JPS60176599A - Selective reagent to a-group streptococcus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to group A Streptococcus (Streptococcus pyogenes, 5trept).
ococcus pyroxenes) f-selective liquid agar medium for rapid detection and presumptive identification.

The genus Streptococcus, a member of the Lactobacillus family, is widely distributed in nature. Some strains of the Streptococcus genus are pathogenic to humans and /''1.bamboo animals, while other strains may generally exist as harmless parasites. Streptococci are known as Substance C. It is classified according to the presence of a type of carbohydrate (Lancefield classification system), and at least 13 groups are known (represented by A to 0, excluding two alphabets). Among these, group A (purulent chain Streptococci) are almost always involved in human infections, although other groups may also be pathogenic in certain settings. Because it is involved in diseases such as fever, glomerulonephritis, pharyngitis, and even sepsis, confirming the presence of GAS is extremely important in diagnosing the disease and selecting the appropriate treatment route. Probably.

According to the present method for detecting GAS = i, the entire specimen is obtained with a cotton or polyester swab in the infected area (eg pharynx). Then, using this swab, GAS
Characteristic complete or °゛β″ that appears around the colony
To detect total hemolysis, inoculate appropriate medium containing mammalian blood. However, throat cultures usually contain other microorganisms that may be more numerous and their proliferation obscures the hemolysis caused by GAS. - The presence of micitracin-resistant bacteria also impedes determination of bacitracin susceptibility on plates inoculated with specimens.

Accordingly, the prior art requires the isolation of β-bacterium as a pure culture 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.

Many antibiotics are known to which GAS is resistant, but GAS
No single antimicrobial agent or combination of antimicrobial agents is known that is completely selective for S. (ie, inhibits the growth of most other microorganisms). Therefore, non-group A streptococci and other microorganisms can grow well on existing selective media, and G
The characteristic β-hemolysis caused by AS can make it extremely difficult to detect. Additionally, false positive results may be obtained if other β-hemolytic bacterial species are present.

Current GAS detection methods are relatively reliable and often 9
Although a precision of better than zero is achieved, it is necessary to isolate β-hemolytic streptococci in pure culture to determine bacitracin susceptibility for presumptive identification. 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 would be identified earlier and therefore appropriate therapeutic reagents to prevent serious sequelae of infection with GAS are: 1) polymyxins or aminoglycosides; 2)
Sulfonamides such as sulfamethoxazole, 3)
trimethoprim, and 4) crystal violet. When this reagent is included in a medium containing mammalian blood, bacteria other than GAS are substantially eliminated, and therefore the β-bath blood surrounding the GAS colonies can be observed without being obscured.

The selective reagents of the present invention provide a substantial improvement in selectivity, so that the presumptive identification of GAS is possible based on the susceptibility to bacitracin (004 unit disk) and the β-bath band surrounding colonies of these microorganisms (GAS). Obtained within 18-24 hours after inoculation.

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, e.g. a specimen collected from a human. This improves GAS detection. This improved selectivity allows determination of bacitracin (004 units) susceptibility or serological tests to be carried out on a second separation lithographic plate. 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 selected reagent is G
When contained in blood-containing culture plates inoculated with human biological specimens containing AS (i7), a very distinct β-hemolytic pattern was observed in the growth areas of GAS, and unrelated growth of other microorganisms was observed in the plates. For this reason, 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 produced by Bacillus polymyxa (Bacj, ll
p, these substances and their sulfates are known to have antibacterial properties. Polymyxin iA, B.

It takes several shapes including C, D, E, M and Pi.

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 tozramycin can be used. These ingredients are known to be effective against coliform bacteria and other Gram-negative bacteria.

Other components of the selection reagent are sulfonamides, such as sulfamethoxazole, sulfadiazine, or α in combination with trimethoprim.
Others exhibit synergistic action against streptococci and have sufficient solubility for use in aqueous media. A preferred sulfonamide is sulfamethoxazole. Sulfonamides are known to inhibit both Durham-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 Durum-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 n<inter> and hexamethyl=p-rosanaline chloride. This ingredient is known to inhibit neomycin-resistant staphylococci.

