Impact of nutritional conditions on colony morphology variants isolated from P. aeruginosa and S. aureus biofilms

In natural habitats, microorganisms are challenged all the time due to stress conditions imposed by the surrounding environment. To adapt to these environmental changes, bacteria alter their physiological and genetic traits. This adaptive behavior may be achieved by phenotype switching. This process consists in a reversible switch of phenotypes, as a mechanism ON/OFF, which occurs at high frequencies than spontaneous mutations. Colony morphology variation is the macroscopic feature of the phenotypic switching. Colony variation may have serious impact on bacterial virulence and antimicrobial resistance potentiating its ability to cause disease. Some colony variants are strongly associated to antibiotic resistance due to their presence in chronic infections despite antibiotic therapy. In cystic fibrosis, the switch of P. aeruginosa from non-mucoid to mucoid morphotype, which overproduce alginate, is a crucial stage to the establishment of this recalcitrant disease. Small colony variants (SCV) are other well-known resistant morphotype. These variants exhibited small size because its slow growth rate, pigmentation, haemolysis, reduced range of carbohydrate utilization and higher resistance to aminoglycosides antibiotics and cell-wall inhibitors. It has been growing the number of studies related with phenotypic switching and colony morphology characterization. However, normally each study reports the use of different solid growth media which makes the comparison between studies inaccurate. In order to clarify the role of nutritional conditions on bacterial colony morphologies and on its populational diversity, P. aeruginosa and S. aureus planktonic and biofilm-growing cells were spread onto the most common solid laboratory media (TSA, MHA, LB agar, MacConkey agar and Columbia horse blood agar). Additionally, the reproducibility of each medium was also inspected. Data showed that P. aeruginosa and S. aureus colony morphotypes are strongly influenced by the plating medium used. The main differences observed were the size, texture and form of colonies. The largest colonies were detected in TSA, MHA and LB agar. Colonies grown on MHA and LB agar were very similar possibly due to their identical nutritional composition. All the solid media tested showed reproducibility between assays except the Columbia horse blood agar which exhibited some inconsistency probably due to the presence of blood in its composition. Amongst the solid media tested, for planktonic and biofilm cultures, TSA gave rise to higher number of colony variants. Phenotype diversity seems to be more influenced by nutritional factors when bacteria derived from biofilms. This study allows concluding that, in contrast to fungi, bacterial colony appearance is influenced by the nutritional conditions of the solid media used to spread the cells. This evidence should be taking into account when important phenomena as phenotypic switching are going to be studied. The data obtained with this preliminary work may question the classification of colony morphotypes used until now.Fundação para a Ciência e Tecnologia (FCT) - Projecto PTDC/SAUESA/6460912006/FCOMP-01-0124-FEDER-007480, bolsa de Doutoramento SFRH/BD/31065/2006European CommunityFEDER - Program COMPET