Abstract:
The invention relates to a novel antibiotic from the macrolactin group and a microbial method for production of macrolactins and a novel isolated  Bacillus subtilis , of application as the producing strain for the known Macrolactin A and the novel derivative.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     Applicants claim priority under 35 U.S.C. §119 of German Application No. 10 2004 046 024.8 filed Sep. 21, 2004. Applicants also claim priority under 35 U.S.C. §365 of PCT/EP2005/054736 filed Sep. 21, 2005. The international application under PCT article 21(2) was not published in English. 
     The present invention relates to a novel antibiotic of the macrolactin family as well as to a microbial method suitable for producing macrolactins and the producer strain thereof. 
     It is acknowledged that bacteria, more particularly pathogenic bacteria, are becoming increasingly resistant to known antibiotics. An example of this phenomenon are staphylococci which, being a cause of nosocomial infections, are also resistant to methicillin and other known antibiotics with the exception of vancomycin and teicoplanin. Emerging in recent years, however, have been staphylococcal strains that are resistant to vancomycin or to the precursors thereof. 
     The object of the present invention is, therefore, the preparation of novel, medically effectual substances, more particularly those, which, having antibacterial activity, can be used as an antibiotic against bacterial and/or eurkaryotic infections. 
     A further object of the present invention is a method for producing substances suitable for pharmaceutical use, an example whereof are antibiotically active substances, and a bacterium that can be employed by the method as a producer of antibiotic substances. 
     THE STATE OF THE ART 
     Known in the art are several macrolactins that include macrolactin A of the chemical formula 8, 14, 16-trihydroxy-24(R)-methyl-oxacyclotetracosa-3(Z), 5(B), 9(E), 11(Z), 17(E), 19(E)-hexaen-2-on. Macrolactin A, which is known to be antiviral and for example, is active against HIV, is cytotoxic. The antibacterial activity of macrolactin A is, on the other hand, relatively weak. 
     JP 9301970 discloses that the derivative macrolactin M is antimicrobially active. 
     Methods for chemically synthesizing macrolactin A are disclosed in Smith et al.,  J. Am. Chem. Soc,  120, 3935-3948 (1998) and in Marino et al.,  J. Am. Chem. Soc.  124, 8, 1664-1668 (2002). 
     OVERVIEW OF THE INVENTION 
     7-O-malonyl macrolactin A is, first of all, highly effective against bacteria and can thus confer antibiotic capability on pharmaceutical compositions. In addition, 7-O-malonyl macrolactin A at low concentrations is less cytotoxic than macrolactin A and is, therefore, especially suitable for use in pharmaceutical compositions. 
     In addition to its general antibacterial effectiveness, 7-O-malonyl macrolactin A is antibiotically active against at least some bacteria that exhibit resistance to known antibiotics or are in and of themselves resistant. In this regard, 7-O-malonyl macrolactin A interestingly enough exhibits, at concentrations below the minimal inhibitory concentration level, strong bacteriostatic activity against at least a number of bacteria from clinical isolates that are resistant to conventional antibiotics, as opposed to the corresponding non-resistant strains. For example, 7-O-malonyl macrolactin A is antibiotically effective against both methicillin-resistant  Staphylococcus aureus  and vancomycin and ampicillin-resistant  Enterococcus faecium.    
     The structural formula for 7-O-malonyl macrolactin A is reproduced hereunder: 
     
       
                 
         
             
             
         
      
     
     The proposed compounds, which are suitable for use as active ingredients in pharmaceutical products, are produced from a strain of  Bacillus subtilis  that was deposited with the Indonesian Center for Biodiversity and Biotechnology under No. ICBB 1582 (also deposited under no. DSM 16696 with the DSMZ, Mascheroder Weg 1, 38124 Braunschweig, date of filing Sep. 6, 2004). This strain was isolated from a soil sample obtained in Takalar, South Sulawesi in Indonesia. 
     The deposited producer strain was characterized as a gram-positive rod (±0.2 μm diameter) capable of motility by means of flagella, forming endospores and in culture (5 g/l yeast extract, 20 g/l tryptone, 5 g/l sodium chloride, 20 g/l glucose, 15 g/l agar) forming opaque, milky-white colonies exhibiting undulating, rough edges. In the API system (Biomerieux), positive reactions were observed for the following: oxidase, ornithine, mannitol, Voges-Proskauer, citrate, TDA and amylohydrolysis, whereas negative reactions were observed for nitrate, lysine, hydrogen sulfide production, glucose, xylose, β-galactosidase, indol and urease. Following the biochemical tests and the homology test of the 16S-RNA sequence searches using the FASTA program, the strain was determined to be  Bacillus subtilis.    
     The present invention also relates to a method employing fermentation for producing the prior art macrolactin A, 7-O-succinyl macrolactin A and 7-O-malonyl macrolactin A. 
     The present invention relates furthermore to the application of 7-O-succinyl macrolactin A to the production of pharmaceutical preparations for medical use in combating antibiotic-resistant, e.g. multiresistant bacteria. 
     It is therefore proposed that pharmaceutical compositions be prepared that comprise at least one of the aforementioned compounds. 
     DETAILED DESCRIPTION OF THE INVENTION 
     7-O-Malonyl Macrolactin A 
     7-O-malonyl macrolactin A is antibiotically active against bacteria, especially gram-positive bacteria and is, in particular, advantageously bacteriostatic at very low concentrations, for example, in the region of 0.05-4 μg/ml. This activity is especially evident against antibiotic-resistant bacteria, which enables 7-O-malonyl macrolactin A to be particularly effective in combating antibiotic-resistant bacteria, including notably gram-positive bacteria, or at least in inhibiting the further proliferation thereof. The bacteriostatic activity of 7-O-malonyl macrolactin A has already been observed at weak concentrations, as compared to the prior art macrolactin A, even where there has been no direct or immediate bactericidal effect at such low concentrations. 
     Yet another advantage of 7-O-malonyl macrolactin A as compared to prior art 7-O-succinyl macrolactin A or macrolactin A is its bacteriostatic activity against antibiotic-resistant bacteria even at very low concentrations. Such antibiotic-resistant bacteria can, for example, be obtained from clinical isolates and be resistant to erythromycin or vancomycin or be multiresistant, examples whereof are  Staphylococcus aureus  (MRSA) or enterococci (VRE) from clinical samples. In addition to its activity against such gram-positive bacteria, 7-O-malonyl macrolactin A is active against gram-negative bacteria, e.g. small colony-forming variants (SCV) of  Burkholderia cepacia . The antibacterial activity of 7-O-malonyl macrolactin A against antibiotic-resistant strains and SCV is more pronounced than against wild-type strains. 7-O-malonyl macrolactin A at higher concentrations also inhibits eukaryotic microorganisms such as pathogenic yeasts, especially  Candida krusei.    
     Due to its bacteriostatic effectiveness, 7-O-malonyl macrolactin A is preferred for employment in the manufacture of pharmaceutical preparations for use against the following medical conditions: bacterial infections; endocarditis; meningitis; osteomyelitis; included are infections caused by toxin-forming pathogens e.g. streptococci and staphylococci, more particularly, toxic shock syndrome (TSS) strains of  Staphylococcus aureus.    
     The proposed compositions can also have application against other medical conditions such as respiratory tract infections caused, in particular, by antibiotic-resistant (e.g. resistant to penicillin) bacteria or bacteria that are difficult to manage with conventional antibiotics, such as, for example, surface-dwelling pathogens. Examples of respiratory tract infections are those caused by extracellular and/or intracellular bacteria, such as, for example, streptococci e.g.  Streptococcus pneumoniae  or  Streptococcus pyogenes.    
     The ability of 7-O-malonyl macrolactin A to inhibit bacterial growth at low concentrations that can be significantly below the MIC is medically significant, in particular, against clinically relevant strains, e.g. those that exhibit resistance. 7-O-malonyl macrolactin A, even at low concentrations, interferes with cell division in both gram-positive and gram-negative bacteria A, which is supported by visual evidence of disrupted septum formation and cell wall alterations using an electron microscope. Thus, for example, antibiotic-resistant enterococci, in particular VRE, staphylococci, in particular MRSA, and  Burkholderia cepacia  SCV in the presence of 7-O-malonyl macrolactin A exhibit a thickened cell wall and/or an anomalous morphology. Interruption of cell division leads to pseudomulticellular accumulations incapable of forming daughter cells. It is presently thought that the reduction of the propagation of bacterial infections by means of 7-O-malonyl macrolactin A is also attributable to the disruption of cell division, since the proliferation of the bacteria can thus be retarded or prevented. 
     7-O-malonyl macrolactin A, which is produced from the  Bacillus subtilis  strain deposited under no. DSM 16696, is precipitated out in the culture broth. 7-O-malonyl macrolactin A, 7-O-succinyl macrolactin A and macrolactin A are extracted from the culture broth by means, for example, of adsorption to a hydrophobic adsorbent resin such as XAD, whereafter said resin is washed with aqueous methanol and eluted with 100% methanol. 7-O-malonyl macrolactin A is then isolated from the resulting eluate. 
     7-O-Malonyl macrolactin A: C 27 H 36 O 8 , M=488.57, UV (MeOH) λ max  (lg ε)=227 nm (4.397), 230 (sh), 260 (4.006). [α] 22   D =−6.2 (c=0.63 in MeOH), MS: (−)-ESI (TOF): m/z (%)=487.2 (100) [M−H] − , 443.2 (44) [M−H—CO 2 ] − , 383.2 (27) [M−H-malonic acid] − . 
     The foregoing compound was identified as a macrolactin type compound from its UV spectrum that was identical to that of macrolactin A. Mass spectrometry indicated a molecular weight of 488, which is 86 absolute mass units higher than the mass observed for macrolactin A. Corresponding to the elimination of one H 2 O from macrolactin A, 7-O-malonyl macrolactin A showed the loss of malonic acid by a fragment ion at a m/z of 383 in the (−)-ESI spectrum. The NMR data for 7-O-malonyl macrolactin A, which are shown in Table 1, were nearly identical to those of macrolactin A. However, compared to macrolactin A, the 7-H signal was shifted about 1.2 ppm downfield as a consequence of the acylation of 7-O. The radical bonded to the 7-O was identified by NMR spectroscopy in comparison with macrolactin A. The sole carboxy group was directly visible in the NMR spectra in dichloromethane-d 4 , and the malonyl residue was clearly indicated by additional carboxy  13 C signals at 166.33 and 169.15 ppm and a methylene  13 C signal at 42.17 ppm. The related methylene  1 H signals were observed as doublet signals at 3.51 and 3.40 ppm (J=15.5 Hz). Only one carboxy- 13 C— signal was observed in methanol-d 4 . 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 NMR data for macrolactin A and 7-O-malonyl macrolactin A in methanol-d 4   
               
