CA1205029A - Selective agent for group a streptococci - Google Patents
Selective agent for group a streptococciInfo
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- CA1205029A CA1205029A CA000467966A CA467966A CA1205029A CA 1205029 A CA1205029 A CA 1205029A CA 000467966 A CA000467966 A CA 000467966A CA 467966 A CA467966 A CA 467966A CA 1205029 A CA1205029 A CA 1205029A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
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Abstract
SELECTIVE AGENT FOR GROUP A STREPTOCOCCI
ABSTRACT OF THE DISCLOSURE
An agent, selective for group A streptococci (GAS) on a culture plate, includes: 1) a polymyxin sulfate, such as colistin sulfate, or an aminoglyco-side; 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.
ABSTRACT OF THE DISCLOSURE
An agent, selective for group A streptococci (GAS) on a culture plate, includes: 1) a polymyxin sulfate, such as colistin sulfate, or an aminoglyco-side; 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
P-7~1 SELECTIVE AGENT FOR GROUP A STREPTOCOCCI
The present invention relates to rapid detection and presumptive identification of group ~ strepto-cocci (Streptococcus p~ ~nes) by means of a selec-tive blood agar medium.
BACKGROUND OF THE INVENTION
__ _ Streptococci, a genus of the family Lactobacil-laceae 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 O with two alphabetic omissions are known. Of these groups, group A ( _ 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 se-lecting 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"
~ ' ~3~
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 lQ pure culture to determine bacitracin susceptibility as an aid to presumptive identifica~ionO 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 bac-terial species are present, false positive results may be obtainedO
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 presump-tive identification. This takes two to-three days ~ P-7~1 longer than the method of the present inventionO
Because of the poor selectivity of existing media, greater skill is required to prevent error in diagno-sis. A more selective medium would reduce interpre-tive errors and would result in earlier .identifica-tion 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 sulfonamid~, such as sulfa-methoxazole~ 3) trimethoprim, and 4) crystal violet.
When the agent is incorporated in a mammalian blood-containing culture m~di.um, bacteria other than GAS
are substantially eliminated, thus, permitting an unobscured observation of the beta hemolysis sur-rounding colonies of GAS. As a result of the sub-stantial 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 hemolytic zones surrounding colonies of these organisms is obtained within 18 to 24 hours after inoculation with the specimen~
DETAILED DESCRIPTION OF THF PREFERRED EMBODIMENTS
_._ __ _ In accordance with the present invention, ~r 120S~2~ P- 741 improved detection of ~AS is achieved with a selec-tion agent that permits selective growth of GAS, (S.
~yogenes), substantially inhibiting the growth of other microorganisms which are likely to be present in a biological specimen, e.g., a specimen taXen from a human. This improved selectivity allows the deter-mination of bacitracin (0.04 units) susceptibility or serological testing o~ the primary isolation plate.
All of these components are individually known for their anti-bacterial properties; however, this cornbination of anti bacterial components has not been previously described, and it is found that the selection agent of the invention gives very signifi-cantly improved selectivity for GAS. In particular, when the selection agent is incorporated into a blood-contaning 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 vexy little extraneous growth from other microorganisms is present on the plate. This also permits, in most casesr the pre-sumptive identification based on bacitracin suscep-tibility and confirmatory identlfication by serologi-cal grouping tests within 24 hours a~ter inocluation with the specimen.
Polymyxins are complexes produced by Bacillus polymyxa, and these complexes and their sulfates are known to have antibacterial properties. Polymyxin takes several forms including A, B, C, D, E, K, M and P. Preferably, polymyxin E sulfate (colistin sul-fate) is one of the components of the selection ~ P-7~l agent. As a substitute for polymyxin sulfate, an aminoglycoside, such as gentamicin, amikacin, neo-mycin or tobramycin can be used. These components are known to be active against coliform bacilli and other Gram-negative bacteria.
Another component of the selection agen~ is a sulfonamide, such as sulfamethoxazole, sulfisoxazole, sulfadiazine, or any other which demonstrates syner-gistic 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. Sulfona-mides 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 selec-tive growth of GAS, but is not effective in in-hibiting staphylococci.
