GB2154606A - Selective agent for group a streptococci - Google Patents

Abstract

An agent, selective for group A streptococci (GAS) on a culture plate, includes: 1) a polymyxin, such as colistin sulfate or an aminoglycoside; 2) a sulfonamide, such as sulfamethoxazole; 3) trimethoprim; and 4) crystal violet. The agent is incorporated in a culture medium containing nutrients for group A streptococci. The medium is inoculated with a biological specimen and incubated. The presence of beta hemolytic colonies on the medium is indicative of GAS in the biological specimen.

Description

SPECIFICATION Selective agent for group a streptococci The present invention relates to rapid detection and presumptive identification of group A streptococci (Streptococcus pyogenes) by means of a selective blood agar medium.

Background of the invention Streptococci, a genus of the family Lactobacillaceae are well distributed in nature. Some strains of streptococci are pathogenic to man and/or animals, whereas others may exist as generally harmless parasites. Streptococci are classified (Lancefield classification system) according to the presence of a type of carbohydrate known as the C substance, and at least 13 groups, designated A through 0 with two alphabetic omissions are known. Of these groups, group A (S. pyogenes) is almost always responsible for human infections, although other groups, in certain circumstances, might also be pathogenic.Group A streptococci (GAS) are responsible for such diseases as scarlet fever, rheumatic fever, glomerulonephritis, pharyngitis and puerperal spesis; hence, identification -of the presence of GAS can be very significant in diagnosing a disease and selecting an appropriate course of treatment.

According to present methods of detection of GAS, a specimen is obtained by means of a cotton or polyester swab applied to the infected area, such as the pharynx. The swab is then used to inoculate a suitable medium containing mammalian blood in order to detect the characteristic complete or "beta" hemolysis which appears around colonies of GAS. However, a throat culture usually contains other microorganisms which may be present in a much greater number so that their growth obscures the hemolysis produced by GAS. The presence of bacteria resistant to bacitracin also prevents the determination of bacitracin susceptibility on the plate inocluated with the specimen. Therefore, prior art methods require isolation of the beta-hemolytic organisms in pure culture to determine bacitracin susceptibility as an aid to presumptive identification.Thus, two or more days may be required before information is available to initiate appropriate therapy.

Although a number of anti-bacterial agents are known to which GAS are resistant, there is no single anti-bacterial agent or known combination of anti-bacterial agents which are fully selective for GAS, i.e., which will inhibit the growth of most other microorganisms. Non-group A streptococci and other microorganisms may, therefore, grow sufficiently on existing selective media so as to make it difficult to detect the characteristic beta hemolysis produced by GAS. Furthermore, if other beta-hemolytic bacterial species are present, false positive results may be obtained.

Current methods for the detection of GAS are relatively reliable, often achieving upwards of 90 per cent accuracy but require isolation in pure culture of the beta hemolytic streptococci in order to determine bacitracin susceptibility for presumptive identification. This takes two to three days longer than the method of the present invention. Because of the poor selectivity of existing media, greater skill is required to prevent error in diagnosis. A more selective medium would reduce interpretive errors and would result in earlier identification and hence earlier institution of appropriate therapy to prevent the serious sequelae of infection by GAS.

Summary of the invention An agent is provided for selectively culturing GAS. The agent is a mixture of 1) a polymyxin or an aminoglycoside, 2) a sulfonamide, such as sulfamethoxazole, 3) trimethoprim, and 4) crystal violet. When the agent is incorporated in a mammalian blood-containing culture medium, bacteria other than GAS are substantially eliminated, thus, permitting an unobscured observation of the beta hemolysis surrounding colonies of GAS. As a result of the substantial improvement in selectivity afforded by the selection agent of the present invention, presumptive identification of GAS based on susceptibility to bacitracin (0.04 unit disc) and beta hemolyticzones surrounding colonies of these organisms is obtained within 18 to 24 hours after inoculation with the specimen.

