Title of Invention	A METHOD OF REMOVAL OF PLASMIDS FROM LIVE AND MULTIPLYING PLASMID CONTAINING E. COLI
Abstract	A method of removal of plasmids from live, multiplying plasmid containing bacteria is disclosed. The method comprises treating the bacteria with silver submicronic particles of a predetermined size and concentration under specific conditions.

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FORM-2 THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES 2003 COMPLETE (See section 10 and rule 13) A CULTURE INDEPENDENT METHOD OF REMOVAL OF PLASMIDS FROM LIVE AND MULTIPLYING PLASMID CONTAINING BACTERIA AGHARKAR RESEARCH INSTITUTE OF MAHARASHTRA ASSOCIATION FOR THE CULTIVATION OF SCIENCE a Society registered under the Societies Act of G. G. Agarkar Road, Pune 411 004, Maharashtra, India THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED. This application claims priority from provisional patent application No.1220/MUM/2006. FIELD OF INVENTION This patent relates to a process for removal of plasmids from bacteria . Particularly, this invention relates to the removal of plasmids from live and multiplying bacteria. BACKGROUND OF THE INVENTION Plasmids are autonomous, self-replicating extra-chromosomal DNA molecules that coexist in the host as a stable component. They are not essential for survival of the bacteria, although they represent an important factor in bacterial evolution by enabling the organism to adapt for the changing environmental conditions. Plasmids confer a wide array of phenotypes on the host like ability to resist toxic substances, heavy metals, antibiotics etc. Both intrinsic and extrinsic properties are encoded by plasmids. Conjugation (F plasmid), resistance to antibiotics, metals (R plasmid), degradation of xenobiotics (TOL plasmid), Bacteriocin production, virulence etc are some extrinsic properties; while the genes that constitute the nature of the replicon (viz., ori, rep, cop/rop, par loci) are part of intrinsic properties. 2 Some plasmids undergo spontaneous segregation and deletion. However, some plasmids are stable and can be maintained through successive generations by being pardoned to each daughter cell during cell division. Most bacterial strains contain multiple plasmids, which might create complications and practical problems in genetic and biochemical studies. Plasmid-free strains are better for genetic manipulation. Some of these well-known problems are: (i) Assignment of a plasmid-encoded characteristic to a particular plasmid, especially in cases of simultaneous loss of more than one plasmid molecule at a time. (ii) Isolation and purification of a single plasmid for genetic and molecular characterization. (iii) Molecular cloning and further characterization of recombinant plasmids. (iv) Commercial production of a single plasmid coded gene product without any interfering effects of other plasmids. Secondly, the phenomenon of plasmid curing can be exploited to restrict the horizontal gene transfer and the subsequent emergence of antibiotic resistance in micro-organisms. It has been established that plasmids are very effective at maintaining and spreading drug resistance among bacteria. Plasmids are present in defined copy numbers in bacterial cells, and their replication largely depends on host-encoded factors. 3 The excessive and not always appropriate use of antibiotics in humans and animal feed, bacterial resistance currently constitutes a major public health crisis. The incidence of occurrence of drug resistant strains particularly the opportunistic human pathogens is the leading cause in many nosocomial infections. The World Health Organization (WHO) reported that drug resistant strains of microbes had a negative impact on their fight against tuberculosis, cholera, diarrhea and pneumonia, which together killed more than ten million people worldwide in 1995. Resistance to antimicrobial agents is a heritable trait thus genes encoding the resistance are either chromosomal or extrachromosomal in location. Generally among clinical isolates, chromosomal drug resistance involves changes in cellular structures that make the organism impermeable to the antibiotic or that render the specific target site indifferent to the presence of the drug. Extrachromosomal drug resistance is due to presence of plasmids (R plasmids). R-plasmid mediated resistance usually involves either a decrease in the permeability of the cell or enzymatic inactivation of the inhibitor. Plasmid curing is an irreversible loss of plasmids from the bacterial cell, leading to loss of plasmid-encoded properties. A variety of agents have been reported which increase the frequency of curing either by enhancing the rate of spontaneous segregation and elimination of plasmids or by enhancing the plasmid-less segregant clones. These agents are called curing agents. To date, a number of physical and chemical agents have been reported for their ability to cure various plasmids. 