Efflux as a Glutaraldehyde Resistance Mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa Biofilms

Abstract

A major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms. P oor control of biofilm growth is a major concern in many industries, including health care, food production, and oil and gas (1-4). For example, medical device-associated biofilms are the source of 60% to 70% of nosocomial infections (5). In the oil and gas industry, biofilms present a serious hazard to infrastructure through corrosion and reduced oil quality (6). Biocides are typically used to control and inactivate the biofilms, but resistance to biocides decreases the efficacy of disinfection. Biocide resistance was historically believed to be rare (7), but numerous reports of microbial biofilms resistant to biocides, including chlorine, quaternary ammonium compounds, and aldehydes (8-12), indicate a more widespread phenomenon. Biofilms are inherently more resistant to biocide treatments (8-12), a feature generally attributed to physical mechanisms, such as limited penetration of biocides through exopolysaccharides, reactivity and absorption of biocides to biofilm matrix, and phenotypic adaptations (13-15). Recent reports suggest that genetic factors may also contribute to biocide resistance; for example, efflux pumps were shown to contribute to biocide resistance, indicating the potential for genetically mediated biocide resistance mechanisms (10, 11). The cross-resistance between biocides and antibiotics provides further evidence that biocide resistance may be mediated by genetic factors (15, 16). The control and design of effective biofilm disinfection strategies require understanding of the mechanisms of action of antimicrobials and biocide resistance of biofilms, specifically, the genetically modulated response of microbial biofilms.Glutaraldehyde...

The Role of Efflux and Physiological Adaptation in Biofilm Tolerance and Resistance

“…In this study, the PA1874 -1877 operon was identified as a novel efflux system potentially involved in biofilm-specific resistance to antibiotics: the expression of PA1874 was found to be 10-fold higher in sessile than in planktonic cells, deletion of the operon only affected susceptibility to tobramycin in biofilms, and overexpression of the operon decreased the susceptibility toward selected aminoglycosides and fluoroquinolones in planktonic cells (27). In addition, in Pseudomonas fluorescens, expression of two efflux systems was up-regulated in glutaraldehydetreated biofilms, and efflux pump inhibitors increased sensitivity of P. aeruginosa and P. fluorescens biofilms to this disinfectant, confirming that not only antibiotic resistance is mediated by efflux (28). Finally, it was observed that planktonic P. aeruginosa cells grown under hypoxic conditions showed an increased expression of MexEF-OprN (29).…”

Nutrient Level Determines Biofilm Characteristics and Subsequent Impact on Microbial Corrosion and Biocide Effectiveness

“…This bacteria has an outer sheath-like structure that might provide physical characteristics that confers resistance to biocides (75). Resistance of some Pseudomonas species to glutaraldehyde has been related to the presence of genetic mechanisms, specifically, the expression of efflux pumps and induction of modulators of biofilm formation (76). Efflux pumps can be induced when microorganisms are under stress conditions which contributes to higher bacterial resistance of stressed populations to antimicrobial substances and could be the reason for the higher increase in the relative on October 11, 2020 by guest…”

Tolerance of Pseudomonas aeruginosa in in-vitro biofilms to high-level peracetic acid disinfection

“…Age related resistance to glutaraldehyde, ahigh level disinfectant also used in endoscope disinfection, has been reported in P. aeruginosa and P. flourescens. (38) Resistance to disinfection may be due to the components of the ECM, which react with the disinfectant before the agent reaches the bacterial cell surface. If this is the case then higher biomass should result in reduced effectiveness of disinfection.…”