Patent Publication Number: US-5529924-A

Title: Legionella specific antibiotic produced by Streptomyces sp AL91 access ion number KCCM 10055

Description:
This application is a divisional of application Ser. No. 08/340,358, filed Nov. 14, 1994, now U.S. Pat. No. ,486,630. 
    
    
     FIELD OF THE INVENTION 
     The present invention is directed to a novel Legionella specific antibiotic AL072, a novel Streptomyces sp. AL91 producing the same, and a process for producing the said antibiotic. 
     BACKGROUND OF THE INVENTION 
     Legionella pneumophila, which is the causal agent of Legionella infections, has been known to cause Legionnaires&#39; disease or Pontiac fever. Since the bacteria was first isolated in Philadelpia, USA, it has been reported that many isolations of a species of Legionella were made from various patients and environments in all parts of the world, including the USA, and 10 or more species were identified. Legionella pneumophila is one of the pathogens capable of causing pneumonia and it is presumed that 5% of the occurences of pneumonia are due to Legionella pneumophila. Legionella pneumophila grows in air-conditioning cooling towers, water service pipes, drain pipes, etc., and infections are believed to occur in respiratory organs by inhalation of contaminated aerosols. In July, 1984, criticism was caused by the surprising fact that ill-defined symptoms of pneumonia which occurred in patients as well as medical teams at intensive care units of Koryo Hospital in Seoul appeared to be caused by Legionella species. 
     According to a result of investigation completed in 1985 by the National Health Institute, at least 90% of air-conditioning cooling towers within Seoul were contaminated with Legionella species and 93% of the isolated Legionella were identified as Legionella pneumophila. An additional investigation completed by the National Health Institute in major cities throughout the country, for example Seoul, Pusan, Daejeon, etc., between June and September, 1988 revealed that 83% of the isolated Legionella were classified to Legionella pneumophila serogroup 1. 
     Macrolide antibiotics, such as erythromycin, and quinolone antibiotics, such as rifampin, are known to be active against Legionella species and have been used for the treatment of the Legionella infections. However, these antibiotics have a wide spectrum of activity against a variety of microbes, in addition to the Legionella species. In this regard, an abuse of such antibiotics for extended periods may not only generate resistance to various microbes but also cause harmful problems such as the collapse of the balance of microbes occurring in a human body. 
     Additionally, there have been serious problems in that chemical agents for disinfecting air-conditioning cooling towers, which are contamination sources of Legionella species, may result in the contamination of the environment and the corrosion of the air-conditioning device. 
     Therefore, the development of novel Legionella specific antibiotics which are specifically active against only Legionella species, is needed and thereby may minimize the undesired problems. We have intensively investigated soil microbes over several years for the purpose of developing such antibiotics. Eventually, we succeeded in isolating a species of Streptomyces producing Legionella specific antibiotics and purifying a novel antibiotic specifically active against only Legionella species, which is designated Antibiotic AL072. 
     SUMMARY OF THE INVENTION 
     Cultivation of the novel microorganism Streptomyces sp. AL91 yields a novel antibiotic substance AL072 which is active exclusively against Legionella species, whose structure is represented as follows: 
    
    
     DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows the ultraviolet absorption spectrum of antibiotic substance AL072. 
     FIG. 2 shows the infrared absorption spectrum of antibiotic substance AL072. 
     FIG. 3 shows the mass spectrum of antibiotic substance AL072 measured by FAB/MS. 
     FIG. 4 shows the 400 MHz proton nuclear magnetic resonance spectrum of antibiotic substance AL072. 
     FIG. 5 shows the 100 MHz carbon nuclear magnetic resonance spectrum of antibiotic substance AL072. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The Microorganism 
     The microorganism used for the production of Legionella specific antibiotic substance AL072 is Streptomyces sp. AL91. The microorganism was deposited at the permanent collection of the Korean Culture Center of Microorganisms, Seoul, Korea, on Jun. 2, 1994 under the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, and a subculture of the organism can be obtained from the repository under the accession number KCCM 10055. In addition to the specific microorganism described and characterized herein, it should be understood that mutants of the microorganism (e.g., mutants produced through the use of X-ray, ultraviolet radiation or nitrogen mustards) can also be cultivated to produce the antibiotic substance AL072. 
     Isolation of Streptomyces sp. AL91 was accomplished by shaking 1.0 g of dried soil sample in 10 ml of sterile distilled water, plating 0.1 ml of dilutions of the soil suspension on a Humic acid-Vitamin agar supplemented with Cycloheximide (50 μg/ml) and Nalidixic acid (200 μg/ml), and cultivating the -plate at the temperature of 28° C. for 14-21 days. The medium was sterilized at 121° C. for 15 minutes before the antifungal agent Cycloheximide and the antibacterial agent Nalidixic acid, seperately sterilzed by filtration, were added. The composition of Humic acid-Vitamin agar is described in Table I. 
     