             
          
           
               
                 Macrolactin A 
                 7-O-malonyl macrolactin A 
               
             
          
           
               
                 H 
                 δ H   
                 m 
                 J [Hz] 
                 C 
                 δ C   
                 m 
                 H 
                 δ H   
                 m 
                 J [Hz] 
                 C 
                 δ C   
               
               
                   
               
             
          
           
               
                  1 
                 — 
                 — 
                 — 
                 1 
                 168.02 
                 s 
                  1 
                 — 
                 — 
                 — 
                  1 
                 167.94 
               
               
                  2 
                 5.58 
                 d 
                 11.33 
                 2 
                 118.00 
                 d 
                  2 
                 5.59 
                 d 
                 11.7 
                  2 
                 118.52 
               
               
                  3 
                 6.67 
                 t 
                 11.71 
                 3 
                 144.96 
                 d 
                  3 
                 6.67 
                 t 
                 11.3 
                  3 
                 144.50 
               
               
                  4 
                 7.26 
                 dddd 
                 15.1, 11.4, 2, 1 
                 4 
                 130.26 
                 d 
                  4 
                 7.25 
                 dd 
                 14.7, 11.7 
                  4 
                 130.79 
               
               
                  5 
                 6.20 
                 m 
                 — 
                 5 
                 142.16 
                 d 
                  5 
                 6.15 
                 dt 
                 15.4, 7.2 
                  5 
                 140.51 
               
               
                  6 
                 2.45 
                 m 
                 — 
                 6 
                 42.84 
                 t 
                  6 
                 2.60 
                 m 
                 5.3 
                  6 
                 40.13 
               
               
                  7 
                 4.29 
                 ddt 
                 4.9, 1.2, 6.8 
                 7 
                 72.33 
                 d 
                  7 
                 5.50 
                 ddd 
                 6.0, 6. 0, 6.0 
                  7 
                 74.72 
               
               
                  8 
                 5.79 
                 dd 
                 15.1, 6.0 
                 8 
                 137.55 
                 d 
                  8 
                 5.75 
                 dd 
                 15.3, 5.5 
                  8 
                 132.06 
               
               
                  9 
                 6.61 
                 ddt 
                 15.2, 11.0, 1.1 
                 9 
                 125.96 
                 d 
                  9 
                 6.71 
                 dd 
                 15.1, 11.3 
                  9 
                 128.09 
               
               
                 10 
                 6.15 
                 t 
                 11.14 
                 10 
                 131.39 
                 d 
                 10 
                 6.13 
                 t 
                 10.2 
                 10 
                 130.91 
               
               
                 11 
                 5.58 
                 ddd 
                 10.5, 8.6, 8.2 
                 11 
                 128.39 
                 d 
                 11 
                 5.63 
                 dt 
                 10.6, 8.4 
                 11 
                 129.78 
               
               
                  12a 
                 2.53 
                 dddd 
                 13.5, 8.2, 7.4, 0.8 
                 12 
                 36.50 
                 t 
                  12a 
                 2.63 
                 m 
                 — 
                 12 
                 36.39 
               
               
                  12b 
                 2.36 
                 dddd 
                 13.5, 7.7, 4.9, 1.1 
                   
                   
                   
                  12b 
                 2.33 
                 ddd 
                 13.0, 7.2, 5.5 
               
               
                 13 
                 3.89 
                 ddt 
                 5.3, 5.1, 6.9 
                 13 
                 69.24 
                 d 
                 13 
                 3.84 
                 ddd 
                 10.6, 6.0, 5.7 
                 13 
                 69.51 
               
