Effect of trypsin, lidase and fluimucil on the growth of <i>Bordetella pertussis</i> biofilms on an abiotic 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

Genetic variability of <i>Bordetella pertussis</i> and its role in vaccine prevention of pertussis

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

CULTIVATION OF BORDETELLA PERTUSSIS BIOFILMS ON ABIOTIC SUBSTRATE

Aim. Selection of optimal conditions for the cultivation of Bordetella pertussis biofilms and comparative assessment of the ability of different strains to form biofilms. Materials and methods. Used a vaccine strain of B. pertussis № 475 and selected from this strain strain № 475а, characterized by high virulence. Cultures of strains grown on dense nutrient medium (DNM) and liquid nutrient medium (LNM) were used as inoculates for biofilms production. The intensity of biofilms formation in round-bottom 96-well polystyrene plates was estimated by staining with 0,1% gentian-violet solution. Results. Daily cultures of the strain № 475 with LNM formed moderate biofilms in the range of doses 5 — 1,25 IOU (international optical units) /ml, in the absence of growth at lower doses. The daily cultures of this strain with the DNM formed a dense biofilms when planting a doses in the range from 10 to 1,25 IOU/ml, moderate from 0,625 to 0,157 IOU/ml and weak biofilms at a dose of 0,079 IOU/ml. Strain № 475а with the DNM formed dense biofilms in the doses range of 10 to 0,04 IOU/ml and only at the dose of 0,02 IOU/ml were formed moderate biofilms. Conclusion. A simple and informative method has been developed to evaluate the ability of B. pertussis strains to form biofilms in polystyrene plates. Cultures obtained with the DNM formed a more significant biofilms than cultures with LNM. Identified high ability to biofilms formation by a selected strain №. 475a, compared to the original strain № 475

The biofilm formation of freshly isolated and vaccine strains of Bordetella pertussis of different serotypes

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

PROFILES OF CYTOKINES IN MICE DURING IMMUNIZATION WITH ADTP-VACCINE WITH ACELLULAR PERTUSSIS COMPONENT

Aim. Study cytokine status in mice immunized with vaccines containing acellular pertussis component. Materials and methods. Vaccines developed in Mechnikov RIVS - acellular pertussis vaccine (aPV) and adsorbed pertussis-diphtheria-tetanus vaccine (aDTaP), containing a complex of protective antigens of pertussis microbe - were used in the study. Fi (CBAxCsyBle) line mice weighing 12 - 14 g were immunized intraperitoneally 3 times at an interval of 7 days with aPV and aDTaP at human immunization dose (0.5 ml), containing 25 pg of pertussis component. Intact mice were used as a control group. Levels of IFN-y, IL-2, IL-4, IL-5, IL-12 cytokines were determined after each immunization in enzyme immunoassay using commercial test-systems from Cusabio (China). Results. An increase of levels of IFN-y, IL-2, IL-5, IL-12 and lack of stimulation of production of IL-4 was established in dynamics of immune response after administration of aPV and aDTaP vaccines. Conclusion. The data obtained indicate that immunization of mice with aPV and aDTaP vaccines resulted in activation of production of cytokines characteristic for immune response during pertussis infection and immunization with whole-cellular aDTP-vaccines

Sensitivity of <i>Bordetella pertussis</i> biofilms to polyvalent pertussis serum Eugene M. Zaytsev

Aim. Study of the sensitivity of Bordetella pertussis biofilms to polyvalent pertussis serum.Materials and methods. The intensity of biofilm formation by strains of B. pertussis in round-bottomed polystyrene 96-well plates in the presence of polyvalent pertussis serum was estimated by staining with 0.1% gentian-violet solution. The serum titer was estimated as a highest dilution, which suppressed the growth of biofilm cultures.Results. Serum titers that completely suppressed the formation of biofilms by the studied strains ranged from  : 1,000 to 1 : 20,000. Vaccine strain No. 475a (serotype 1.2.3) was characterized by the highest sensitivity to serum, the titer of which was 1 : 20,000. Vaccine strain No. 305 (serotype 1.2.0) and strains isolated from whooping cough patients in the period from 2001 to 2010: No. 287 (serotype 1.0.3), No. 178 (serotype 1.2.0), No. 317 (serotype 1.2.3) were sensitive to serum in the titers 1 : 1,000-1 : 2,000. Vaccine strain No. 703 (serotype 1.0.3) was resistant to all serum dilutions studied. When sowing supernatants from wells with biofilms on a dense nutrient medium, the growth of colonies typical for B. pertussis was observed. Similar results were obtained when seeding the supernatants of the cultures from the wells with no biofilm.Conclusion. These data indicate that B. pertussis strains are heterogeneous in sensitivity to pertussis serum. The growth of colonies typical for B. pertussis when culture supernatants are sown on a dense nutrient medium, in the absence of biofilms in the wells, indicates that the biofilm formation is suppressed by inhibiting the adhesion of microbial cells on the substrate, and not due to the bactericidal action of the serum