@phdthesis{Rachid2000,
  author    = {Rachid, Shwan},
  title     = {Molecular investigation of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1882},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2000},
  abstract  = {Biofilm production is an important step in the pathogenesis of S. epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin (PIA). The PIA represents a sugar polymer consisting of ß-1,6 linked N-acetyl glucosaminoglycans and mediates the intercellular adherence of the bacteria to each other and the accumulation of a multilayered biofilm. Epidemiological and experimental studies strongly suggest that PIA-production and subsequently biofilm formation contributes significantly to the virulence of specific S. epidermidis strains. This work aimed on the investigation of external factors regulating the ica expression in S. epidermidis. For this purpose, a reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from E. coli was constructed and integrated into the chromosome of an ica positive S. epidermidis clinical isolate. The reporter gene fusion was used to investigate the influence of external factors and of sub-MICs of different antibiotics on the ica expression. It was shown that the S. epidermidis biofilm formation is growth phase dependent with a maximum expression in the late logarithmic and early stationary growth phase. The optimal expression was recorded at 42 °C at a neutral pH ranging from 7.0 to 7.5. The glucose content of the medium was found to be essential for biofilm formation, since concentrations of 1.5 to 2 per cent glucose induced the ica expression. In addition, external stress factors as high osmolarity (mediated by 3 to 5 per cent sodium chloride), and sub-lethal concentrations of detergents, ethanol, hydrogene peroxide, and urea significantly enhanced the biofilm production. Subinhibitory concentrations of tetracyline, the semisynthetic streptogramin quinupristin/dalfopristin and the streptogramin growth promoter virginiamycin were found to enhance the ica expression 8 to 11-fold, respectively, whereas penicillin, oxacillin, gentamicin, clindamycin, vancomycin, teicoplanin, ofloxacin, and chloramphenicol had no effects. A weak induction was recorded for sub-MICs of erythromycin. Both quinupristin/ dalfopristin and tetracyline exhibited a strong postexposure effect on the S. epidermidis ica expression, respectively, even when the substances were immediately removed from the growth medium. The results were confirmed by Northern blot analysis of the ica transcription and quantitative analysis of biofilm formation in a colorimetric assay. Expression of the icaprom::lacZ reporter gene plasmid in Bacillus subtilis and S. epidermidis revealed that the ica induction by sub-MICs of streptogramins and tetracycline might depend on unidentified regulatory elements which are specific for the staphylococcal cell. In contrast, the activation by external stress signals seems to be mediated by factors which are present both in Staphylococci and in Bacillus subtilis. Construction and analysis of an agr-mutant in a biofilm-forming S. epidermidis strain excluded the possibility that the Agr-quorum-sensing system significantly contributes to the ica expression in the stationary growth phase. However, clear evidence was provided that in S. aureus the ica transcription depends on the expression of the alternative transcription factor sigmaB, which represents a global regulator of the stress response in S. aureus as well as in B. subtilis. For this purpose, a sigB knockout mutant had been constructed in a biofilm-forming S. aureus. This mutant showed a markedly decrease of the ica transcription and biofilm-production, whereas a complement strain carrying the sigB gene on an expression vector completely restored the biofilm-forming phenotype of the S. aureus wild type. Southern blot analysis indicated that the the sigB gene is also present in S. epidermidis and Northern analyses of the sigB and the ica transcription revealed that both genes are activated under identical conditions (i. e. in the stationary growth phase and by external stress factors) suggesting a similar regulatory pathway as in S. aureus. However, since neither in S. aureus nor in S. epidermidis the ica promoter has obvious similiarities to known SigB-dependent promotoer sequences it is tempting to speculate that the ica activation is not directely mediated by SigB, but might be indirectely controlled by other SigB-dependent regulatory elements which remain to be elucidated.},
  subject      = {Staphylococcus epidermidis},
  language  = {en}
}