Inhibitory Effect of Biocides on the Viable Masses and Matrices of Staphylococcus aureus and Pseudomonas aeruginosa Biofilms

Abstract

Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides.Microbial communities irreversibly attached to a surface and encapsulated in a self-produced polymeric matrix are known as biofilms. A particular characteristic is their extreme resistance to antimicrobial treatment (6). This resistance is mediated by several mechanisms that can act together: (i) poor penetration or inactivation of antimicrobials in the matrix, (ii) an altered bacterial metabolic state, (iii) the formation of persister cells, and (iv) resistance induced by the antimicrobial itself following the use of sublethal concentrations and the upregulation of efflux pumps (2, 7). Hence, biofilms are hard to eradicate and are claimed to be responsible for up to 60% of all infections in humans (1, 5). Staphylococcus aureus and Pseudomonas aeruginosa are notorious biofilm producers, the first being nosocomial and responsible mainly for medical device-associated infections (13, 28) and the latter being an opportunistic pathogen causing life-threatening infections mainly in cystic fibrosis patients (13,19).Looking at the high biofilm-related morbidity and mortality, the antibiofilm properties of antimicrobials have been studied extensively, with a main focus on the activity of antibiotics (1,17,20,26). However, a recent study indicated that the prospect of using solely antibiotics to achieve complete biofilm destruction is limited, since the biofilm matrix persists (25). As their mechanisms of action are not limited to the bacterial metabolism, biocides should also be considered as valuable...

Effect of Chlorhexidine and Sodium Hypochlorite on Staphylococcus aureus Biofilm

“…Similar results were observed with concentrations of 0.2% (p value 0.001), 0.15% (p value 0.000) and 0.075% (p value 0.000), this indicate inhibitory effect of chlorhexidine on bioflim formation of Staphylococcus aureus is affected by time of exposure. This result agree with another study conducted in Belgium by Toté et al [13] which showed that longer contact time generally increase the antibiofilm activity of chlorhexidine.…”

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

“…[9][10][11] The fact that the increase or maintained levels of the EPS content could take place after exposure to penicillin G and ciprofloxacin, which display two completely different mechanisms of actions on S. aureus biofilms, inclines us to hypothesize that this may be a more general protective response against chemical insults. Our results also support the previous views proposed by Toté et al 10 using a completely different method of EPS staining and other bacterial strains.…”

“…Thus, antibiotics can kill biofilm cells but cause other effects on the EPS fraction. It has been suggested that surfaces in which the matrix has been left, can be recolonized more rapidly by biofilm-forming bacteria, [9][10][11] causing infection reappearance. In two previous contributions it has been demonstrated that some antibiotics (that is, vancomycin), at millimolar concentrations, decrease the bacterial viability without having a significant effect on the matrix.…”

“…In two previous contributions it has been demonstrated that some antibiotics (that is, vancomycin), at millimolar concentrations, decrease the bacterial viability without having a significant effect on the matrix. 10,11 In such studies, along with the conventional resazurin assay, a method to estimate the matrix content was used, based on the staining with dimethyl methylene blue, a cationic dye that stains the polysaccharide intercellular adhesin in Gram-positive biofilms. 9 Given the tremendous implications of those mentioned studies for the critical assessment of the long chemotherapeutical effects of conventional antibiotics, we decided, in the present contribution, to further explore the effects of antibiotics on the matrix, viability and biomass of S. aureus biofilms.…”

Effects of sub-MIC antibiotic concentrations on biofilm production of Salmonella Infantis

“…The MBEC assay was developed for rapid and reproducible antimicrobial susceptibility testing for bacterial biofilms. It has been reported that MBEC values are greater than MIC against isolated strains from Salmonella infections [30,31]. In the present study, at first, we determined the MBEC levels of seven different antibiotics on S. Infantis serovar biofilms.…”