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Interference with cell signaling, cell: a potential therapeutic approach against Vibrio species

Vijaya Baskar. Veera P 1 * 2 ravi.A

1. Dr. GRDC Department of Biotechnology, Coimbatore.
2. Department of Biotechnology, Alagappa University, Karaikudi.

ABSTRACT
Bacterial biofilms are sessile communities at high cell density that are ubiquitous in natural, medical, and engineering environments, which are fascinating because they are primitive tissue with a chemical advanced communications network. Currently, there are a number of explosives biofilm research, mostly with the ultimate goal of preventing biofilm control or eradication.The this research was to study cell signaling in principle, algae epibionts bacterial organisms, 54 bacterial isolates were conducted on 20 species of algae. Among the 54 isolates, including 17 producers, some 17 of them still inducing strains of 20 isolates were normal with no signs of activity. The 17 producers and 17 strains were subjected to cross-species induction of induction by quorum sensing principle and found that only three of them responded induction of production among species of antibacterial compounds against strains respective inducer. The three producers and inducer have been identified through analysis biochemical and surprising that all three producing strains belonging to the genus Pseudomonas and tensions have lead belongs to the genus Vibrio. Floating obtain a mixed culture Pseudomonas and Vibrio shows antibiotic activity against various species of Vibrio have been isolated from other sources. As Vibrio cholerae Vibrio main movie sediments epibiotics algae. The results clearly showed the Pseudomonas including algae have been fouling potential production capacity consisting of antibiotics against a wide range of species of Vibrio by quorum sensing.

* Author for correspondence by e-mail: vijay10bas@yahoo.co.in

INTRODUCTION
We live in a microbial world. About 71% of the surface of this planet is covered by seawater One milliliter of seawater contains 103 typical cells by fungi, 106 bacteria, viruses and 107 including pathogens that cause global mortality and microbes that activate the surface fouling of the Interior (Rheinheimer, 1992). Thus, plants and marine animals are constantly exposed to high concentrations of potentially dangerous microbes. These micro-organisms in nature are also planktonic life free lifestyle in sea water can exist as organisms that live on surfaces and non-living epibionts. Among living organisms, algae and invertebrates that serve as substrate for the development of algae fouling organisms are known to release a large amount of organic carbon in the environment that provide a habitat rich in nutrients for microorganisms such as bacteria. The bacteria are usually considered independent of unicellular organisms. A cell performs all the tasks of feeding, locomotion, reproduction, respiration and other processes required to maintain a living organism. There are several kinds of bacteria such as movies Elementary forming bacteria, the bacteria in the sediment, symbiotic bacteria and bacteria fouling in various aquatic organisms. Environment sea surface is a site of intense composition of living space by a variety of organisms. The bacteria are generally recognized as primary colonizers of this habitat and are capable of forming a biofilm on the surface and marine invertebrates and algae (Bryers, et al., 1982). The bacteria also can be abundant on the surface of some algae as epibionts important organization. In many cases, people specific bacteria found, with the changes that occur throughout the year or the life of algae from the surface. This symbiotic relationship algae-bacteria is in most cases, the bacteria in the work of algae fouling a protective role through the release of secondary metabolites in the surrounding seawater to help prevent clogging of the extent of surface. fouling bacteria are attracting attention as a source of new natural products. The bacteria in the larvae certain crustaceans protects against fungal infections by producing antimicrobial compounds simple. Bacteria isolated from the tunicate surface prevented an agreement larvae of barnacles and tunicates exposed to biofilm bacteria in Petri (Evelyn et al., 2001).
The algae secrete secondary metabolites to prevent dirt and grazing. Also fouling bacteria in macro-algae can also produce antifouling compounds that work together with the compounds derived from seaweed to protect the surface of algae. Studies recently highlighted an important role epibiont bacteria colonizing the surface of algae and composed of free anti-fouling. For antibiotics in recent years 50 have revolutionized medicine, providing cures for life-threatening illnesses in the past. However, bacterial strains that have emerged recently are almost immune to antibiotics such as multidrug resistance, derived mainly through the misuse of antibiotics, is now recognized as a problem global health. In this situation, it is clear that new kinds of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites showing antibacterial properties. The first antibiotic from a marine bacterium has been identified and characterized in 1966. In addition, bacteria in a biofilm on the surface marine organisms have been documented to contain a greater proportion of bacteria to antibiotics that some producers in other marine environments (Burgess et al., 1999). fouling marine bacteria associated with an area of nutrient-rich algae have also shown that the antibacterial secondary metabolites that prevent competitors solution potential. Recently, many of the antibiotics of a new novel, such as phenazine, thiomarinol acid, phenazine-1-carboxylic 1-2-n-hydroxyphenazine heptylquinol-4-one, 2-n-4-one, pyolipic nonylquinol, loloatins, agrochelin, sesbanimides, indomycione indomycione pelagiomicins and have been identified in various bacteria marine fouling organisms. In particular, Pseudomonas species may produce antibiotics and other bioactive substances. For example, Pseudoalteromonas rubra Pseudoalteromonas aurantia and have been reported in bacteria to produce antibiotics. The phenomenon of higher agencies using its associated microflora to produce beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). Study bacteria isolated from the surfaces of algae indicates that the incidence of this habitat-producing strains was 20%, while that of sea water was only a small percentage. In addition, some bacteria that previously did not produce all the active compounds were found in the production of metabolites such when exposed other bacterial species in extracellular chemical or other bacteria. Bacteria can also produce antimicrobial compounds when they feel the presence of competition agencies. But few attempts have been made to study chemical communication among different species of bacteria or how it might affect. Secretion of antimicrobial compounds (Al-Mearns Spragg et al., 1998). bacterial communication by chemical signals specific function is simply called quorum sensing. In a population bacterial receives information from the environment and creates an appropriate response (Hiroaki and Kristina. 2003). The term "quorum sensing" describes the ability of a microorganism to perceive and respond to the diffusible signal molecules. Bacterial cells sense their population density through a sophisticated mobile communication system cell and activate the expression of specific genes. Tne first set of density-dependent regulation has been studied in detail with the luminescence of Photobacterium fischeri (Formerly known as Vibrio fischeri name) by Bassler et al., 1997. Finally, they found that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamide or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible for the induced luminescence agent in the broth. Following this large number of researchers have confirmed that in Gram-negative acyl-homoserine lactone system is responsible for communication between cells.
In Gram-positive bacteria and the molecules of peptides derived from the signal peptide seems be the main mode of communication. High cell density in marine bacteria can produce enzymes, surfactants, toxins, antibiotics and communication signals chemistry. fouling marine bacteria are also known to produce compounds active against drug-resistant pathogens to the hospital by the induction method of the cross of the same species. On the basis of tests described by a process of Austin (Austin and Billaud 1990) test has been developed in which marine bacteria are challenged when exposed to bacteria on Earth before assaying antimicrobial compounds. Thus, in this present investigation aims to explore the possibility of algae fouling organism bacteria to produce compounds for bacterial quorum sensing.

MATERIALS AND METHODS:

Specimen Collection:

Algae samples were collected in the Gulf of Mannar Marine Biosphere Reserve and identified to species level by CMFRI newsletter (14) as follows:

Table 1. List of species of algae collected in this study

NAME OF SPECIES
Halimeda gracilis Chlorophyceae
Ulva lactuca Chlorophyceae
Chlorophyceae tenunis Microdictyon
Chlorophyceae hornemonii Chondrococcus
Chlorophyceae Enteromorpha intestinalis
Caulerpa cupressoides Chlorophyceae
Caulerpa racemosa Chlorophyceae
Dictyota Phaeophyceae dichotoma
Turbinaria ornata Phaeophyceae
Padina gymnospora Phaeophyceae
Sargassum cinearifolium Phaeophyceae
Phaeophyceae batryensis Dictyota
MSF Sargassum Phaeophyceae
Hypnea musciformis Rhodophyceae
Dendroides Acanthophora Rhodophyceae
Jania rubens Rhodophyceae
Hypnea valentiae Rhodophyceae
Hypnea pannosa Rhodophyceae
Hypnea ESPERI Rhodophyceae
Spicifera Acanthophora Rhodophyceae

Isolation of epiphytic bacteria
Algae samples were washed carefully with sterile water to remove bacteria from low flow / particles. algal fronds were washed with sterile swabs for bacterial epiphytes. Epiphytes body swab bacteria were inoculated in a sterile peptone broth (50% seawater) and incubated at 28 ° C in an incubated shaker (220 rpm) overnight. After the period incubation, the cultures were diluted in series to a 10-8 rich concentration and 200 microliters of each diluted sample is transferred to nutrient agar plate (50% water sea). The plates were incubated at 28 ° C for 5 days and overcrowded plates with colonies were selected. In the crowded plates these colonies, which showed the signal inhibition zone around the margins to the nearby village have been selected and considered the producer strain. The nearby settlements were treated as sensitive induction of the strain. Both producers and stumps were removed several times to induce obtain a pure culture. The pure culture have been properly labeled and subjected a quorum-sensing analysis.

Quorum sensing
NUMBER experience 1
In this study, the producer and the strains were a reaction inductor cross namely the production of antibiotic compounds by quorum sensing. Fully three groups of cultures were maintained as follows (with a witness).

A. Living cells of the producer and inducer strains
B. Living cells of strain that produce only
C. Living cells of strain induced only

In the culture system A 200ul of 16 hours old broth culture of the producer and inducer strains were added to 15 ml of nutrient broth.

In the system culture of strain B 200ul 16 hours old producer was inoculated alone.

In the culture system 200ul C 16 hours strain was inoculated alone induce age.

All Cultures were incubated at 28 ° C for 5 days. After the period of incubation, the cultures were centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and subjected to test bacterial strain with the respective inducer.

Experiment 2 NUMBER
In this experiment, culture supernatant was obtained by the procedure described in Experiment 1. 50 ml of supernatant was mixed with an equal volume of 80% methanol and 1% acetic acid mixture and stir well to a file. Finally, methanol and acetic acid fractions were collected and concentrated by evaporation in a water bath at 55 ° C. The residues were resuspended in 600 microliters viscous colloidal 50% methanol and used for antibacterial dosing of the test against different.

Test organisms:
1. Vibrio epiphytic algae
2. Primary Vibrio Film
3. Vibrio sediment
4. pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella sp. and Proteus

Agencies tests have been Vibrio species isolated from epiphyles algae, biofilm, sediment and fish using a balloon-TCBS (Media Hi) pathogenic bacteria collected in the clinical laboratories.

ANTIBIOTIC TRIALS
antibiotic activity was performed in duplicate method that uses a standard paper disc assay media also. And 10mm diameter test wells have been made on marine agar plates and the plates were buffered with 16 hours old stump inducer. For these wells 200ul of the cell-free supernatant were added to each well. In the test drives Watmann paper filter paper No. 1 (6 mm in diameter) were saturated with 200ul of the cell-free supernatant. Dlaced discs were impregnated in the center of the plate printed with the test organisms. The plates are incubated at 37 ° C overnight to observe the zone of inhibition. The zone of inhibition was measured as the distance from the edge of the paper disk on the edge of the clear zone and expressed in mm.

Of bacterial identification
Organizations have responded to the single quorum sensing processes have been identified by biochemical analysis below.

Colony morphology, Gram stain, motility test, oxidase test, catalase test, indole production, methyl red test, Voges Proskauer test citrate use test, triple sugar iron test nitrate reduction test, lactose fermentation, urease test
test starch hydrolysis, protein hydrolysis test, test for hydrolysis of lipids, oxidation / fermentation test, the concentration of salt (0%, 3%, 5% 7%, 10%), TCBS, growth temperature, 42 ° C and 47 ° C

All of the above biochemical tests were carried out following the standard methodology contained in the microbiological laboratory Manual 3.Cappuccino James (1999).

Results and discussion:

Quorum Sensing / CROSS SPECIES ANALYSIS INDUCTION
In this research isolates were obtained totally 54 species of algae. Among the 54 strains, 17 are forced to producers, the rest are 17 induction of other ethnic groups 20 isolates is normal and shows no sign of activity (Table.2).

a) Among these 17 producers strain 6 strains were isolated from musiformis Hypnea. Edulis Gracillaria 6, 4 and 1 of sediments Ulva lactuca.
b) These strains 17-6 lead musiformis strains were isolated from Hypnea, 6 edulis Gracillaria, 4 and 1 of sediments Ulva lactuca.