                       TABLE I                                                     
______________________________________                                    
Humic acid              1.0    g                                          
(dissolved in 10 ml of 0.2N NaOH)                                         
Na.sub.2 HPO.sub.4      0.5    g                                          
KCl                     1.71   g                                          
MgSO.sub.4.7H.sub.2 O   0.05   g                                          
FeSO.sub.4.7H.sub.2 O   0.01   g                                          
Thiamin-HCl             0.5    mg                                         
Vitamin B.sub.2         0.5    mg                                         
Niacin                  0.5    mg                                         
Pyridoxine-HCl          0.5    mg                                         
Inositol                0.5    mg                                         
Pantothenic Acid Ca-salt                                                  
                        0.5    mg                                         
p-Aminobenzoic Acid     0.5    mg                                         
Biotin                  0.25   mg                                         
Cycloheximide           50     mg                                         
Nalidixic acid          200    g/mL                                       
Agar                    20     g                                          
Distilled water (pH 7.0)                                                  
                        1      L                                          
______________________________________                                    
 
    
     Characteristics of Streptomyces sp. AL91 KCCM 10055 
     Morphology: Spores are formed by spiral, branched chains. The surface of spores is smooth. 
     Biochemical characteristics: The ability to liquefy gelatin is negative and the ability to degrade starch is positive. 
     Cultural characteristics: 
     
         __________________________________________________________________________
               Color of                                                   
                       Reverse                                            
                              Soluble                                     
Media     Growth                                                          
               aerial mycelium                                            
                       color  pigments                                    
__________________________________________________________________________
Trypton-yeast                                                             
          good brown   brown  brown                                       
extract agar                                                              
Yeast extract-                                                            
          good white   brown   --                                         
malt extract                                                              
agar                                                                      
Oat meal  good pale orange                                                
                        --     --                                         
extract agar   or white                                                   
Inorganic salt-                                                           
          good white    --     --                                         
starch agar                                                               
Glycerol- good dense yellow                                               
                       dense yellow                                       
                               --                                         
asparagine agar                                                           
Peptone-yeast                                                             
          poor dense brown                                                
                       brown  dense brown                                 
extract-iron                                                              
agar                                                                      
Tyrosine agar                                                             
          good white-brown                                                
                       dense brown                                        
                               --                                         
Bennett&#39; medium                                                           
          good white   yellow-brown                                       
                               --                                         
__________________________________________________________________________
 
    
     Carbon utilization: 
     Positive: D-Glucose, sucrose, D-xylose, D-mannitol, D-fructose, rhamnose, raffinose, cellulose. 
     Negative: L-Arabinose, I-inositol. 
     Susceptibility to antibiotics: 
     
         ______________________________________                                    
                        Size of ring occured                              
            Concentrations                                                
                        by inhibition of growth                           
antibiotics (μg/mL)  (mm, in diameter)                                 
______________________________________                                    
Carbenicillin                                                             
            100         --                                                
Chloramphenicol                                                           
            30          27.7                                              
Neomycin    30          12.0                                              
Nalidixic acid                                                            
            30          --                                                
Vancomycin  30          22.0                                              
Clindamycin 2           --                                                
Ampicillin  10          12.0                                              
Kanamycin   30          17.0                                              
Tetracycline                                                              
            30          17.0                                              
Cephalothin 30          24.0                                              
Erythromycin                                                              
            15          40.0                                              
Rifampin    5           --                                                
Gentamycin  10          10.0                                              
Streptomycin                                                              
            10          17.0                                              
______________________________________                                    
 
