JPH0456600B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to group A Streptococcus (Streptococcus pyogenes,
The present invention relates to the rapid detection and presumptive identification of Streptococcus pyrogenes) using selective blood agar media. The genus Streptococcus, a genus of the Lactobacillus family, is widely distributed in nature. Some strains of the genus Streptococcus are pathogenic to humans and/or animals, while other strains may generally exist as harmless parasites. Streptococci are classified according to the presence of a type of carbohydrate known as Substance C (Lansfield classification system), and there are at least 13 groups (A to O, excluding two alphabets).
) is known. Among these, A
Streptococcus pyogenes (Streptococcus pyogenes) are almost always involved in human infections. However, other groups may also be pathogenic in certain circumstances. Group A Streptococcus (hereinafter referred to as
GAS (abbreviated as GAS) is involved in diseases such as scarlet fever, rheumatic fever, glomerulonephritis, pharyngitis, and sepsis, so confirming the presence of GAS is important in diagnosing the disease and providing appropriate treatment. This will be extremely important when choosing a route. According to the present method for detecting GAS, a specimen is obtained with a cotton or polyester swab in the infected area (eg, pharynx). This swab is then used to inoculate an appropriate medium containing mammalian blood in order to detect the characteristic complete or "β" hemolysis that appears around GAS colonies. However, throat cultures usually contain other microorganisms that may be present in greater numbers, so their growth is
Obscure the hemolysis caused by GAS. The presence of bacitracin-resistant bacteria also impedes determination of bacitracin susceptibility in plates inoculated with specimens. The prior art therefore required the isolation of β-hemolytic bacteria as pure cultures to determine bacitracin susceptibility as an aid to presumptive identification. Therefore, it may take two or more days to obtain information to begin appropriate treatment. Although there are many known antibiotics to which GAS is resistant, there are no single antibiotics or combinations of antibiotics that are completely selective for GAS (i.e. inhibit the growth of most other microorganisms). It has not been done. Therefore, non-group A streptococci and other microorganisms may also grow well on existing selective media, making it difficult to detect the characteristic β-hemolysis caused by GAS. Additionally, false positive results may be obtained if other β-hemolytic bacterial species are present. Although current GAS detection methods are relatively reliable, often achieving an accuracy of >90%, β-hemolytic streptococci are isolated in pure culture to determine bacitracin susceptibility for presumptive identification. There is a need. This takes 2-3 days longer than the method of the invention. Due to the poor selectivity of existing media, considerable skill is required to avoid errors in diagnosis. A more selective medium would result in less estimation error and be confirmed faster, thus increasing GAS
Appropriate treatment would be started earlier to prevent serious sequelae of infection due to A reagent for selectively culturing GAS was provided by the present invention. This reagent is a mixture of (1) a polymyxin or aminoglycoside, (2) a sulfonamide such as sulfamethoxazole, (3) trimethoprim, and (4) crystal violet. When this reagent is included in a culture medium containing mammalian blood, bacteria other than GAS are substantially eliminated;
β-hemolysis around GAS colonies can be observed without blurring. The selective reagents of the present invention provide a substantial improvement in selectivity, so that the presumptive identification of GAS based on susceptibility to bacitracin (0.04 unit disc) and the β-hemolytic zone surrounding the colonies of these microorganisms is possible after specimen inoculation. 18
Obtained within ~24 hours. According to the present invention, a reagent that selectively grows GAS (Streptococcus pyogenes) and substantially inhibits the growth of other microorganisms likely to be present in a biological specimen, such as a specimen collected from a human. According to
GAS detection is improved. This improved selectivity allows determination of bacitracin (0.04 units) sensitivity or serological testing on primary separation plates. Although all of these components were individually known for their antimicrobial properties, this combination of antimicrobial components has not been previously described, and the selection reagent of the present invention exhibits a very significantly improved selectivity for GAS. It was found to give. Especially when the selection reagent is included in a blood-containing culture plate inoculated with a human biological specimen containing GAS, a very distinct β-
A hemolytic pattern is observed and very little extraneous growth of other microorganisms is observed on the plates. Therefore, in most cases, presumptive identification based on bacitracin susceptibility and confirmatory identification based on serological classification tests can be made within 24 hours after specimen inoculation. Polymyxins are multiple substances produced by Bacillus polymyxa, and these substances and their sulfates are known to have antibacterial properties. Polymyxins take several forms including A, B, C, D, E, K, M and P. Preferably polymyxin E sulfate (colistin sulfate) is used as a component of the selection reagent. As an alternative to polymyxin sulfate, aminoglycosides such as gentamicin, amikacin, kanamycin, neomycin or tobramycin can be used. These ingredients are known to be effective against coliform bacteria and other Gram-negative bacteria. Other components of the selection reagent exhibit synergistic activity against alpha-streptococci in combination with sulfonamides, such as sulfamethoxazole, sulfisoxazole, sulfadiazine, or trimethoprim, and are sufficient for use in aqueous media. Others are soluble. A preferred sulfonamide is sulfamethoxazole. Sulfonamides are known to inhibit both Gram-positive and Gram-negative bacteria, but streptococci are largely resistant. The other component of the selection reagent is trimethoprim or trimethoprim lactate. Although trimethoprim is effective in controlling both Gram-positive and Gram-negative bacteria, it is synergistic in combination with sulfonamides, so many microorganisms that are not inhibited by either agent are inhibited when both are present. Ru. A combination of sulfamethoxazole and trimethoprim has previously been used in selective growth media for GAS, but was not effective in inhibiting staphylococci. The final ingredient is crystal violet (also known as gentian violet). This is a mixture of penta and hexamethyl-p-rosanaline chloride. This ingredient is known to inhibit neomycin-resistant staphylococci. Selective reagents according to the present invention generally have about 30.0 to 50.0