The final component i5 crystal violet also known as gentian violetD which is a mixture of penta~ and hexamethyl p-rosanaline chloridesO This component is known to inhibit neomycin resistant staphylococci.
~ P-741 The selection agent according to the invention typi~ally comprises between about 30.0 and abou~ 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 sulfonarnide, i.e., pr~ferably between abou~ 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 se~ection 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 J with the casein digest being present in amounts of from about 10 to about 20, preferably 1~, gm per liter and soybean meal digest being present in amounts of from about 3 to ab~ut 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.
~2~ P-741 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 prema-ture hemolysis of the blood~
Mammalian blood, which is present in amounts of from about 4.0 to abo~t 10.0 volume per cent, is derived from a variety of mammalian sources, in-cluding 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 charac-teristic hemolysis in the present of such org~nisms. The occurrence of bet~a hemolysis around colonies growing on a culture plate having the selection agent that substantially inhibits the growth of other hemolytic microorg2nisms 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 ab.sence of beta hemolytic colonies in a region containing bacitracin, an antibiokic produced by a member of the Bacillus subtilis group. Bacitra-cin~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 oE commercially available ~its.
Culture media containing the selection agent provide exceptional inhibition of normal microorga nismst especially the viridans streptococci, which are the predominant organisms present in most throat culturesO 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.y~, 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:
Per Liter of Ingredient Purified Water ~ . _ Pancreatic Digest oE Casein 15.0 g Papaic Digest o~ Soybean Meal 500 g Sodium Chloride 5~0 g Agar15.0 g Colistin Sulfate 15.0 mg Sulfamethoxazole 23.75 mg Trimethoprim 1.25 mg Crystal Violet 0.2 mg Sheep Bl03d~ Defibrinated50.0 mL
~2~ P-7~1 The ingredients, excluding the sheep blood, may be conveniently mixed together to produce a homo-genous 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 ~he defibrirlated sheep blood is added. The medium is mixed by agitation and 18 to 20 rnl 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 (cello-phane, polyethylene, NylonR, etc.), and are stored at 2 to 8C 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 2S bacitracin disc is placed a few cen~imeters away Erom the cut~ but still in the heaviest inoculated area.
The plate is then incubated at 35 ~ 2C in an atmo sphere of 3 to 8% carbon dioxide for 18 to 24 hours.
Upon examination, the beta hernolytic GAS colo-nies on the non-selective blood agar plate are ~ Z~ P-741 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 suppres-sion of the normal throat flora a~d permits unob-scured observation o 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.
Exam~le 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~l) C(2) D(3) E(4) Colistin Sulfate ~ - - +
Sulfamethoxazole ~ +
Trimethoprim Crystal Violet Neomycin Sulfate - ~ +
Nalidixic Acid - - + ~ _ ~1) Neomycin Blood agar
The present invention relates to rapid detection and presumptive identification of group ~ strepto-cocci (Streptococcus p~ ~nes) by means of a selec-tive blood agar medium.
BACKGROUND OF THE INVENTION
__ _ Streptococci, a genus of the family Lactobacil-laceae 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 O with two alphabetic omissions are known. Of these groups, group A ( _ 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 se-lecting 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"
~ ' ~3~
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 lQ pure culture to determine bacitracin susceptibility as an aid to presumptive identifica~ionO 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 bac-terial species are present, false positive results may be obtainedO
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 presump-tive identification. This takes two to-three days ~ P-7~1 longer than the method of the present inventionO
Because of the poor selectivity of existing media, greater skill is required to prevent error in diagno-sis. A more selective medium would reduce interpre-tive errors and would result in earlier .identifica-tion 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 sulfonamid~, such as sulfa-methoxazole~ 3) trimethoprim, and 4) crystal violet.
When the agent is incorporated in a mammalian blood-containing culture m~di.um, bacteria other than GAS
are substantially eliminated, thus, permitting an unobscured observation of the beta hemolysis sur-rounding colonies of GAS. As a result of the sub-stantial 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 hemolytic zones surrounding colonies of these organisms is obtained within 18 to 24 hours after inoculation with the specimen~
DETAILED DESCRIPTION OF THF PREFERRED EMBODIMENTS
_._ __ _ In accordance with the present invention, ~r 120S~2~ P- 741 improved detection of ~AS is achieved with a selec-tion agent that permits selective growth of GAS, (S.