    
     Production of the Antibiotic substance AL072 
     Streptomyces sp. AL91 KCCM 10055 was grown on a nutrient agar containing the components listed in Table II for 3 days. The cultures were inoculated into 200 mL of a liquid medium containing the components listed in Table III and cultivated at the temperature of 28° C. under aerobic conditions for 3 days. Subsequently, the culture solution was inoculated into 6 L of a liquid medium containing the components listed in Table IV and cultivated at the temperature of 28° C. under aerobic conditions for 5 days. The antibiotic AL072 was isolated and purified from the final culture solution by the procedures described below. 
     
                       TABLE II                                                    
______________________________________                                    
Sucrose                20.0   g                                           
Glucose                10.0   g                                           
Corn steep liquor      5      mg                                          
Yeast extracts         4.9    g                                           
Soybean flour          20.0   g                                           
CaCO.sub.3             4.0    g                                           
NaCl                   2.0    g                                           
K.sub.2 HPO.sub.4      0.05   g                                           
Agar                   15     g                                           
Distilled water (pH 7.3)                                                  
                       1      L                                           
______________________________________                                    
 
    
     
                       TABLE III                                                   
______________________________________                                    
Glucose                 1     g                                           
Soluble starch          24    g                                           
Peptone                 3     g                                           
Malt extract            5     g                                           
CaCO.sub.3              4     g                                           
Distilled water (pH 7.0)                                                  
                        1     L                                           
______________________________________                                    
 
    
     
                       TABLE IV                                                    
______________________________________                                    
Sucrose                20.0   g                                           
Glucose                10.0   g                                           
Corn steep liquor      5      mL                                          
Yeast extract          4.9    g                                           
Soybean flour          20.0   g                                           
CaCO.sub.3             4.0    g                                           
NaCl                   2.0    g                                           
K.sub.2 HPO.sub.4      0.05   g                                           
Distilled water (pH 7.3)                                                  
                       1      L                                           
______________________________________                                    
 