Selective reagents according to the invention generally have about 300-500 mp/
1 (medium). Sulfonamides are generally the largest antimicrobial component by weight, generally around 15-30 mg/l (medium)
used in amounts of Polymyxin sulfate is about 10.0-2
0. 'O1 is preferably used in an amount of 15.0 m9/l (medium). If an aminoglycoside is used instead of polymyxin, it is present in an amount of about 50-30, om9/l, depending on the aminoglycoside used. Trimethoprim is used in an amount of about 5 parts by weight of the sulfonamide, ie preferably about 0.74 to about 1.5 m9/l (medium). Crystal violet is about 01 to about 0.3
m91 l (medium) is used.

The medium to which the selection reagent is added includes an organic nitrogen source such as pton, mammalian blood, salt, and a gelling agent, preferably agar. Rpton is present in an amount of about 15 to about 30 P/l. Preferably, the protein is a pancreatic juice digest of casein and a papain digest of soybean porridge, and the casein digest is about 10 to about 20, preferably 15 g/l.
and the soybean porridge digestate is present in an amount of about 3 to about 8, preferably 51/l. Agar provides a gel of moderate hardness in amounts of about 13 to about 20, preferably 15 Lt/e.

Sodium chloride is about 2 to about 8 y-/l, preferably about 5
It is supplied in an amount of g-/l. 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 40 to about 100 volumes, can be obtained from a variety of mammalian sources, including sheep and rabbits. Blood is defibrinated before being used in the culture medium to prevent blood clotting. As mentioned above, blood is an indicator for hemolytic microorganisms such as GAS, and in the presence of such microorganisms a characteristic blood flow occurs. The occurrence of β-bath blood 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

Additional evidence for the presence of GAS on blood-containing culture plates containing the selection reagents described above is provided by Bacillus subtilis.
The absence of β-hemolytic colonies in the region containing bacitracin, an antibiotic produced by bacteria of the 5 ubtilis group. Bacitracin-impregnated disc (0
, 04 units) is commercially available for use on culture plates to apply the 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 with 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 Pseudomonas aeruginosa are also improved. Moreover, the high 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 can be performed substantially faster than is possible with prior art formulations (i.e., in 18-24 hours compared to 42-72 hours).

The invention will now be described in more detail by way of specific embodiments.

Example 1 Kanakura's Group A Streptococcus selective medium was prepared with the following ingredients.

A homogeneous powder was prepared by appropriately mixing components other than sheep blood. The powdered medium was hydrated with distilled or deionized water 11 and boiled until all ingredients were dissolved and the medium became clear. The medium was then cooled to 45-50 r in a water bath and defibrinated sheep's milk was added. Mix the medium by stirring thoroughly and use standard bacteriological methods for 18-20 min.
ml! f1.00 nm-s) was dispensed into IJ dishes. After this medium gels, place the dish in a suitable container.
For example, put it in a tube of plastic film (cellophane, polyethylene, nylon, etc.) and store it at 2 to 8C until use.
Saved to. The plates can be stored in this manner for 12 weeks.

Throat swab specimens obtained from patients (later shown to contain GAS-2) were plated on the plates and cytoselective blood agar plates described above according to routine microbiological procedures. The inoculum was then spread with a sterile (flamed) biological inoculation loop to obtain dispersed colonies. Several incisions were made in the agar with a loop in the most heavily inoculated area, and a 004 unit bacitracin disc was placed several centimeters away from the full incision, but in the most heavily inoculated area. Next, carbon dioxide gas 3 at a flat plate total of 35±2C
In an atmosphere of ~8% for 18-24 hours.

Upon examination, the β-hemolytic GAS colonies on non-selective blood agar plates were difficult to distinguish from the dense growth of normal throat biphases present on the medium. Susceptibility to bacitracin disc was also difficult to determine. However, with the plate medium containing the selective reagent, both normal throat phases were excellently suppressed, and the β-bath blood of GAS was observed without being greatly suppressed. 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 A medium (Al Gold) prepared in Example 1 above, similar to the medium (B-E ). Media B-E are commercially available.