~yogenes), substantially inhibiting the growth of other microorganisms which are likely to be present in a biological specimen, e.g., a specimen taXen from a human. This improved selectivity allows the deter-mination of bacitracin (0.04 units) susceptibility or serological testing o~ the primary isolation plate.
All of these components are individually known for their anti-bacterial properties; however, this cornbination of anti bacterial components has not been previously described, and it is found that the selection agent of the invention gives very signifi-cantly improved selectivity for GAS. In particular, when the selection agent is incorporated into a blood-contaning 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 vexy little extraneous growth from other microorganisms is present on the plate. This also permits, in most casesr the pre-sumptive identification based on bacitracin suscep-tibility and confirmatory identlfication by serologi-cal grouping tests within 24 hours a~ter inocluation with the specimen.
Polymyxins are complexes produced by Bacillus polymyxa, and these complexes and their sulfates are known to have antibacterial properties. Polymyxin takes several forms including A, B, C, D, E, K, M and P. Preferably, polymyxin E sulfate (colistin sul-fate) is one of the components of the selection ~ P-7~l agent. As a substitute for polymyxin sulfate, an aminoglycoside, such as gentamicin, amikacin, neo-mycin or tobramycin can be used. These components are known to be active against coliform bacilli and other Gram-negative bacteria.
Another component of the selection agen~ is a sulfonamide, such as sulfamethoxazole, sulfisoxazole, sulfadiazine, or any other which demonstrates syner-gistic 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. Sulfona-mides 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 selec-tive growth of GAS, but is not effective in in-hibiting staphylococci.
The final component i5 crystal violet also known as gentian violetD which is a mixture of penta~ and hexamethyl p-rosanaline chloridesO This component is known to inhibit neomycin resistant staphylococci.
~ P-741 The selection agent according to the invention typi~ally comprises between about 30.0 and abou~ 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 sulfonarnide, i.e., pr~ferably between abou~ 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 se~ection 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 J with the casein digest being present in amounts of from about 10 to about 20, preferably 1~, gm per liter and soybean meal digest being present in amounts of from about 3 to ab~ut 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.
~2~ P-741 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 prema-ture hemolysis of the blood~
Mammalian blood, which is present in amounts of from about 4.0 to abo~t 10.0 volume per cent, is derived from a variety of mammalian sources, in-cluding 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 charac-teristic hemolysis in the present of such org~nisms. The occurrence of bet~a hemolysis around colonies growing on a culture plate having the selection agent that substantially inhibits the growth of other hemolytic microorg2nisms 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 ab.sence of beta hemolytic colonies in a region containing bacitracin, an antibiokic produced by a member of the Bacillus subtilis group. Bacitra-cin~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 oE commercially available ~its.
Culture media containing the selection agent provide exceptional inhibition of normal microorga nismst especially the viridans streptococci, which are the predominant organisms present in most throat culturesO 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.y~, 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:
Per Liter of Ingredient Purified Water ~ . _ Pancreatic Digest oE Casein 15.0 g Papaic Digest o~ Soybean Meal 500 g Sodium Chloride 5~0 g Agar15.0 g Colistin Sulfate 15.0 mg Sulfamethoxazole 23.75 mg Trimethoprim 1.25 mg Crystal Violet 0.2 mg Sheep Bl03d~ Defibrinated50.0 mL
~2~ P-7~1 The ingredients, excluding the sheep blood, may be conveniently mixed together to produce a homo-genous 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 ~he defibrirlated sheep blood is added. The medium is mixed by agitation and 18 to 20 rnl 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 (cello-phane, polyethylene, NylonR, etc.), and are stored at 2 to 8C 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 2S bacitracin disc is placed a few cen~imeters away Erom the cut~ but still in the heaviest inoculated area.
The plate is then incubated at 35 ~ 2C in an atmo sphere of 3 to 8% carbon dioxide for 18 to 24 hours.
Upon examination, the beta hernolytic GAS colo-nies on the non-selective blood agar plate are ~ Z~ P-741 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 suppres-sion of the normal throat flora a~d permits unob-scured observation o 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.