    
     After cultivation was completed, 6 L of the culture solution was mixed with an equivalent amount of isopropyl alcohol by stirring. After standing over night, the mixture was centrifuged and the resulting supernatant was taken. The supernatant was filted through the passage of diatomaceous earth and the filtrate was then concentrated under reduced pressure to remove the isopropyl alcohol. The resulting concentrate was three times extracted with ethylacetate, which was then removed under reduced pressure. The residue was dissolved in 50% isopropyl alcohol and the resulting solution was then concentrated under reduced pressure to remove the isopropyl alcohol. The residual aqueous solution was passed on a column filled with octadecyl silica gel and the passed solution was discarded. Legionella specific antibiotics adhered to the ODS resin were eluted with 70% ethyl alcohol and the elutes were then concentrated under reduced pressure to dryness. After the dried concentrates were dissolved in 80% isopropyl alcohol, preparative high pressure liquid chromatography (206 nm) on silica gel, eluting with acetonitrile-distilled water, 68:32 at the rate of 30 mL/min, gave crude Leginella specific antibiotic substance AL072. Subsequently, the crude antibiotics were concentrated under a reduced pressure to dryness, the dried concentrates were dissolved in 50% isopropyl and the resulting solution was again concentrated under reduced pressure. The isopropyl alcohol was removed and the residue was three times extracted with chloroform. The chloroform was removed and the residue was dissolved in 80% isopropyl alcohol. High pressure liquid chromatography, run in the same conditions as used in the first chromatography, gave pure antibiotic AL072. 
     Physico-chemical properties of Antibiotic substance AL072 
     1. Thin-layer chromatography was conducted on Merck &amp; Co. No. 5642 HPTLC silica gel plate with the development of chloroform-methyl alcohol, 19:1. Antibiotic AL072 samples were spotted on the silica gel plate and developed in a sealed container. After development, an aqueous 10% sulfuric acid solution containing 5% ammonium molybdate and eerie ammonium sulfate was sprayed on the silica gel plate which was then heated to 10013 for 10 minutes to develop a colour. As a result of this, the Rf value of antibiotic AL072 was 0.58. 
     2. Antibiotic AL072 is soluble in alcohols such as methyl alcohol, ethyl alcohol, isopropyl alcohol, etc., and organic solvents such as ethyl acetate, chloroform, etc., whereas it is not or poorly soluble in water. 
     3. Antibiotic AL072 is very stable in aqueous solutions with pH values between pH 2 and pH 12. The activity of antibiotic AL072 is unchanged by heat of 9513 for 1 hour. 
     4. As a result of ultraviolet spectrometry, antibiotic AL072 in methyl alcohol is shown to have a maximum absorption in 242 nm (FIG. 1). 
     5. The infrared absorption spectrum of antibiotic AL072 in potassium bromide using Bruker FT-IR spectrometer is shown in FIG. 2. 
     6. The proton nuclear magnetic resonance spectrum of antibiotic AL072 in CDCl 3  using Bruker ARX400 spectrometer is shown in FIG. 4. H-NMR δ(400MHz): 0.86(3H,m); 0.87(3H,m); 0.88(3H,m); 0.91(3H,m); 1.29(43H,m); 1.61(4H,m); 2.03(2H,m); 2.31(4H,m); 2.75(1H,m); 3.62(1H, dd, 12Hz, 6Hz); 3.72(1H,dd,4Hz, 12Hz); 3.96(1H,m); 4.14(2H,m); 5.35(4H,m) ppm. 
     7. The carbon nuclear magnetic resonance spectrum of antibiotic AL072 in CDCl 3  using Bruker ARX400 spectrometer is shown in FIG. 5. C-NMR δ(100MHz): 12.01(q); 14.67(q); 19.85(q); 23.23(q); 23.28(q); 25.34(t); 25.36(t); 26.29(q); 27.78(t); 27.84(t); 27.87(t); 28.10(t); 28.63(d); 29.72(t); 29.75(t); 29.82(t); 29.93(t); 30.02(t); 30.17(t); 30.26(t); 30.29(t); 20.33(t); 30.36(t); 30.38(t); 30.62(t); 30.69(t); 32.20(t); 24.79(t); 34.81(t); 35.08(d); 37.33(t); 39.75(t); 63.90(t); 25.95(t); 70.96(d); 128.50(d); 128.70(d); 130.60(d); 30.70(d); 174.90(s); 181.00(s)ppm. 
     Biological Activity of Antibiotic substance AL072 
     The effects of antibiotic substance AL072 obtained according to the above procedures on growth of various microbes were shown in Table V below. It is evident that while the antibiotic substance AL072 is powerfully active against Legionella pheumophila, it is not or only slighly active against the other microbes tested. 
     
                       TABLE V                                                     
______________________________________                                    
                     Diameter of ring                                     
                     produced by inhibition                               
Microorganism        of growth (mm)                                       
______________________________________                                    
Legionella pneumophila ATCC 33152                                         
                     108                                                  
Staphylococcus aureus                                                     
                     11                                                   
Streptococcus pyogenes                                                    
                     --                                                   
Streptococcus        20                                                   
Streptococcus agalactiae                                                  
                     --                                                   
Streptococcus equi   21                                                   
Streptococcus durans 24                                                   
Listeria monocytogenes                                                    
                     12                                                   
Corynebacterium diphtheriae                                               
                     9                                                    
Bacillus subtilis    8                                                    
Bacillus megaterium  6                                                    
Escherichia coli     6                                                    
Citrobacter freundii --                                                   
Salmonella typhimurium                                                    
                     14                                                   
Shigella flexneri    --                                                   
Klebsiella pneumoniae                                                     
                     --                                                   
Klebsiella oxytoca   --                                                   
Klebsiella aerogenes --                                                   
Enterobacter cloacae --                                                   
Enterobacter aerogenes                                                    
                     9                                                    
Serratia marcescens  --                                                   
Proteus mirabilis    --                                                   
Proteus vulgaris     12                                                   
Proteus rettgeri     --                                                   
Proteus morgani      --                                                   
Pseudomonas aeruginosa                                                    
                     --                                                   
Pseudomonas cepacia  --                                                   
______________________________________