Dynamics of the Action of Biocides in Pseudomonas aeruginosa Biofilms

Abstract

The biocidal activity of peracetic acid (PAA) and benzalkonium chloride (BAC) on Pseudomonas aeruginosa biofilms was investigated by using a recently developed confocal laser scanning microscopy (CLSM) method that enables the direct and real-time visualization of cell inactivation within the structure. This technique is based on monitoring the loss of fluorescence that corresponds to the leakage of a fluorophore out of cells due to membrane permeabilization by the biocides. Although this approach has previously been used with success with various Gram-positive species, it is not directly applicable to the visualization of Gram-negative strains such as P. aeruginosa, particularly because of limitations regarding fluorescence staining. After adapting the staining procedure to P. aeruginosa, the action of PAA and BAC on the biofilm formed by strain ATCC 15442 was investigated. The results revealed specific inactivation patterns as a function of the mode of action of the biocides. While PAA treatment triggered a uniform loss of fluorescence in the structure, the action of BAC was first localized at the periphery of cell clusters and then gradually spread throughout the biofilm. Visualization of the action of BAC in biofilms formed by three clinical isolates then confirmed the presence of a delay in penetration, showing that diffusion-reaction limitations could provide a major explanation for the resistance of P. aeruginosa biofilms to this biocide. Biochemical analysis suggested a key role for extracellular matrix characteristics in these processes.The control of microbial surface contamination is a major concern in terms of public health. Pseudomonas aeruginosa is a Gram-negative bacterium that is well known to be involved in a large number of human infections (14, 30). Numerous outbreaks have been linked directly to its presence on medical equipment (11,15,16,25). The persistence of this bacterium in the environment can be attributed to its ability to form biofilms that increase its resistance to disinfection treatments. Numerous studies have indeed reported the high resistance of P. aeruginosa biofilms (compared to their planktonic counterparts) to numerous biocides, including chlorine, quaternary ammonium compounds, and aldehydes (5,10,13,26). Although the precise mechanisms underlying this resistance remain unclear, it appears to be a multifactorial process that is primarily related to the physiological and structural characteristics of the biofilm. It is now generally accepted that biofilms constitute heterogeneous structures that group subpopulations with distinct physiological states and resistance phenotypes (28).Data on biocide reactivity within these heterogeneous structures could provide a clearer understanding of the mechanisms involved in biofilm resistance and ultimately facilitate the development of new and more efficient treatments. Recently, a noninvasive technique based on confocal laser scanning microscopy (CLSM) was developed and used to investigate spatial and temporal patterns of antimicrobial...

Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

“…A direct-interference mechanism preventing water and organic-solvent penetration has been demonstrated for the BslA protein, which forms a hydrophobic raincoat on the inner layer of B. subtilis colonies (46)(47)(48)(49), as well as curli fibers in E. coli colonies or extracellular DNA in P. aeruginosa submerged biofilms (50,51). Biocide molecules can be delayed by the biofilm polymer matrix (52) or diffuse easily into the biofilm but strongly react with organic material, such as the oxidizing agent PAA (53,54). Aldehydes, such as OPA, act as cross-linking agents reacting against proteins, DNA, and RNA (55).…”

Is peracetic acid suitable for the cleaning step of reprocessing flexible endoscopes?

“…Artificial P. aeruginosa biofilms resisted treatment with various biocidal agents including peracetic acid, compared with their planktonic counterparts [150][151][152] . Biofilms composed of E. coli [152,153] , S. aureus [152,154,155] , Mycobacterium fortuitum [156] or Listeria monocytogenes [157] also resisted treatment with diverse biocides compared with planktonic cells.…”

“…Bacteria in mature (old) biofilms were more resistant to killing than those in young biofilms [153,158,159] . An older biofilm of P. aeruginosa required up to 20-fold higher concentrations of peracetic acid (0.2%) to be eradicated, compared with their planktonic counterparts (0.01%) [151] . Similar results were found with an E. coli biofilm and peracetic acid/H2O2 [153] .…”

“…Especially "build-up" biofilms mimicking repeated endoscope reprocessing cycles exhibited a significantly higher survival rate than 'traditional' biofilms [158] . The mechanisms underlying disinfectant-resistant phenotypes appear to be multifactorial [133,148,151,153,164] .…”

Quillaja saponaria Saponins with Potential to Enhance the Effectiveness of Disinfection Processes in the Beverage Industry

“…An essential step to improve the disinfection process is therefore the loosening or eradication of biofilm structures. One common and useful method is the mechanical disruption of biofilm matrices [33]. However, this method is effective only on easily-accessible surfaces.…”