Exam~le 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~l) C(2) D(3) E(4) Colistin Sulfate ~ - - +
Sulfamethoxazole ~ +
Trimethoprim Crystal Violet Neomycin Sulfate - ~ +
Nalidixic Acid - - + ~ _ ~1) Neomycin Blood agar
(2) Neomycin/Nalidixic Blood Agar
(3) CNA Agar
(4) SXT Blood Agar ~ P-741 The concentrations of each cornponent 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 literO The concentration of nalidixic acid is 15 mg per liter in medium C an~ 10 mg per liter in medium D.
The media are evalua~ed 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.
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 streptococciXXXX - X X XXXX
Inhibition of viridàns streptococci XXXX - - - XXXX
Inhibition of other beta~ or non hemolytic streptococci XXX - X X XXX
Inhibitio~ of Neisseria XXXX XXXX XXXX XXXX XX
Inhibition of Pseudomonas XXXX - XX XXXX XX
.
Inhlbition of other gram negative species XXXX XXXX XXXX XXXX XXXX
Inhibition of 5. aureus XXXX XXXX XXXX X XX
XXXX EXCELLENT
XXX GOOD
XX FAIR
X TRACE
- NONE
~2~5~ P-~41 The results, as set forth in the above table, illustrate the surprising improvement of the selec-tion agent of ~he present invention over selection agents known in the prior art.
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 wi th the procedure described in Example 1. Inocu lated media are then incuba~ed for 18 ~o 24 hours at 35 -~ 2~C in an atmosphere of 3 to 8% carbon dioxide.
Of 460 random throat cultures, 117 are positive for G~S with the media containing the selective agent of the invention (,A), 130 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 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-se~
lective control ~13-Although the invention has been described in terms of certain preferred e~bodiments, modiications obvious to one with ordinary skill in the art may be made without departin~ from the scope of the inven-tion. 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.
The media are evalua~ed 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.
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 streptococciXXXX - X X XXXX
Inhibition of viridàns streptococci XXXX - - - XXXX
Inhibition of other beta~ or non hemolytic streptococci XXX - X X XXX
Inhibitio~ of Neisseria XXXX XXXX XXXX XXXX XX
Inhibition of Pseudomonas XXXX - XX XXXX XX
.
Inhlbition of other gram negative species XXXX XXXX XXXX XXXX XXXX
Inhibition of 5. aureus XXXX XXXX XXXX X XX
XXXX EXCELLENT
XXX GOOD
XX FAIR
X TRACE
- NONE
~2~5~ P-~41 The results, as set forth in the above table, illustrate the surprising improvement of the selec-tion agent of ~he present invention over selection agents known in the prior art.
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 wi th the procedure described in Example 1. Inocu lated media are then incuba~ed for 18 ~o 24 hours at 35 -~ 2~C in an atmosphere of 3 to 8% carbon dioxide.
Of 460 random throat cultures, 117 are positive for G~S with the media containing the selective agent of the invention (,A), 130 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 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-se~
lective control ~13-Although the invention has been described in terms of certain preferred e~bodiments, modiications obvious to one with ordinary skill in the art may be made without departin~ from the scope of the inven-tion. 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 (13)
1. A group A streptococcus selection agent comprising a mixture of (1) a sulfonamide, (2) trimethoprim; (3) a component selected from the group consisting of polymyxin and an aminoglycoside; and (4) crystal violet.
2. An agent according to Claim 1 wherein said sulfonamide is selected from the group consisting of sulfamethoxazole, sulfisoxazole and sulfadiazine.
3. An agent according to Claim 1 wherein said component(s) is colistin sulfate.
4. An agent according to Claim 1 wherein said component(s) is an aminoglycoside selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin or neomycin.
5. A medium for the selective culturing of group A streptococcus comprising a nutrient for supporting growth of group A streptococci and in-cluding a selective agent active against microorga-nisms other than Group A Streptocci comprising comprising a mixture of (1) a sulfonamide; (2) trimethoprim; (3) a component selected from the group consisting of a polymyxin and an aminoglycoside; and (4) crystal violet.