               
                 14 
                 1.65 
                 m 
                 — 
                 14 
                 43.92 
                 t 
                 14 
                 1.66 
                 m 
                 — 
                 14 
                 43.84 
               
               
                 15 
                 4.34 
                 dt 
                 6.3, 6.3 
                 15 
                 69.83 
                 d 
                 15 
                 4.39 
                 dt 
                 6.3, 6.3 
                 15 
                 69.77 
               
               
                 16 
                 5.60 
                 dd 
                 15.1, 6.5 
                 16 
                 135.23 
                 d 
                 16 
                 5.60 
                 dd 
                 15.1, 6.4 
                 16 
                 135.32 
               
               
                 17 
                 6.21 
                 dd 
                 15.5, 10.9 
                 17 
                 131.21 
                 d 
                 17 
                 6.21 
                 dd 
                 15.1, 10.6 
                 17 
                 131.27 
               
               
                 18 
                 6.09 
                 dd 
                 14.9, 10.4 
                 18 
                 131.72 
                 d 
                 18 
                 6.10 
                 dd 
                 15.1, 10.6 
                 18 
                 131.78 
               
               
                 19 
                 5.69 
                 ddd 
                 14.7, 7.2, 6.8 
                 19 
                 135.13 
                 d 
                 19 
                 5.69 
                 ddd 
                 14.9, 7.0, 6.8 
                 19 
                 135.10 
               
               
                  20a 
                 2.23 
                 ddt 
                 14.2, 7.1, 6.8 
                 20 
                 32.98 
                 t 
                  20a 
                 2.23 
                 td 
                 14.0, 6.8 
                 20 
                 33.03 
               
               
                  20b 
                 2.14 
                 ddt 
                 14.2, 6.4, 7.1 
                   
                   
                   
                  20b 
                 2.15 
                 td 
                 14.4, 7.2 
               
               
                 21 
                 1.54 
                 m 
                 — 
                 21 
                 25.65 
                 t 
                 21 
                 1.54 
                 m 
                 — 
                 21 
                 25.81 
               
               
                  22a 
                 1.68 
                 m 
                 — 
                 22 
                 36.01 
                 t 
                  22a 
                 1.70 
                 m 
                 — 
                 22 
                 36.08 
               
               
                  22b 
                 1.61 
                 m 
                 — 
                   
                   
                   
                  22b 
                 1.62 
                 m 
                 — 
               
               
                 23 
                 5.05 
                 ddq 
                 7.3, 4.5, 6.2 
                 23 
                 72.21 
                 d 
                 23 
                 5.05 
                 ddq 
                 4.5, 7.1, 6.1 
                 23 
                 72.37 
               
               
                 24 
                 1.29 
                 d 
                 6.04 
                 24 
                 20.11 
                 q 
                 24 
                 1.30 
                 d 
                 6.0 
                 24 
                 20.14 
               
               
                     1′ 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                     1′ 
                 — 
                 — 
                 — 
                     1′ 
                 169.64 
               
               
                     2′ 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                     2′ 
                 2.90 
                 m 
                 —(br) 
                     2′ 
                 44.74 
               
               
                     3′ 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                     3′ 
                 — 
                 — 
                 — 
                     3′ 
                 n/o 
               
               
                   
               
               
                   1 H at 600 MHz:  13 C at 150 MHz; (a) from the HMQC-NMR spectrum. 
               
               
                 n/o = not observed due to signal scattering. 
               
               
                 The multiplicity of carbon signals were captured from the DEPT and HMQC spectra. 
               
             
          
         
       
     
     Macrolactin A 
     Macrolactin A: C 24 H 35 O 5 , M=402.53, UV (Methanol): λ max  (lg ε)=227 nm (4.537), 261 (4.146) [Lit.: 227 (4.691), 261 (4.272)]. [α] 22   D =−10.7 (c=0.68 in MeOH) [Lit.: −9.6 [c=1.86 in MeOH]. MS: (−)-ESI (TOF): m/z (%)=401.2 (38) [M−H] − , 437.2 (100) [M+Cl] − , 803.4 (63) [2M−H] − ; (−)-DCI (isobutane): m/z (%)=402 (100); (+)-DCI (isobutane): m/z (%)=349 (56) [M+H-3H 2 O] + , 367 (100); [M+H-2H 2 O] + , 385 (75) [M+H-2H 2 O] + ; EI (200° C.): m/z (%)=255 (100), 273 (72), 348 (18), 366 (68), 384 (60), 400 (5.9), 402 (2.5). The NMR data are shown in Table 1. 
     7-O-Succinyl Macrolactin A 
     C 28 H 38 O 8 , M=502.60: UV (MeOH): λ max  (lg ε)=227 nm (4.596), 259 (4.192) [Lit.: 229 (4.57), 261 (4.18)]. [α] 22   D =−19.9 (c=0.7 in MeOH) [Lit.: −9.6 [c=0.18 in MeOH]. MS: (−)-ESI (TOF): m/z (%)=501.3 (100) [M−H] − , 117.0 (12) [Bernstein acid-H] − ; (−)-DCI (isobutane): m/z (%)=502.7 (100); 484 (44), 402 (18), 384 (68), 366 (26), 117 (20). 
     The  1 H NMR data in CDCl 3  were found to be identical to those described by Jaruchoktaweechai et al. J. Nat. Prod. 63, (7), 984-986 (2000). 
     The chemical properties of the three aforementioned macrolactin compounds, which are produced from  Bacillus subtilis  DSM 16696, are summarized in Table 2, which is reproduced hereunder: 
     
       
         
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 7-O-malonyl- 
                 7-O-succinyl- 
               
               
                 Property 
                 Macrolactin A 
                 macrolactin A 
                 macrolactin A 
               
               
                   
               
             
             
               
                 Molecular formula 
                 C 24 H 35 O 5   
                 C 27 H 36 O 8   
                 C 28 H 38 O 8   
               
               
                 Molecular weight 
                 402.53 
                 488.57 
                 502.60 
               
               
                 UV (MeOH) 
                 227 (4.537) 
                 227 (4.397), 230 (sh) 
                 227 (4.596) 
               
               
                 [λ max  (lg ε] 
                 261 (4.146) 
                 260 (4.006) 
                 259 (4.192) 
               
               
                 [α] 22     D     
                 −10.7 (0.68) 
                 −6.2 (0.63) 
                 −19.9 (0.7) 
               
               
                 (c in MeOH) 
               
               
                 (−)-MS-ESI-TOF 
                 401.2 (38) [M − H] −   
                 487.2 (100) [M − H] −   
                 501.3 (100) [M − H] −   
               
               
                 [m/z (%)] 
                 437.2 (100) [M + Cl] −   
                 443.2 (44) [M − H—CO 2 ] −   
                 117.0 (12) 
               
               
                   
                 803.4 (63) [2M − H] −   
                 383.2 (27) [M-H − Malonic acid] −   
                 [Bernstein acid − H] −   
               
               
                   
               
             
          
         
       
     
