Abstract:
Pathogenic microorganisms such as Staphylococcus aureus are differentiated and identified by observing the selective inhibition of the microorganism which occurs when it is contacted with Alphazurine A dye.

Description:
The present invention relates to a device and procedure for the clinical differentiation and identification of specific pathogenic microorganisms. More particularly the present invention is directed to a device and procedure for distinguishing and identifying Staphylococcus aureus in clinical environments. 
     BACKGROUND OF THE INVENTION 
     The staphylococci are by far the commonest cause of skin infections such as boils, abscesses, carbuncles and similar suppurative processes in man. They are primarily significant as pathogens. They can be grown on culture media, such as agar or meat extract media, and the individual colonies are Circular with entire edges. The pathogenic forms, that is, those isolated from suppurative processes, usually are Staphylococcus aureus. 
     Staphylococci ferment mannitol with the formation of acid, the latter being detected by a decrease in pH of the media, as evidenced by color change of a pH indicator such as brom thymol blue, brom cresol purple, or phenol red. 
     Pathogenic staphylococci, that is, the Staphylococcus aureus, also cause clotting of decalcified blood plasma due to the production of staphylocoagulase, an active clotting agent. There is a high correlation between staphylococcal virulence for man and staphylocoagulase production. Coagulase-positive bacteria are usually virulent. 
     Plasma clotting (i.e., staphylocoagulase activity) can be demonstrated by mixing a bacterial culture with decalcified plasma, human or rabbit, incubating the plasma and observing clotting. The simple slide method is often used for qualitative purposes; the bacteria are suspended in a drop of plasma and observed on a slide for clumping. 
     In the treatment of wounds and similar skin lesions it is often desirable, and sometimes necessary, to identify the microorganisms in the wound or lesion in an agar medium and the microorganisms developed therein. It is therefore desirable to be able to identify Staphylococcus aureus promptly and accurately so that appropriate treatment can be instituted without delay. For this reason it is desirable to have a culture medium, and a sensitivity paper disc which inhibits the growth of Staphylococcus aureus and allows the rapid growth of most other microorganisms and thus allows rapid and accurate identification of coagulase positive organisms, mainly Staphylococcus aureus if present in the wound or lesion. 
     As discussed above, two of the most widely used tests for identifying staphylococci are the coagulase production test and the mannitol fermentation test. Most strains of Staphylococcus aureus isolated from human lesions are coagulase-positive and mannitol-positive. Coagulase-negative and mannitol-negative staphylococci, i.e. S. epidermidis and S. saprophyticus are most frequently considered to be less pathogenic. Positive reactions to both the coagulase production and mannitol fermentation tests are an indication of the pathogenicity of the staphylococci. Negative reactions to both tests indicate that the isolate has less clinical significance. Classical tests for coagulase production are performed routinely by the so-called &#34;tube&#34; method which measures free coagulase production or by the so-called &#34;slide&#34; technique which measures bound coagulase either on or in the cell wall of the organism. The tube method and the slide technique are described in the publications J. Bact. 41:431-440 (1941) and Medical Microbiology, T. Cruickshank, pages 137-138, published by Williams and Wilkins Company, 11th edition, 1965, respectively. In these tests, both rabbit plasma and human plasma have been used as the substrate. 
     Mannitol Salt Agar is used as a selective medium for the isolation of pathogenic staphylococci. This medium is descried in J. Bact. 50:201-203 (1945). Due to the presence of 7.5% sodium chloride therein, growth of most bacteria, other than staphylococci, is inhibited on this medium. When the medium is inoculated with a material containing staphylococci and incubated for a period of 36 hours at a temperature of 37° C., mannitol-fermentation staphylococci grow luxuriantly, surrounded by yellow zones. In contrast, mannitol-non fermentation staphylococci produce small colonies, surrounded by red or purple zones. Positive identification of an unknown specimen as pathogenic can require as long as five days when conventional coagulase production and mannitol fermentation tests are employed. 
