1 Center for Biofilm Engineering, 366 EPS Building PO Box 173980, Montana State University-Bozeman, MT 59717, USA
2 Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, USA
Correspondence
B. Purevdorj-Gage
laura_p{at}erc.montana.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In previous experiments (Purevdorj-Gage & Stoodley, 2004) we reported hollow areas inside clusters of non-mucoid P. aeruginosa PAO1 grown in flow cells on LuriaBertani (LB) medium. We hypothesized that these hollow mounds were linked to an active dispersal process. It was the goal of this work to quantify the progression and dimensions of biofilm colonies as well as to document the dispersion of cells from the microcolonies in the flowing system. Biofilms of non-mucoid P. aeruginosa PAO1 wild-type, isogenic rhamnolipid-deficient strain PAO1-
rhlA, PAO1-
lasI
rhlI null mutant JP2 strain and cystic fibrosis (CF) clinical isolate P. aeruginosa FRD1 were grown in a once- through flow system. A digital time lapse imaging microscope and a scanning laser confocal microscope were used for visualization and quantification. The effects of rhamnolipid and quorum sensing (QS) deficiency on seeding dispersal were of particular interest, since these factors are actively involved in P. aeruginosa biofilm structural development (Davies et al., 1998
) as well as in maintaining the void channels within the biofilms (Davey et al., 2003
).
Since cells in the periphery of microcolonies became highly agitated as a prelude to seeding dispersal, we also investigated various aspects of motility by performing swimming, swarming and twitching assays on LB agar using each of the four strains. To determine the potential of biofilm viscosity to affect motility of single cells within the biofilm matrix, we measured the viscoelasticity of PAO1 and FRD1 microcolonies using rheometry.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Reactor sterilization.
The reactor system was autoclaved at 121 °C for 15 min. The sterility of the reactor system was confirmed by plating 0·1 ml aliquots of effluent onto LB agar (LA). Pure culture and conversion of mucoidy (for P. aeruginosa FRD1) flow cell experiments were confirmed through visual examination of colonies grown by plating 0·1 ml aliquots of effluent onto solid LA on a daily basis. Effluent from the PAO1-rhlA and PAO1-
lasI
rhlI biofilms was plated onto LA plates and LA plates with appropriate selective antibiotics to confirm integrity of the mutations. There was no statistical difference between the c.f.u. counted between LA and LA plates with selective markers (all P values>0·05).
Inoculum and medium.
The reactor containing full-strength LB was inoculated with 1 ml of an overnight full-strength LB 37 °C shake flask culture of micro-organisms. The flow system was allowed to sit for 30 min, to permit attachment, before switching to continuous culture. The system was then switched to continuous culture mode by delivering LB to the mixing chamber via a peristaltic pump (Cole Parmer #7553-80). Effluent samples were taken periodically to monitor the detached population and to confirm culture purity.
Microscopy.
The developing biofilm was visualized in situ by using transmitted light and x5, x10 and x50 objective lenses with an Olympus BH2 microscope. Images were collected using a COHU 4612-5000 CCD camera and captured with a Scion VG-5 PCI framestone board. Scion Image software was used to collect time-lapse sequences and for image enhancement and analysis. A 1 mm graticule with 10 µm divisions was used to calibrate length measurements. The biofilm thickness and surface area coverage were measured on each day at five random locations in the biofilm area for each flow cell experiment (Stoodley et al., 1999). The diameters of individual microcolonies were also measured using Scion Image. We estimated that for the particular settings of the light microscope, we could confidently resolve individual cell clusters down to diameters of approximately 10 µm.
Rheometry experiments.
To investigate the role of biofilm viscosity on seeding dispersal, PAO1 and FRD1 biofilms were grown on LA plates for 48 h at 36 °C. The colonies were aseptically scraped off the agar surface and used for rheometry measurements according to Towler et al. (2003). Two individual agar plates were used for each individual measurement. A total of eight replicate measurements from PAO1 and seven from FRD1 were performed for statistical comparisons. The viscosity was measured from creep tests using a TA Instruments AR 1000 Rheometer (info{at}tainst.com) in which the resultant strain in response to an applied shear stress of 0·5 or 1 Pa was measured over time.
