Lipova I1,3 Klochko V.2,3

D.K. Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine

1Department of physiology of industrial microorganisms

2Department of antibiotics,

3National Technical University of Ukraine “I. Sikorsky Kyiv Polytechnic Institute”


 Batumin by its chemical structure has no analogues among the existing antibiotics and has high and selective antistaphylococcal action (Klochko et al., 2008). This antibiotic has been isolated from the strain of new species Pseudomonas batumici at the D.K. Zabolotny Institute of Microbiology and Virology NAS of Ukraine (Kiprianova et al., 2011). Complete genome sequencing revealed an operon comprising 28 genes responsible for batumin biosynthesis (Klochko et al., 2016).

Previous studies of P. batumici fermentation broth were mainly focused on batumin as a target compound or on phenazine-1-carboxylic acid as a byproduct of biosynthesis. The aim of our study was to analyze physicochemical and biological properties of P. batumici byproducts of biosynthesis and further biological properties of batumin.

P. batumici UCM B-321 was grown in a synthetic liquid media containing glucose, urea, potassium orthophosphate, and a number of trace elements, 100 mL of synthetic broth in 750 mL flasks for 96 hours at 25°C and 220 rpm. The resulting fermentation broth of P. batumici UСM B-321 was previously extracted three times with chloroform in a ratio of 2:1, respectively. The extract was dried over anhydrous sodium sulfate and evaporated. Separation of the extracted substances was performed by thin layer chromatography (TLC) on silica gel plates (Merck, USA) in the benzene-isopropanol system (5:1). Identification and calculation of retention factor (Rf) of spots was held in iodine vapor. The disc-diffusion method with Staphylococcus aureus B-918, Escherichia coli B-926, Bacillus subtilis B-901, Candida albicans Y-2681 was used for bioautography. Inhibition zones in mm were measured after 24 hours. Physicochemical characterization of the obtained compounds was performed by measuring the absorption spectra. Molecular weights were identified using LC/MS method (Agilent 1200, USA).

There were revealed 4 spots in iodine vapor with the following retention factors: Rf1=0.41; Rf2=0.37; Rf3=0.32 and Rf4=0,28. Each substance showed antibiotic activity against S. aureus B-918, inhibition zones were: 25±2, 35±3, 24±3, 23±3 mm, respectively and on B. subtilis B-901 with inhibition zones: 23±2, 31±3, 20±2, 16±2 mm. The second compound also showed moderate activity (16±2) against E. coli B-926, whereas to other three substances E. coli B-926 was resistant. C. albicans Y-2681 was resistant to all 4 compounds. Absorption spectrums (in nm) were following: 231, 231, 228, 229. Molecular weights for substances were: 505, 548, 548, 550, respectively. Substance №2 had the most significant antistaphylococcal activity, moderate activity against E. coli, and by its physicochemical properties was identified as batumin. First substance was identified previously as descarbamoyl batumin. Taking into account chemical properties of batumin, we suggest that the third substance is an enolic form of batumin. Molecular weight of the fourth molecule was slightly higher; it may be a related compound with a methyl group instead of methylene group of batumin molecule. For the first time, batumin was tested on 2 clinical strains of Acinetobacter spp. using disc-diffusion method and has shown significant activity: inhibition zones were 25±3 mm.

Revealed biological activity of batumin allows it to be considered as a perspective drug for the treatment of skin infections caused by staphylococci or Acinetobacter spp.