     Medical Application of 7-O-Succinyl Macrolactin A Against Antibiotic-Resistant Bacteria 
     7-O-succinyl macrolactin A, like the above-described 7-O-malonyl macrolactin A, exhibits vigorous activity against antibiotic-resistant bacteria, such as, e.g. multiresistant bacteria, and particularly against bacteria which, for example, are resistant to one of a group comprising vancomycin, erythromycin, methicillin or ampicillin. 
     The present invention therefore also enables the employment of 7-O-succinyl macrolactin A in the manufacture of pharmaceutical preparations that are effective against antibiotic-resistant bacteria. In view of the striking bacteriostatic effectiveness of low concentrations (sub-MIC, i.e. in the region of the MBC) of 7-O-succinyl macrolactin A and of 7-O-malonyl macrolactin A against antibiotic-resistant bacteria, such pharmaceutical preparations can contain 7-O-succinyl macrolactin A and 7-O-malonyl macrolactin A alone or in combination. The combination of 7-O-succinyl macrolactin A with 7-O-malonyl macrolactin A is especially preferred, since each can be bacteriostatically active against specific antibiotic-resistant bacteria. 
     Production Method Using Fermentation 
     The proposed  B. subtilis  DSM 16696 strain can be used in the production of 7-O-malonyl macrolactin A, 7-O-succinyl macrolactin A and/or macrolactin A. 
     Described in the following examples is a fermentative production method employing a shake culture. Other prior art submersion processes in fermenters can also be employed for large-volume cultivation. This includes batch, fed batch and continuous fermentation processes. 
    
    
     
       The invention will next be described in greater detail with the aid of examples. The figures show the following: 
         FIG. 1A  shows the kinetics of growth for  Staphylococcus aureus  (#) without (0) and with 1 μg/ml (1) or 4 μg/ml (4) 7-O-malonyl macrolactin A; 
         FIG. 1B  shows the kinetics of growth for  Staphylococcus aureus  (methicillin-resistant, from a clinical isolate, MRSA 3) without (0) and with 1 μg/ml (1) or rather, 4 μg/ml (4) 7-O-malonyl macrolactin A; 
         FIG. 1C  shows the kinetics of growth for  Enterococcus faecalis  ATCC 29212 without (0) and with 4 μg/ml (4) or rather, 16 μg/ml (16) 7-O-malonyl macrolactin A; 
         FIG. 1D  shows the kinetics of growth for  Enterococcus faecium  (VRAR E315, vancomycin-resistant, ampicillin-resistant, clinical isolate) without (0) and with 4 μg/ml (4) or rather, 16 μg/ml (16) 7-O-malonyl macrolactin A; 
         FIG. 1E  shows the kinetics of growth for  Candida krusei  DSMZ6128 without (0) and with 32 μg/ml (32) or rather, 128 μg/ml (128) 7-O-malonyl macrolactin A; 
         FIG. 1F  shows the kinetics of growth for  Burkholderia cepacia  SCV 141 (clinical isolate) without (0) and with 32 μg/ml (32) or rather, 128 μg/ml (128) 7-O-malonyl macrolactin A; 
         FIGS. 2   a ),  b ) and  c ) are electron microscope photographs of  S. aureus  MRSA 3 after 4 hours growth without antibiotic and d), e) and f) in the presence of 16 μg/ml 7-O-malonyl macrolactin A; 
         FIG. 3   a ) to  c ) are electron microscope photographs of  E. faecium  VRAR E315 after 4 hours growth without antibiotic and d), e) and f) in the presence of 16 μg/ml 7-O-malonyl macrolactin A; 
         FIG. 4   a ) to  c ) are electron microscope photographs of  Burkholderia cepacia  SCV after 4 hours growth without antibiotic and d), e) and f) in the presence of 128 μg/ml 7-O-malonyl macrolactin A; 
         FIG. 5A  shows the inhibition of the proliferation of mouse cells L-929 by means of 7-O-malonyl macrolactin A (●), 7-O-succinyl macrolactin A (▾) and macrolactin A (▪), and; 
         FIG. 5B  shows the inhibition of the proliferation of human epithelial cells (HeLa, ▾) and mouse cells L-929 (●) by means of 7-O-malonyl macrolactin A. 
     
    
    
     EXAMPLE 1 
     Cultivation of  Bacillus subtilis  for the Production of 7-O-Malonyl Macrolactin A 
     Producer strain DSM 16696 was injected into a liquid culture medium comprising 5 g/l yeast extract, 20 g/l tryptone, 5 g/l sodium chloride and 5 g/l glucose at a pH of 7. Present in the culture medium during cultivation in a shake culture (120 Upm) for more than 7 days at 30° C. was 4% by weight of the hydrophobic adsorbent resin Amberlite XAD-16 (Röhm und Haas, Germany). 
     Following extraction by decanting, the adsorbent resin was washed in a column containing 50% aqueous methanol, the adsorbed compounds were next eluted with methanol and then concentrated by means of evaporation in a rotary evaporator to 1/100 of the culture volume. The methanol was removed from the extract by means of evaporation, and the residual aqueous mixture extracted 4 times with the aid of ethyl acetate. The ethyl acetate was removed under reduced pressure yielding approx. 300 mg of oily residue from a total culture of 4 l, to which the adsorbent resin had been added. Said residue was re-suspended in methanol, and the solution extracted 4 times with the same volume of n-heptane to remove lipophilic products and contaminants. 
     EXAMPLE 2 
     Cultivation of  Bacillus subtilis  for the Production of 7-O-Malonyl Macrolactin A 
     For production purposes, the producer strain was used to inoculate a medium OM, which, having a pH of 7, is advantageous for the production of macrolactins, since employment of the same process sequence yielded an isolate of greater purity. 
     OM medium is obtained by autoclaving 1.0 g starch, 1.0 g glucose, 1.0 g peptone and 1.5 g yeast extract per 980 ml water and by adding 10 ml/l solution A (5 g/l KH 2 PO 4 , 5 g/l K 2 HPO 4 , autoclaved), 10 ml/l solution B (17 g/l MgSO 4 , 1.0 g/l NaCl, 0.7 g/l MnSO 4 , 0.06 g/l CuSO 4 , autoclaved), 1 ml/l solution C (7 g/l FeSO 4 .7H 2 O, 22 g/l Na 3 -citrate.3H 2 O, 2.0 g/l ammonium citrate, 7.5 g/l Na-thioglycalase, 33 g/l Na2-succinate.6H 2 O, sterile-filtered) and 1 ml/l solution D (100 mg/l biotin, 350 mg/l nicotinic acid amide, 300 mg/l thiamine.HCl, 200 mg/l p-amino benzoic acid, 100 mg/l pyridoxal hydrochloride, 100 mg/l Ca-panthothenate, 50 mg/l vitamin B 12 , sterile filtered). 
     EXAMPLE 3 
     Purification of Macrolactins by Means of Liquid Chromatography 
     Extracts and fractions from examples 1 and 2 were analyzed by means of reversed-phase liquid chromatography (RP-HPLC) using a Nucleosil 100-5 C18 column (125/2 mm, Macherey Nagel). Used for detection were a 320-600 nm UV-diode array detector and an external light scatter detector (PL•ELS-1000, Polymer Laboratories). Employed as solvents were A: 0.5% acetic acid/water and B: 0.5% acetic acid/methanol at a flow rate of 0.3 ml/min. The macrolactins were separated by means of preparatory RP-HPLC using a Nucleosil 100-7 C18 column (250/21 mm, Macherey-Nagel) and a 51% to 56% gradient of solvent A and solvent B at a flow rate of 30 ml/min, with UV detection at 280 nm. Between 40 and 60 mg extract in 0.2 ml methanol was used for injection purposes. 
     The purification of macrolactins was accomplished in the following manner: preparatory RP-HPLC was employed using a Nucleosil 100-7 C18 column (250/21 mm, Macherey-Nagel, Düren, Germany) with the following solvent gradients: solvent A (0.5% acetic acid/51% aqueous methanol) and solvent B (0.5% acetic acid/56% aqueous methanol): solvent B from 0 to 100% for over 60 minutes at a flow rate of 30 ml/min. UV detection was at 280 nm. 40 to 60 mg extract in 0.2 ml methanol was used for injection. Each of the macrolactins (5 to 7 mg) was then purified using chromatography in a LH-20 column (760/25 mm, the solvent used being methanol/dichloromethane (1:1) at a flow rate of 5 ml/min.) with an application quantity of between 5 and 7 mg. 
     Between 4 and 6 mg macrolactin A, 5 and 7 mg 7-O-malonyl macrolactin A and 6 and 8 mg 7-O-succinyl macrolactin A were isolated from a 4 l culture using preparatory RP-HPLC and subsequent purification by means of LH-20 chromatography. 
     The macrolactin compounds isolated from the proposed  Bacillus subtilis  strain yielded the following data upon analysis: Macrolactin A, the molecular ion m/z at 402 and the UV absorptions at 227 and 261 nm enabled identification of the compound as macrolactin A or one of the 10E isomers thereof. The latter was ruled out by its optical rotation of [α] 22   D =−138, compared to a result of −10 for macrolactin A. A comparison of the  1 H and  13 C-NMR data for the well-defined spectra of macrolactin A in methanol d 4  are shown in Table 2. The signals were identified by means of  1 H,  1 H-COSY and  1 H—,  13 C-HMBQ spectra. 
     EXAMPLE 4 
     Antimicrobial Effect 
     The agar diffusion method employing Mueller-Hinton (MH) agar (Difco Laboratories) was used to determine effectiveness against bacteria and yeasts. Sterile paper disks (Schleicher &amp; Schüell, Germany) were seeded with 10 μl crude extract or with a solution of purified macrolactin (final compound concentration on the disk 50 μg) and then placed on the surface of MH-agar plates that had been seeded beforehand with a suspension of overnight culture of the test microorganisms at a concentration of 10 5  cells/ml. Following incubation for 18 hours at 37° C., the diameter of the zones of inhibition of growth around the disks was measured. 
     The crude extract of Example 1 exhibited full bactericidal activity with a completely clear aureola about the disk. It is thought that this result arises from the cumulative action of the macrolactins so isolated. 
     7-O-malonyl macrolactin A exhibited antibacterial activity against gram-positive bacteria, such as, for example,  Burkholderia cepacia , gram-negative reference and clinical isolates with an effectiveness comparable to or better than that obtainable with erythromycin. A notable advantage conferred by the novel capabilities of 7-O-malonyl macrolactin A and 7-O-succinyl macrolactin A, in particular against methicillin or ampicillin, vancomycin and/or erythromycin-resistant bacterial isolates is that said compounds also exhibit antibacterial activity against antibiotic-resistant isolates. The findings are set out in Table 3 hereunder: 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Comparison of the antibacterial activity in vitro 
               
