Bacteria are able to adapt to several environmental stresses such as the presence of antimicrobial molecules
and, as consequence, bacterial resistance may increase with increasing exposure to antimicrobials. The most
impressive mechanism of the bacterial mode of life is their grow as part of a sessile community referred to as
biofilm [1]. Biofilm formation is an important aspect of many bacterial diseases, especially those related with
medical devices [2]. When biofilms are identified as the cause of infection, treatment becomes very difficult
since bacteria within biofilms demonstrate peculiar features, that confer them increased resistance to biocides.
The adaptive response to antimicrobial stresses of sessile bacteria is more effective than the corresponding
planktonic populations. Adaptive resistance to antimicrobials has been widely reported in planktonic state and
characterized in terms of phenotypic traits and proteomic analysis [3,4]. Concerning biofilm adaptation, the
response of the biofilm-entrapped cells to chemical stress conditions is not yet well studied. This work aimed to
examine whether exposure of Pseudomonas aeruginosa biofilms to benzalkonium chloride (BC) and ciprofloxacin
(CIP) during a laboratory adaptation process could induce any proteomic alterations in the outer membrane
(OM) of the biofilm cells. Biofilms were formed in 6-well plates for 24 h being after submitted to the presence of
324 mg/L of BC and 6.0 mg/L of CIP, during 13 days. The obtained biofilm-cells were separated from the biofilm
matrix and the OM proteins extracted. Protein patterns were analyzed by 2-DE and gels by Progenesis SameSpot
software. Protein spots from the bacterial populations were considered to display significant quantitative
differences if they fulfilled the following criteria: p values ≤ 0.05 (t-test); detection threshold, average volume ≥
20 (n = 3); differential tolerance, fold change ≥ 2. Excised spots from three different gels of each adapted
bacteria were identified by LC-MS/MS. Biofilm proteome analysis showed that P. aeruginosa adaptation to BC
and CIP changed the expression of six proteins. The biofilm exposure to both antimicrobials generated common
down-regulation of three proteins: GroEL, major capsid protein and putative tail sheath protein, revealing a
possible similar stress response. The type 4 fimbrial biogenesis outer membrane protein PilQ precursor was
over-expressed only in biofilms submitted to BC, while the probable bacteriophage protein and the hypothetical
protein PA0537 were overexpressed in CIP exposed biofilms. When bacteria are within biofilms and exposed to
chemical stress, the regulation of OM proteins expression can contribute to increase the biofilm resistance. The
proteins involved in adhesion, oxidative stress response, as well as in synthesis of lipopolysaccharide, were upor
down-regulated in adapted P. aruginosa biofilms. These acquired proteomic profiles may be associated with
antimicrobial tolerance
