Combination of selected enzymes with cetyltrimethylammonium bromide in biofilm inactivation, removal and regrowth

Abstract

Enzymes are considered an innovative and environmentally friendly approach for biofilm control due to their lytic and dispersal activities. In this study, four enzymes (β-glucanase, α-amylase, lipase and protease) were tested separately and in combination with the quaternary ammonium compound cetyltrimethylammonium bromide (CTAB) to control flow-generated biofilms of Pseudomonas fluorescens. The four enzymes caused modest reduction of biofilm colony forming units (CFU). Protease, β-glucanase and α-amylase also caused modest biofilm removal. CTAB combined with either β-glucanase or α-amylase increased biofilm removal. Its combination with either β-glucanase or protease increased CFU reduction. However, CTAB-protease combination was antagonist in biofilm removal. Long-term effects in biofilm mass reduction were observed after protease exposure. In contrast, biofilms treated with β-glucanase were able to regrow significantly after exposure. Moreover, short-term respirometry tests with planktonic cells were performed to understand the effects of enzymes and their combination with CTAB on P. fluorescens viability. Protease and lipase demonstrated antimicrobial action, while α-amylase increased bacterial metabolic activity. The combination of CTAB with either protease or α-amylase was antagonistic, decreasing the antimicrobial action of CTAB. The overall results demonstrate a modest effect of the selected enzymes in biofilm control, either when applied alone or each one in combination with CTAB. Total biofilm removal or CFU reduction was not achieved and, in some cases, the use of enzymes antagonized the effects of CTAB. The results also propose that complementary tests, to characterize biofilm integrity and microbial viability, are required when someone is trying to assess the role of novel biocide - enzyme mixtures for effective biofilm control.

Removal of Salmonella enterica serovar Typhimurium and Cronobacter sakazakii biofilms from food contact surfaces through enzymatic catalysis

“…However, when the biofilms of both bacteria were treated with the enzymatic product, the microbial load decreased significantly (2–2.5 log CFU cm −2 of reduction) compared to the biofilm growth achieved in 48 hr. Our results indicate a detachment of the cells adhered to the stainless steel caused by the enzymatic treatment, as reported by other authors (Araújo et al, ; Lequette, Boels, Clarisse, & Faille, ; Molobela, Cloete, & Beukes, ). The effectiveness of different enzymes on the detachment of different microorganisms in biofilms has also been thoroughly studied.…”

“…sakazakii biofilms compared with at 45°C, which were similar to previous results obtained in this study. Furthermore, the figures obtained in this study were higher than those obtained by Araújo et al (), who reported that enzymatic treatments with a single enzyme, including protease, β‐glucanase and α‐amylase caused a modest elimination of P . fluorescens biofilms with a reduction ranging from 1 to 1.5 log CFU cm −2 .…”

“…Our results indicate a detachment of the cells adhered to the stainless steel caused by the enzymatic treatment, as reported by other authors (Araújo et al, 2017;Lequette, Boels, Clarisse, & Faille, 2010;Molobela, Cloete, & Beukes, 2010). The effectiveness of different enzymes on the detachment of different microorganisms in biofilms has also been thoroughly studied.…”

“…However, the temperature played an important role in the enzymatic treatment of the surfaces, with a greater, albeit nonsignificant (p > .05), effectiveness at 50 C for the C. sakazakii biofilms compared with at 45 C, which were similar to previous results obtained in this study. Furthermore, the figures obtained in this study were higher than those obtained by Araújo et al (2017), who reported that enzymatic treatments with a single enzyme, including protease, β-glucanase and α-amylase caused a modest elimination of F I G U R E 3 Digital images obtained by DEM using the Live/Dead BacLight viability assay of C. sakazakii on (a) stainless steel and (d) polystyrene surfaces; Biofilms on stainless steel surfaces treated with water at 50 C for 30 min on (b) stainless steel and (e) polystyrene surfaces; and biofilms after preventive enzymatic OES on (c) stainless steel and (f) polystyrene surfaces. Magnification: 40×.…”

“…In this study, the enzymatic formula contained different kinds of enzymes to potentiate the detachment action, taking the different components that a biofilm matrix can be formed of into account and taking special note of the various matrices formed by the different bacterial species. The effectiveness of different enzymatic formulas in disintegrating mono‐ or multispecies microbial biofilms has been a subject of study (Araújo et al, ; Lequette et al, ; Molobela et al, ). To this effect, Rodríguez‐López, Puga, Orgaz, and Cabo () focused on the effect of different enzymatic formulations, including DNAase, cellulase, and pronase, in combination with benzalkonium chloride, on mixed biofilms formed by L .…”