Detailed description of the preferred embodiments In accordance with the present invention, improved detection of GAS is achieved with a selection agent that permits selective growth of GAS, (S. pyogenes), substantially inhibiting the growth of other microorganisms which are likely to be present in a biological specimen, e.g., a specimen taken from a human. This improved selectivity allows the determination. This improved selectivity allows the determination of bacitracin (0.04 units) susceptibility or serological testing on the primary isolation plate. All of these components are individually known for their anti-bacterial properties; however, this combination of anti-bacterial components has not been previously described, and it is found that the selection agent of the invention gives very significantly improved selectivity for GAS.In particular, when the selection agent is incorporated into a blood-containing culture plate on which a human biological specimen containing GAS is inoculated, very clear beta hemolysis patterns are observed in the region of GAS growth, and very little extraneous growth from other microorganisms is present on the plate. This also permits, in most cases, the presumptive identification based on bacitracin susceptibility and confirmatory identification by serological grouping tests within 24 hours after inocluation with the specimen.

Polymyxins are complexes produced byBacilluspolymyxa, and these complexes and their sulfates are known to have the antibacterial properties. Polymyxin takes several forms including A, B, C, D, E, K, M and P.

Preferably, polymyxin E sulfate (colistin sulfate) is one of the components of the selection agent. As a substitute for polymyxin sulfate, an aminoglycoside, such as gentamicin, amikacin, neomycin ortobramycin can be used. These components are known to be active against coliform bacilli and other Gram-negative bacteria.

Another component of the selection agent is a sulfonamide, such as sulfamethoxazole, sulfisoxazole, sulfadiazine, or any other which demonstrates synergistic activity in combination with trimethroprim against alpha Streptococcus and is sufficiently soluble to be used in an aqueous culture medium. The preferred sulfonamide is sulfamethoxazole. Sulfonamides are known to inhibit both Gram-positive and Gram-negative bacteria, but streptococci are mostly resistant.

Another component of the selection agent is trimethoprim or trimethoprim lactate. Trimethoprim is effective in inhibiting both Gram-positive and Gram-negative bacteria, but in combination with a sulfonamide, it exhibits a synergistic effect, so that many organisms that are not inhibited by either alone are inhibited when both are present. The combination of sulfamethoxazole and trimethoprim has been used previously in culture media for selective growth of GAS, but is not effective in inhibiting staphylococci.

The final component is crystal violet also known as gentian violet, which is a mixture of penta- and hexamethyl p-rosanaline chlorides. This component is known to inhibit neomycin-resistant staphylococci.

The selection agent according to the invention typically comprises between about 30.0 and about 50.0 mg per liter of a culture medium. The sulfonamide is generally the antibacterial component of greatest amount by weight, typically being used in amounts of between about 15 and about 30 mg per liter of the culture medium. Polymyxin sulfate is used in amounts of between about 10.0 and 20.0, preferably 15.0 mg per liter of culture medium. If an aminoglycoside is substituted for the polymyxin, it is present at between about 5.0 and 30.0 mg per liter, depending on which aminoglycoside is used. Trimethoprim is used at about 5 per cent of the weight of the sulfonamide, i.e., preferably between about 0.74 and about 1.5 mg per liter of culture medium. Crystal violet is used at between about 0.1 and about 0.3 mg per liter of culture medium.

The culture medium to which the selection agent is added includes an organic nitrogen source, such as peptones, mammalian blood, salt and a gelling agent, preferably agar. Peptones are present in amounts of about 15 to about 30 gm per liter. The preferred peptones are pancreatic digest of casein and a papaic digest of soybean meal, with the casein digest being present in amounts of from about 10 to about 20, preferably 15, gm per liter and soybean meal digest being present in amounts of from about 3 to about 8, preferably 5, gm per liter. Agar in amounts of from about 13 to about 20, preferably 15, gm per liter provides a gel of appropriate firmness.

Sodium chloride is provided in amounts of from about 2 to about 8 gm per liter and preferably about 5 gm per liter. The salt concentration is important for proper osmolality of the gel, preventing premature hemolysis of the blood.

Mammalian blood, which is present in amounts offrom about 4.0 to about 10.0 volume per cent, is derived from a variety of mammalian sources, including sheep and rabbit. The blood is defibrinated for use in a culture medium to prevent clotting. The blood, as stated above, is an indicator for hemolytic organisms, such as GAS, producing characteristic hemolysis in the present of such organisms. The occurence of beta hemolysis around colonies growing on a culture plate having the selection agent that substantially inhibits the growth of other hemolytic microorganisms is a strong indication of the presence of GAS.

