Mar 9, 2004

Green and red fluorescent protein vectors for use in biofilm studies of the intrinsically resistant Burkholderia cepacia complex

Journal of Microbiological Methods
Kerry L TomlinH Ceri

Abstract

Cystic fibrosis isolates of the Burkholderia cepacia complex (BCC) have demonstrated a propensity to associate intimately with Pseudomonas aeruginosa in mixed community biofilms, which may impact on their overall pathogenicity during infection of the lungs in cystic fibrosis. Here, we describe the construction and use of novel green and red fluorescent protein expression vectors suitable for labeling biofilm cells of multi-resistant clinical isolates of the BCC for microscopic analysis of both single species biofilms and mixed community associations with P. aeruginosa. Antimicrobial susceptibility testing established that tetracycline and/or trimethoprim were suitable selective agents for widespread use in BCC. The green and red fluorescent protein genes, driven by constitutively active promoters, were cloned into two mobilizable plasmids pBBR1MCS-3 and pBBR1Tp, carrying tetracycline and trimethoprim resistance cassettes, respectively. The fluorescence of transformed BCC and P. aeruginosa planktonic cells was detectable using fluorescence microscopy and/or fluorometry. The plasmids were stable in the absence of selection for at least 3 days in planktonic and biofilm cultures, and fluorescence was still visible in a 4-day glass ...Continue Reading

  • References25
  • Citations31

References

Mentioned in this Paper

Tetracycline Antibiotics
Pseudomonas Infections
Tetracyclines
Luria
Shuttle Vectors
Fluorometry
Pseudomonas aeruginosa (antigen)
Cross-talk
Lung
Gentamicins

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