[25] Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa
Publisher Summary
This chapter discusses that Biofilms of Pseudomonas aeruginosa have been intensively studied during the last decade. Pseudomonas aeruginosa offers several advantages as a model organism in biofilm research. This gram-negative bacterium is well characterized with respect to its molecular genetics, biochemistry, and physiology; it is also of hygienic relevance as an opportunistic pathogen, because biofilms harboring this bacterium in technical water systems and on medical devices may be a source of human infections. The extracellular polymeric substances (EPS) of P. aeruginosa predominantly consist of different polysaccharides and proteins. Mucoid variants of P. aeruginosa are characterized by an overproduction of the viscous exopolysaccharide alginate, resulting in the production of large slimy colonies, when the bacteria are cultivated overnight on common agar-based media. Alginates from P. aeruginosa are high molecular weight unbranched copolymers consisting of (1→ 4)-linked uronic acid residues of β-D-mannuronate and α-L-guluronate. The formation of biofilms is regarded as part of a natural “life cycle” of mucoid P. aeruginosa. Alginates represent major components of the EPS of mucoid P. aeruginosa and have been implicated in the development as well as the maintenance of the mechanical stability of biofilms formed by P aeruginosa on living and abiotic surfaces. In this chapter, methods of isolation and biochemical characterization are described for both whole EPS and alginate from mucoid strains of P. aeruginosa grown as a confluent bacterial lawn on agar media as simple in vitro model systems for bacterial biofilms.
References (39)
- Sherbrock-CoxV. et al.
Carbohydr. Res.
(1984) - RossN.W. et al.
FEMS Microbiol. Lett.
(1991) - MartyN. et al.
FEMS Microbiol. Lett.
(1992) - MayerC. et al.
Int. J. Biol. Macromol.
(1999) - LowryO.H. et al.
J. Biol. Chem.
(1951) - SmithP.K. et al.
Anal. Biochem.
(1985) - BradfordM.M.
Anal. Biochem.
(1976) - FrolundB. et al.
Wat. Res.
(1996) - BlumenkrantzN. et al.
Anal. Chem.
(1973) - Filisetti-CozziT.M.C.C. et al.
Anal. Biochem.
(1991)
J. Biol. Chem.
FEMS Microbiol. Lett.
J. Biol. Chem.
Mol. Aspects Med.
Polymer
Carbohydr Res.
Science
Cited by (145)
Contemporary strategies and approaches for characterizing composition and enhancing biofilm penetration targeting bacterial extracellular polymeric substances
2024, Journal of Pharmaceutical AnalysisExtracellular polymeric substances (EPS) constitutes crucial elements within bacterial biofilms, facilitating accelerated antimicrobial resistance and conferring defense against the host's immune cells. Developing precise and effective antibiofilm approaches and strategies, tailored to the specific characteristics of EPS composition, can offer valuable insights for the creation of novel antimicrobial drugs. This, in turn, holds the potential to mitigate the alarming issue of bacterial drug resistance. Current analysis of EPS compositions relies heavily on colorimetric approaches with a significant bias, which is likely due to the selection of a standard compound and the cross-interference of various EPS compounds. Considering the pivotal role of EPS in biofilm functionality, it is imperative for EPS research to delve deeper into the analysis of intricate compositions, moving beyond the current focus on polymeric materials. This necessitates a shift from heavy reliance on colorimetric analytic methods to more comprehensive and nuanced analytical approaches. In this study, we have provided a comprehensive summary of existing analytical methods utilized in the characterization of EPS compositions. Additionally, novel strategies aimed at targeting EPS to enhance biofilm penetration were explored, with a specific focus on highlighting the limitations associated with colorimetric methods. Furthermore, we have outlined the challenges faced in identifying additional components of EPS and propose a prospective research plan to address these challenges. This review has the potential to guide future researchers in the search for novel compounds capable of suppressing EPS, thereby inhibiting biofilm formation. This insight opens up a new avenue for exploration within this research domain.
