Aim. To study the sensitivity of biofilms of vaccine and freshly isolated strains of Bordetella pertussis to antibiotics.Materials and methods. Vaccine and freshly isolated strains of B. pertussis were used. Cultures of strains grown on dense nutrient medium were used as inoculate for biofilms production. The intensity of biofilm formation in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution. The following antibiotics were used in experiments: penicillins (ampicillin), cephalosporins (ceftriaxone), aminoglycosides (gentamicin), macrolides (erythromycin).Results. The highest resistance to antibiotics was demonstrated by the vaccine strain No. 305 and freshly isolated strain No. 211, sensitive only to erythromycin. Vaccine strain No. 703 was sensitive to gentamicin and ampicillin and showed resistance to erythromycin and ceftriaxone. Vaccine strain No. 475 was sensitive to all tested antibiotics. The Tohama 1 strain was resistant to ampicillin and sensitive to other antibiotics. Freshly isolated strains No. 178 and No. 162 were resistant to ceftriaxone and sensitive to gentamicin, erythromycin and penicillin. Minimal inhibitory concentrations of tested antibiotics ranged from 0.2 μg/ml to 5.0 μg/ml.Conclusion. These data indicate the heterogeneity of vaccine and freshly isolated strains of B. pertussis in sensitivity to antibiotics. The greatest activity was shown by erythromycin, which suppressed the growth of biofilms of 6 out of 7 strains. The least effective was ceftriaxone, which suppressed the growth of biofilms of only 2 strains.
In many countries of the world despite the extensively vaccination against pertussis has increased the incidence of the whooping cough in all age group of the population. The MLST, MLVA and CGH investigations revealed the differences in genotypes between the vaccine strains B.pertussis and the circulating isolates B.pertussis in consequence of adaptation of the bacterium B.pertussis to the immunized hosts. It is lead to waning immynity and outbreak of incidence of pertussis. The mutations in the genes encoding the major virulence factors, the allelic polimorfism and decreasing the genome size of B.pertussis strains are the basis of the B.pertussis adaptation to the immunized hosts and dependent on the type of the vaccine used for immunization Programme. In countries that use acellular pertussis vaccine for vaccination programme the dominant isolates genotypes are: ptxА1-ptxС2- ptxР3-prn2- tcfA2-1-fim3-2 и ptxА1- ptxС2- ptxР3-prnА2- tcfA2- fim2-1- fim3-1, and that use the cellular pertussis vaccine the dominant isolates genotypes are ptxА1-ptxС1- ptxР1-prn1- tcfA2- fim2-2 fim3-1 и ptxА1- ptxС1- ptxР1- prn2- tcfA2- fim2-1- fim3-1. The constant monitoring of the genotypes of isolates B.pertussis is necessary to reveal the dominant genotypes and include them in the national immunization programme in combination with vaccine strains B.pertussis.
Aim. To study the formation of biofilms by freshly isolated and vaccine strains of Bordetella pertussis of different serotypes.Materials and methods. The intensity of biofilm formation by B. pertussis strains in 96-well round-bottom polystyrene plates by using three sowing doses of microbial cells (1,25 IOU/ml, 2,5 IOU/ml and 5,0 IOU/ml) was estimated by staining with 0,1% gentian-violet solution. The results were interpreted after measuring the optical density (OP) of the colored solvent at a wavelength of 600 nm.Results. The highest intensity of biofilm formation was found in the newly isolated strain No. 211 and vaccine strain No. 475, both belonging to serotype 1.2.3. Cultures of these strains formed dense biofilms at all sowing doses of microbial cells. Certain differences in the intensity of biofilm formation were found between freshly isolated and vaccine strains of serotypes 1.2.0 and 1.0.3, especially when using a sowing dose of 5,0 IOU/ml. Freshly isolated strains at this dose formed dense biofilms, while two of the three vaccine strains formed moderate biofilms, and one strain was dense.Conclusion. The revealed differences between the strains of B. pertussis in the intensity of biofilm formation may be associated with the peculiarities of the expression of agglutinogens, as well as other surface structures of microbial cells involved in the adhesion process on the substrate.
Aim. Study the effect of trypsin, lidase (hyaluronidase) and fluimucil (N-acetyl-L-cysteine) on the growth of biofilms of Bordetella pertussis strains on the abiotic substrate. Materials and methods. In the experiments, the strains of the main B. pertussis serotypes isolated in the Russian Federation from whooping cough patients in 20012010 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, lidase and fluimucil in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution. Results. Trypsin suppressed the growth of biofilms and destroyed the formed biofilms. Lidase suppressed the growth of biofilms less actively, without affecting the formed biofilms. Fluimucil did not affect both the growth of biofilms and the formed biofilms. The growth of colonies typical for B. pertussis was noted when planting fluids from cultures in the presence of preparations, as well as from culture control wells on a dense nutrient medium. Conclusion. The different effect of the drugs studied by us may be related to the different quantitative content of targets for trypsin (proteins), lidase (mucopolysaccharides, containing uronic acids), fluimucil (acid mucopolysaccharides) in the biofilm matrix. The growth of the typical morphological properties of the colony of B. pertussis during the sowing of culture seedlings on a dense nutrient medium testifies to the destruction of the biofilm matrix by trypsin and lidase in the absence of influence on plankton cells.
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.
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