Additional confirmation of the presence of GAS on a blood culture plate containing the selection agent is the absence of beta hemolytic colonies in a region containing bacitracin, an antibiotic produced by a member of the Bacillus subtilis group. Bacitracin-impregnated discs (0.04 units) are commercially available for placement on culture plates to apply the antibiotic in limited regions of the plates. Furthermore, definitive serological identification can be obtained on a culture plate containing the selection agents by means of commercially available kits.

Culture media containing the selection agent provide exceptional inhibition of normal microorganisms, especially the viridans streptococci, which are the predominant organisms present in most throat cultures.

While inhibition of viridans streptococci is accomplished with a mixture of sulfamethoxazole and trimethoprim alone, the selection agent of the invention also provides improved inhibition of staphylococci, other streptococci and Gram-negative bacteria, including neisseria species and Pseudomonas aeruginosa. In addition, the highly selective nature of the selection agent of the invention permits the presumptive identification of GAS (through the appearance of hemolytic colonies on the plate and the absence of hemolytic colonies in the region of a bacitracin disc) and definitive serological grouping substantially earlier than is possible with prior art formulations, e.g., 18 to 24 hours as compared to 42 to 72 hours.

The invention will now be described in greater detail by means of specific examples.

EXAMPLE 1 A group A streptococcus selective medium is prepared having the following ingredients: PerLiterof Ingredient Purified Water Pancreatic Digest of Casein 15.0 g Papaic Digest of Soybean Meal 5.0 g Sodium Chloride 5.0 g Agar 15.09 Coliston Sulfate 15.0 mg Sulfamethoxazole 23.75 mg Trimethoprim 1.25 mg Crystal Violet 0.2 mg Sheep Blood, Defibrinated 50.0 mL The ingredients, excluding the sheep blood, may be conveniently mixed together to produce a homogenous powder. The powdered medium is hydrated with one liter of distilled or deionized water and boiled until all ingredients are dissolved and the medium is clear. The medium is then cooled to 45 to 50"C in a water bath, and the defibrinated sheep blood is added.The medium is mixed by agitation and 18 to 20 ml is dispensed into 100 mm Petri dishes according to standard bacteriologic practice. After this medium is gelled, the dishes are placed in a suitable container, such as a sleeve of plastic film (cellophane, polyethylene, NylonR, etc.). and are stored at 2 to 8 C until ready for use. The plates may be stored in this manner for up to 12 weeks.

A throat swab specimen from a patient who was subsequently shown to contain GAS is inoculated onto the above described plated medium and a plate of non-selective blood agar, according to routine microbiological procedure. The inoculum is then spread out with a sterile (flamed) bacteriological inoculating loop so as to obtain isolated colonies. In the area of heaviest inoculation, several cuts are made into the agar with the loop and a 0.04 unit bacitracin disc is placed a few centimeters away from the cuts but still in the heaviest inoculated area. The plate is then incubated at 35 + 2"C in an atmosphere of 3 to 8% carbon dioxide for 18to 24 hours.

Upon examination, the beta hemolytic GAS colonies on the non-selective blood agar plate are difficult to distinguish from the heavy growth of normal throat flora also present on the medium. Sensitivity to the bacitracin disc is also difficult to determine. The plated medium containing the selective agent, however, provides excellent suppression of the normal throat flora and permits unobscured observation of the beta hemolysis of GAS. A zone of inhibition surrounding the bacitracin disc is clearly evident, thus resulting in the presumptive identification of the beta hemolytic growth as GAS within only 18 to 24 hours after inoculation with the specimen.

EXAMPLE 2 The medium (A) prepared in Example 1 above is compared with media (B-E) in an identical manner except with or without certain selective components as indicated by the pluses or minuses in the table below. The media B-E are commercially available.

A B {Z) C ,2) D /3) Colistin Sulfate + - - + Sulfamethoxazole + - - - + Trimethoprim + - - - + Crystal Violet + + Neomycin Sulfate - - + Naíidixic Acid - + + - (1) Neomycin Blood agar (2) Neomycin/Nalidixic Blood Agar (3) CNAAgar (4) SXT Blood Agar The concentrations of each component where present are equal to the amounts as specified in Example 1 above. The concentration of neomycin sulfate in media B and C is 30 mg per liter. The concentration of nalidixic acid is 15 mg per liter in medium C and 10 mg per liter in medium D.

