The percent pyocyanin inhibition of both mannitol and medium 5294 extracts was estimated at 250-1000 µg/ml…………………69 Table 2.6: Effect of sponge-associated Bacillus spp. The percent pyocyanin inhibition of both mannitol and medium 5294 extracts was estimated at 250-1000 µg/ml………70 Table 2.7: Effect of sponge-associated Bacillus spp.
1
Introduction
Marine bacteria such as the actinomycetes associated with sponges show great promise in this field (Saurav et al., 2016). Marine sponges and related bacteria are very promising in this field (Ellithey et al., 2014; Indraningrat et al., 2016).
The sponge microbiome
- Phylum Porifera
- Sponge-associated bacteria
- Sponge-associated bacterial secondary metabolite production
These classes are distinguished by tissue, skeletal (spicule) and developmental characteristics (Renard et al., 2014). While some symbionts can reside on the outer layers of the fungus (Selvin et al., 2010;
Quorum sensing inhibition (QSI) as an alternative to antimicrobial therapies
- Quorum sensing
- Mechanisms of quorum sensing inhibition to target virulence
- Inhibiting biosynthesis of AHLs
- Interfering with signal dissemination – Quorum quenching
- Blocking of receptors
The first QSI acylase discovered was VAI-C from Variovorax paradoxus (Castillo-Juárez et al., 2017). In addition, long-chain AHL (10 or more carbon atoms in length) can be used to effectively inhibit short-chain AHL receptors ( Romero et al., 2012 ).
Quorum sensing inhibition and Pseudomonas aeruginosa
- Pseudomonas aeruginosa: An introduction
- Quorum sensing systems of Pseudomonas aeruginosa
- Pseudomonas aeruginosa biofilm production
- Quorum sensing inhibition of Pseudomonas aeruginosa
- Targeting P. aeruginosa virulence factor production
- Targeting P. aeruginosa QS systems
The release of eDNA is also mediated through QS-controlled quinolone production (Castillo-Juárez et al., 2017). The production of rhamnolipids is instrumental in the formation of Pseudomonas biofilms (Moradali et al., 2017).
Sponges and sponge-associated bacteria as inhibitors of quorum sensing and biofilm
Pseudoalteromonas, Micrococcus, Vibrio, Bacillus and Phaeobacter have already been shown to be efficient producers of QSI compounds (Hollants et al., 2013), with more than 3% of the Bacillus genome dedicated to the production of secondary metabolites (Bastos et al., 2013). al., 2013). Although these bacteria have not been identified and their Gram designation is unknown, Gram-positive marine bacteria such as Bacillus cereus, although not as abundant as Gram-negative bacteria (Martin et al., 2014), have also been shown to inhibit QS. , often through the production of lactonases (Fetzner, 2015).
Sponge-associated bacteria as inhibitors of HIV-1
- Classes of antiviral compounds isolated from marine sources
- Sponge-derived antiviral compounds
- Sponge-associated bacteria-derived antiviral compounds
- Targeting HIV-1 reverse transcriptase enzymes
Nucleosides mycalamide A and B with activity against HSV and corona viruses were also isolated from marine fungi (Sagar et al., 2010). Several avarol derivatives, such as avarone, have since been isolated from other marine sponges such as Dysidea cinerea (Sagar et al., 2010).
Rationale of study
Mushrooms have shown strong QSI potential in the past, but recently it has been shown that the metabolites responsible for this are often produced mainly by SPAB and not by the mushroom itself (Sun et al., 2015). Pseudomonas aeruginosa is an important candidate for QSI due to its prolific resistance mechanisms and prevalence in the hospital environment (Castillo-Juárez et al., 2017).
Hypotheses
Objectives
Aims
Key questions to be answered
Thesis Layout and Chapter Design
Effect of quorum quenching lactonase in clinical isolates of Pseudomonas aeruginosa and comparison with quorum sensing inhibitors. Presence of quorum-sensing inhibitor-like compounds from bacteria isolated from the brown alga Colpomenia sinuosa. Marine organisms as a source of extracts to disrupt bacterial communication: Bioguided isolation and identification of quorum sensing inhibitors from Ircinia felix.
Exploiting disruptive quorum sensing strategies in Gram-negative bacteria to improve environmental applications. One such alternative is the use of anti-virulence strategies such as quorum sensing inhibition (QSI).
Introduction
Quorum sensing inhibition (QSI) has gained popularity as one such alternative therapeutic option (García-Contreras, 2016), with the Gram-negative acylhomoserine lactone QSI being the most studied (Rekha et al., 2016). Marine microorganisms have gained interest as they gain momentum as a promising source of biologically active compounds (Abbamondi et al., 2014). Marine species have evolved QSI enzymes and mechanisms as a result of competition for limited habitats and nutrients (Grandclément et al., 2016).
