Title:
METHOD OF SCREENING FOR ANTICAPSULAR SUBSTANCES
United States Patent 3793151
Abstract:
A method of screening for compounds which inhibit the formation of hyaluronic acid capsules implicated in the virulence of certain pathogenic microorganisms.
US Patent References:
Preparation for typing of staphylococci and process therefor
Schwartz - June 1963 - 3094466
Preparation for typing of staphylococci and process therefor
Schwartz - June 1963 - 3094466
Application Number:
05/270098
Publication Date:
02/19/1974
Filing Date:
07/10/1972
View Patent Images:
Export Citation:
Primary Class:
Other Classes:
435/32, 435/235.100
International Classes:
C12Q1/02; C12K1/04
Field of Search:
195/13.5R
Primary Examiner:
Tanenholtz, Alvin E.
Assistant Examiner:
Hensley, Max D.
Attorney, Agent or Firm:
Woodard, Weikart, Emhardt & Naughton
Parent Case Data:
This is a continuation of application Ser. No. 8,736, filed Feb. 4, 1970, now abandoned.
Claims:
I claim
1. A method of screening for compounds which inhibit the formation of hyaluronic acid capsules on pathogenic microorganisms comprising:
2. A method in accord with claim 1 wherein the hyaluronic acid capsule-forming microorganism is Streptococcus pyogenes.
3. A method in accord with claim 2 wherein the hyaluronic acid capsule-forming microorganism is Streptococcus pyogenes ATCC 12384 and the bacteriophage is phage OX240, ATCC 12384B.
4. A method in accord with claim 2 wherein said test plate contains from 0.5 to 1.5 ml. of a cell suspension of Streptococcus pyogenes having an O.D. of 0.3 at 540 mμ.
5. A method in accord with claim 3 wherein said test plate contains from 110 to 135 international units of hyaluronidase.
6. A method in accord with claim 5 wherein the phage stock has a titer of at least 1 × 109 plaque-forming units.
1. A method of screening for compounds which inhibit the formation of hyaluronic acid capsules on pathogenic microorganisms comprising:
2. A method in accord with claim 1 wherein the hyaluronic acid capsule-forming microorganism is Streptococcus pyogenes.
3. A method in accord with claim 2 wherein the hyaluronic acid capsule-forming microorganism is Streptococcus pyogenes ATCC 12384 and the bacteriophage is phage OX240, ATCC 12384B.
4. A method in accord with claim 2 wherein said test plate contains from 0.5 to 1.5 ml. of a cell suspension of Streptococcus pyogenes having an O.D. of 0.3 at 540 mμ.
5. A method in accord with claim 3 wherein said test plate contains from 110 to 135 international units of hyaluronidase.
6. A method in accord with claim 5 wherein the phage stock has a titer of at least 1 × 109 plaque-forming units.
Description:
BACKGROUND OF THE INVENTION
Virulence may be defined as the degree of pathogenicity within a group of microorganisms. In some instances, the ability of a pathogenic microorganism to cause a disease or infection can be partially related to the production of specific cellular structures or products, hereinafter referred to as "virulence factors." Examples of such virulence factors are capsules, certain enzymes, toxins, and antigens. Any treatment of such a pathogenic microorganism, which would in some way alter either the biosynthesis or biological function of a virulence factor, may in fact have therapeutic utility.
Streptococcus zooepidemicus and Streptococcus pyogenes, members of the Pyogenic group of Streptococci, both produce a capsular structure composed of hyaluronic acid at the outer surface of their cell walls. This capsule has been implicated in the virulence of the latter microorganism by rendering it less susceptible to phagocytosis and destruction when phagocytized: (Faley et al., "Studies on the Pathogenicity of Group A Streptocci," J. Exp. Med., 110: 603-605 (1959); Rothbard, J. Exp. Med. 88: 325 (1948); Kass and Seastone, "The Role of Mucoid Polysaccaride (hyaluronic acid) in the Virulence of Group A Hemolytic Streptococci," J. Exp. Med., 79: 319-329 (1944). Such encapsulated cells are also resistant to infection and lysis by bacteriophage, hereinafter referred to as "phage."
It has been found that a strain of S. pyogenes which produces a hyaluronic acid capsule, a phage specific for this strain, and an appropriate positive control, which in this case is the enzyme hyaluronidase, can be employed to detect the activity of, and hence screen for, substances which inhibit the synthesis of said capsule. Such substances, hereinafter referred to as anticapsular compounds, do not usually exhibit classical antibacterial activity, and thus are not detected in conventional antibiotic screens currently employed by those skilled in the art.
