A study on surface immunogens of biofilm-associated Staphylococcus aureus in mice
Abstract
Staphylococci are the most important of all the pathogens causing clinical and subclinical bovine mastitis worldwide. In past two decades, role of ‘biofilms’ in persistence of these organisms in the host has clearly been established, but how immune system sees the biofilm-forming organisms in the infected udder remains largely unanswered. The present study was therefore conducted with the objectives to determine the immunogens specifically associated with biofilm-forming S. aureus phenotype and to assess their role in protection against biofilm-forming pathogen in the mouse model of mastitis.
S. aureus isolate from a clinical bovine mastitis case was grown as planktonic and biofilm producing bacteria under different conditions. Biofilm production by S. aureus isolate was confirmed by appearance of black colonies on Congo red agar plate, Gram staining, biofilm quantitation and scanning electron microscopy. Extracellular polysaccharide (EPS) and cell membrane (CM)
-xxviiantigens
were extracted from the biofilm phenotype and CM antigens from the planktonic. Polypeptide profiles were determined by SDS-polyacrylamide gel electrophoresis. Several up- and down-regulated proteins in biofilm-forming S. aureus CM profile were visible. Different groups of mice (n=8/group) were inoculated with these antigens using Freund’s incomplete adjuvant (FIA) and CpG motifs-containing chromosomal DNA as adjuvant. Sequential sera samples at day 0, 14, 28, 42, 56 were collected and kinetics of humoral immune response was determined by indirect ELISA. Antigen profiles of CM were determined by immunoblotting using sera collected above. Mice immunized twice four-weeks apart were challenged with 106 CFUs of biofilm-forming S. aureus by intra-mammary route after >8 weeks in all groups for determining protection levels.
Antibody (Ab) levels in biofilm-associated CM-CpG group were higher at most days than those in CM-FIA group. IgM titre, but not IgG rose in EPS groups. Planktonic CM-CpG induced lower Ab levels than CM-FIA did. Biofilm-associated CM immunogens induced lower Ab levels than planktonic CM, irrespective of adjuvant used. However, these differences did not correlate with protection against challenge. In immunoblotting, seven major antigens specific to biofilm CM were revealed. Mice immunized with biofilm-associated CM with both adjuvants withstood challenge showing 100% protection, whereas planktonic CM immunized group with both adjuvants also withstood challenge showing nearly 100% protection. ELISPOT assay detected the presence of B lymphocyte clones secreting specific antibodies against various antigens used in the study. Based on these findings, use of biofilm-associated S. aureus surface immunogens as vaccine for control of mastitis was envisaged.