Bacillus subtilis species, related to the sponge Aplysina aerophoba, produce a host of antimicrobial compounds effective against multidrug-resistant Staphylococcus aureus (Bramhachari et al., 2016) while Bacillus spp. Bacillus species are also known producers of AHL-degrading lactonase enzymes, which hydrolyze the AHL lactone ring ( Zhang and Li, 2016 ; Guendouze et al., 2017 ).
Methods and Materials
- Primary QSI screening and maintenance of cultures
- Preparation of ethyl acetate culture extracts
- Qualitative QSI screening
- Molecular identification of isolates
- Genomic DNA isolation
- Antimicrobial testing of bacterial extracts using agar-well diffusion method
- Screening of extracts against P. aeruginosa virulence factor production
- Inhibition of pyocyanin production
- Inhibition of pyoverdine production
- Inhibition of LasB production
- Qualitative inhibition of casein hydrolysis
- Quantitative inhibition of casein hydrolysis
- Qualitative inhibition of rhamnolipid production
- Quantitative inhibition of rhamnolipid production
- Inhibition of swimming and swarming motilities
- Inhibition of biofilm production
- Statistical analyses
Pseudomonas aeruginosa ATCC 27853 was then exposed to different concentrations of ethyl acetate extracts (0 – 1000 μg/ml) at 37. Proteolytic activity was defined as the difference between the absorbance at 450 nm of the treatments and the blank. We then compared the proteolytic activity of treated and untreated samples (Nicodème et al., 2005).
After an overnight incubation at 37 °C, the diameter of the clear zone around the bacterial colony was measured as evidence of rhamnolipid production using a UV transilluminator (Senturk et al., 2012). Swimming diameters were read every 24 hours to determine the effect of the extracts on swimming motility over time.
Results
- Sponge-associated bacterial inhibition of AHL reception in bio-sensing mutant
- Sponge-associated bacterial extracts continue to display QSI activity against C
- Antimicrobial testing of sponge-associated Bacillus spp. extracts against
- Bacillus spp. extracts and P. aeruginosa pyocyanin production
- Bacillus spp. extracts and P. aeruginosa pyoverdine production
- Bacillus spp. extracts and P. aeruginosa elastase production
- Bacillus spp. extracts and P. aeruginosa protease production
- Bacillus spp. extracts and P. aeruginosa rhamnolipid production
- Bacillus spp. extracts and P. aeruginosa swimming motility
- Bacillus spp. extracts and P. aeruginosa swarming motility
- Bacillus spp. extracts and P. aeruginosa initial biofilm formation
- Bacillus spp. extracts and P. aeruginosa mature biofilm formation
- Sponge-associated Bacillus spp. extracts as inhibitors of specific P. aeruginosa QS
No resulting cell death (>40%) was observed after treatment with any of the sponge-associated Bacillus spp. No extract was observed to inhibit growth and 10% DMSO without bacterial extract was found to increase the production of protease P. No extract was observed to inhibit growth and 10% DMSO without bacteria The extract was found to cause a non-significant (p > 0.05) inhibition P.
No growth inhibition was observed as a result of any of the extracts, unless indicated (Table 2.14), and 10% DMSO without bacterial extract actually. The mannitol extract of the same organism (SP1-AB4) resulted in the highest inhibition of initial biofilm formation, 73.58%.
Discussion
Even with the complexity of the systems involved in production of the sponge-associated Bacillus spp. extracts were able to inhibit pyocyanin production to some extent. This was consistent with the results of a similar. test performed by Singh et al. after exposure of Pseudomonas to 0.1 mg/ml of their bacterial extract. 2012) observed a maximum protease inhibition of 33% after exposure of P. aeruginosa to 1 ml of their crude Bacillus spp. extracts, from the sponge-associated bacterial extracts B. These results were comparable to those obtained by Gutiérrez-Barranquero et al. 2018), who noted that most of their marine sponge-associated bacterial extracts were capable of a qualitative inhibition in swarming, with three of the most promising extracts being Bacillus spp-derived.
When screening against initial biofilm production of Bacillus spp. extracts resulted in a ≥40% reduction in initial biofilm formation. Due to the variety of QSI compounds discovered to date, and the promising activity of the sponge-associated Bacillus spp.
Effects of Pseudomonas aeruginosa quorum-sensing molecules on organismal growth, elastase B production, and primary adhesion to hydrogel contact lenses. Herbs, spices and medicinal plants used in Spanish traditional medicine can reduce the virulence of Pseudomonas aeruginosa in a quorum sensing-dependent manner. Antibiofilm and quorum sensing inhibitory potential of Cuminum cyminum and its secondary metabolite methyl eugenol against gram-negative bacterial pathogens.
In vitro study of aggregation phenomena and antimicrobial activity of pyocyanin produced by Pseudomonas aeruginosa isolated from various human infections. Anti-quorum sensing and anti-biofilm activity of Delftia tsuruhatensis extract by attenuating quorum-sensing controlled virulence factor production in Pseudomonas aeruginosa.