The 17 strains were named

STAIN OF PRODUCERS
Bra + + + BRB BRC, + BRD, BRE + MRB + musiformis Hypnea
SCM + + Gcc + GCB, GCD +, + + GCF edulis Gracillaria CME
U1 + U2 + U3 + U4 + Ulva lactuca
SA + sediment

Inductor STRAIN
Bra-,-BRB BRC, BRD, Bre-FRC-musiformis Hypnea
ACG, BCG-gcc-GCD-CME-GCF-Gracillaria edulis
U1, U2, U3, U4, Ulva lactuca
SA-sediments

In this experiment 17 producers and the inductor voltages only 3 of them responded to quorum sensing principle. (+ BRB / BRB "), (GCC + / GCC) and (+ SA / SA-)
Table 2: Results of analysis of quorum sensing in bacteria isolated from algal epibionts.
algal sample group of producers Inductor Cross inductor agency producer species supernatant Cross-species test with inductor Area Clearance (mm)
Hypnea musiformis
1. Bra +
2. BRB +
3. BRC +
4. BRD +
5. BRE +
6. Bra-MRB +
BRB-
BRC-
BRD-
Bre-
MRB-Br A + / A-Br
H. B + / B-Br
H. C + / C-Br
Br J + / D-Br
E + H. / Br-E
Br + F / H F-Bra-
B-Br
BRC-
BRD-
Bre-
BRF-NIL
39
NIL
NIL
NIL
NIL
Gracillaria
edulis 7. SCM +
8. BCG +
9. GCC +
10. GCD +
11. CME +
12. GCA-GCF +
GCB-
GCC-
GCD
CME-
GCF GC-A + / GCA-
Gc B + /-GCB
C + gc / gcc "
D + gc / GCD
E + GC / CME
Gc + F / GCF-GCA-
GCB-
GCC-
GCD
CME-
GCF-NIL
NIL
26
NIL
NIL
NIL
Ulva lactuca
13. U1 +
14: U2 +
15. + U3
16. U1-U4 +
U2-
U3
U4-U1 + /-U1
U2 + /-U2
U3 + /-U3
U4 + /-U4-U1
U2-
U3
U4-NIL
NIL
NIL
NIL
Sediment 17. SA + SA-SA + / SA-SA-28

c) The normal bacteria isolated 20 strains of 20 species of seaweed were crossed with bacteria land such as E. coli, Staphylococcus aureus separately

This experiment showed no inhibition zones

bacterial identification
Producer strains 3 and 3 said that induce quorum sensing principles have only been subjected to biochemical tests for identification. The results revealed all-producing Pseudomonas strains showed sings SPS and inducer strains showed signs of Vibrio MSF. Thus, according to the results of all strains producers appears to be a Pseudomonas MSF, while all the tension inducing belongs to the genus Vibrio.

In this research, it aims to produce a quorum algal epibionts antibiotics detection principle. The bacteria isolated from algal epibionts were identified as Pseudomonas and Vibrio species of algae and musiformis edulis Gracillaria Hypnea. In this study, Pseudomonas strain acts as a producer and as an inducer Vibrio strain. Recent findings indicate that algal epibionts with the potential to control metabolic activity of competition authorities. Allison and others. 1998 reported that many strains of bacteria in the attachment to a surface to produce exopolysaccharides or exopolypeptides. In addition, it has been suggested that mediation can exopolysaccharides of bacterial adhesion to the surface and induce metabolic changes.

The results suggest Vanderivere and Kirchman 1993 that the addition of larger areas by adding sand will result in exopolymer synthesis by cell system for high-density dependent. Similarly bacterial organisms attached to the surface of algae shows the change in gene expression may be due to the reaction, the highly competitive environment. When the cell density increases competition for space and nutrients are also increased. Thus, existing bacteria have been forced to protect yourself in this competitive environment. Normally in this condition bacteria are activated to induce the expression of certain genes hidden in the genetic material by quorum sensing. Detection quorum is active principles were written (autoinducer) of the bacterial cell to promote the expression of a particular gene of the bacteria hidden in a state another.

