Direct Evaluation of Pseudomonas aeruginosa Biofilm Mediators in a Chronic Infection Model

Abstract

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilms in vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcus pneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterial and host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilm and/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivobis-(3,5)-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing-in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes should be examined in multiple infection systems to fully understand their role in disease.The formation of biofilms facilitates chronic bacterial infections and reduces the efficacy of antimicrobial therapy (20, 39). The Gram-negative pathogen Pseudomonas aeruginosa is a model organism for biofilm studies and causes both acute and chronic infections by exploiting deficiencies in host immunity. P. aeruginosa is thought to exist as a biofilm during infections of the cystic fibrosis (CF) airway (5, 48), in acute burn wounds (45), and in chronic suppurative otitis media (13). Biofilm formation in P. aeruginosa is regulated by a complex network of signals that includes small molecules, two-component systems, small RNAs, and nutritional cues (27). As a result of these signals, a matrix that consists predominately of polysaccharides and extracellular DNA is formed (1,17,25,33,35,52). While P. aeruginosa biofilms have been studied extensively in vitro, there have been few studies characterizing biofilm formation in vivo (31,45,48,53).The chinchilla model has been widely used to study Haemophilus influenzae and Streptococcus pneumoniae otitis media infections, establishing that these pathogens form biofilms in vivo (3,15,19,24,26,42). Addressing biofilms in the context of an intact immune system and the complex environmental and structural features present in a mammalian host is necessary to determine if in vitro phenotypes are relevant to clinical infections. We used a chinchilla otitis media model to characterize P. aeruginosa biofilm formation and then focused on three systems involved in pathogenesis and/or biofilm formationbis-(3Ј,5Ј)-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing-in order to understand their contribution to P. aeruginosa infection.The intracellular second messenger c-di-GMP positively regulates aggregation and biofilm formation in P. aeruginosa (22,30,51) and mediates ...

Which Bacterial Biofilm Exopolysaccharide Is Preferred, Psl or Alginate?

“…P. aeruginosa with psl and pel genes deleted lost their ability to form biofilms altogether, indicating the importance of these exopolysaccharides in early stages of biofilm formation. These data show that alginate, Psl, Pel, and extracellular DNA interactively contribute to some aspect of P. aeruginosa biofilm architecture (16). The current state of knowledge indicates that Psl and Pel are likely involved with the initial stages of biofilm development (or acute stages of infection), whereas alginate is the "stress" response polysaccharide associated with chronic stages of infection.…”

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…Small-colony variants generated in vitro as well as obtained from clinical isolates contained mutations that upregulate the activity of the DGC TpbB (YfiN), suggesting a key role of this enzyme (193,245). The importance of c-di-GMP for enhanced persistence of P. aeruginosa has also been demonstrated in a chinchilla model of middle ear infection (355).…”

Nitric Oxide: A Key Mediator of Biofilm Dispersal with Applications in Infectious Diseases

“…For instance, 2-aminoimidazole derivatives targeting QS have been designed and found to disperse established biofilms [40,41]. Proof of concept studies showed that in vivo manipulation of c-di-GMP levels can effectively clear (by decreasing c-di-GMP) or prolong (by increasing c-di-GMP) Pseudomonas aeruginosa infections in murine models [42,43]. Modification of the BdcA protein to enhance its c-di-GMP binding, thus reducing the intracellular c-di-GMP concentration, caused nearly complete removal of biofilms via dispersal in vitro [44].…”