4 Physical agents that promote plasmid loss: 1. Heat e.g. 42° C (temperature non-permissive for growth, normally) 2. UV exposure. Chemical agents that promote plasmid loss: 1. Intercalating dyes such as acriflavine, acridine orange, ethidium bromide and quinacrine inhibits plasmid replication and have been successfully used in curing bacteria of plasmids. 2. Antibiotics such as Coumermycin and novobiocin inhibit DNA gyrase-dependent supercoiling of plasmids. 3. Plasmids containing cells are possibly more sensitive to detergents such as SDS (sodium dodecyl sulphate) because of plasmid-specified pili on cell surface. 4. Inorganic metal chelates/complexes. In a method reported by Sinha (1989), strains of lactic streptococci are grown overnight at 32°C in an unbuffered medium (Ml7) and held at the same temperature for an extended period (for strain C2 in Ml7- broth it was 0.3% at 96 h incubation, whereas under the same conditions it was 68% for strain ML3). Under these conditions of extended incubation period, it was argued that the acid environment induces loss of plasmid DNA of different sizes. Further, on repetition of process of growth followed by extended incubation at 32°C, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. 5 Laxmi and Polasa (1991) report the loss of plasmids pBR322 and pBR329 from Escherichia coli strain HB101, on treatment with cis-dichlorodiamine platinum(II) chloride Cis-DDP, a novel curing agent. The MIC values for these compounds were in the range 200-400 |ag/ml. On treatment subinhibitory concentrations of these compounds, plasmids were cured with 100% frequency. Kunte et al, reported that plumbagin and juglone exhibit high MIC values, eliminate 3 plasmids coding for resistance to Hg and Ag and Nm,Cp,Ct and the curing frequency was 60 to 80%. Phthiocol and lawsone eliminated only one plasmid coding resistance to Nm,Cp and Ct with 20% efficiency. The copper compound of juglone was effective in elimination of same 3 plasmids with a curing frequency of 40 to 60%. Brandi et al, (2000) report the effect of sub-inhibitory concentrations of trovafloxacin, on Escherichia coli cells to maintain three different types of plasmids viz., (pT713, pJEL144 and pRK2) which carry the same resistance gene (ampR). The capability of curing agent was investigated by a number of approaches, including the quantification of the loss of plasmid-borne functions and of plasmid DNA by quantitative PCR. At MIC (15 ng/ml) curing efficiency was 50%. The drug is a recently developed fluoroquinolone molecule, and curing was not due to DNA supercoiling. Mesas et al (2004) reported that when the wild-type strains of Oenococcus oeni were grown in lOmL of MRST in the presence of acriflavine or acridine orange (2.5-80 fig/mL) at 37 °C for 4 weeks, the highest curing agent concentrations that still allowed visibly detectable growth were 2.5 |ng/mL 6 of acriflavine or 20 |ig/mL of acridine orange for O. oeni RSI, and 10 ug/mL of acriflavine or 10 ug/mL of acridine orange for O. oeni RS2. For pRS2 the curing efficiency was 31.2% with acridine orange and 62.5% with acriflavine. In case of pRSl it was 18% with acriflavine. Bharati and Ploasa (1991) reported the susceptibility of broad-host range vector plasmids belonging to Inc PI and Q groups in Escherichia coli to various curing agents. Plumbagin and SDS eliminated RP4 (Inc PI group) plasmid whereas pKT231 (Inc Q) and pRK2013 (having ColEl replicon) were eliminated by hexamine ruthenium (III) chloride, alpha-santonin, coumermycin A 1 and cis-dichloro diamine platinum (II). The curing activity of these agents was specific. Plasmid pKT231 was cured (90%) by alpha-santonin, whereas the curing was 20% in case of pRK2013, the broad host range plasmids in E.coli. Mansi El-Mansi et al (2000) report sodium dodecyl sulphate (SDS) to be very effective in curing the indigenous plasmid from Klebsiella pneumoniae with a relatively high frequency (6.25 XI0-4). Spengler and Molnar (2003) report the positive effect of proton pump inhibitor l-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone on plasmid curing by 9-aminoacridine and two phenothiazines. Doxycycline resistance of tested enteric bacteria was the target of 'curing' in the presence of promethazine and trifluoperazine. Curing % was 10-26 % for promethazine and trifluopiperazine. 