The Role of Quorum Sensing in Biofilm Development - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 101

environment dictates bacterial behavior, so applying the QS rules of engagement
seen in planktonic cell cultures to all populations is intrinsically flawed. Therefore,
it is important that investigators study biofilms in as close to their native state as
possible. In addition, most of the studies discussed above were based on the
behavior displayed by strains harboring mutations in different QS regulators. As
these QS regulators can control a significant portion of the transcriptome, teasing
out exactly what role cell-to-cell communication plays can be challenging. As
Parsek and Greenberg noted, “Perhaps the best way to evaluate the role of quorum
sensing is to monitor the signaling process in situ in a developing biofilm of a wild-
type strain and determine if the onset of quorum sensing corresponds to any
discernible transition in development, such as changes in structure or an increase
in antimicrobial tolerance” (Parsek and Greenberg 2005 ). The methods described
below have been developed to meet this goal.
4.1 QS Monitor Strains
Probably the most straightforward way to determine when and where QS is
involved in biofilm formation is to use QS reporter strains to monitor the activity
of QS in situ. De Kievit et al. used this approach to study the roles of the las and rhl
QS systems in biofilm formation by P. aeruginosa (De Kievit et al. 2001 ). Biofilms
grown in vitro with P. aeruginosa strains harboring lasI and rhlI transcriptional
fusions to green fluorescent protein (GFP) were imaged after 4, 6, and 8 days of
growth in polycarbonate flowcells. Prior to imaging, biofilm cells were stained with
propidium iodide-Syto85 so that all cells could be visualized in the red spectra,
while only those producing GFP could be visualized in the green. The investigators
found lasI expression was maximal at 4 days and then decreased over time, while
rhlI expression was more stable but occurred in fewer cells. They also found that
bacterial cells closest to the polycarbonate surface maximally expressed both genes
and expression of the QS genes decreased with increasing biofilm height. This study
clearly showed that QS occurred in biofilms, but the percentage of cells expressing
lasI or rhlI was very limited, both in number and in the area of the biofilm in which
they resided.
Similar strategies have been used to monitor QS in P. aeruginosa lung infections
in animal models and measure the effects of QS inhibiting agents in situ (Hentzer
et al. 2003 ; Wu et al. 2004 ). Yarwood et al. also used a GFP-fusion strain to
examine the contribution of the agr QS system to S. aureus biofilm formation
in vitro (Yarwood et al. 2004 ). The investigators found that agr 's influence on
biofilms was highly dependent on growth conditions, with agr expression enhanc-
ing biofilm development under some conditions and having no effect or inhibiting
biofilm development under other conditions. They also noted that Agr-dependent
expression occurred in patches within cell clusters in the biofilm and sometimes
inversely correlated with detachment of cells from the biofilm.