     A solid growth medium which is suitable for the visual identification of pathogenic staphylococci from initial cultures is described in the publication Am. J. Clin. Path. 32:192-194 (August 1959). Some measure of success has been achieved using this medium. However, it has been found that other bacteria which produce coagulase, grow on this medium. 
     DESCRIPTION OF THE PRIOR ART 
     U.S. Pat. No. 4,035,238 to Meyer et al describes a broth medium for the detection of Staphylococcus aureus which employs mannitol to promote growth of the microorganisms and potassium tellurite to inhibit growth of gram-negative organisms. 
     U.S. Pat. No. 3,597,321 to Kronish et al describes a diagnostic composition for differentiating staphylococci which detects the fermentation of mannitol and an agent which detects production of coagulase. 
     U.S. Pat. No. 3,416,998 to Streitfeld describes dried transparent agar sheets for detecting microorganisms such as Staphylococcus aureus. 
     U.S. Pat. No. 3,278,393 to Bahn et al describes selective culture mediums for distinguishing Staphylococcus aureus and Pseudomonas aeruginosa from non-pathogenic microorganisms. 
     Green, Floyd J. The Sigma-Aldrich Handbook of Stains, Dyes and Indicators 1990 Published by Sigma-Aldrich Corporation Page 87. 
     Loos, W. E., and Jorgensen, 1985, Page 145 and 147. In E. H. Lennette, Al Balows, W. H. Hauser, Jr., H. J. Shadomy. (ed) Manual of Clinical Microbiology. 4th ed., American Society for Microbiology, Washington, D.C. 
     DETAILED DESCRIPTION OF THE INVENTION 
     In accordance with the present invention Staphylococcus aureus is clinically identified and differentiated from other staphylococci by selectively inhibiting growth of the microorganisms in a culture medium by incorporating into the medium Alphazurine A dye. 
     Alphazurine A which is a bright greenish-blue anionic triphenylmethane dye with a high affility for proteinaceous substrates and a weak attraction to cellulosic materials. 
     This invention is based on the discovery that Alphazurine A dye inhibits the growth of Staphylococcus aureus when incorporated in a culture media and that Staphylococcus aureus is sensitive to a paper disc containing Alphazurine A on a culture plate, hereafter described as the Alphazurine effect. All staphylococci exhibiting the Alphazurine A effect were coagulase positive, representing an additional diagnostic characteristic for the identification for pathogenic staphylococci. The ability to clot plasma is the most widely used and generally accepted criterion for the frankly pathogenic Staphylococci, for example, Staphylococcus aureus in humans. Alphazurine A effect correlates 100% with positive coagulation tests. 
     Most advantageously in accordance with the invention, the Alphazurine dye is impregnated onto an absorbent material such as sterile paper to form &#34;sensitivity discs&#34; which are then placed on the surface of inoculated media and incubated to develop the culture. Where the inoculum is Staphylococcus aureus, the growth of the organism around the disc is inhibited, whereas other microorganisms are not so inhibited and generally flourish. 
     When Alphazurine A dye is added to Petri plates containing cultures of Staphylococcus aureus, Staphylococcus saprophyticus and Staphylococcus epidermidis respectively, growth of S. aureus is inhibited while growth was not inhibited of S. saprophyticus and S. epidermidis. 
     Only one Staphylococcus, Staphylococcus warneri showed the Alphazurine A effect. Staphylococcus warneri, is a questionable or uncommon pathogen, and does not enter into the diagnosis of Staphylococcus aureus clinically. 
     All strains or Staphylococcus aureus which showed the alphazurine effect, were coagulase positive whereas all Staphylococci not showing the Alphazurine A effect were coagulase negative. 
     Alphazurine A sensitivity to Staphylococcus aureus can also be elicited on blood agar plates. As a result, a diagnosis of Staphylococcus aureus in accordance with the invention can be made on the primary inoculum from the infected site, which permits an early diagnosis. 
     One hundred twenty-six cultures of Staphylococcus aureus were studied and all showed Alphazurine sensitivity. Thirty-eight cultures of Staphylococcus epidermidis and twenty-eight cultures of Staphylococcus saprophyticus showed no sensitivity to Alphazurine A. 
     Thirty-eight cultures of gram negative bacteria also showed no Alphazurine A effect. 
    
    
     The following examples are illustrative of the invention: 
     Alphazurine A was impregnated in paper discs. Mueller-Hinton agar media in culture plates was swabbed with a suspension in water of Staphylococcus aureus. Alphazurine A sensitivity disc was placed on the surface of the inoculated media and incubated at 37° C. for 24 hours. Staphylococcus aureus was sensitive to Alphazurine A and prevented the growth of Staphylococcus aureus in a zone around the sensitivity disc. 
     In addition to Mueller-Hinton media other culture media as Mannitol salt agar, Trypticase soy agar and Trypticase soy agar with 5% sheep blood (Becton-Dickinson Microbiology Systems, Cockeysville, Md.) were used, and when streaked with Staphylococcus aureus showed a zone of inhibition of growth around Alphazurine A disc. 
     The use of Alphazurine discs, Mannitol salt agar, and Novobiocin resistance permitted the presumptive differential diagnosis of the most important Staphylococcus pathogens in humans. The following procedures were followed and results obtained: 
     PREPARATION OF ALPHAZURINE A CULTURE MEDIA 
     
         ______________________________________The formula is as follows:______________________________________1.     Brain Heart Infusion Agar                        52. grams2.     Distilled Water       1000 ml3.     Alphazurine A         200 mgm  (Aldrich Chemical Company  Milwaukee, Wisconsin)______________________________________ 
    
     Brain heart infusion agar was added to distilled water and boiled for one minute. The media was cooled to approximately 50° C and Alphazurine A added. The media was agitated to insure complete solution of the dye. The media was poured into plastic Petri plates. 
     PREPARATION OF ALPHAZURINE A SENSITIVITY DISC 
     Alphazurine A, 700 mg, was dissolved in 100 ml of sterile distilled water. Sterile paper 1/4 inch discs were soaked for 2 hours in the dye solution. The excess solution was decanted and the discs dried at room temperature. 
     METHODS AND RESULTS 
     ALPHAZURINE A CULTURE MEDIA 
     A suspension of Staphylococci in a concentration of 0.5 McFarland unit was made in distilled water. The bacterial suspension was streaked on the Alphazurine A culture media plate with a swab, and the plate incubated for 24 hours at 37° C. 
     Staphylococcus aureus was inhibited or did not grow on the Alphazurine A culture plate, whereas Staphylococcus epidermidis and Staphylococcus saprophyticus grew profusely. 
     ALPHAZURINE A SENSITIVITY DISCS 
     Staphylococcus aureus suspension in distilled water in a concentration of 0.5 McFarland unit was made. The bacterial suspension was streaked on a Mueller-Hinton agar plate. After five minutes, and Alphazurine A disc was placed on the surface of the media, pressed down, and the plate inoculated at 37° C. for 24 hours. Staphylococcus aureus was sensitive to the Alphazurine A and produced a zone of inhibition of the bacteria of 10 to 16 mm in diameter. The zone was clear and sharply demarcated. 
     Staphylococcus saprophyticus was resistant. 
     Staphylococcus epidermidis produced a zone of inhibition of bacterial growth to a small degree. The zone of inhibition was 7 mm or less and the zone of demarcation was feathered and not sharp. 
     
         ______________________________________SUMMARY OF TEST RESULT    Staphyl-    ococcus Staphylococcus                        Staphylococcus    Aureus  Epidermidis Saprophyticus______________________________________Alphazurine A      +         -           -SensitivityUtilization of      +         -           variableMannitol(acid production)Novobiocin -         -           +Resistance______________________________________