Contains mg/(medium). Sulfonamides are generally the largest antimicrobial component by weight, generally around 15-30mg/
(medium) amount used. Polymyxin sulfate is about 10.0 to 20.0, preferably 15.0 mg/(medium)
used in amounts of If an aminoglycoside is used in place of polymyxin, it is present in an amount of about 5.0-30.0 mg/depending on the aminoglycoside used. Trimethoprim is about 5 of the sulfonamides.
% by weight, i.e. preferably about 0.74 to about 1.5 mg/
(medium) amount used. Crystal violet is used in an amount of about 0.1 to about 0.3 mg/(medium). The medium to which the selection reagent is added includes an organic nitrogen source such as peptone, mammalian blood, salt, and a gelling agent, preferably agar. Peptone is approx.
Present in an amount of 15 to about 30g/. Preferred peptones are pancreatic juice digests of casein and papain digests of soybean porridge, with casein digests ranging from about 10 to about
20, preferably 15 g/, and the soybean porridge digest is present in an amount of about 3 to about 8, preferably 5 g/. Agar is about 13 to about 20, preferably 15g/
amount gives a gel of moderate hardness. Sodium chloride is provided in an amount of about 2 to about 8 g/, preferably about 5 g/. Salt concentration is important for proper osmolarity of the gel and prevents premature hemolysis of the blood. Mammalian blood present in an amount of about 4.0 to about 10.0% by volume is obtained from a variety of mammalian sources including sheep and rabbits. Blood is defibrinated before being used in culture media to prevent clotting. As mentioned above, blood is an indicator for hemolytic microorganisms such as GAS, and characteristic hemolysis occurs in the presence of this type of microorganism. β-hemolysis occurs around microbial colonies growing on culture plates containing selective reagents that substantially inhibit the growth of other hemolytic microorganisms.
This strongly indicates the existence of GAS. onto blood-containing culture plates containing the above-mentioned selection reagents.
Additional evidence for the presence of GAS is the absence of β-hemolytic colonies in areas containing bacitracin, an antibiotic produced by bacteria of the Bacillus subtilis group. Bacitracin-impregnated discs (0.04 units) are commercially available to be placed on culture plates to apply this antibiotic to limited areas of the plate. Furthermore, commercially available kits allow serological confirmation identification to be carried out in media containing the above-mentioned selection reagents. Media containing selective reagents provide excellent inhibition of common microorganisms, especially mutant strains of Streptococcus, which are the predominant microorganisms present in most throat cultures. Although suppression of streptococcal mutants is also achieved using a mixture of sulfamethoxazole and trimethoprim alone, the selective reagents of the present invention inhibit staphylococci, other streptococci, and gram-negative bacteria, as well as Neisseria spp. (neisseria) species and S. aeruginosa are also improved. Furthermore, the high degree of selectivity of the selection reagents of the present invention allows for the presumptive identification of GAS (by the presence of hemolytic colonies on the plate and the absence of hemolytic colonies in the area of the bacitracin disk) and serological confirmation classification substantially faster than possible with prior art formulations (i.e., compared to 42-72 hours).
(in 18-24 hours). The invention will now be described in more detail by means of specific embodiments. Example 1 A group A streptococcus selection medium containing the following components was prepared. Ingredients Content per 1 unit of purified water Pancreatic juice digest of casein 15.0g Papain digest of soybean porridge 5.0g Sodium chloride 5.0g Agar 15.0g Colistin sulfate 15.0mg Sulfamethoxazole 23.75mg Trimethoprim 1.25mg Crystal violet 0.2mg Sheep Blood (defibrinization treatment) 50.0ml Components other than sheep blood were appropriately mixed to prepare a homogeneous powder. The powdered medium was hydrated with distilled or deionized water and boiled until all ingredients were dissolved and the medium became clear. The medium was then cooled to 45-50°C in a water bath and defibrinated sheep blood was added. The medium was mixed by agitation and 18-20 ml was dispensed into 100 mm Petri dishes using standard bacteriological methods. After this medium gels,
The dishes were placed in a suitable container, such as a plastic film (cellophane, polyethylene, nylon, etc.) cylinder, and stored at 2-8°C until use. The plates can be stored in this way for 12 weeks. Throat swab specimens obtained from patients (later shown to contain GAS) were inoculated onto the plates and non-selective blood agar plates described above following microbiological routine processing. The inoculum is then spread with a sterile (flamed) biological inoculation loop;
Dispersed colonies were obtained. Several incisions were made in the agar with a loop over the most heavily inoculated area, and a 0.04 unit bacitracin disc was placed several centimeters from the incision, but still in the most heavily inoculated area. The plates were then incubated for 18-24 hours at 35±2° C. in an atmosphere of 3-8% carbon dioxide. For testing, β on non-selective blood agar plates is
- Hemolytic GAS colonies were difficult to distinguish from the dense growth of normal throat bacteria present on the medium. Susceptibility to bacitracin disc was also difficult to determine. However, the plate culture medium containing the selective reagent was excellent in suppressing common throat bacteria, and β-hemolysis of GAS was observed without being significantly inhibited. An inhibition zone surrounding the bacitracin disk was clearly visible and β-hemolytic proliferation of GAS could be presumptively identified only 18-24 hours after specimen inoculation. Example 2 The medium (A) produced in Example 1 above is shown in the table below.
Also, similar media (B-
Compared with E). Medium BE is commercially available.

