5/20/2019

Interference With Cell-to-cell Signaling: a Potential Therapeutic Approach Against Vibrio Species 

Vijaya baskar .P 1* AND Veera ravi.A 2 1. Department of biotechnology Dr. G.R.D.C.S, Coimbatore. 2. Department of biotechnology, Alagappa University, karaikudi.


*Corresponding author e-mail: vijay10bas@yahoo.co.in INTRODUCTION We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread mortalities and microbes that initiate fouling of host surfaces (Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, 'reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).
 




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Mearns - Spragg, A., K. G. Boyd, and M. O. Hubble. 1997. Antibiotics from surface associated marine bacteria. Proceedings of the fourth underwater science symposium. The society for underwater Technology, London: 147 - 157.

ANTIBIOTIC ASSAYS Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37??C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

Bassler, B.L.1999. Curr.Opin.Microbiol.2: 582-587.* Bernan, V. S., M. Greenstein, and W. M. Maiese. 1997. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43: 57 - 89.

Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

Korber, D. R., J. R.Lawrence, H. M. Lappin-scott, and J. W. Costerton. 1995. Growth of microorganisms on surfaces. In. Microbial. Biofilm, Lapp in - Scott. Cambridge, UK: Cambridge university press.

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/BrB-), (GcC+/GcC) and (SA+/SA-)

illaud, A. C, and B. Austin. 1990. Inhibition of the fish pathogen, Serratia liquefaciens by an antibiotic producing isolates of planococcus recovered from sea water. Journal of

Lemos, M. L, A. E. Toranzod, and L. J. Barja. 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecology. 11: 149-163.

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

Rheinheimer, G.1992.Aquatic microbiology (Wiley, New York), 3rd Ed. Stead, P., B. A. M. Rudd, H. Bradshaw, D. Nobel, and M. J. Dawson. 1996. Induction of phenazine biosynthesis in cultures Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone. FEMS Microbiology Letters 140: 15-22.

MATERIALS AND METHODS: SAMPLE COLLECTION: Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

Ines, M. M., B. Karaigher, U. Cepon, and I. Mahne. 2003. Variability of the Quorum sensing system in natural isolates of Bacillus sp., Food technol. Biotechnol. 41: 23 - 28.

Hypnea musiformis 1. BrA+ 2. BrB+ 3. BrC+ 4. BrD+ 5. BrE+ 6. BrF+ BrA- BrB- BrC- BrD- BrE- BrF- Br A+ /Br A-

Holmstrom, C, and S. Kjelleberg. 1994. The effect of external biological factors on, settlement of marine invertebrates and new antifouling technologies. Biofouling. 8: 147-160.

Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

Microdictyon tenunis Chlorophyceae Chondrococcus hornemonii Chlorophyceae Enteromorpha intestinalis Chlorophyceae Caulerpa cupressoides Chlorophyceae

Seaweeds itself secretes secondary metabolites to prevent fouling and grazing. In addition to that epibiotic bacteria on macro algae can also produce antifouling compounds that work in concert with the seaweed derived compounds to protect the seaweed surface. Recent studies have highlighted important roles of epibiotic bacteria colonizing the surface of seaweeds and releasing antifouling compounds. For the past 50 years antibiotics have revolutionized medicine by providing cures for formerly life threatening diseases. However, strains of bacteria have recently emerged that are virtually unresponsive to antibiotics such multidrug resistance, arising mainly through antibiotic misuse, is now recognized as a global health problem. In this situation, it is clear that new classes of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites that display antibacterial properties. The first antibiotic from a marine bacterium was identified and characterized in 1966. In addition, bacteria in biofilm on the surface of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Burgess, et al., 1999). Marine epibiotic bacteria, associated with nutrient-rich algal surfaces have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors. Recently a lot new novel antibiotics such as Phenazine, thiomarinol, phenazine-1-carboxylic acid, 1-hydroxyphenazine 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one pyolipic, loloatins, agrochelin, sesbanimides, pelagiomicins, indomycione and indomycione have been identified from various marine epibiotic bacterial organisms. In particular, some species of the genus Pseudomonas produce both antibiotics and several other bioactive substances. For example, Pseudoalteromonas rubra and Pseudoalteromonas aurantia have been reported to be antibiotic producing bacteria. The phenomenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). A study of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% whereas that from sea water was only a few percent. In addition, some bacteria that previously did not produce any active compounds have been found to be producing such metabolites when they are exposed to other bacterial species or extra cellular chemical from other bacteria. Bacteria may also produce antimicrobial compounds when they sense the presence of competing organisms. However, few attempts have been made to study such chemical communication between different bacterial species or how this might affect. The secretion of antimicrobial compounds (Mearns-Spragg, et al., 1998). Bacterial communication by the chemical signals for specific function is simply known as Quorum sensing. In which a bacterial population receives input from the environment and elicits an appropriate response (Hiroaki and Kristina. 2003). The term "quorum sensing" describes the ability of a microorganism to perceive and response to diffusible signal molecules. Bacterial cells sense their population density through a sophisticated cell to cell communication system and trigger expression of particular genes. Tne first system of density-dependent regulation was studied in detail with the luminescence of Photobacterium fischeri (formerly known as Vibrio fischeri) by Bassler et al., 1997. Eventually, they discovered that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamid or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible the agent in the broth that induced luminescence. Followed by this many researchers have confirmed that in Gram negative bacteria acyl-homoserine lactone is responsible for the cell to cell communication system.