Motility assay.
Sterile LA plates with varying concentrations of agarose [0·3 % for swimming (Kohler et al., 2000), 1·3 % for swarming (Aendekerk et al., 2002
), 1·0 % for twitching assay (Semmler et al., 1999
), 1·5, 3 and 5 % for rheometry experiments] were prepared. Equal concentrations of bacterial cultures (OD600) were stab-inoculated into the agar plates with a sterile toothpick. The plates were inverted and incubated at 36 °C for 16 h. The diameter of the zone of spreading from the inoculation point was then measured. Twelve replicate measurements were performed for each strain tested.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
Involvement of the QS system
In comparison to a wild-type biofilm, PAO1-lasI
rhlI cells covered only about 40 % of the surface during the first 2 days (P<0·05). However, by day 4 the surface coverage was statistically similar to that measured in the PAO1 biofilm (P>0·05). Throughout the experimental run time there were no differences between wild-type and PAO1-
lasI
rhlI biofilm thicknesses (P>0·05). On day 3 a total of 97 clusters with a median diameter of 250±150 µm were observed, of which none developed hollow structures during the full 5 days of growth (Fig. 2d
). By the end of the experiment the biofilm covered the entire surface and thickness measurements were comparable to the wild-type biofilm (Fig. 1
).
Motility assay in P. aeruginosa FRD1, PAO1 wild-type and its isogenic mutants
In addition to twitching (Semmler et al., 1999) and swimming (Kohler et al., 2000
), swarming in P. aeruginosa has recently been discovered as a novel mode of coordinated propagation on a semisolid medium (Kohler et al., 2000
) and Aendekerk et al. (2002)
have utilized LB medium solidified with 1·3 % agarose to test the swarming ability of various isogenic mutants in comparison to the PAO1 wild-type strain. Since coordinately moving cells were observed during the process of seeding dispersal, we investigated the ability of P. aeruginosa PAO1 isogenic
rhlA and
lasI
rhlI mutants, as well as mucoid strain FRD1, for twitching, swimming and swarming motility on LB agar, which to our knowledge has not been reported previously (medium composition is known to greatly affect the motility of P. aeruginosa; Rashid & Kornberg, 2000
). All strains except FRD1 were able to form twitching, swimming and swarming colonies similar to previous descriptions (Semmler et al., 1999
; Aendekerk et al., 2002
) (data not shown).
Material properties of PAO1 and FRD1 biofilms
On 0·3 % agar (viscosity 3·1x105 Pa s1) the swimming motility zone measured in PAO1-fliM (Klausen et al., 2003
) and FRD1 strains was significantly less than that of PAO1 and PAO1-
rhlA (all P values>0·05; data not shown) and none of the strains was able to spread over the plate surface with an agar concentration of
1·5 % (viscosity 1·4x106 Pa s1). Since agar viscosity has an important role in motility of our test strains, we wished to determine if the increased viscosity associated with FRD1 may partly explain its deficiency in swimming motility. We performed creep curve tests to determine the difference in biofilm viscosity between mucoid and non-mucoid P. aeruginosa by culturing FRD1 and PAO1 biofilms on the surface of agar plates. Contrary to our expectations, we found that the FRD1 colonies were significantly less viscous (4·8x105±0·02x105 Pa s1, n=7) compared to the PAO1 colonies (5·7x105±0·2x105 Pa s1, n=8) (P=0·014).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Role of rhamnolipid surfactant production in seeding dispersal
PAO1-rhlA biofilms also formed hollow structural mounds similar to those in the PAO1 biofilm and were able to detach via the seeding dispersal process (Fig. 2b
). The structural dimensions of these hollowed clusters, including the wall thickness, were statistically comparable to those measured in PAO1 biofilms (P>0·7; data not shown). Although both PAO1 and PAO1-
rhlA biofilms covered the entire surface of the glass flow cells, the PAO1-
rhlA strain resulted in a significantly thicker biofilm by the end of the experimental run, consistent with the findings of Davey et al. (2003)
. While the hollow differentiated mounds persisted in PAO1, the hollow colonies of the PAO1-
rhlA biofilm differentiated earlier in biofilm growth and by day 4 formed a flat homogeneous biomass. This observation can be explained by the findings of Davey et al. (2003)
where rhamnolipid was important in maintaining the non-colonized channels surrounding biofilm macrocolonies. The transient nature of differentiation and dispersal in PAO1-
rhlA illustrates the importance of frequent monitoring.