In situ solid-state fabrication of Z-Scheme BiVO<inf>4</inf>/g-C<inf>3</inf>N<inf>4</inf> heterojunction photocatalyst with highly efficient-light visible activity and their antibacterial properties against bacterial pathogens
2024, Journal of Molecular StructureHere, BiVO4/g-C3N4 composite photocatalyst with various weight percent of BiVO4 were synthesized by in-situ solid-state fabrication via calcination process. Photocatalyst was characterized by various analytical techniques (XRD, SEM, TEM, UV–vis-DRS, and TGA). The SEM and TEM results showed that composites materials BiVO4 dispersed uniformly on the surface of g-C3N4 sheets, revealing that g-C3N4 sheets was probably a promising support for in-situ growth of nano-size materials. The achieved intimate interfacial contact between BiVO4 and g-C3N4 promoted the charge transfer and inhibited recombination rate of photogenerated electron-hole pairs, which significantly improved the photocatalytic activity. Remarkably, catalytic efficiency of BiVO4/g-C3N4 composites has been demonstrated, via photodegradation of organic pollutants such as rhodamine-B (RhB) and ciprofloxacin (CIP). The 5 wt.% BiVO4/g-C3N4 showed excellent photocatalyst activity and potentially degraded RbB (98 %) and CIP (95 %) in 75 min of irradiation time in the presence of visible light. Moreover, radical trap experiment indicated that the –•O2 and •OH acted as main reactive species for photocatalytic degradation. In addition, BiVO4/g-C3N4 nanocomposite showed strong antibacterial potential against bacterial pathogens; E. coli ATCC-15224, Staphylococcus aureus MTCC-3160 and Pseudomonas aeruginosa PAO1. Studies revealed that depended on time and BiVO4/g-C3N4 concentration, bacterial growth, and viability (CFU count) was consistently declined. The increasing doses of BiVO4/g-C3N4 significantly (p ≤ 0.05) inhibited the virulence factors like pyoverdin, pyocyanin, elastase, exoprotease, rhamnolipid, and swimming motility of P. aeruginosa PAO1 by 76.9, 49.0, 71.1, 53.3, 89.5, and 60 %, respectively. Furthermore, biofilms formed by E. coli, S. aureus and P. aeruginosa PAO1 were maximally inhibited at 10 µgmL−1 BiVO4/g-C3N4 to a maximum of 67, 56 and 56 %, respectively. Also, exopolysaccharide (EPS) releasing ability of isolates were suppressed by BiVO4/g-C3N4. The fluorescently labelled probe DCFH-DA staining of bacterial isolates showed nanocomposite-dependent increase in ROS production and superoxide radicals. The photodegradation of RhB and (CIP) recommends that synthesized BiVO4/g-C3N4 nanocomposite have strong photocatalytic properties. Summarily, newly synthesized BiVO4/g-C3N4 photocatalyst can be employed as an alternative to remediate the environmental pollution as well as a therapeutic agent for topical administration in controlling the pathogenic infections.
Pseudomonas stutzeri improves the tolerance of Lemna minor to Cu(OH)<inf>2</inf> nanopesticide by regulating the uptake of copper, antioxidant defense mechanisms, and the expression of metacaspase-1, chlorophyllase, and stress-responsive genes
2023, Plant Physiology and BiochemistryThis study investigated the effect of Pseudomonas stutzeri inoculation on Lemna minor treated with Cu(OH)2 nanopesticide (NP). The results showed that P. stutzeri inoculation increased the relative growth rate (RGR) in NP-treated plants. Although chlorophyll and carotenoid contents decreased significantly in NP-treated plants, P. stutzeri inoculation led to an increase in chlorophyll and carotenoid contents in NP-treated plants. Copper (Cu) content increased with increasing NP concentration, but it decreased significantly in the presence of P. stutzeri. NP treatment caused increased H2O2 and TBARS levels, as well as proline levels. However, P. stutzeri inoculation led to decreased H2O2 and TBARS levels and increased SOD, POX, GST, GR, GPX, and DHAR activities. The expression of genes encoding SOD, GST, metacaspase-1, and chlorophyllase was upregulated by NP treatment alone. Additionally, when plants were inoculated with P. stutzeri, the expression of these genes was further enhanced. In conclusion, P. stutzeri inoculation had a positive effect on the growth and antioxidant system of L. minor treated with NP as it enhanced RGR, increased chlorophyll and carotenoid contents, and decreased Cu content and oxidative stress. These findings suggested that P. stutzeri has the potential to promote aquatic plant growth and counteract the negative impacts of NP on these plants.
