Gram Negative Bacterial Biofilm Formation and Characterisation of Extracellular Polymeric Substances

Gram negative bacteria such as Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Citrobacter freundii are often associated with multiple drug resistance and the generation of nosocomial infections. In the current study several clinical strains of theses bacteria (Ps 1, Ps 3, Ps 5, St 18, St 51, St 53 and C. freundii) and two culture collection strains Ps 10421 and St 9203 were evaluated for their ability to generate biofilms and the characteristics of the associated extracellular polysaccharides they produced. The ability of these strains to develop biofilms on a range of media and with a number of carbon sources was investigated. A range of mineral media employing glucose, ethanol and glycerol were developed in such a way as to ensure they did not contain compounds that interfere with extracellular polysaccharide analysis allowing a more in depth analysis of the extracellular polysaccharide generated by the bacteria under investigation. Following an assessment of the biofilm forming potential of all the strains under consideration, three were singled out for particular attention, i.e. Ps 3, St 53 and C. freundii strain isolated during this investigation. Two of strains were chosen for the strength of their biofilm forming potential (Ps 3 and St 53), on the other hand C. freundii was chosen because the scientific literature contains very little published information regarding its extracellular polysaccharide and its biofilm forming characteristics. These bacteria were able to produce biofilm on both hydrophobic (plastic) and hydrophilic (glass) surfaces. In order to get a broader understanding of the biofilm forming capabilities of these bacteria their whole genomes were sequenced and subsequently published. These genomes demonstrated that St 53 and C. freundii both contained the pgaABCD which is known to be associated with biofilm formation. Whilst Ps 3 contains a full complement of pel (PA3058-PA3064), psl (PA2231-2245) and alginate biosynthesis operons (PA3540-3548) related to biofilm formation. In addition all three species contained genes associated with virulence, pathogenicity and antibiotic resistance. The generation and extraction of extracellular polymeric substance generated by these three bacteria underwent a period of optimisations which included an optimisation of both the media and the growth conditions and the extraction process. In particular the use of trichloroacetic acid (TCA) was found to be critical with 0-5% TCA considered optimum for the removal of proteins prior to polysaccharide extraction. This is far less than has been previously employed in studies on lactic acid bacteria, however when used with the Gram negative bacteria investigated here, high levels of TCA degraded the polysaccharide that was being generated preventing its extraction in the quantities required for analysis. Analysis of the polysaccharides produced by St 53, Ps 3, and C. freundii, all demonstrated typical NMR spectra associated with bacterial extracellular polysaccharide. However, the NMR spectra from these polysaccharides also contained peaks typical of the presence of dextran. The use of a fungal dextranase confirmed the presence of a dextran like polymer in the polysaccharide generated by these bacteria. This indicated that all three of these bacteria generated complex polysaccharides with at least two components one mannose rich and the second a dextran like glucose rich polymer. This is the first report of a dextran being associated with the EPS of these bacteria and suggests that the Pel polysaccharide of P. aeruginosa is a dextran. Investigation of bacterial pathogenicity focussed on Ps 3 since P. aeruginosa is the most pathogenic of the three species investigated. The culture collection strain Ps 10421 failed to produce outer membrane vesicles (OMV) without antibiotic treatment, however Ps 3 generated OMV under normal growth conditions generating more when grown on ethanol rather than glucose. In order to investigate the impact of ethanol vs glucose grown culture a wax worm pathogenicity model was employed. This model revealed that ethanol grown cells were more pathogenic than glucose grown cells. This difference could be attributed to the effects of type of carbon sources that induce virulence genes to generate more toxins. Transcriptomic analysis of Ps 3 grown with ethanol vs growth on glucose revealed large differences in gene expression but no definitive evidence of which cellular processes were responsible for this enhanced pathogenicity associated with grown on ethanol.