The media are evaluated with specific cultures to determine relative performance with respect to the recovery of GAS and the inhibition of non-GAS present in normal throat specimens. Results are indicated in Table 1.

TABLE 1 A B C D E Recovery of GAS XXX XXXX XXXX XXXX XXX Beta-hemolysis of GAS XXX XXXX XXXX XXXX XXX Inhibition of group B streptococci XXX - X X XXX Inhibition of groups C and G streptococci XXXX - X X XXXX Inhibition of viridans streptococci XXXX - - - XXXX Inhibition of other beta, or non hemolytic streptococci XXX - X X XXX Inhibition of Neisseria XXXX XXXX XXXX XXXX XX Inhibition of Pseudomonas XXXX - XX XXXX XX Inhibition of other gram negative species XXXX XXXX XXXX XXXX XXXX Inhibition of S. aureus XXXX XXXX XXXX X XX XXXX EXCELLENT XXX GOOD XX FAIR X TRACE NONCE The results, as set forth in the above table, illustrate the surprising improvement of the selection agent of the present invention over selection agents known in the prior art.

EXAMPLE 3 In a study involving human throat specimens, the selective medium of the invention (medium A in Example 1) and SXT Sheep Blood Agar (medium E in Example 2) are compared with non-selective sheep blood agar (control). Throat swab specimens are inoculated onto each of the three media in accordance with the procedure described in Example 1. Inoculated media are then incubated for 18 to 24 hours at 35 + 2"C in an atmosphere of 3 to 8% carbon dioxide. Of 460 random throat cultures, 117 are positive for GAS with the media containing the selective agent of the invention (A), 100 with SXT Blood Agar (E) and only 84 with the non-selective control as illustrated in the table below.

A E Control Cultures positive for GAS at 24 hours 103 80 32 Cultures positive for GAS at 48 hours 14 20 52 TOTAL 117 100 84 Furthermore, of the 117 cultures positive for GAS on the selective medium of the invention (A), 103 are presumptively identified as GAS by bacitracin susceptibility within 24 hours. This represents a significant improvement as compared to 80 of 100 on SXT Blood Agar (E) and only 32 of 84 on the non-selective control Although the invention has been described in terms of certain preferred embodiments, modifications obvious to one with ordinary skill in the art may be made without departing from the scope of the invention.

For example, while the invention sets forth four groups of components to be included in a medium, it is contemplated that additional components might be added, further enhancing medium selectivity.

Various features of the invention are set forth in the following claims.

Claims (11)

1. A group A streptococcus selective agent comprising a mixture of (1) a sulphonamide, (2) trimethoprim; (3) polymyxin and/or an aminoglycoside; and (4) crystal violet.

2. An agent according to claim 1, wherein the sulphonamide is selected from sulphamethoxazole, sulphisoxazole and sulphadiazine.

3. An agent according to claim 2, wherein the sulphonamide is sulphamethoxazole.

4. An agent according to claim 1,2 or 3, wherein the trimethoprim is present as the lactate.

5. An agent according to any preceding claim, which contains the aminoglycoside.

6. An agent accordingly to claim 5 wherein the aminoglyoside is selected from gentamicin, amikacin, kanamycin, tobramycin and neomycin.

7. An agent according to any preceding claim, which contains the polymyxin.

8. An agent according to claim 7, wherein the polymyxin is present as colistin sulphate.

9. A medium for the selective culturing of group A streptococcus comprising a nutrient for supporting growth of group A streptococci and including a selective agent active against microorganisms other than Group A Streptocci, the selective agent being as defined in any proceeding claim.

10. A method of selectively identifying group A streptococci by the detection of beta hemolytic colonies susceptible to low concentrations of bacitracin the method comprising: inoculating a medium as defined in claim 9 by streaking with a biological specimen suspected of containing group A streptococci; incubating the inoculated medium for a period sufficient to obtain visible growth of group A streptococci and selectively inhibit the growth of substantially all other microorganisms present in the specimen ; and examining the medium for the presence of beta hemolytic colonies.

11. A method according to Claim 10 including applying a bacitracin-impregnated disc onto a portion of the streaked area of the medium and detecting the presence or absence of hemolysis in the area, the absence of hemolysis near the disc indicating the organism to be group A streptococcus.