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Introduction
Sponge-associated actinobacteria fall into three orders: Actinomycetales (the most abundant), Rubrobacteriales and Acidimicrobiales (Valliapan et al., 2014). They can then be further grouped into 39 families and 112 genera, of which 102 belong to the order Actinomycetales (Valliapan et al., 2014). Streptomyces is the most prominent Actinomycete genus, with 30% of sponge-associated bacteria-derived compounds originating from this genus (Indraningrat et al., 2016).
Quorum sensing inhibition (QSI) is enabled by small molecules and enzymes such as acylases and lactonases (Guendouze et al., 2017). Streptomyces and Rhodococcus species are known producers of AHL acylases that can degrade AHL chains having more than six acyl chains (Banerjee and Ray, 2016; Devaraj et al., 2017).
Methods and Materials
- Antimicrobial testing of bacterial extracts using agar-well diffusion method
- Screening of extracts against P. aeruginosa virulence factor production
- Statistical analyses
Wells were filled with 5 and 10 mg of the 20 Actinomycete bacterial extracts (mannitol and medium 5294) and the plates were incubated at 37 °C for 24 h, after which they were examined for the presence of zones of inhibition (El-Naggar and Abdelwahed, 2014 ). Proteolytic activity was defined as the difference between the absorbance at 450 nm of the treatments. After an overnight incubation at 37 °C, the diameter of the clear zone around the bacterial colony was measured as evidence of rhamnolipid production using a UV transilluminator light box (Senturk et al., 2012).
For this assay (Z-)-4-bromo-5-(bromoethylene)-2(5H)-furanone was used as a positive control at concentrations corresponding to 10% of the tested bacterial extracts. Data were obtained after assays were performed in duplicate or triplicate, in order to ensure the validity of the results.
Results
- Antimicrobial testing of sponge-associated Actinomycete extracts against
- Actinomycete extracts and P. aeruginosa pyocyanin production
- Actinomycete extracts and P. aeruginosa pyoverdine production
- Actinomycete extracts and P. aeruginosa elastase production
- Actinomycete extracts and P. aeruginosa protease production
- Actinomycete extracts and P. aeruginosa rhamnolipid production
- Actinomycete extracts and P. aeruginosa swimming motility
- Actinomycete extracts and P. aeruginosa swarming motility
- Actinomycete extracts and P. aeruginosa initial biofilm formation
- Actinomycete extracts and P. aeruginosa mature biofilm formation
- Sponge-associated Actinomycete extracts as inhibitors of specific P. aeruginosa QS
No cell death (>40%) was observed after treatment with any of the sponge-associated Actinomycete extracts (Supp. S3.4). No growth inhibition was observed as a result of any of the extracts, and 10% DMSO without bacterial extract was found to increase the production of P. No growth inhibition was observed as a result of any of the extracts, unless indicated (Table 3.11). and 10% DMSO without bacterial extract actually resulted in an increase in initial biofilm formation (Supp.
Overall three of the Actinomycete extracts resulted in a ≥40% reduction in initial biofilm formation, reaching a maximum inhibition of 53.16% (Table 3.12). No growth inhibition was observed due to either extract, and 10% DMSO without bacterial extract was found to result in a significant mean decrease of 12% (p < 0.05) in mature biofilm formation (Supp.
Discussion
These results were lower than those of Chang et al. 2017), who observed a pyoverdine inhibition of 55.7% with marine Rhizobium extracts. This result was only slightly below that of Chang et al. aeruginosa with a 5% aqueous Rhizobium extract. Furthermore, rhamnolipids increase the solubility of PQS while inhibiting normal tracheal ciliary function (Müller et al., 2015).
The inhibition observed as a result of extract SP2-AB6 (5294) was more similar to those obtained by Singh et al. 2017), who achieved an 85% rhamnolipid inhibition after treatment with P. This was similar to the results observed by Otton et al. 2017), who observed a 30% decrease in initial biofilm formation.
Antimicrobial proteins have also demonstrated extensive QSI capabilities, particularly anti-biofilm activities as a result of rhamnolipid inhibition and binding of eDNA (Algburi et al., 2017). Originally identified from a Streptomyces isolate in 1940, the QSI activity of this metabolite had been neglected until recently and has since been shown to be effective against P. The fungal-associated Actinomycete extracts tested in this study have shown hold enormous promise as quorum-sensing inhibitors, and as such should be further studied to identify the active agents as well as to determine their future clinical potential.
Stigmatellin Y - an anti-biofilm compound from Bacillus subtilis BR4 may interfere with the PQS-PqsR-mediated quorum sensing system in Pseudomonas aeruginosa. Disruption of N-acyl-homoserine-lactone-specific signaling and virulence in clinical pathogens by marine sponge bacteria.
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- Introduction
- Methods and materials
- Ethyl acetate extract preparation
- HIV-1 retroviral transcriptase (RT) assay
- Statistical analysis
- Results
- Discussion and conclusion
- References
- References
Solvent 10% DMSO (B) had a negligible effect on cell growth, as well as pyocyanin and pyoverdine production (p . > 0.05).