6. A medium according to Claim 5 wherein said sulfonamide is selected from the group consisting of sulfamethoxazole.
7. A medium according to Claim 5 wherein said component 3 is colistin sulfate.
8. A medium according to Claim 5 wherein said component 3 is an aminoglycoside selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin and neomycin.
9. A method of selectively identifying group A
streptococci by the detection of beta hemolytic colonies susceptible to low concentrations of baci-tracin comprising providing a medium including a nutrient for Group A streptococci and a selection agent comprising a mixture of (1) a sulfonamide; (2) trimethoprim; (3) a component selected from the group consisting of a polymyxin and an aminoglycoside; and (4) crystal violet;
incubating said medium by streaking with a biological specimen suspected of containing group A
streptococci;
incubating said medium for a period sufficient to obtain visible growth of group A streptococci and selectively inhibit the growth of substantially all other microorganisms present within said specimen;
and examination of the medium for the presence of beta hemolytic colonies.
streptococci by the detection of beta hemolytic colonies susceptible to low concentrations of baci-tracin comprising providing a medium including a nutrient for Group A streptococci and a selection agent comprising a mixture of (1) a sulfonamide; (2) trimethoprim; (3) a component selected from the group consisting of a polymyxin and an aminoglycoside; and (4) crystal violet;
incubating said medium by streaking with a biological specimen suspected of containing group A
streptococci;
incubating said medium for a period sufficient to obtain visible growth of group A streptococci and selectively inhibit the growth of substantially all other microorganisms present within said specimen;
and examination of the medium for the presence of beta hemolytic colonies.
10. A method according to Claim 9 wherein said sulfonamide is selected from the group consisting of sulfamethoxazole, sulfisoxazole and sulfadiazine.
11. A method according to Claim 9 wherein said component 3 is colistin sulfate.
12. A method according to Claim 9 wherein said component 3 is an aminoglycoside selected from the group consisting of gentamicin, amikacin, kanamycin, tobramycin and neomycin.
13. A method according to Claim 12 including applying a bacitracin impregnated disc onto a portion of the streaked area of said medium and detecting the presence or absence of hemolysis in said area, the absence of hemolysis near the disc indicating the organism to be group A streptococcus.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US58194384A | 1984-02-21 | 1984-02-21 | |
| US581,943 | 1990-09-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1205029A true CA1205029A (en) | 1986-05-27 |
Family
ID=24327209
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000467966A Expired CA1205029A (en) | 1984-02-21 | 1984-11-16 | Selective agent for group a streptococci |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JPS60176599A (en) |
| CA (1) | CA1205029A (en) |
| DE (1) | DE3505311A1 (en) |
| GB (1) | GB2154606B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003290377A1 (en) * | 2003-01-03 | 2004-07-29 | Marton Milankovits | Pharmaceutical compositions comprising an antibacterial agent nd antifungal agent and a nitroimidazole for the treatment and prevention of genitourinary infections and their extragenital complications |
| SK287315B6 (en) | 2006-06-02 | 2010-06-07 | Biotika, A. S. | A method for polymyxin B isolation from fermented soil |
| SK287293B6 (en) | 2006-06-15 | 2010-05-07 | Biotika, A. S. | A method for fermentation of polymyxin B by means of productive microorganism Bacillus polymyxa |
| JP5118336B2 (en) * | 2006-11-28 | 2013-01-16 | 日水製薬株式会社 | Enterococci detection medium |
-
1984
- 1984-11-16 CA CA000467966A patent/CA1205029A/en not_active Expired
- 1984-11-27 GB GB08429863A patent/GB2154606B/en not_active Expired
-
1985
- 1985-01-11 JP JP314985A patent/JPS60176599A/en active Granted
- 1985-02-15 DE DE19853505311 patent/DE3505311A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0456600B2 (en) | 1992-09-08 |
| GB2154606B (en) | 1987-03-25 |
| JPS60176599A (en) | 1985-09-10 |
| DE3505311A1 (en) | 1985-12-19 |
| GB2154606A (en) | 1985-09-11 |
| DE3505311C2 (en) | 1987-05-07 |
| GB8429863D0 (en) | 1985-01-03 |
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