Role of global cell signalling in seeding dispersal
In comparison to PAO1 biofilms, the efficiency with which PAO1-lasI
rhlI was able to colonize the surface area was retarded by up to 2 days. However, unlike Davies et al. (1998)
, the PAO1-
lasI
rhlI biofilms were able to differentiate into mature microcolonies in our flow cell system as early as day 2 of the experimental run. This observation was consistent with our previous findings where we demonstrated that homoserine lactone was not required for biofilm formation in which we also utilized LB as a growth substrate (Purevdorj et al., 2002
). A total of 97 individual biofilm microcolonies with diameters statistically comparable to PAO1 (P>0·05; except on day 4 where the PAO1-
lasI
rhlI clusters were larger than those in PAO1, P<0·05) were observed in our flow system, none of which developed hollow mounds. The inability of the PAO1-
lasI
rhlI biofilm to seed cannot be attributed to the lack of motility since the three types of motility were not abolished in PAO1-
lasI
rhlI on LB agar (data not shown). This suggests involvement of QS in the differentiation process, possibly by sensing cell density and nutrient depletion within the periphery of the biofilm clusters.
Clinical significance of seeding dispersal
It is well known that chronically infected CF patients frequently harbour mucoid variants of P. aeruginosa (Høiby et al., 2001). We wished to investigate the clinical relevance of this dispersal phenomenon by analysing biofilms grown from the CF isolate, mucoid P. aeruginosa FRD1. Interestingly in all three replicate flow cell experiments, FRD1 biofilms did not form hollow structures, despite the fact that the mean cluster size, as well as general biomass accumulation, did not significantly differ between the two biofilms (all values P>0·05). Initially we speculated that the inability of FRD1 to undergo differentiation and dispersal could be due to its high viscosity. In general FRD1 biofilms are commonly perceived to be a more viscous counterpart of the wild-type strain due to its voluminous slimy nature. However, the rheometry results showed that FRD1 biofilms were less viscous than those of the environmental non-mucoid strain PAO1 (P=0·014), which ruled out this hypothesis. Whether the inability of FRD1 to undergo differentiation and seed is due to abolished motility and/or genetic regulation needs to be addressed in future studies. Regardless, the active seeding dispersal process we describe here may not be the method utilized in mucoid variants of clinical CF isolates, but rather a transmission mechanism utilized by environmental strains of P. aeruginosa.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bryers, J. D. (1988). Modeling biofilm accumulation. In Physiological Models in Microbiology, vol. 2, pp. 109144. Edited by M. J. Bazin & J. I. Prosser. Boca Raton, FL: CRC Press.
Characklis, W. G. (1990). Biofilm processes. In Biofilms, pp. 195231. Edited by W. G. Characklis & K. C. Marshall. New York: Wiley.
Davey, M. E., Caiazza, N. C. & O'Toole, G. A. (2003). Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185, 10271036.
Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295298.
Høiby, N., Krogh Johansen, H., Moser, C., Song, Z., Ciofu, O. & Kharazmi, A. (2001). Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of growth. Microbes Infect 3, 2335.[CrossRef][Medline]
Kaplan, J. B., Ragunath, C., Ramasubbu, N. & Fine, D. H. (2003a). Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous -hexosaminidase activity. J Bacteriol 185, 46934698.
Kaplan, J. B., Meyenhofer, M. F. & Fine, D. H. (2003b). Biofilm growth and detachment of Actinobacillus actinomycetemcomitans. J Bacteriol 185, 13991404.
Klausen, M., Aaes-Jorgensen, A., Molin, S. & Tolker-Nielsen, T. (2003). Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol Microbiol 50, 6168.[CrossRef][Medline]
Kohler, T., Curty, L. K., Barja, F., van Delden, C. & Pechere, J. C. (2000). Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182, 59905996.