               
                 of macrolactins and conventional antibiotics 
               
             
          
           
               
                   
                 Inhibition zone (mm) 
               
             
          
           
               
                   
                   
                   
                 7-O- 
                 7-O- 
                   
                   
                   
               
               
                   
                   
                   
                 Malonyl- 
                 Succinyl- 
               
               
                   
                 Crude 
                 Macro- 
                 macrolactin 
                 macrolactin 
                 Erythro- 
                 Vanco- 
                 Ampi- 
               
               
                 Test strain 
                 Extract 
                 lactin A 
                 A 
                 A 
                 mycin 
                 mycin 
                 cillin 
               
               
                   
               
             
          
           
               
                 
                   S. aureus 
                 
                 28 
                 18 
                 26 
                 18 
                 30 
                 20 
                 30 
               
               
                 DSMZ 
               
               
                 1104** 
               
               
                   S. aureus **, #   
                 32 
                 25 
                 30 
                 22 
                 40 
                 27 
                 41 
               
               
                 MRSA 2 
                 24 
                 27 
                 40 
                 37 
                 0 
                 24 
                 0 
               
               
                 MRSA 3 
                 28 
                 35 
                 41 
                 38 
                 0 
                 24 
                 0 
               
               
                 
                   E. faecalis 
                 
                 18 
                 0 
                 25 
                 12 
                 25 
                 24 
                 32 
               
               
                 ATCC 29212 
               
               
                 
                   E. faecalis 
                 
                 15 
                 0 
                 15 
                 0 
                 0 
                 0 
                 35 
               
               
                 VRAS E305 
               
               
                 
                   E. faecium 
                 
                 40 
                 0 
                 15 
                 20 
                 25 
                 0 
                 0 
               
               
                 VRAR E315 
               
               
                 
                   B. cepacia 
                 
                 40 
                 0 
                 15 
                 0 
                 0 
                 not 
                 0 
               
               
                 SCV* 141 
                   
                   
                   
                   
                   
                 determined 
               
               
                   
               
               
                 *SCV = Small Colony Variant 
               
               
                 MRSA = Methicillin-resistant  S. aureus   
               
               
                 VRAS = Vancomycin-resistant, ampicillin-sensitive 
               
               
                 VRAR = Vancomycin-resistant, ampicillin-resistant 
               
               
                 **= Control strains, methicillin-sensitive 
               
               
                   # = own collection 
               
               
                 10 μl crude extract and 50 μg macrolactin were spread on a disk. 78 μg erythromycin, 30 μg vancomycin and 10 μg ampicillin were contained on a disk (prepared disks). 
               
             
          
         
       
     
     The data shown in Table 3 indicate that 7-O-succinyl macrolactin A exhibits no activity against, for example, the erythromycin-resistant strains  E. faecalis  VRAS E305 and  B. cepacia  SCV* 141 but that only 7-O-malonyl macrolactin A produced an inhibition zone due to antibacterial activity. 
     By contrast, both 7-O-succinyl macrolactin A and 7-O-malonyl macrolactin A exhibit antibacterial activity against the multiresistant enterococci, as is indicated, for example, at staphylococci MRSA 2 and MRSA. In contrast to the macrolactin A, which also exhibits activity, 7-O-succinyl macrolactin A and 7-O-malonyl macrolactin A have, when applied as proposed, the advantage of having low cytotoxicity, a property that renders these compounds in the low concentration region of the MBC suitable for use as antibacterial ingredients, and especially as bacteriostatic ingredients. 
     The data shown in Table 3 also suggest that 7-O-malonyl macrolactin A has markedly stronger antibacterial activity against all test bacteria compared to macrolactin A. The sole exception is  E. faecium  VRAR E315, against which the antibacterial activity of 7-O-malonyl macrolactin A is slightly weaker than that of 7-O-succinyl macrolactin A. It is therefore preferred, in one embodiment of the invention, that the antibacterially active pharmaceutical composition comprise 7-O-malonyl macrolactin A, optionally in combination with 7-O-succinyl macrolactin A. 
     The proposed compound 7-O-malonyl macrolactin A, in particular, demonstrated capability to inhibit the growth of methicillin-resistant  Staphylococcus aureus  (MRSA 2 or 3, clinical isolate) and vancomycin-resistant  Enterococcus  (VRE). 7-O-malonyl macrolactin A was the most active of the three macrolactin compounds produced from the proposed  Bacillus subtilis  strain. Use of the agar diffusion procedure permitted observation of a broad aureola of bacterial growth inhibition around the disk. Inhibition of the bacterial growth of  Staphylococcus  was, however, incomplete, and small colonies were evident within this zone. 
     The  B. cepacia  strain used, which is a clinical isolate from a cystic fibrosis patient, forms small colonies (SCV 141). This isolate exhibits the typical characteristics of bacteria in combination with mucoidal bacteria in a biofilm and which, due to higher antibiotic resistance, has considerable clinical relevance for cystic fibrosis patients in particular. In this comparison test, 7-O-malonyl macrolactin A alone exhibited antibacterial activity against the isolate of  B. cepacia , in tandem with characteristics that suit it to medical application. 
     EXAMPLE 5 
     Minimal Inhibition Concentrations of 7-O-Malonyl Macrolactin A 
     Although the minimal inhibition concentrations (MIC) of 7-O-malonyl macrolactin A for staphylococci was greater than 128 μg/ml, concentrations of between 1 and 4 μg/ml permitted pronounced inhibition of bacterial growth for the reference strain ( S. aureus  #, non-methicillin-resistant), or rather, for the MRSA strains. The MIC value for 7-O-malonyl macrolactin A against  Enterococcus faecalis  ATCC 29212 exceeded 128 μg/ml and bacterial growth was markedly inhibited at the sub-MIC value of 4 μg/ml. 7-O-malonyl macrolactin A, although exhibiting activity at MIC values above 128 μg/ml against VRE strains, noticeably inhibited bacterial growth at 0.06 or 4 μg/ml for the strains  Enterococcus faecalis  E305, vancomycin-resistant/ampicillin-sensitive (VRAS) or rather,  Enterococcus faecium  B315, vancomycin-resistant/ampicillin-resistant (VRAR). 
     7-O-malonyl macrolactin A was not effective against some of the gram-negative bacteria tested by the inventors. Inhibition of the growth of clinical isolates of gram-negative bacteria was observed, e.g. for  Burkholderia cepacia  SCV 141 at concentrations of 32 μg/ml, however, such activity was not observed against wild-type strain  Burkholderia  cepacia 139, for which the MIC value exceeded 128 μg/ml. These results indicate, however, that due to its effectiveness even against clinically relevant gram-negative strains, in particular those having resistance to at least one antibiotic, 7-O-malonyl macrolactin A is suitable for medical purposes. 
     Inhibition activity against  Candida  spp. was also noted. 
     The marked inhibition of bacterial growth, which was observed at sub-MIC concentrations of 7-O-malonyl macrolactin A, indicates that this compound, even at very low concentrations, is bacteriostatically active against at least some of the test strains. 
     Even comparing the antimicrobial activity of 7-O-malonyl macrolactin A with that of reference compounds in liquid culture suggests that the minimal required bacteriostatic concentrations (MBC) at which pronounced inhibition of bacterial growth was observed, was very low for 7-O-malonyl macrolactin A. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Antimicrobial activity of 7-O-malonyl macrolactin A and reference 
               
               
                 compounds against clinical isolates and reference strains 
               
             
          
           
               
                   
                 7-O-malonyl- 
                 Vanco- 
                 Ampi- 
                 Erythro- 
                 Genta- 
                   
               
               
                   
                 macrolactin A 
                 mycin 
                 cillin 
                 mycin 
                 mycin 
                 MCZ 
               
             
          
           
               
                 Strain 
                 MIC 
                 MBC 
                 MIC 
               
               
                   
               
             
          
           
               
                   S. aureus **, #   
                 &gt;128 
                 1 
                 0.125 
                 0.06 
                 0.06 
                 0.25 
                 ND 
               
               
                 MRSA 2 
                 &gt;128 
                 4 
                 2 
                 &gt;128 
                 &gt;128 
                 128 
                 ND 
               
               
                 MRSA 3 
                 &gt;128 
                 4 
                 1 
                 64 
                 &gt;128 
                 128 
                 ND 
               
               
                 
                   E. faecalis 
                 
                 128 
                 4 
                 2 
                 0.5 
                 2 
                 32 
                 ND 
               
               
                 ATCC 29212 
               
               
                 
                   E. faecalis 
                 
                 &gt;128 
                 0.06 
                 &gt;128 
                 0.5 
                 128 
                 64 
                 ND 
               
               
                 VRAS E305 
               
               
                 
                   E. faecium 
                 
                 &gt;128 
                 4 
                 &gt;128 
                 &gt;128 
                 2 
                 64 
                 ND 
               
               
                 VRAR E315 
               
               
                 
                   B. cepacia 
                 
                 &gt;128 
                 — 
                 ND 
                 &gt;128 
                 &gt;128 
                 &gt;128 
                 ND 
               
               
                 WT139 
               
               
                 
                   B. cepacia 
                 
                 128 
                 32 
                 ND 
                 128 
                 128 
                 &gt;128 
                 ND 
               
               
                 SCV 141 
               
               
                 
                   Candida 
                 
                 128 
                 — 
                 ND 
                 ND 
                 ND 
                 ND 
                 2 
               
               
                 
                   parapsilosis 
                 
               
               
                 DSM5784 
               
               
                 
                   Candida krusei 
                 
                 &gt;128 
                 32 
                 ND 
                 ND 
                 ND 
                 ND 
                 2 
               
               
                 DSM 6128 
               
               
                 
                   Candida 
                 
                 &gt;128 
                 — 
                 ND 
                 ND 
                 ND 
                 ND 
                 2 
               
               
                   albicans  DSM 
               
               
                 11225 
               
               
                   
               
               
                 Concentrations are expressed in μg/ml. 
               
               
                 ND = not determined 
               
               
                 MIC = minimal inhibitory concentration 
               
               
                 MBC = minimal bacteriostatic or fungistatic concentration 
               
               
                 MCZ = miconazol, which is a fungicide 
               
             
          
         
       
     
     The data given in Table 4 suggest that 7-O-malonyl macrolactin A, at low concentrations is active in the same order of magnitude, as prior art antibiotics. This suggests that it is not necessary that the concentration of 7-O-malonyl macrolactin A be at MIC, although it is necessary that the minimal bacteriostatic concentration be reached for strong bacteriostatic activity to occur. 7-O-malonyl macrolactin A exhibits, moreover, activity against pathogenic yeasts, an example whereof is  Candida.    
     EXAMPLE 6 
     Biocidal Activity of 7-O-Malonyl Macrolactin A Against Pro- and Eukaryotic Microorganisms 
     Investigation of the bacteriostatic activity of 7-O-malonyl macrolactin A required testing the effects of sub-MIC concentrations. The kinetics of growth for  Staphylococcus aureus  (#), shown in  FIG. 1A , indicates that 7-O-malonyl macrolactin A kills off this bacterium within the first 4 hours following dosage, even at concentrations far below the MIC. The subsequent cultivation time indicates that the growth of this bacterium is reduced by a factor of 10. The activity of 7-O-malonyl macrolactin A against MRSA 3 ( FIG. 1B ) becomes apparent only following prolonged cultivation, even though the growth of this bacterium strain after 4 hours is almost completely inhibited. 24 hours after treatment, the titre of viable cells was reduced by a factor of 100 as compared to the control without 7-O-malonyl macrolactin A. 
     Four hours following cultivation, 7-O-malonyl macrolactin A inhibited the growth of  Enterococcus faecalis  ATCC 29212; in this case, the titre of viable cells remained below that of the untreated control culture throughout the final phase of the cultivation period ( FIG. 1C ). 
     Four hours following cultivation in the presence of 7-O-malonyl macrolactin A, the growth of  Enterococcus faecium  E315, (vancomycin-resistant, ampicillin-resistant), was almost completely inhibited ( FIG. 1D ). 
     It is interesting to note that 7-O-malonyl macrolactin A exhibits stronger bacteriostatic activity against antibiotic-resistant strains isolated from clinical patients than against non-resistant strains. It was also observed that the live cell count did not rise substantially during cultivation in the presence of 7-O-malonyl macrolactin A. Particularly interesting is a comparison of  FIG. 1A  with  FIG. 2B , which clearly indicates that the bacteriological activity of 7-O-malonyl macrolactin A against the antibiotic-resistant MRSA 3 is of a magnitude equal to or greater than the activity against reference strain  S. aureus   # , which is not antibiotic resistant. 
     7-O-malonyl macrolactin A exhibits antibiotic activity even against the eukaryotic microorganism  Candida krusei  ( FIG. 1E ) and against  Burkholderia cepacia  SCV 141 ( FIG. 1F ) which, in and of itself, is resistant to prior art antibiotics and inhibits growth up to a factor of 10 at concentration of 128 μg/ml for  C. krusei  and 32 μg/ml for  B. cepacia.    
     In general, investigations of the inhibitory activity of 7-O-malonyl macrolactin A did not indicate dosage dependency where two sub-MIC concentrations were used in experiments. 
     EXAMPLE 7 
     Disruption of Cell Division of Bacteria by Means of 7-O-Malonyl Macrolactin A 
     Investigation of bacterial strains from clinical isolates which, in accordance with Example 5, in shake cultures are in the presence of sub-MIC concentrations of 7-O-malonyl macrolactin A and undergo pronounced growth inhibition, indicates that 7-O-malonyl macrolactin A interferes with the process of cell division. It is thought that 7-O-malonyl macrolactin A inhibits the division of bacterial cells, which interrupts the proliferation thereof. This investigation suggests that 7-O-malonyl macrolactin A is more effective in this role than the prior art 7-O-succinyl macrolactin A or macrolactin A. 
     The antibiotic activity of 7-O-malonyl macrolactin A becomes apparent at concentrations far below the MIC, which suggests that use of sub-MIC dosages may provide effective concentrations for medical purposes. In particular, in combination with the weakly cytotoxic activity of 7-O-malonyl macrolactin A, primarily at low concentrations, this property confers the advantages of achieving antibiotic effect at low dosages. 
     Electron microscope photographs of the clinical isolate of  Staphylococcus aureus  (MRSA 3, methicillin-resistant) in  FIG. 2  illustrate the activity of 7-O-malonyl macrolactin A.  FIGS. 2   a ) to c) show untreated shake cultures following incubation for 4 hours and  FIGS. 2   d ) to f) show the same cultures but with the addition of 16 μg/ml 7-O-malonyl macrolactin A. This concentration, although clearly sub-MIC, strongly inhibits growth, which can also be observed in  FIG. 1B  for concentrations of 1 and 4 μg/ml. 7-O-malonyl macrolactin A influences cell division in MRSA 3, which is evident from the altered division planes. The untreated cells shown in  FIGS. 2   a ) to  c ) exhibit division planes that appear as a light-shaded transverse wall as indicated in  FIGS. 2   b ) and  c ) by the white arrowheads. The treated MRSA 3, on the other hand, exhibits disrupted cell division. The large black arrowheads ( FIGS. 2   e ) and  f )) indicate the division planes, while the small black arrowheads indicate an asymmetrical initiation of cell division. 
     The effect of 7-O-malonyl macrolactin A on cell division can also be observed from the electron microscope photographs of  Enterococcus faecium  (VRAR=vancomycin-resistant, ampicillin-resistant, E315, clinical isolate) shown in  FIG. 3 . The division planes of the untreated cells after 4 hours growth without antibiotic appear as light regions in  FIGS. 3   a ),  b ) and  c ). In  FIGS. 3   d ) to  f ), which show initiations with 16 μg/ml 7-O-malonyl macrolactin A, there are no fully formed division planes evident, but anomalous, asymmetrical cell division is being initiated (indicated by large black arrowheads). In this example, however, no fully formed cell division planes are evident. The initiation of asymmetrical cell division is indicated by the small black arrowheads. One visible result of disrupted cell division ( FIG. 3   f ), is a pseudomulticellular chain (indicated by a white star in  FIG. 3   d ), which also does not exhibit a complete septum in the plane and in which, in the case of the untreated cells ( FIGS. 3   a ) and  b )), the cell division is visible as a light-shaded region. 
     The electron microscope photographs moreover elucidate the bacteriostatic effect of 7-O-malonyl macrolactin A against gram-negative bacteria, an example whereof is the cell division of  Burkholderia cepacia . Thus,  FIGS. 4   a, b  and  c  show  Burkholderia cepacia  SCV (clinical isolate) in a shake culture without the addition of antibiotic ingredients (control) and  FIGS. 4   d, e  and  f  after 4 hours growth in a shake culture in the presence of 128 μg/ml 7-O-malonyl macrolactin A. In the control culture, white arrowheads point to the cell division planes and the transverse walls in connection therewith. In the case of the gram-negative cells treated with 7-O-malonyl macrolactin A, ( FIGS. 4   e  and  f ) black arrows indicate the cell division planes and the unnatural protuberances. In the case of the cells treated with 7-O-malonyl macrolactin A, anomalously sized cells were also evident, as indicated in  FIG. 4   d  by a black arrow. 
     EXAMPLE 8 
     Activity of 7-O-Malonyl Macrolactin A Against Microorganisms, Especially Against Gram-Negative Bacteria and Eukaryotic Microorganisms 
     In one example of its activity against eukaryotic microorganisms, e.g. yeast, 7-O-malonyl macrolactin A was tested against  Candida krusei  DSMZ 6128. The results, shown in  FIG. 2   e , indicate some inhibitory activity. 7-O-malonyl macrolactin A also exhibits inhibitory activity against the gram-negative bacterium  Burkholderia cepacia  SCV 141 ( FIG. 2   f ). 
     EXAMPLE 9 
     Cytotoxic Activity of 7-O-Malonyl Macrolactin A, 7-O-Succinyl Macrolactin A and Macrolactin A Against Animal Cells 
     The cytotoxicity of 7-O-malonyl macrolactin A was compared to that of 7-O-succinyl macrolactin A and macrolactin A in vitro with respect to the inhibition of the proliferation of L929 mouse fibroblast cells or the human epithelial cell line HeLa. 
     For the cell culture, the HeLa cells were cultivated in low-glucose DMEM medium (Gibco), and the L929 cells in high-glucose DMEM medium. Both were supplemented with 10% by volume foetal calf serum (Gibco) at 37° C. in an atmosphere containing 5% CO 2 . The cells were extracted from stock cultures by means of trypsinization with EDTA (HeLa) or without EDTA (L929), then counted and diluted to 2×10 5  cells/ml. For the tests, microtitre plates (Nunc.) comprising 96 depressions were plated out in serial dilution without or in the presence of the test compounds or methanol. Determination of morphological alterations of the cells was carried out by means of phase contrast microscopy after 1, 2 and 5 days incubation. 
     After 5 days cultivation, cell counts were determined by the CyQUANT cell proliferation test (Molecular Probes), which is a highly sensitive microtitre plate test based on fluorescence. The test employs the dye CyQUANT, which, by binding to cellular nucleic acids, enhances fluorescence, which can then be measured by fluorescein excitation. The emission of fluorescence by the dye-nucleic acid complex correlated linearly with the cell count. For test purposes, the residue was carefully removed after 5 days incubation, the cells washed with PBS (phosphate buffered saline solution), the buffer removed and the cells frozen at −80° C. For the test, the cells were thawed at ambient temperature and lysated in a buffered solution containing the dye CyQUANT, in accordance with manufacturer&#39;s instructions. Fluorescence was measured by means of a fluormetric microtitre-plate reader (Titertex Fluorskan II) (excitation at 480 nm, emission at 520 nm). The absorption values were used to calculate the percentage of cellular proliferation in medium both alone and in the presence of serial dilutions of methanol and the macrolactin compounds. As illustrated in  FIG. 5A  or  5 B, after a cell count of ca. 10 4 −2×10 5 /ml had been reached, the DMEM medium was replaced by fresh medium, containing concentrations of 7-O-malonyl macrolactin A or, as shown in Table 5, of various macrolactins. Proliferation was measured by means of the fluorescence-based test CyQUANT for determining viable cell counts. Table 5 illustrates the relative inhibition of proliferation as compared to a control culture comprising fresh medium without the addition of a macrolactin. 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Inhibition of proliferation by means of macrolactins 
               
             
          
           
               
                   
                 Compound concentration μg/ml 
               
             
          
           
               
                 Compound 
                 Cells 
                 125 
                 56.5 
                 31.25 
                 15.6 
                 7.8 
                 3.9 
               
               
                   
               
               
                 7-O-malonyl macrolactin A 
                 HeLa 
                 100%  
                 93% 
                 62% 
                  0 
                 0 
                 0 
               
               
                 7-O-malonyl macrolactin A 
                 L929 
                 84% 
                 86% 
                 80% 
                 32% 
                 0 
                 0 
               
               
                 7-O-succinyl macrolactin A 
                 L929 
                 87% 
                 83% 
                 55% 
                  0 
                 0 
                 0 
               
               
                 7-O-macrolactin A 
                 L929 
                 83% 
                 83% 
                 81% 
                 38% 
                  29% 
                  23% 
               
               
                   
               
             
          
         
       
     
       FIG. 5A  clearly shows that macrolactin A (▪), like 7-O-succinyl macrolactin A (▾), more strongly inhibits the proliferation of mouse cells L292 than 7-O-malonyl macrolactin A (●). 
       FIG. 5B  shows that the proliferation of human cells, shown here in the example of HeLa cells (▾), is inhibited to a lesser extent by 7-O-malonyl macrolactin A than that of mouse fibroblast cells L-929 (●). It is interesting that, at low concentrations, inhibition of the proliferation of human cells is weaker than that of mouse cells. At higher concentrations, however, 7-O-malonyl macrolactin A was cytotoxic for both the human epithelial test cells and the fibroblast cells. 
     It has already been demonstrated that macrolactin A, in addition to its antibacterial capability, exhibits cytotoxic and antiviral activity. The proposed isolated 7-O-malonyl macrolactin A at higher concentrations exhibits higher cytotoxicity than the other reference test compounds. 
     At a concentration of 15.6 μg/ml, 7-O-malonyl macrolactin A failed to inhibit the proliferation of HeLa cells. However, the fibroblast cell line was approx. 32% inhibited at the same concentration. Overall, 7-O-malonyl macrolactin A exhibits stronger inhibiting activity against the tested fibroblast cells than 7-O-succinyl macrolactin A, and for these two compounds no further inhibition activity was observed at a concentration of 7.8 μg/ml. Compared to macrolactin A, 7-O-malonyl macrolactin A at higher concentrations up to the test concentration of 15.6 μg/ml exhibited similar or slightly reduced inhibition activity against proliferation of the test fibroblast cells, but at concentrations of 7.8 and 3.9 μg/ml exhibited no further inhibition or proliferation as compared to macrolactin A. 
     Since the concentration levels above which cytotoxic activity becomes apparent for 7-O-malonyl macrolactin A exceed that at which marked bacteriostatic activity becomes evident, especially against antibiotic-resistant bacteria, 7-O-malonyl macrolactin A can be used in the production of pharmaceutical preparations for medical use. One notable advantage of 7-O-malonyl macrolactin A as compared to 7-O-succinyl macrolactin A or macrolactin A is its increased antimicrobial activity, especially at low concentrations, coupled with weak cytotoxicity against human cells, particularly in comparison with macrolactin A. 
     Microscopic investigation of the effect of 7-O-malonyl macrolactin A on human epithelial cells HeLa and the L929 mouse fibroblast cells revealed that the human cell line is subjected to less interference when treated with 7-O-malonyl macrolactin A than are those of the mouse. The antiproliferative activity is due to a toxic effect since the morphology of the treated cells was round. Proliferation of the control cells treated with methanol was not hindered. 
     Quantification of the cytotoxic effect of 7-O-malonyl macrolactin A using the CyQUANT test demonstrated that the compound was, at a concentration of 31.25 μg/ml, capable of inhibiting the proliferation of HeLa cells. The cells treated with a 62.5 μg/ml concentration of 7-O-malonyl macrolactin A exhibited practically complete inhibition of growth. 
     Translated Captions for the Figures 
     
         
           FIG. 1A  y-axis: Log CFU (Colony Forming Units) Cells/ml
       x-axis: Time (HRS)   
     
           FIG. 1B  y-axis: Log CFU (Colony Forming Units) Cells/ml
       x-axis: Time (HRS)   
     
           FIG. 1C : y-axis: Log CFU (Colony Forming Units) Cells/ml
       x-axis: Time (HRS)     
           FIG. 1D : y-axis: Log CFU (Colony Forming Units) Cells/ml
       x-axis: Time (HRS)     
           FIG. 1E : y-axis: Log CFU (Colony Forming Units) Cells/ml
       x-axis: Time (HRS)     
           FIG. 1F : y-axis: Log CFU (Colony Forming Units) Cells/ml
       x-axis: Time (HRS)     
           FIG. 5A : y-axis: % Inhibition
       x-axis Concentration μg/ml     
           FIG. 5B : y-axis: % Inhibition
       x-axis Concentration μg/ml