Linking patterns of physical and chemical organic matter fractions to its lability in sediments of the tidal Elbe river
2023, Applied GeochemistryDegradability of organic matter in river sediments differs in relation to origin and age. In order to explain previously observed spatial patterns of organic matter degradability and stabilization, this study investigated sediment organic matter (SOM) properties along a tidal Elbe river transect using dissolved organic matter (DOM) fractions, density fractions, carbon stable isotopes and thermometric pyrolysis (Rock-Eval 6©). These properties were linked to SOM decay rates and biological indicators such as chlorophyll a and silicic acid in the water phase, and sediment-bound extracellular polymeric substances (EPS), microbial biomass and oxygen consumption. Sediment source gradients were established using the concentration of Zn in the fraction < 20 μm as proxy.
The specific Zn concentration showed that the most upstream location was nourished primarily by upstream fluviatile sediments while the other locations carried a downstream signature. The upstream location was also characterised by the highest concentrations of chlorophyll a, microbial biomass, silicic acid, EPS, humic acids and hydrophilic DOM, the most negative δ13C signature and by the highest oxygen consumption rate, with decreasing trends towards downstream locations. This trend was also evident in the decreasing SOM lability from upstream to downstream, an increasing share of total SOM found in the acid-base-extractable fractions and a decreasing share of carbon in the light density fractions. Thermometric pyrolysis revealed the highest H-index (easily degradable SOM) for the most upstream location and the ratio of the I-index (immature SOM) to the R-index (refractory SOM) to correlate positively with measured SOM decay rates.
This study suggests that spatial patterns of SOM degradability can be explained by a source gradient, with young organic matter entering the system from upstream from predominantly biogenic sources, while downstream sources (North Sea sediment) deliver more refractory SOM that is stabilized in organo-mineral associations to a higher extent. In the investigated sediments, dissolved organic matter represented 0.23–1.20% of the total organic carbon (TOC) from anaerobically degradable SOM, while 4.10–11.46% TOC was liberated as CO2 and CH4 after long-term incubation (250 days). Thermometric pyrolysis is shown to serve as a useful proxy for SOM degradability in river sediments, with the Hydrogen-Index (HI) correlating well with degradability and the relationship between the I-index and R-index changing consistently towards lower I-indices and higher R-indices with an increasing degree of SOM stabilization.
Biofilm control strategies in food industry: Inhibition and utilization
2022, Trends in Food Science and TechnologyBiofilms are microorganisms community embedded in the self-secreted extracellular polymeric substances, which can provide microbes with strong tolerance and favorable living environments. As an important physiological indicator, biofilm formation could not only induce various food safety problems but also regulate the accumulation of different bio-products.
This review aims to summarize the regulation strategies of controlling biofilm in food process and enhancing production of different bioproducts by utilizing biofilm. Therefore, research papers published during the past decades were summarized and discussed.
Mature biofilm consists of exopolysaccharides, extracellular DNA and proteins. Three processes of biofilm formation can be utilized for biofilm regulation. Inhibiting biofilm formation and eradicating mature biofilm are commonly used for solving food related problems. Among them, ultrasound, ultraviolet, magnetic fields with magneto-materials, different chemicals addition such as amino acids and vitamins, and many other inhibitors present extraordinary biofilm inhibition ability. However, several biofilm utilization techniques exhibit potential application value for enhancing food additives and biochemicals production, including providing proper culture environments, metabolic engineering, using biofilm devices and bio-electrochemical system. To further explore the effects of biofilm regulation in food industry and exploit more regulation strategies for productivity improvement, more efforts should be paid on in the future.
Pseudomonas aeruginosa (P. aeruginosa) contamination poses challenges to the food industry. Total flavonoids of Potentilla Kleiniana Wight et Arn (TFP) are poorly understood for their antibiofilm effects against P. aeruginosa. Therefore, the inhibitory effects of TFP against planktonic cells were determined by agar diffusion, microtiter plate and time-kill curve assays. The mechanism of inhibition is evaluated by changes in membrane (potential, permeability and damage), cell motility and virulence factors production. Morphological changes were verified by transmission electron microscopy (TEM). A modified microtiter-plate assay determined its inhibitory effects against biofilm cells. The inhibitory mechanism was studied by changes in extracellular polymeric substances (EPS) and cell surface hydrophobicity. These inhibition effects were verified by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Finally, TFP was applied to stainless steel surfaces to evaluate its potential application in food production. TFP damaged P. aeruginosa cell membrane integrity, inhibited bacterial motility, virulence factors, surface hydrophobicity and colony counting on stainless steel. These results provide evidence for the utilization of TFP as a novel natural bioactive preservative in food processing.