Vandevivere, P., and D. L. Kirchman.1993. Attachment stimulates exopolysaccharide synthesis by a bacterium. Appl. Environ. Microbiol. 59: 3280-3286.

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We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.

U1+, U2+, U3+, U4+ Ulva lactuca SA+ Sediment INDUCER STRAIN BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

GcD- GcE- GcF- NIL NIL 26 NIL NIL NIL Ulva lactuca 13. U1+ 14: U2+ 15. U3+ 16. U4+ U1-

Burgess, J. G., E. M. Jordan, M. Bregu, and A. Mearns-spragg. 1999. Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70: 27-32.

Burgess, J. G., E. M. Jordan, M. Bregu, and A. Mearns-spragg. 1999. Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70: 27-32.

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

All the 17 strains were named as PRODUCERS STAINS BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

Bainton, N. J., P. Stead, and S.R. Chhabra. 1992. N-(3-oxohexanoyl)-L-homoserine Lactone regulated carbapenem antiobiotic production in Erwinia carotovora. Biochem. J. 288: 997-1004.

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing - controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I - generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199. Msadek, T. 1999. When the going gets tough: survival strategies and environmental signaling network in Bacillus subtilis. Trends Microbiol. 7: 201-207.

Trischman, J.A., D.M.Tapiolas, P.R.Jensen, R.Dwight, W.Fenical, T.C.Mckee, C.M.Ireland, T.J.Stout, J.CIarely. 1994. Salinamide - A and salinamide - B - anti -inflammatory depsipeptides from a marine streptomycete. J.Am.Chem.Soc. 116: 757 -758.

Burgess, J. G., M. Elizabeth, M. Bregu, A. Meams-Sprugg, and K. G. Boyd. 1999 Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70. 27-32.

NIL NIL NIL NIL Gracillaria edulis 7. GcA+ 8. GcB+ 9. GcC+ 10. GcD+ 11. GcE+ 12. GcF+ GcA- GcB-

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a) Among these 17 producers strain 6 strains were isolated from Hypnea musiformis. 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from Sediment.

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing - controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I - generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

Patterson, G. L., and C. Ml. Bolis. 1997. Fungal cell - wall poly sacchraides elicit an antifungal secondary metabolite in the Cyanobacterium Scytonema ocellatum. J. Phycology. 33: 54 - 60.

All the above mentioned biochemical tests were performed by following standard methodology given in the Microbiological Laboratory Manual by James 3.Cappuccino (1999).

Evelyn, A., Y. Liming, K.G. Boyd, P. C. Wright, and J. G. Burgess. 2001. The symbiotic role of marine microbes on living surfaces. Hydrobiologia. 461: 37 - 40

Boyd, K.G., A. Mearns -Soragg, G. Briedley, K. Hatzidimitriou, A. Rennie, M. Bregu, M. 0. Hubble, and J. G. Burgess. 1998. Antifouling potential of epiphytic marine bacteria from the surface of marine algae. Plouzane, France: 128 -136.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

GcC- GcD- GcE- GcF- Gc A+/GcA- Gc B+/GcB- Gc C+/GcC- Gc D+/GcD- Gc E+ /GcE- Gc F+/GcF- GcA- GcB- GcC-

U1+, U2+, U3+, U4+ Ulva lactuca SA+ Sediment INDUCER STRAIN BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

A. Live cells of producer and inducer strains B. Live cells of producer strain alone C. Live cells of inducer strain alone In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

Liming, Y., K. G. Boyd, and D. R. Adams. 2003. Biofilm specific cross species induction of antimicrobial compounds in Bacilli. AppI.Envi.Microbiol, 69:3719-3727.

The test organisms Vibrio species were isolated from seaweed as epiphyles, biofilm, sediment and puffer fish by using TCBS medium (Hi media) The pathogenic bacteria were collected from clinical laboratories.

Meains - Spragg, A., M. Bregu, K. G. Boyd, and J. G. Burgess. 1998. Cross species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria. Letters in Applied Microbiology. 27: 142-146.

EXPERIMENT NUMBER 2 In this experiment, culture supernatant was obtained as per the procedure given in the experiment 1. 50ml of supernatant was mixed with equal volume of 80% methanol and 1% acetic acid mixture and it was shaked thoroughly in a separating funnel. Finally the methanol and acetic acid fractions were collected and concentrated by evaporation using water bath at 55??C. The viscous colloidal residues were resuspended in 600 microlitre of 50% methanol and it was used for antibacterial assay against different test organism.


Zhang, L., P. J. Murphy, and A. Kerr. 1993. Agrobacterium conjugation and gene regulation by /V-acyl-L-homoserine lactones. Nature 362: 446 - 448.



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