【table】

Table: The concentrations of each component, if present, are equal to the amounts specified in Example 1 above. The concentration of neomycin sulfate in medium B and C is 30 mg/. The concentration of nalidixic acid is 15 mg/in medium C and 10 mg/in medium D. These media were evaluated for specific cultures to determine their relative performance in GAS recovery and inhibition of non-GAS present in common throat specimens. The results are shown in Table 1.

[Table] ×××× Excellent ××× Good ×× Fair × Trace amount - None The results shown in the above table demonstrate the surprising properties of the selective reagent of the present invention compared to the selective reagents known in the prior art. Show the improvements that need to be made. Example 3 Comparison of selective media of the invention (Medium A from Example 1) and SXT sheep blood agar (Medium E from Example 2) with non-selective sheep blood agar (control) in a study using human throat specimens. did. Throat swab specimens were inoculated into each of the three media as described in Example 1. The inoculated medium was then incubated at 35±2° C. in an atmosphere of 3-8% carbon dioxide for 18-24 hours. As shown in the following table, 117 of the 460 random throat cultures were obtained by medium containing the selection reagent of the invention (A), 100 by SET blood agar (E), and only 84 by non-selection. It was positive for GAS by standard control.

[Table] Hot culture

Claims (1)

Claims: 1. A group A Streptococcus selection reagent comprising a mixture of: 1 (a) a sulfonamide; (b) trimethoprim; (c) a component selected from the group consisting of polymyxins and aminoglycosides; and (d) crystal violet. 2 Sulfonamide is sulfamethoxazole,
The reagent according to claim 1, selected from the group consisting of sulfisoxazole and sulfadiazine. 3. The reagent according to claim 1, wherein component (c) is colistin sulfate. 4 component (c) is an aminoglycoside selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin and neomycin;
A reagent according to claim 1. 5. A mixture comprising a nutrient source that supports the growth of group A streptococci, and further comprising: (a) a sulfonamide; (b) trimethoprim; (c) a component selected from the group consisting of polymyxins and aminoglycosides; and (d) crystal violet. comprising a selective reagent having activity against microorganisms other than group A streptococci consisting of
A medium for selectively culturing group A streptococci. 6 Sulfonamide is sulfamethoxazole,
The medium according to claim 5, which is selected from the group consisting of sulfisoxazole and sulfadiazine. 7. The medium according to claim 5, wherein component (c) is colistin sulfate. 8. The medium according to claim 5, wherein component (c) is selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin and neomycin. 9 a nutrient source for group A streptococci and a selective reagent consisting of a mixture of (a) sulfonamides; (b) trimethoprim; (c) a component selected from the group consisting of polymyxins and aminoglycosides; and (d) crystal violet. inoculating the medium by smearing a biological specimen suspected of containing group A streptococci; incubating for a period sufficient to selectively inhibit the growth of substantially all other microorganisms present; and testing the medium for the presence of β-hemolytic colonies; A method for selective identification of group A streptococci by detecting susceptible β-hemolytic colonies. 10. Claim 9, wherein the sulfonamide is selected from the group consisting of sulfamethoxazole, sulfisoxazole and sulfadiazine.
The method described in section. 11. The method according to claim 9, wherein component (c) is colistin sulfate. 12. The method according to claim 9, wherein component (c) is an aminoglycoside selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin and neomycin. 13 A patent comprising placing a bacitracin-impregnated disk over a portion of a smeared area of culture medium and detecting the presence or absence of hemolysis in the area, the absence of hemolysis in the vicinity of the disk indicating that the microorganism is group A streptococcus. The method according to claim 12.