Life Science Journal 2013;10(6s) http://www.lifesciencesite.com 356 Growth in biofilm enhances potential to form new biofilm by Pseudomonas aeruginosa Ashfaque Hossain Department of Microbiology, College of Medicine, University of Hail, GPO Box-2440, Hail, Saudi Arabia. a.hussain@uoh.edu.sa Abstract: Biofilm production potential of clinical isolates of Pseudomonas aeruginosa were investigated in this study. Isolates from blood and wound produced biofilm but wound isolates produced relatively higher amounts. Sequential passage of the P. aeruginosa strains in biofilm culture in trypticase soy broth (TSB) resulted in gradually increasing amount of biofilm production the by isolates whereas passage of the same isolates in planktonic culture did not result in enhanced biofilm production. Passage induced enhanced biofilm production reached maximum level at passage 3 (P-3) for the strains B-6, W-2 and W-14 and at passage 4 (P-4) for the strain B-9. These values were 64.7% 83.4% and 75.6% and 72.8 % increased biofilm production by the biofilm passaged strains in comparison to their plantonic counterparts. Ferric ammonium citrate (FAC) which inhibits biofilm production by P. aeruginosa, was only partially effective in reducing biofilm production by bacteria passaged in biofilm. On the other hand, FAC efficiently reduced biofilm production by cultures which were grown in planktonic state. Taken together, the results of this study indicate that growth of P. aeruginosa in biofilm enhances its potential to form new biofilm. [Hossain, A. Growth in biofilm enhances potential to form new biofilm by Pseudomonas aeruginosa. Life Sci J 2013;10(6s):356-359] (ISSN:1097-8135). http://www.lifesciencesite.com . 54 Key Words: Biofilm, Pseudomonas aeruginosa, Ferric Ammonium Citrate, Clinical Isolate. 1. Introduction Biofilm is defined as a structured community of bacterial cells enclosed in a self-produced extracellular polymeric matrix (EPS) and adherent to an inert or living surface (Costerton et al., 2001). The EPS consists mainly of carbohydrate, protein and DNA (Branda et al., 2005; Mulchay et al., 2008). Biofilm impacts human lives in many different ways as they can form in medical, industrial and environmental settings (Daniel et al., 2011) Bacteria form biofilm when they transit from free floating (planktonic) state to a lifestyle in which they attach to a surface. The alternative lifestyle as a biofilm facilitates bacteria to survive in adverse environments. Bacterial cells residing in biofilm states are physiologically diverse are and less susceptible to antibacterial action of the antibiotics and clearance by immune system and hence, it is considered as a virulence attribute (Stickler, 1999; Stewart and Franklin, 2008). Pseudomonas aeruginosa (PA) is an opportunistic human pathogen that causes wide ranging infections in immunocompromised hosts and individuals with cystic fibrosis (Costerton et al., 2001). It also causes bacteremia and wound infections and often form biofilm during infection (Fergie et al., 1994; Harrison- Balistra et al., 2003). The high degree of genomic flexibility and remarkable capacity to phenotypic adaptation of P. aeruginosa contributes to its ability for growth and persistence under various environmental and clinical settings (Wehmhoner et al., 2003). P. aeruginosa is considered as the best model pathogen to investigate biofilm production in clinical settings. (McDougald et al., 2008). Various factors influence biofilm production by P. aeruginosa strains. These includes growth conditions, motility, cell surface properties, extracellular enzymes and various physicochemical stress factors, including various antibiotics (Mah et al., 2008; O’Toole et al., 2000 ). However, whether growth of bacteria in biofilm influences its potential to form new biofilm has not been investigated. This study was designed to investigate whether growth P. aeruginosa in biofilm has any influence on formation of new biofilm. Results obtained in this study show that in comparison to their isogenic planktonic counterparts, bacteria grown in biofilm has enhanced potential to form new biofilm. 2.Materials and Methods Bacterial strains and biofilm assay: P. aeruginosa strains were obtained from King Khaled General Hospital, Hail, Saudi Arabia. Trypticase soy broth (TSB) and trypticase soy agar (TSA) plates were used to culture of bacteria as needed. Biofilm assay: Biofilm formation by P. aeruginosa strains was examined by crystal violet staining procedure (Moskowitz et al., 2004). Overnight cultures of bacteria in TSB was diluted 1:100 in 3 ml of freshTSB contained in glass tubes and allowed to grow at 37 C in a static condition for 24 hours. Biofilms attached to the glass tubes were washed to remove inbound bacteria and stained with 1% (w/v) crystal violet for 10 min at room temperature. After washing with water, the stained biofilms were dissolved in 100% ethanol and the absorbance at 570 nm was determined