However, if M is also present, the concentrations of each component are equal to the amounts specified in Example 1 above. The concentration of neomycin sulfate in media B and C is 30 μ/l. The concentration of nalidixic acid was 15 mq/L in medium C and LOm9/L in medium Dvc.
It is l.

One particular culture was evaluated to determine the relative performance of these media in terms of GAS recovery and inhibition of non-GAS present in common throat specimens.

The results are shown in Table 1.

The results shown in the table above demonstrate the surprising improvement that the selection reagents of the present invention have over selection reagents known in the prior art.

Example 3 In a study using human throat specimens, a selective medium of the invention (Medium A of Example 1) and SXT sheep blood agar (Medium E of Example 2) were combined with a non-selective sheep blood agar (control).
compared with. Throat swath specimens were inoculated into each of the three media as described in Example 1. The inoculated medium was then incubated at 35±2C in an atmosphere of 3 to 8 g carbon dioxide for 18
~24 hour incubation was complete. 46 as shown in the following table
Of the 0 random throat cultures, 117 were treated with medium (A) containing the selection reagent of the invention and 100 were treated with S]1iET.
Blood agar (E)K and only 84 species were ten against GAS' by the non-selective control.

In addition, 103 of the 117 cultures that were positive for GAS in the selective medium (A) of the present invention were identified as putatively GAS by bacitracin sensitivity within 24 hours. This is done on SXT blood agar (1 in El).
This represents a significant improvement over the putative identification of 80 out of 00 species and only 32 out of 84 species in the non-selective control.

Although the invention has been described in terms of certain preferred embodiments,
Modifications apparent to those skilled in the art may be made without departing from the scope of the invention. For example, although the present invention has been illustrated with four groups of components contained in a medium, it is contemplated that additional components may be added to further enhance the selectivity of the medium.

Various features of the invention are set out in the claims.

Patent applicant Becton Dickinson & Company 0 − + (5 others)

Claims (1)

[Claims] (11fa) Sulfonamide; (b) Trimethoprim;
(C) a component selected from the group consisting of polymyxins and aminoglycosides; and (d) a group A Streptococcus selection reagent comprising a mixture of crystal violet. (2) The reagent according to claim 1, wherein the sulfonamide is selected from the group consisting of sulfamethoxazole, sulfisoxazole and sulfadiazine. (3) The reagent according to claim 1, wherein the component (cl) is colistin sulfate. (4) The component (C) is an aminoglycoside selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin, and neomycin. , the reagent according to claim 1. (5) A nutrient source that supports the growth of group A streptococci, further [(a) sulfonamide r (b)) rimethoprim; fc) polymyxin and aminoglycoside. A medium for selectively culturing group A streptococci, comprising a component selected from the group consisting of; ) The medium according to claim 5, wherein the sulfonamide is selected from the group consisting of sulfamethoxazole, sulfisoxazole and sulfadiazine. (7) The medium according to claim 5, wherein component c) is colistin sulfate. The medium according to claim 5. (8) The medium according to claim 5, wherein the ingredients (cl) are selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin and neomycin. (9) A nutrient source for group A streptococci and (al sulfone (b) trimethoprim; (a component selected from the group consisting of "cbt" remixin and an aminoglycoside; and fd) a selection reagent consisting of a mixture of crystal violet; inoculating the biological specimen by smearing the medium; resulting in macroscopic growth of group A streptococci and selectively inhibiting the growth of substantially all other microorganisms present in the specimen; and inspecting the medium for the presence of beta-hemolytic colonies; group A streptococci by detecting all beta-hemolytic colonies susceptible to low concentrations of bacitracin; (10) The sulfonamide is sulfamethoxazole,
10. The method of claim 9, wherein the compound is selected from the group consisting of sulfisoxazole and sulfadiazine. (11) The method according to claim 9, wherein the component (cl) is colistin sulfate. +121 The component (C) is gentamicin, amikacin,
10. The method according to claim 9, wherein the aminoglycoside is selected from the group consisting of kanamycin, toframycin and neomycin. (13) placing a bacitracin-impregnated disk over a portion of the smeared area of the medium and detecting the presence or absence of hemolysis in the area, the absence of hemolysis near the disk indicating that the microorganism is group A streptococcus; , claim 1
The method described in Section 2.