Biofilm

Biofilm: Role of alg and amp genes

A natural mode of bacterial growth is the formation of organized biofilm communities on surfaces, held together by a matrix composed of EPS (3). The formation of an EPS matrix may play an important role in establishing a sustainable biofilm. In recent years, it has become apparent that predicting actual bacterial behavior based on experiments done in liquid suspension in test tubes (planktonic form) may not be reliable. As a result many laboratories have begun to investigate how cells can coordinate their activities and build the complex structures that are found in mature biofilms. Biofilm cells typically have very slow growth rates relative to those that are grown in planktonic mode. The difference in physiology of the bacteria in these two modes of growth contributes to the difference in their response to various environmental stressors (2, 3).

P. aeruginosa has emerged as a model organism to study the role of exopolysaccharide, in particular alginate, in biofilm formation. This is due to the fact that the complex genetics of alginate production in planktonic form has been worked out in great details. Mathee et al established that repeated exposure of a P. aeruginosa biofilm in vitro to activated polymorphonuclear leukocytes (PMNs), or to low-levels of hydrogen peroxide, can give rise to mucoid variants that overproduce exopolysaccharide, with defects in mucA gene, mimicking that seen in vivo (8). Previous analysis of alginate gene expression in biofilm has been done by mainly looking at expression of alginate genes, algC and algD, upon early attachment of mucoid strains to inert surfaces. The product of the algC gene besides its role in alginate synthesis is involved in LPS synthesis and rhamnolipid production (11). Thus, the detection of the activation of algC gene on surface could reflect activation of any of the three pathways. The algD gene promoter drives the 18-kb alginate biosynthetic operon, a tightly regulated operon in nonmucoid cells (completely off), and expressed only in alginate-producing strains. Thus, activation of this promoter should indicate expression of alginate genes. Hoyle et al., using a mucoid strain, showed that algD expression was enhanced in surface-attached cell vs free-floating planktonic form with transient production of matrix exopolysaccharide, following adherence (6). The studies used a mucoid strain for analysis that should have the promoters constitutively expressed. These studies did not address the possibility that the transient activation/expression may reflect switching off of the unstable alginate phenotype. The present study aims at systematically elucidating if alginate production plays any role in biofilm formation by comparing a prototrophic nonmucoid P. aeruginosa PAO1 with its isogenic mucoid variant PDO300 and an isogenic algD deletion derivative (WFPA14) that is incapable of producing alginate.

The biofilm mode of growth appears to contribute the increased resistance to antibiotics (4, 7, 9, 10). The production of alginate, to generate a firm biofilm further protects the cells from the destructive antipseudomonal molecules, such as carbenicillin and titarcillin (12). It has been demonstrated that tobramycin resistance of P. aeruginosa is increased 20 to 100-fold for biofilms relative to equivalent planktonic counterparts (9). In addition, Giwercman et al (5) demonstrated that pipercillin and imipenem were able to induce beta-lactamase production in biofilm and remain associated longer in biofilm than planktonic cells. Coquet et al show that there is significant enhancement of beta-lactamase induction (1). Interestingly, no studies to date have looked at the effect the antibiotics on the expression of genes involved in beta-lactamase production, namely the amp genes. Non-destructive on-line biofilm studies will be used to address the contribution of these genes in antibiotic resistance.

Mathee Publications:

a. Mathee, K., O. Ciofu, C. Sternberg, P. W. Lindum, J. I. Campbell, P. Jensen, A. H. Johnsen, M. Givskov, D. E. Ohman, S. Molin, N. Hoiby, and A. Kharazmi. 1999. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145:1349-1357.

b. Mathee, K., A. Kharazmi, and N. Høiby. 2002. Role of Exopolysaccharide in Biofilm Matrix Formation: The Alginate Paradigm. In R. J. C. McLean and A. W. Decho (ed.), Molecular Ecology of Biofilms, 1st ed. Horizon Press, UK.

c. A.S. Plata, G. Narasimhan, D. E. Ohman, M. Hentzer, S. Molin, A. Kharazmi, N. Høiby, K. Mathee. Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation.

References Cited:

1.
Coquet, L., G. A. Junter, and T. Jouenne. 1998. Resistance of artificial biofilms of Pseudomonas aeruginosa to imipenem and tobramycin. J Antimicrob Chemother 42:755-760.

2.
Costerton, J., P. Stewart, and E. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science 284:1318-1322.

3.
Costerton, J. W., K. J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial biofilms in nature and disease. Annu Rev Microbiol 41:435-464.

4.
Duguid, I. G., E. Evans, M. R. Brown, and P. Gilbert. 1992. Effect of biofilm culture upon the susceptibility of Staphylococcus epidermidis to tobramycin. J Antimicrob Chemother 30:803-810.

5.
Giwercman, B., E. T. Jensen, N. Høiby, A. Kharazmi, and J. W. Costerton. 1991. Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm. Antimicrob Agents Chemother 35:1008-1010.

6.
Hoyle, B. D., L. J. Williams, and J. W. Costerton. 1993. Production of mucoid exopolysaccharide during development of Pseudomonas aeruginosa biofilms. Infect. Immun. 61:777-780.

7.
Kunin, C. M., and C. Steele. 1985. Culture of the surfaces of urinary catheters to sample urethral flora and study the effect of antimicrobial therapy. J Clin Microbiol 21:902-908.

8.
Mathee, K., O. Ciofu, C. Sternberg, P. W. Lindum, J. I. Campbell, P. Jensen, A. H. Johnsen, M. Givskov, D. E. Ohman, S. Molin, N. Hoiby, and A. Kharazmi. 1999. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145:1349-1357.

9.
Nickel, J. C., I. Ruseska, J. B. Wright, and J. W. Costerton. 1985. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother 27:619-624.

10.
Nickel, J. C., J. B. Wright, I. Ruseska, T. J. Marrie, C. Whitfield, and J. W. Costerton. 1985. Antibiotic resistance of Pseudomonas aeruginosa colonizing a urinary catheter in vitro. Eur J Clin Microbiol 4:213-218.

11.
Olvera, C., J. B. Goldberg, R. Sanchez, and G. Soberon-Chavez. 1999. The Pseudomonas aeruginosa algC gene product participates in rhamnolipid biosynthesis. FEMS Microbiol. Lett. 179:85-90.

12.
Rolinson, G. N. 1998. Forty years of beta-lactam research. J Antimicrob Chemother 41:589-603.

Students and Postdoctoral fellows involved:

Post-doc 1. Dr. DeEtta Mills

Graduate Students 2. Dr. Shalaka Indulkar
3. Dr. Suriya Jayawardena
4. Mr. Kok-Fai Kong