Synergistic Effect of Enzyme Preparations and Gentamycin on Biofilms of <i>Bordetella pertussis</i>

Abstract

Relevance. An increase in the incidence of whooping cough, a high proportion of severe forms of the disease, and a decrease in the sensitivity of circulating strains of B. pertussis to antibiotics require the development of more effective etiotropic therapies, including those capable of influencing biofilm forms of the whooping cough pathogen, which differ from planktonic cells by increased resistance to the host immune system and antibacterial drugs.Аim of the work is to study the effect of trypsin and lidase in combination with gentamycin on the growth of biofilms of Bordetella pertussis strains on an abiotic substrate.Materials and methods. In the experiments B. pertussis strains isolated in the Russian Federation from whooping cough patients in 2001‒2010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin (10 mcg/ml), lidase (20 IU/ml), gentamycin (2.0 mg/ml, 0.4 mg/ml and 0.08 mg/ml) and their combinations in roundbottomed polystyrene 96­well plates was evaluated by staining with 0.1% gentian­violet solution.Results. Gentamycin partially suppressed the formation of biofilms and caused partial destruction of the formed biofilms in the absence of growth of microbial colonies when sowing supernatants from biofilm cultures on a dense nutrient medium. The minimum suppressive concentration of gentamycin (MSC) was 2 mg/ml. Trypsin completely suppressed the growth of biofilms and caused the complete destruction of the formed biofilms. Lidase also suppressed the growth of biofilms, but less effectively affected the formed biofilms. The growth of colonies typical of B. pertussis was noted when sowing supernatants from biofilm cultures in the presence of trypsin and lidasе on a dense nutrient medium. Trypsin in combination with all the studied concentrations of gentamycin completely suppressed the growth of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused complete destruction of biofilms in the absence of microbial growth on a dense nutrient medium. Lidase in combination with all the studied concentrations of gentamycin also suppressed the formation of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused partial destruction of the formed biofilms in the absence of microbial growth on a dense nutrient medium.Conclusion. The synergistic effect of the combination of trypsin and lidase with gentamycin on growing and formed biofilms of B. pertussis strains was revealed. The combined use of trypsin or lidase with gentamicin reduced its MSC for growing biofilms by 25 times. The most pronounced effect on the formed biofilms was the combination of trypsin with gentamycin at a concentration of 2 mg/ml, which caused their complete destruction and death of planktonic cells. The effect of the combination of lidase with gentamycin on the formed biofilms was less pronounced.

Keywords

• b. pertussis strains
• biofilms
• planktonic cells
• trypsin
• lidase
• gentamycin