7 In another study, Molnar et al (2003) report the antiplasmid effect of promethazine in mixed bacterial cultures. For E.coli K12 LE140 plasmid elimination was 0.3% at 80 mg/1 promethazine at 23°C and 0.22% for S. epidermidis. At 37°C these values were 77.6% for E. Coli and 60.8% for S. epidermidis (promethazine 60 mg/1). The % was highest at 39°C with 51.8 for Bacillus cereus, 40 mg/1 promethazine and 100 and 97% respectively for E. coli and S. epidermidis at 100 and 80 mg/1 promethazine respectively. Cofre and Sanchez-Rivas (1983) report the plasmid curing after protoplast cell wall regeneration in B. subtilis: enhanced curing with multi-plasmid strains, absence of physical loss, and greater plasmid-membrane binding in multi-plasmid strains than in a single plasmid strain. Curing efficiency ranged from 20-55% depending on strain and characteristic tested. US application No. 2003130169 discloses methods of treatments to remove plasmids using a composition containing apramycin, tobramycin, paramomycin I, kanamycin B and derivatives thereof which mimic plasmids in compatibility. Physical plasmid curing agents such as Heat and UV irradiation often give unpredictable results. These techniques may or may not work for removal of the plasmids. Moreover, these physical techniques can not be employed in any therapeutic application as they will be extremely harmful to the recipient. Most of chemicals agents presently being used for plasmid curing or removal are hazardous, toxic and potent mutagens. Thus special care is required while handling and disposal of such chemicals. These chemical 8 agents are sensitive to changes in pH and temperature and may not work under broad range of pHs and temperatures due to denaturation. Further many of the chemicals and other substances used in the prior art processes are expensive, need special storage conditions and are not easily available. Therefore, there is a requirement for an alternative, safe method for curing plasmids. OBJECTS OF THE INVENTION An object of this invention is provide an inexpensive, safe and easy to use substance for complete removal of plasmids from bacteria without causing any side effects to the bacteria or the user handling the substance. DESCRIPTION OF THE INVENTION According to this invention, there is provided a culture independent method of removal of plasmids from live, and multiplying plasmid containing bacteria comprising the following steps:- -Preparing an aqueous first suspension of submicronic silver particles; - estimating the MIC of the silver particles for the bacteria to determine the inhibitory concentration of the particles suspension for the bacteria; - Adding in a reaction vessel, the first suspension and growth medium of the bacteria to obtain a second suspension containing sub-MIC concentration of silver particles; 9 - Introducing the bacteria in the reaction vessel under conditions favouring the multiplication of the bacteria, for 12 to 48 hrs, to obtain subsequent generations of the bacteria; - testing the bacterial generations for absence of plasmids to obtain a generation of plasmid free bacteria; and - separating the plasmid free bacteria from the growth media/silver particles suspension mixture by a known method. Typically, the particle size of silver is in the range of 1 to 999nm and preferably . in the range of 5 to 80nm. Typically, the concentration of silver particles in the first suspension is in the range of about 50 mg/L to about 500 mg/L. Preferably, the concentration of silver particles in the second suspension is in the range of about 0.1 mg/L to about 50 mg/L. In accordance with a preferred embodiment of the invention, the concentration of silver particles in the second suspension is in the range of about 25 to 50 % of the MIC value for the bacteria. Typically, the reaction vessel is shaken during multiplication of the bacteria. Typically, the MIC for the bacteria is estimated by double dilution method. 10 Typically, the testing of the generation is done by gel electrophoresis Typically, the bacteria are Gram negative bacteria Typically, the testing of the generation is done by using at least one antibiotic as a marker Typically, the first suspension is prepared by a method selected from a group of methods consisting of physical, chemical, biological methods and combinations thereof. Preferably, the first suspension contains biostabilized silver submicronic particles. Typically, the bacteria are treated by the second suspension at temperatures ranging from 5 to 90 C, preferably 20 to 45 C. Typically, the bacteria are treated by the second suspension at pH ranging from 1 to 13, preferably 6 to 8. The expression culture in accordance with this invention means a suspension of bacteria in a growth media. The expression culture-independent in accordance with this invention means that the method does not depend on the type of bacteria that is to be treated and the type of plasmid present in the bacteria. For instance, 11 the method can be extended to aerobic, microaerophillic , anaerobic, Gram positive , Gram-negative , Gram-variable bacteria. Similarly, the process can be extended to bacteria containing plasmid of any molecular weight