The anticapsular compounds detected in the screening process of this invention inhibit the synthesis of the hyaluronic acid capsule of S. pyogenes. Cells of S. pyogenes thus treated are significantly more susceptible than encapulated cells to in vitro phagocytosis by human leukocytes. Administering anticapsular compounds to a suitable host may allow the host's phagocytic system to more readily destroy the infecting S. pyogenes, as well as enhance the activity of conventional antibiotics against such organisms. Such compounds may also be useful in treating conditions in man wherein an increased production of hyaluronic acid is implicated. Such conditions include Marfan's syndrome, Hurler's syndrome, myxedema, osteroarthritis, arthro-acteoonychodysplasia, and neuropathy.
SUMMARY OF THE INVENTION
This invention relates to a method of screening for anticapsular compounds. As used herein, an anticapsular compound is one which inhibits the formation of hyaluronic acid capsules of pathogenic microorganisms wherein the capsule is implicated in the virulence of the microorganisms. The screen process of this invention utilizes a capsule-forming, pathogenic microrganism or bacterium, a bacteriophage to which the organism is susceptible only when non-encapsulated, and a suitable positive control which removes the hyaluronic acid capsule but does not inhibit either the bacterium or the bacteriophage, to screen for anticapsular compounds.
Generally speaking, filtered fermentation broths or solutions of organic compounds are screened for anticapsular activity as follows. Assay plates are prepared containing a culture of the hyaluronic acid capsule-forming Streptococcus, and a stock solution of the particular bacteriophage which will infect and lyse the organism only when the organism is not encapsulated. Control plates contain no bacteriophage. Filter paper discs, dipped in the sample to be screened, are applied to the plates, and zones of lysis of bacterial growth are observed and recorded after a suitable incubation period. Samples producing zones of inhibition on the control plates are considered to exhibit classical antibiotic activity and are discarded. Samples producing zones of lysis on test plates, but no zones of growth inhibition on control plates are considered to have anticapsular activity.
As will be apparent to those skilled in the art, by appropriate modification, the method of this invention can be used as a means of assaying such compounds during production, isolation, purification, and like procedures.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The screening method of this invention can be more fully understood by the following example, which shall be considered the preferred embodiment of this invention. According to the preferred embodiment of this invention, filtered fermentation broths and solutions of organic compounds are screened for anticapsular activity as follows.
A single-colony isolate from Streptococcus pyogenes ATCC 12384 is used as the test organism. The organism is a Group A Streptococcus, which, under appropriate conditions, produces a hyaluronic acid capsule.
The test organism is cultured and maintained by routine procedures well known in the art in a culture medium of Difco Brain Heart Infusion (BHI) broth containing 5 percent by volume sterile horse serum. Maximum capsule formation is usually observed after the cultures have been incubated for approximately 2.5 to 3.5 and preferably 3 hours at from 35° to 39° c. and preferably 37° C. After the incubation period, the cultures are adjusted to an O.D. of 0.2 to 0.3, preferably 0.25 at 540 mμ for use in plating.
A phage was isolated, hereinafter referred to as phage X240, ATCC No. 12384B, by methods well known in the art, which was capable of infecting and lysing only the non-encapsulated Streptococcus pyogenes host cells. Sewage samples were plated on double layered agar plates prepared according to the method described by Adams, Bacteriophage, p. 450, Interscience Publishers, Inc., New York (1959), containing from 0.2 to 0.4 ml. of the above cell suspension of the bacterial host and from 110-135 international units of hyaluronidase in the soft agar layer. Plaque picks were made according to methods well known in the art, and a phage stock having a titer of 1 × 10 9 plaque-forming units/ml. was prepared by repeated propagation as hereinbelow described.
The bacteriophage so selected was progated on double-layered agar plates containing preferably 0.3 ml. of the above-prepared culture of Streptococcus pyogenes ATCC 12384, 125 international units of hyaluronidase, (commercially available from Nutritional Biochemicals), in physiological saline and 0.1 ml. of phage stock having a titer of at least 1 × 10 9 plaque-forming units per ml. in the soft agar layer. The above concentrations gave confluent lysis after incubation overnight at 37° C. The soft agar layers were then harvested in 2.5 ml./plate of BHI broth, centrifuged, and the supernatant filtered through a sterile, membrane filter having a pore size of 0.45μ. The resulting phage stock has a titer of approximately 1 × 10 9 plaque-forming units per ml. as determined by titration of several dilutions of the phage stock on plates prepared as above for propagation of the bacteriophage.
Test and control plates are then prepared for screening. The test plates contain approximately 1.0 ml. of the above-prepared Streptococcus pyogenes cell suspension, having an O.D. of 0.25 at 540 mμ, 0.2 ml. of above-prepared phage stock having a titer of approximately 1 × 10 9 plaque-forming units per ml., and from 10 to 14 and preferably 12 ml. of BHI agar containing 0.8 percent by weight of agar and 5 percent by volume of sterile horse serum in a 100 mm. square phage plate. Control plates, prepared as above, contain no bacteriophage.
After the agar had solidified, small (approximately 6.35 mm. diameter) filter pads are dipped into the samples to be screened for anticapsular activity and applied to each plate. If the compounds to be tested are organic solutions, rather than filtered fermentation broths, the discs are dipped into the organic solutions and air-dried before they are applied to the test and control plates to minimize the solvent effects. As a positive control, additional pads dipped in a hyaluronidase solution, prepared as above, are also applied to each plate. The test and control plates are then incubated at temperatures of from 28° to 37° C., preferably 30° C., for from 15 to 18 hours, preferably for about 16 hours. The plates are then observed for zones of inhibition.
After the test and control plates have been incubated for an appropriate period of time, the plates are observed, and the results from corresponding samples on tests and control plates are compared. Samples producing zones of inhibition on the control plates are considered to be antibiotic in nature and are discarded. Samples producing zones of inhibition on the test plate but no zones of inhibition in the control plates are considered to be anticapsular in nature, and are subjected to a confirming screening procedure using larger filter pads on control and test plates for greater ease and accuracy in reading the zones. The pads dipped in hyaluronidase, which serve as a positive control, will give zones of inhibition on the test plate and no zones of inhibition on the control plates. Such results provide evidence that only the non-encapsulated bacteria are susceptible to infection and lysis by the bacteriophage. Such positive control readings are essential to the interpretation of the zones of lysis observed on test plates.
Virulence may be defined as the degree of pathogenicity within a group of microorganisms. In some instances, the ability of a pathogenic microorganism to cause a disease or infection can be partially related to the production of specific cellular structures or products, hereinafter referred to as "virulence factors." Examples of such virulence factors are capsules, certain enzymes, toxins, and antigens. Any treatment of such a pathogenic microorganism, which would in some way alter either the biosynthesis or biological function of a virulence factor, may in fact have therapeutic utility.
Streptococcus zooepidemicus and Streptococcus pyogenes, members of the Pyogenic group of Streptococci, both produce a capsular structure composed of hyaluronic acid at the outer surface of their cell walls. This capsule has been implicated in the virulence of the latter microorganism by rendering it less susceptible to phagocytosis and destruction when phagocytized: (Faley et al., "Studies on the Pathogenicity of Group A Streptocci," J. Exp. Med., 110: 603-605 (1959); Rothbard, J. Exp. Med. 88: 325 (1948); Kass and Seastone, "The Role of Mucoid Polysaccaride (hyaluronic acid) in the Virulence of Group A Hemolytic Streptococci," J. Exp. Med., 79: 319-329 (1944). Such encapsulated cells are also resistant to infection and lysis by bacteriophage, hereinafter referred to as "phage."
It has been found that a strain of S. pyogenes which produces a hyaluronic acid capsule, a phage specific for this strain, and an appropriate positive control, which in this case is the enzyme hyaluronidase, can be employed to detect the activity of, and hence screen for, substances which inhibit the synthesis of said capsule. Such substances, hereinafter referred to as anticapsular compounds, do not usually exhibit classical antibacterial activity, and thus are not detected in conventional antibiotic screens currently employed by those skilled in the art.
The anticapsular compounds detected in the screening process of this invention inhibit the synthesis of the hyaluronic acid capsule of S. pyogenes. Cells of S. pyogenes thus treated are significantly more susceptible than encapulated cells to in vitro phagocytosis by human leukocytes. Administering anticapsular compounds to a suitable host may allow the host's phagocytic system to more readily destroy the infecting S. pyogenes, as well as enhance the activity of conventional antibiotics against such organisms. Such compounds may also be useful in treating conditions in man wherein an increased production of hyaluronic acid is implicated. Such conditions include Marfan's syndrome, Hurler's syndrome, myxedema, osteroarthritis, arthro-acteoonychodysplasia, and neuropathy.
SUMMARY OF THE INVENTION
This invention relates to a method of screening for anticapsular compounds. As used herein, an anticapsular compound is one which inhibits the formation of hyaluronic acid capsules of pathogenic microorganisms wherein the capsule is implicated in the virulence of the microorganisms. The screen process of this invention utilizes a capsule-forming, pathogenic microrganism or bacterium, a bacteriophage to which the organism is susceptible only when non-encapsulated, and a suitable positive control which removes the hyaluronic acid capsule but does not inhibit either the bacterium or the bacteriophage, to screen for anticapsular compounds.
Generally speaking, filtered fermentation broths or solutions of organic compounds are screened for anticapsular activity as follows. Assay plates are prepared containing a culture of the hyaluronic acid capsule-forming Streptococcus, and a stock solution of the particular bacteriophage which will infect and lyse the organism only when the organism is not encapsulated. Control plates contain no bacteriophage. Filter paper discs, dipped in the sample to be screened, are applied to the plates, and zones of lysis of bacterial growth are observed and recorded after a suitable incubation period. Samples producing zones of inhibition on the control plates are considered to exhibit classical antibiotic activity and are discarded. Samples producing zones of lysis on test plates, but no zones of growth inhibition on control plates are considered to have anticapsular activity.
As will be apparent to those skilled in the art, by appropriate modification, the method of this invention can be used as a means of assaying such compounds during production, isolation, purification, and like procedures.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The screening method of this invention can be more fully understood by the following example, which shall be considered the preferred embodiment of this invention. According to the preferred embodiment of this invention, filtered fermentation broths and solutions of organic compounds are screened for anticapsular activity as follows.
A single-colony isolate from Streptococcus pyogenes ATCC 12384 is used as the test organism. The organism is a Group A Streptococcus, which, under appropriate conditions, produces a hyaluronic acid capsule.
The test organism is cultured and maintained by routine procedures well known in the art in a culture medium of Difco Brain Heart Infusion (BHI) broth containing 5 percent by volume sterile horse serum. Maximum capsule formation is usually observed after the cultures have been incubated for approximately 2.5 to 3.5 and preferably 3 hours at from 35° to 39° c. and preferably 37° C. After the incubation period, the cultures are adjusted to an O.D. of 0.2 to 0.3, preferably 0.25 at 540 mμ for use in plating.
A phage was isolated, hereinafter referred to as phage X240, ATCC No. 12384B, by methods well known in the art, which was capable of infecting and lysing only the non-encapsulated Streptococcus pyogenes host cells. Sewage samples were plated on double layered agar plates prepared according to the method described by Adams, Bacteriophage, p. 450, Interscience Publishers, Inc., New York (1959), containing from 0.2 to 0.4 ml. of the above cell suspension of the bacterial host and from 110-135 international units of hyaluronidase in the soft agar layer. Plaque picks were made according to methods well known in the art, and a phage stock having a titer of 1 × 10 9 plaque-forming units/ml. was prepared by repeated propagation as hereinbelow described.
The bacteriophage so selected was progated on double-layered agar plates containing preferably 0.3 ml. of the above-prepared culture of Streptococcus pyogenes ATCC 12384, 125 international units of hyaluronidase, (commercially available from Nutritional Biochemicals), in physiological saline and 0.1 ml. of phage stock having a titer of at least 1 × 10 9 plaque-forming units per ml. in the soft agar layer. The above concentrations gave confluent lysis after incubation overnight at 37° C. The soft agar layers were then harvested in 2.5 ml./plate of BHI broth, centrifuged, and the supernatant filtered through a sterile, membrane filter having a pore size of 0.45μ. The resulting phage stock has a titer of approximately 1 × 10 9 plaque-forming units per ml. as determined by titration of several dilutions of the phage stock on plates prepared as above for propagation of the bacteriophage.
Test and control plates are then prepared for screening. The test plates contain approximately 1.0 ml. of the above-prepared Streptococcus pyogenes cell suspension, having an O.D. of 0.25 at 540 mμ, 0.2 ml. of above-prepared phage stock having a titer of approximately 1 × 10 9 plaque-forming units per ml., and from 10 to 14 and preferably 12 ml. of BHI agar containing 0.8 percent by weight of agar and 5 percent by volume of sterile horse serum in a 100 mm. square phage plate. Control plates, prepared as above, contain no bacteriophage.
After the agar had solidified, small (approximately 6.35 mm. diameter) filter pads are dipped into the samples to be screened for anticapsular activity and applied to each plate. If the compounds to be tested are organic solutions, rather than filtered fermentation broths, the discs are dipped into the organic solutions and air-dried before they are applied to the test and control plates to minimize the solvent effects. As a positive control, additional pads dipped in a hyaluronidase solution, prepared as above, are also applied to each plate. The test and control plates are then incubated at temperatures of from 28° to 37° C., preferably 30° C., for from 15 to 18 hours, preferably for about 16 hours. The plates are then observed for zones of inhibition.
After the test and control plates have been incubated for an appropriate period of time, the plates are observed, and the results from corresponding samples on tests and control plates are compared. Samples producing zones of inhibition on the control plates are considered to be antibiotic in nature and are discarded. Samples producing zones of inhibition on the test plate but no zones of inhibition in the control plates are considered to be anticapsular in nature, and are subjected to a confirming screening procedure using larger filter pads on control and test plates for greater ease and accuracy in reading the zones. The pads dipped in hyaluronidase, which serve as a positive control, will give zones of inhibition on the test plate and no zones of inhibition on the control plates. Such results provide evidence that only the non-encapsulated bacteria are susceptible to infection and lysis by the bacteriophage. Such positive control readings are essential to the interpretation of the zones of lysis observed on test plates.