Nivens, D. E., Ohman, D. E., Williams, J. & Franklin, M. J. (2001). Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J Bacteriol 183, 10471057.
Ochsner, U. A., Fiechter, A. & Reiser, J. (1994). Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 269, 1978719795.
Ohman, D. E. & Chakrabarty, A. M. (1981). Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33, 142148.[Medline]
Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 57565767.
Picioreanu, C., van Loosdrecht, M. C. & Heijnen, J. J. (2001). Two-dimensional model of biofilm detachment caused by internal stress from liquid flow. Biotechnol Bioeng 72, 205218.[CrossRef][Medline]
Purevdorj, B., Costerton, J. W. & Stoodley, P. (2002). Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 68, 44574464.
Purevdorj-Gage, B. & Stoodley, P. (2004). Hydrodynamic considerations of biofilm structure and behavior. In Microbial Biofilms, pp. 160173. Edited by M. A. Ghannoum & G. O'Toole. Washington, DC: American Society for Microbiology.
Rashid, M. H. & Kornberg, A. (2000). Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97, 48854890.
Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. & Davies, D. G. (2002). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184, 11401154.
Semmler, A. B., Whitchurch, C. B. & Mattick, J. S. (1999). A re-examination of twitching motility in Pseudomonas aeruginosa. Microbiology 145, 28632873.[Medline]
Stewart, P. S. (1993). A model of biofilm detachment. Biotechnol Bioeng 41, 111117.[CrossRef]
Stewart, P. S., McFeters, G. A. & Huang, C. T. (2000). Biofilm formation and persistence. In Biofilms II: Process Analysis and Application, pp. 373405. Edited by J. D. Bryers. New York: Wiley-Liss.
Stoodley, P., Lewandowski, Z., Boyle, J. D. & Lappin-Scott, H. M. (1999). Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid-shear: an in situ investigation of biofilm rheology. Biotechnol Bioeng 65, 8393.[CrossRef][Medline]
Stoodley, P., Wilson, S., Hall-Stoodley, L., Boyle, J. D., Lappin-Scott, H. M. & Costerton, J. W. (2001). Growth and detachment of cell clusters from mature mixed-species biofilms. Appl Environ Microbiol 67, 56085613.
Stoodley, P., Sauer, K., Davies, D. G. & Costerton, J. W. (2002). Biofilms as complex differentiated communities. Annu Rev Microbiol 56, 187209.[CrossRef][Medline]
Tolker-Nielsen, T., Brinch, U. C., Ragas, P. C., Andersen, J. B., Jacobsen, C. S. & Molin, S. (2000). Development and dynamics of Pseudomonas sp. biofilms. J Bacteriol 182, 64826489.
Towler, B. W., Rupp, C. J., Cunningham, A. B. & Stoodley, P. (2003). Viscoelastic properties of a mixed culture biofilm from rheometer creep analysis. Biofouling 19, 279285.[CrossRef][Medline]
Van Loosdrecht, M. C. M., Eikelboom, D., Gjaltema, A., Mulder, A., Tijhuis, L. & Heijnen, J. J. (1995). Biofilm structures. Water Sci Technol 32, 3543.
Van Loosdrecht, M. C. M., Picioreanu, C. & Heijnen, J. J. (1997). A more unifying hypothesis for the structure of microbial biofilms. FEMS Microb Ecol 24, 181183.[CrossRef]
Webb, J. S., Thompson, L. S., James, S., Charlton, T., Tolker-Nielsen, T., Koch, B., Givskov, M. & Kjelleberg, S. (2003). Cell death in Pseudomonas aeruginosa biofilm development. J Bacteriol 185, 45854592.
Wilson, S., Hamilton, M. A., Hamilton, G. C., Schumann, M. R. & Stoodley, P. (2004). Statistical quantification of the detachment rates and size distribution of cell clumps from wild type (PAO1) and cell signaling mutant (JP1) Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 70, 58475852.
Received 2 August 2004;
revised 9 December 2004;
accepted 3 February 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |