Phenotypic profiling of several strains of Pseudomonas aeruginosa : identification of potential virulence determinants

Numerous studies have described that bacteria display higher resistance to antimicrobials when growing in biofilm mode of growth, however the phenotypic and virulence factors changes associated with this increased resistance are still poorly understood. The goal of this study was to enlarge the knowledge about the augmented biofilmrelated resistance of Pseudomonas aeruginosa characterizing phenotypically P. aeruginosa biofilms formed by different strains in order to find whether these bacterial biofilms display a common profile. This bacterium is a common opportunistic pathogen that is often related with persistent infections that cause high rates of patient morbidity and mortality due to its intrinsic resistance to several antimicrobials and great adaptive resistance to environmental stimuli. The experimental analysis consisted of a phenotypical examination comprising biofilm formation ability, susceptibility to antibiotics (ciprofloxacin and colistin), expression of several virulence factors (motility, toxin production, and slime production), and phenotypic switching. To best replicate real-world circumstances this work comprised the study of several P. aeruginosa strains: four reference strains (PAO1, ATCC 10145, CECT 111, and PA14) and three clinical isolates (PAI1, PAI2, and PAI3). Biofilms are often related with augmented resistance to antimicrobial agents and are usually associated with chronic infections, which are difficult to eradicate. Therefore, in this study, antibiotic susceptibility was tested and related with P. aeruginosa strains biofilm-forming ability. Results showed that an improved biofilm-forming ability does not seem to be related with biofilm resistance since the seven P. aeruginosa strains showed identical resistance and susceptibility profiles against ciprofloxacin and colistin, respectively, with the exception of PAI3 which exhibited higher resistance to ciprofloxacin. P. aeruginosa motility has been pointed out as a relevant factor for adhesion and biofilm development and also increased virulence. Thus, the swimming, swarming and twitching abilities of P. aeruginosa were evaluated. P. aeruginosa strains presented specific ability to move along surfaces. However, it was not found a clear relationship between higher motility and biofilm-forming ability and increased resistance. Similarly, slime production also contributes to biofilm development and is often implied in increased biofilm-resistance. Considering the results obtained, it was not found a viii Phenotypic profiling of several strains of Pseudomonas aeruginosa: identification of potential virulence factors evident relationship between slime production and both biofilm-forming ability and increased resistance. The ability to produce and release extracellular toxins by P. aeruginosa is frequently related with higher levels of bacterial pathogenicity which can lead to severe consequences and even death. Among the several extracellular toxins, hemolysin and pyocyanin production were assessed in this study. Each P. aeruginosa strain displayed a different expression of these virulence factors. It was not possible to conclude if any P. aeruginosa strain was more virulent than other based only on these results. Phenotypic switching is an usual adaptive mechanism used by P. aeruginosa to increase their diversity and overcome exposure to stress conditions and environmental changes. Because colony morphology variation is the most visible characteristic of phenotypic switching, both planktonic and biofilm colonies were observed in order to establish where there is a relationship between bacterial mode of growth and colony morphology and also biofilm resistance. It was observed that phenotypic switching occurred when the P. aeruginosa strains changed their way of growth to biofilm-form. The rising of small-colony morphotypes was recorded for some P. aeruginosa strains (PAO1, ATCC 10145, PA14, PAI1, and PAI3) when growing in biofilm-form, a morphotype usually associated with biofilm resistance. However it was not found a clear relationship with the rising of these small-colony variants and biofilm resistance and further studies are needed to better understand and establish the relationship of biofilm resistance and these morphotype variants. This work contributed to a further understanding of P. aeruginosa biofilm characteristics, including resistance features, virulence factors expression and phenotypic variation. It was concluded that there is not a general P. aeruginosa biofilm profile. Although resistance and susceptibility profiles against ciprofloxacin and colistin, respectively, of all strains were very similar, all virulence factors evaluated showed to be variable characteristic among all strains tested. In addiction it was not verified direct correlations between all virulence factors studied. Because there was recorded a variability in the virulence factors studied among all strains, no strain was considered more virulent than other given strain. Furthermore, results also show that there is an urgent necessity to reexamine the current clinical guidelines. These guidelines should regard phenotypic switching, intrinsic