Title of Invention

A CYCLIC LIPOPOLYPEPTIDE OF BACILLUS SUBTILIS SSP SUBTILIS (VCRC B4721) WITH POTENTIAL TO KILL MOSQUITO STAGES"

Abstract

This invention relates to a product from a bacterium, Bacillus subtilis ssp. subtilis (VCRC B471) isolated from soil samples, useful for controlling mosquitoes by killing their immature stages and adults. The product is obtained based on subculturing of the bacterium on nutrient yeast salt medium agar (NYSMA), inoculating slant (NYSM) culture thus obtained into NYSM broth and incubating for obtaining seed, inoculating NYSM broth with seed thus obtained, followed by centrifuging the culture for separation of bacterial cells and cell free culture medium, separation of the bacterial metabolite from the cell free culture medium by acid precipitation and its lyophilisation. The product thus obtained and/ or made into suitable formulation has the potential to kill mosquito larvae, pupae and adult. The product incorporated into vaporizing mat or liquid vaporizer or aerosol has the potential to kill adult mosquitoes.

Full Text

FIELD OF INVENTION This invention relates to a mosquitocidal product developed from the metabolite(s) present in the cell free culture filtrate of a bacterium, B. subtilis ssp. subttlis (VCRC B-471). Bacterial agents are being used for the control of mosquitoes. However, all the bacterial agents known so far, except for Pseudomonas fluorescens (a non spore forming bacterium), kill only the larval stages of mosquitoes. The present product from the bacterium kills pupal (a non-feeding stage) as well as adult stages, in addition to the larval stages of mosquitoes. BACKGROUND OF THE INVENTION Even after many decades of using insecticides, mosquito transmitted diseases such as malaria, filariasis, dengue and viral encephalitis are not controlled effectively because of the ability of the mosquito vectors to adapt to new environment by developing resistance towards insecticides applied in public health programmes. In order to overcome these problems, Bacillus thuringiensis var. israelensis and Bacillus sphaericus are being used as biolarvicides for the control of vector mosquitoes. However, there are some reports of resistance development among the vector population to biolarvicides such as B. sphaericus also. It has become important to search for newer bacterial agents with wide spectrum of activity against various stages of mosquitoes. The present product has got the potential towards achieving this goal and the product is from the metabolite secreted extracellularly by the bacterium, Bacillus subtilis ssp. subtilis, which is to be separated and processed for mosquito larvicidal, pupicidal and adulticidal activity. Since the product kills adult mosquitoes it could be considered for use as a personal protection measure in the form of vaporizing mat, liquid vapourizers and insecticidal aerosols to protect humans against mosquito vector. PRIOR ART Bacillus thuringiensis ssp. israelensis (Bti) is a gram-positive spore forming bacterium, producing insecticidal protein toxins (endotoxins) during sporulation. The endotoxins are highly toxic to larval stages of mosquitoes. Bti has been studied in the laboratory and in the field against different species of Culex, Anopheles and Aedes and proved to be a larvicidal biocontrol agent and not effective against pupae. Bacillus sphaericus (Bs) is gram-positive spore forming bacterium, producing insecticidal protein toxins during sporulation. The target spectrum of Bs is restricted to mosquitoes, and its highest activity is against Culex and certain Anopheles species (Balaraman et al., 1987). This biocontrol agent is also active against larvae and not against pupae. Recently, two B. subtilis strains active against third instar larvae of Cx. quinquefasciatus has been reported (Das and Mukherjee, 2006). The mosquito larvicidal activity was reported to be by the cyclic lipopeptides (CLPs) secreted by them. For the past 30 years the term "biological control" pertaining to mosquito was restricted to the control of larval stages of mosquitoes. The first bacterium known to exhibit mosquito pupicidal activity, through its metabolite, is a gram-negative bacterium, Pseudomonas fluorescens isolated indigenously. A formulation was developed from the metabolite(s) of P. fluorescens Migula strain (VCRC B426) and tested against 4th-instar larvae and pupae of three species of vector mosquitoes viz. An. stephensi Liston, Cx. quinquefasciatus Say and Ae. aegypti (L). The larvae and pupae of An. stephensi were found to be the most susceptible followed by those of Cx quinquefasciatus and Ae. aegypti, and the dosage requirement for pupal mortality was less than that required for larval mortality. The LCso dosage requirements for larvae of these mosquito species were, respectively, 70.4, 511.5 and 757.3 ug protein ml(-1), whereas for pupae they were, respectively, 2.0, 9.4 and 19.2 ug protein ml(-1) The lethal fraction was purified from the culture broth and its molecular mass, as determined by high performance liquid chromatography, was found to be 44 kDa (Prabakaran et al., 2003) SUMMARY OF THE INVENTION The present invention relates to a metabolite secreted extra-cellularly by an indigenously isolated bacterium, Bacillus subtilis ssp. subtilis. Product from the said extra-cellular metabolite has the ability to kill the larval, pupal (a non-feeding stage) and adult stages of mosquitoes. The method involves growing the bacterium in nutrient yeast salt medium broth for 24-72 h at 50-300 rpm and at 25°C-35°C, centrifugation of the culture, and separation of the culture filtrate, precipitating the metabolite present in the culture filtrate by hydrochloric acid treatment, neutralization with NaOH and lyophilizing it to obtain the product and making into larvicidal/pupicidal/adulticidal formulations. STATEMENT OF INVENTION The production strain, Bacillus subtilis ssp. subtilis (VCRC B471) was isolated from soil samples. The product from the extra-cellular metabolite produced by the bacterial strain is useful in controlling the immature and adult stages of mosquitoes. The method of production of the extra-cellular metabolite and its processing to the product and formulated products involves: 1. sub culturing of the bacterium from lyophilized stock culture vials on nutrient yeast salt medium agar plates 2. inoculating loopful of culture in 10 ml of nutrient yeast salt medium broth and incubating for 18-24 h. at 50-300 rpm and at 25°C-35°C. (seed) 3. inoculating 600 ml of nutrient yeast salts medium broth with 1-5% seed culture and incubating as mentioned above for 24—72 h. 4. centrifuging the culture 5. precipitating the metabolites with hydrochloric acid, neutralization with NaOH and lyophilizing it to obtain the product (active ingredient) for mosquito control. This product as active ingredient is incorporated into vaporizing mat, liquid vaporizers and aerosol to use against adult mosquitoes as personal protection measures to prevent mosquito bites and prepared into liquid or wettable powder formulations to kill larval and pupal stages. In detail the process for the product involves the following steps: a. Identification of the bacterial strain through molecular typing using 16S rRNA gene sequence confirmed the production strain as Bacillus subtilis ssp. subtilis (VCRC B471) and the sequence is now available hi GeneBank (Ace. No. DQ 133460) (SEQ.ID.No.1). b. subculturing the bacterial strain, Bacillus subtilis ssp. subtilis on nutrient yeast salts medium agar (NYSMA) slant culture c. inoculating 10 ml of NYSM broth with one standard microbiological loop of NYSM slant culture obtained with step 'b' and incubating on a rotary shaker for 18-24 h. at 50-300 rpm and at 25°C-35°C to obtain seed, d. inoculating 600 ml of NYSM broth with 1-5% seed culture and incubating as mentioned above for 24-72 h to obtain seed for fermentor, e. inoculating NYSM broth in fermentor (60 lit) with 1-5% seed obtained by step (d) and fermentation at 25 - 35° C for 24 - 72 h f. centrifuging the culture obtained by step (e), precipitation of the filtrate of step (f) using hydrocholoric acid, neutralization with NaOH and lyophilization of the metabolite for obtaining the desired product, g. bioassay of the product against larval, pupal and adult stages of mosquitoes such as Cx. quinquefasciatus, An. stephensi and Ae. aegypti, which showed larvicidal, pupicidal and adulticidal activity h. characterization of the product by column chromatography OBJECTS OF THE INVENTION Object of this invention is to provide a novel mosquitocidal product from the bacterium Bacillus subtilis ssp. subtilis for use to kill larval and pupal stages of mosquitoes in breeding habitats and to kill adult mosquitoes in the households. DESCRIPTION OF INVENTION The production strain VCRC B471 was isolated from soil samples and identified as belonging to the Bacillus subtilis group by following standard microscopy and biochemical methods and by molecular typing as described below: 1. Molecular identification of the strain: The bacterial strain was grown on nutrient agar plate for 12 h. A loopful of cells was taken in 0.1 ml of water and the mixture was frozen at -70°C for 20 min. and transferred to boiling water for 10 min. to lyse the cells. The resultant cell lysate was centrifuged and 1µl of the supernatant was used as DNA sample for PCR. The 693 bp fragment corresponding to an internal portion of the "Bacillus subtilis group" 16S rRNA numbering positions 544-1136 were PCR amplified using two oligonucleotide primers, namely BsubSF (SEQ.ID.N0.2) and Bsub3R (SEQ.ID.NO.3). The polymerase chain reaction mixture comprised 3.5 mM MgCh, 200 µM of each dNTP's (Promega, Madison, USA), 0.4 µM of each primer (Metabion, Bangalore), 2.5µl of Taq buffer and 1.25 units of Taq polymerase (AmpliTaq Gold, Applied Biosystems, New Jersey, USA). To this mixture was added 1 µl of the DNA template. The control tube was added with 1 ul deinnised water in place of DNA sample and the reaction mixture in the tubes were made up to 25 ul volume using deionized water. The reaction was amplified in a Biorad Thermal Cycler. PCR conditions were: • denaturation at 94°C for 12 min. • followed by 30 cycles of denaturation at 95°C for 0.5 min • annealing at 65°C for 2 min and extension at 72°C for 2 min • a final extension step at 72°C for 7 min. A portion (8 ul) of each amplification product was checked by electrophoresis on 1.5% agarose gel containing ethidium bromide to verify the size and purity of product. The rest of the sample was purified by loading on to a Qiagen gel elution kit. The purified sample was subjected to sequencing and the DNA sequences obtained were blasted and the sequences were found to belong to Bacillus subtilis 16S rRNA. This confirms the identity of the isolate VCRC B471 as B. subtilis. Sequencing of the 16S rRNA region and construction of the phylogenetic tree using MEGA 3.1 showed that this strain belonged to B. subtilis ssp. subtilis. The sequences have been submitted to Genebank (Accession number: DQ 133460). 2. Bacterial culture and the Product: Flask culture of B.subtilis (VCRC B471): The production strain, Bacillus subtilis VCRC B471was was sub cultured from lyophilized stock cultures on nutrient yeast salt medium agar plates followed by inoculating a loopful of culture in 10 ml of nutrient yeast salt medium broth and incubating for 18-24 h. at 50-300 rpm and at 25°C - 35°C. (seed); inoculating 600 ml of nutrient yeast salts medium broth with 1-5% seed culture and incubating as mentioned above for 24-72 h.; centrifuging the culture, precipitating the metabolites, neutralization with NaOH and lyophilizing it to obtain the product for mosquito control. This product as active ingredient is formulated into vaporizing mat, liquid vaporizers and aerosol to achieve mosquito control. Production of the mosquitocidal product hi fermentor : First Stage Seed: A loopful of culture transferred to 10 ml of NYSM broth taken in a boiling tube and incubated on a rotary shaker at 50-300 rpm at 25-35°C for 18-24 h. Second Stage Seed: One to five ml of first stage seed was transferred to 100 ml of NYSM broth held in 500 ml Erlenmeyer flask and incubated on a rotary shaker at 50-300 rpm at25-35°C forl8-24h. Third Stage Seed: Thirty to fifty ml of second stage seed was transferred to 600 ml of NYSM broth held in 2000 ml Erlenmeyer flask and incubated on a rotary shaker at 50- 300 rpm at 25-3 5°C for 18 - 24h. Production: Sixty liters of production medium (NYSM) was charged into a 100 L Bioreactor and sterilized at 121°C /15psi for 20 min. Then the third stage seed was inoculated at 2-5% level (v/v) and the fermentation was carried out for 24-72 hr under the following conditions: Temperature-25-35°C, pH-6-7.0, dissolved oxygen 10- 40% saturation and agitation 50-300 rpm. Foam was controlled with silicone antifoam. Separation of the mosquitocidal product: Fermentation was stopped when the culture sporulated completely, usually after 24-72 hr. Cell harvesting was carried out by using continuous centrifuge (CEPA). The cell free supernatant was subjected to acid precipitation by using HC1 at 4°C for overnight. Then the precipitate formed was centrifuged at 15000 X g for 20 min and the dark brown precipitate obtained was neutralized with NaOH and lyophilized. The lyophilized powder is the crude compound. Further purification will be done by using Sephadex LH 20 column and Thin layer chromatography. 3. Characteristics of the product: The mosquitocidal metabolite was subjected to IR, NMR, MALDI and LC-MS/MS analysis. The IR spectrum showed a strong band characteristic of peptides at 3385 cm"1 resulting from the N-H stretching mode. At 1651 cm-1, the stretching mode of C=O bond was observed and at 1 507 cm-1 the deformation mode of NH combined with C-N stretching mode occurred and the spectrum is similar to the spectrum of surfactin (Fig. 1). The spectrum obtained with 1H-NMR for the lipoprotein was found to be similar to surfactin (Fig. 2). The MALDI spectrum showed mass of the molecule as 1059 Da (Fig. 3), which is also found to be similar to the mass of surfactin. LC- MS/MS analysis of the mosquitocidal metabolite was found to share the conserved domain of lipoprotein of Bacillus. The matched sequence from the similarity hits was "ALYAALGG". Protein with this sequence is homologous to surfactin molecule. 4. Bioassay of the product: Testing the metabolite(s) for larvicidal/pupicidal activity Fifty numbers of larvae/pupae were released into disposable bioassay cups containing 250 ml of chlorine free tap water. Initially, dosages of 1-100 mg of the lyophilized acid precipitate was added to the bioassay cups and from this rough concentrations, dosages for LC50 determination were fixed. For LC50 determination, 5 dosages were selected such that 2 dosages gave below 50% mortality and 2 dosages yielded above 50% mortality. Each dosage had 4 replicates. After 24 h of treatment, mortality was recorded and the data was analysed by probit regression analysis. The experiment was repeated thrice. 5. Testing of the product for adulticidal activity Vaporizing mat : Fifty numbers of Anopheles stephensi fed females were released into fibre glass chamber of size 45 cm x 30 cm x 45 cm. Vapourising mat impregnated with the active ingredient was inserted into the insect mat machine and was switched on. Mosquitoes were exposed to the vapour for 30 min. and the mat machine was switched and the knock-down effect was recorded by counting the number of mosquitoes falling down to the floor of the cage. Aerosol: Adulticidal activity test was conducted in a Peet Grady Chamber measuring 200 x 200 x 2000 cm. A total of 50 laboratory cultured sucrose fed adult female mosquitoes of Anopheles stephensi were released into the chamber. The product was sprayed into the chamber by using a ULV applicator. The knock down effect was observed after spraying for 2 min. and allowing the mosquitoes to remain inside the chamber for 1 hr. Distinguishing property of the present product B. thuringiensis and B. sphaericus were able to control only the larval stages of mosquitoes. Though they are of biological origin, currently, they are applied to mosquito breeding habitats as spores and crystals. Resistance to B. sphaericus has been reported already. Cyclic lipopeptides of B. subtilis reported by Das & Mulerjee (2006) was effective only against the larval stages of mosquitoes. None of the gram positive mosquitocidal bacteria known till date are reported to produce mosquito pupicidai and adulticida! toxins. The pupicidai bacterium, P. fluorescens reported earlier was a gram negative bacterium and it has got limitations to be used as a biocontroi agent common to any gram negative bacterium. The new agent reported here ie. VCRC B471, is a gram positive, spore forming bacterium. Maintenance of the strain is not difficult since the spores can survive in any adverse condition. The toxin produced by B471 was found to be a surfactin like molecule. The distinguishing property is its activity on all the stages of mosquitoes ie, larvae, pupae & adult. Till date there is no report of any microbial candidate producing mosquito adulticidal compounds. WE CLAIM: 1. A cyclic lipopeptide of Bacillus subtilis subsp. subtilis effective against larva, pupa and adult stages of mosquito. 2. A lipopeptide as claimed in claim 1 , wherein the peptide is 1 059Da by weight. 3. A lipopeptide as claimed in claim 1, wherein the peptide is larvicidal, pupicidal and adulticidal against mosquitoes such as Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti 4. A process for producing the mosquitocidal lipopeptide as claimed in claim 1, comprising the following steps: a. identification of the bacterial strain through molecular typing, as Bacillus subtilis ssp. subtilis (VCRC B471) b. subculruring the bacterial strain, Bacillus subtilis ssp. subtilis on nutrient yeast salts medium agar (N YSMA) slant culture c. inoculating NYSM with culture obtained with step (b) d. Incubating the inoculated NYSM of step (c) on a rotary shaker e. inoculating NYSM in fermentor with seed obtained by step (d) and fermentation at 25 - 35° C for 24 - 72 h f. centrifuging the culture obtained by step (e), g. precipitation of the filtrate of step (f) h. lyophilization of the metabolite of step (g) for obtaining the desired product. i. characterization of the product obtained from step (h) 5. A process as claimed in claim 4, wherein the precipitation of the metabolite is by acidification using hydrochloric acid and neutralization with sodium hydroxide. 6. A process as claimed in claim 4, wherein the lipopeptide is characterized through column chromatography 7. A cyclic lipopeptide of B. subtilis ssp. subtilis effective against larvae, pupae and adult stages of the mosquito and a process for producing the lipopeptide substantially described herewith forgoing description, examples, diagrams

Documents:

544-del-2008-Abstract-(15-01-2013).pdf

544-del-2008-Abstract-(28-08-2014).pdf

544-del-2008-abstract.pdf

544-del-2008-Claims-(15-01-2013).pdf

544-del-2008-Claims-(28-08-2014).pdf

544-del-2008-claims.pdf

544-DEL-2008-Correspondence Others-(14-03-2012).pdf

544-del-2008-Correspondence Others-(19-04-2012).pdf

544-del-2008-Correspondence Others-(28-08-2014).pdf

544-del-2008-Correspondence-others (29-05-2008).pdf

544-del-2008-Correspondence-others-(15-01-2013).pdf

544-del-2008-correspondence-others.pdf

544-del-2008-Description (Complete)-(28-08-2014).pdf

544-del-2008-description (complete).pdf

544-del-2008-drawings.pdf

544-del-2008-form-1.pdf

544-del-2008-Form-18 (29-05-2008).pdf

544-del-2008-form-2.pdf

544-del-2008-form-3.pdf

544-del-2008-form-5.pdf

544-del-2008-GPA-(19-04-2012).pdf