Approaches for the control of the attachment of Pseudomonas aeruginosa and Burkholderia cepacia to abiotic surfaces

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The attachment of Pseudomonas aeruginosa and Burkholderia cepacia to surfaces is an important pre-cursor to potential negative outcomes, such as. biofouling and diseases, associated with these bacteria. The attachment of these bacteria to surfaces is linked to factors such as surface structures such as ligands and flagella, hydrophobicity and polysaccharides. Studies on different bacterial species have shown that these factors and attachment to surfaces are controllable by methods such as shear force, temperature manipulation and bioactivity. This study used four separate approaches in an attempt to control the attachment of P. aeruginosa and B. cepacia to abiotic surfaces. The first of these aimed to determine effects of shear force on 6 strains each of Pseudomonas aeruginosa and Burkholderia cepacia with respect to hydrophobicity and attachment to 4 different abiotic surfaces (glass, stainless steel, polystyrene and Teflon®). The second of these examined the effects of crude aqueous asam gelugor (Garcinia atroviridis) extract on the hydrophobicity and attachment of the both the bacteria species to the same abiotic surfaces. In the third of these an attempt was made to disrupt the fliC gene encoding for flagella in P. aeruginosa using the Targetron® system to study its role in attachment to abiotic surfaces. A fourth investigation examined the effect of temperature on the attachment of 2 strains each of P. aeruginosa and B. cepacia to glass and stainless steel. Results of the first part of the study showed that shear force was able to modulate the hydrophobicity and attachment of both P. aeruginosa and B .cepacia. A significant (p < 0.05) reduction in hydrophobicity of all strains of P. aeruginosa tested (17-36%) and B. cepacia MS 5 (20%) were apparent after shearing. A significant (p < 0.05) decrease in attachment of some P. aeruginosa (0.2-0.5 log cfu/cm2) and B. cepacia (0.2-0.4 log cfu/cm2) strains to some surface types were also apparent after shearing. Data from SDS-PAGE analysis indicated that shear force removed proteinaceous surface structures that are important for attachment. Results of the second part of the study showed that an aqueous G. atroviridis extract was able to modulate the hydrophobicity and attachment of both P. aeruginosa, to a greater extent, and B.cepacia, to a lesser extent. Upon application of the extract, all 6 strains of P. aeruginosa showed a significant (p < 0.05) decrease in hydrophobicity (~ 27.74 - 52.95 %), while 3 out 6 B. cepacia strains showed significant (p < 0.05) increase in hydrophobicity (~23.96-32.17 %). A significant (p < 0.05) decrease in attachment of some P. aeruginosa (0.3-1.0 log cfu/cm2) and B. cepacia (0.2-0.9 log cfu/cm2) strains to some surface types were also apparent after application of the extract. Notably, attachment of 3 B. cepacia strains to polystyrene increased (~0.8 log cfu/cm2) significantly (p < 0.05) after application of extract. Chemical analysis indicated that the extract may have damaged the surface proteins of the cells and in doing so changed their ability to attach to abiotic surfaces. In both the shear force and G. atroviridis extract parts of this study, hydrophobicity was found to be positively correlated to attachment with R values ranging from 0.5-0.9. The study of gene disruption using the Targetron® system produced a modified recombinant PbL1 plasmid that flanks the fliC gene in P. aeruginosa. However, after transformation of P. aeruginosa, low colony counts (~30cfu/ml) were apparent and colony PCR showed either no bands or bands that were >98% identical to positive control. The inability to obtain mutants may be due gene essentiality and plasmid temperature sensitivity. The fourth part of the study showed that attachment of P. aeruginosa to glass (~ 6.5 logcfu/cm2 ) and stainless steel (~6.9logcfu/cm2) and their total polysaccharides (6.57 GEµg/ml) were significantly (p 0.5) between temperature, attachment and polysaccharide production indicated that P. aeruginosa may produces more polysaccharides at lower temperatures and that increases attachment to surfaces. No differences in attachment were observed for B. cepacia across all temperatures but 1 strain produced significantly (p < 0.05) more (6.57 GEµgml-1) polysaccharide at 4°C. This study demonstrated that shear force, crude aqueous G. atroviridis extract and temperature were able to modulate attachment of some strains of P. aeruginosa and B. cepacia to some abiotic surfaces. As these methods are still somewhat rudimentary they may not be ready to be applied practically at a larger scale. Since they are potentially safer as compared to conventional methods, however, further research into other parameters and optimization may advance these methods to practically control bacterial attachment.