Quorum sensing is usually focused on bacteria growing in a homogenous environment. However, few studies have attempted to study this principle in a heterogeneous environment as well. In this research, we tried to study both homogeneous and heterogeneous environments. In the above we have isolated the eipbionts strain producing algae and shows inhibitory activity against the organism to induce them fouling algae. Later producing strains of algae epibionts, were treated by different Vibrio organisms different environment. The results of this study shows that the producer of pressure, are able to secrete antibiotic compounds, not only in their competing natural resources in their habitat, but also for pathogens that reside in a remote environment related.

In Gram-negative AHSL is an active ingredient Detection of quorum. Our producer is also the strain was identified as Pseudomonas MSF. Thus, in these organizations is also active AHSL needs. The cell-cell signaling mechanism may require the import of the signal and interactions with intracellular effectors or signal transducer system two components of information across the membrane. In V. harveyi density DNA detection device has two independent detection system density, and each is composed of a couple of cars inductive sensors, a system consists of sensors I and -1 The system consists of two sensors 2 and IA-2. The two densities – detection system is redundant, since a null mutation in both systems only the results of the hidden gene expression (Bassler, et al., 1999.).

The earlier Genetic analysis revealed the Pseudomonas Pseudomonas consist of two quorum sensing systems Las Rh1R R1-I and L and R are linked to the genes and I, and more Lux R homologue third recent advances to a cluster quorum sensing – controlled genes (QSC) were detected. The R is a transcriptional regulator that responds mainly to the I The – Rh1R generated signal and regulate transcription is more responsive to the signal generated by the BSR. In Pseudomonas Aurigin, low population density Las give me a level of Basel 3-O-C12-HSL. As density increases, 3-0 C12-HSL is based on a critical concentration, which interacts with Lasro. The complex R-3-0-012-HSL activates transcription of a number of genes [Whileley, et al., 1999].

We suggest that the top of these mechanisms in Pseudomonas quorum sensing in principle could occur in this study as well. This leads to the bacteria Pseudomonas fouling on algae that secrete compounds active against certain species of Vibrio competitor.

In this paper, the 54 fully isolates were selected from 20 different species of algae, 34 species have been showing signs of quorum sensing or 17 producers and 17 strains of inducer, but when these agencies the issue of quorum sensing principle in mixed culture responded only 3. Thus, this study reveals about 17% of the bacterial species isolated from algae and sediment have responded to quorum sensing. According to the results bacteria isolated from the surfaces of marine algae indicate that the incidence of strains producing this habitat has been 20%, while that of sea water was only a small percentage. In the present study also reveals the more or less the same Pseudomonas spp. Was observed. Our results also show a result of Kell et al. 1995, Stead et al., 1996, said the MSF Pseudomonas culture supernatant known to contain AHL induces the production of phenazine antibiotics. In this study for lack of time, have not attempted to identify the active compound secreted by Pseudomonas quorum sensing, which may leave room for other intensive investigations in the future.

To conclude this debate, quorum sensing is more widely distributed in the bacterial population, and was previously thought (in Gram-positive, Gram negative bacterial communication). ongoing trials for antimicrobial activities are inadequate because some bacteria Antibiotic production may require presence of other bacterial species. The results have important implications for the discovery of new antimicrobial compounds from marine bacteria and may allow the development new methods of detection of new compounds active against multidrug-resistant bacteria.

CONCLUSION:

The present study was quorum sensing principle among bacterial organisms fouling algae. In recent decades there were no new findings of a new class of antibacterial compounds were identified. However, pathogens are much higher rates of development, including antibiotic-resistant potential. So There is an urgent need Discover the new novel antibiotic compounds. Its inhabitants, like the marine microorganisms, algae, invertebrates, etc, act as a source of undepleted the wide range of natural products, including algae act as a potential source of antibiotic compounds. At present, the induction of intra-species / Total quorum sensing attracts the attention of researchers in the search for new drugs against multiple new micro-organisms. Thus, this study is to determine the ability of bacterial organisms fouling algae to produce new drugs against animal and plant pathogens

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About the Author

P.VIJAYA BASKAR
PHD SCHOLAR
DEPT OF BIOTECHNOLOGY
DR.G.R.D.C.S
COIMBATORE

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