according to claim 10, wherein the glycopeptide antibacterial agent is vancomycin. 12. An antimicrobial drug susceptibility test method comprising inoculating a medium containing an especially large amount of nutrients that assist the synthesis of cell walls and containing an antimicrobial agent with Staphylococcus aureus and observing cell proliferation. 13. The test method according to claim 12, wherein the antimicrobial agent is a glycopeptide antibacterial agent. 14. The test method according to claim 13, wherein the glycopeptide antibacterial agent is vancomycin. 15. The method according to claim 13, wherein the observation of cell proliferation is the presence or absence of proliferation after a certain period of culture. 16. The method for detecting 13 or 14, wherein the observation of cell proliferation is the time required until the proliferation is detected. 17. A medium for detecting the biological activity of a microorganism, which does not contain a part of nutrients necessary for the growth and division of the microorganism, and contains a large amount of nutrients for assisting cell wall synthesis. 18. A medium for detecting the physiological activity of a microorganism, comprising a medium capable of causing cell growth and an antibacterial agent or a chemical substance that inhibits cell growth added. 19. A medium for testing antimicrobial susceptibility, which is particularly rich in nutrients that assist in the synthesis of cell walls and contains an antimicrobial.