and encoding any trait. Still further, the process can be extended to bacteria that multiply at different pHs and temperatures. Conventional plasmid removing agents are often pH specific and /or temperature specific. The process can be used only for removing plasmids from live bacteria that are capable of multiplying and therefore not suitable for dead or dormant bacteria. The first suspension is prepared by a method selected from a group of methods consisting of physical, chemical, biological methods and combinations thereof. Preferably, the first suspension contains biostabilized silver submicronic particles. Silver nanoparticles preparations used in the present invention were • Extracellularly synthesized silver nanoparticles, biogenic nanoparticles from (yeast MKY 3) i.e. Issatchenkia orientalis • Silver nanoparticles prepared by using plant extracts • Electrically generated silver ions reduced in presence of citrate with glycerol/Polyinyl pyrrolidone as capping agents 12 • Chemically reduced silver (borohydrate as reductant) stabilized by biomolecules (dextran, fungal polysaccharides as capping agent) • Or any other known method. Size of the silver particles is in the range of 1 to 999 nm. Preferably, the particle size of silver is in the range of 5 to 80 nm. Minimum inhibitory concentration (MIC), in microbiology, is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. MICs can be determined by broth dilution methods usually following the guidelines of a reference body such as the CLSI, BSAC or EUCAST. Another, more modern method is the E-test method using strips of a gradient of antibacterial agent concentration. In the present invention a preferable method of estimating MIC of silver particles for a bacterium is the double dilution method in which the suspension of silver particles having known concentration of the particles is diluted serially in the growth medium, each dilution is placed in a different well of a multiwell microtitre plate. The bacteria are allowed to multiply and the minimum dilution at which inhibition of visible bacterial growth occurs is recorded as MIC in terms of silver concentration in mg/1. Thereafter, the concentration of the silver particles suspension is adjusted to bring it below the MIC, typically the concentration in the growth 13 medium is adjusted to 25% to 50% of the MIC to remove plasmids within 12 to 48 hours. The growth media used in accordance with this invention include any known growth media suitable for multiplication of the bacteria from which the plasmid is to be removed and typically includes Luria broth, Nutrient broth, and MacConkey's broth. For carrying out the method in accordance to this invention, the vessels used include multiwell microtitre plates, Erlenmeyer flasks, test tubes, reactors, fermenters and any other types of vessels that can be sterilized using standard microbiological techniques. In the present invention glass tubes are used for carrying out the reaction. Typically Luria broth is taken in the test tubes and the silver particle suspension is mixed therewith to get desired sub-MIC concentration. Bacteria having plasmids are put in the test tubes and the tubes are typically put on a shaker at temperatures ranging preferably from 20 - 45 C. The bacteria are allowed to multiply under these conditions, typically for 12-48 hrs. The subsequent generations of the bacteria are tested for the presence of plasmids. The testing is effected by drawing a sample of the generation, extracting the plasmid DNA by a known DNA extraction method such as alkaline lysis method, lysozyme and boiling method, lithium chloride method well known in the art of molecular biology and loading the extracted DNA on an agarose gel. The DNA loaded agarose 14 gel is subjected to electrophoresis to determine the presence or absence of plasmid band. The reaction in the vessel is terminated as soon as at least two generations of plasmid free bacteria are seen. Thereafter, the plasmid free bacteria are separated from the growth media silver particles suspension mixture by a known technique such as centrifugation, membrane filtration and any other method well known in the art of microbiology. At present it is difficult to pin point the exact mechanism by which plasmid removal is effected by silver particles whereas it has no apparent effect on chromosomal DNA. It is likely that since chromosomal DNA is around 1000 times larger than the plasmid DNA it could be refractile or less sensitive to the action of the silver particles. The probable mechanisms of plasmid removal could be as follows: It is known that certain curing agents induce generation of superoxide anions. These superoxide anions act on plasmid DNA to degrade it physically. In order to find out whether a similar mechanism is responsible for action of silver particles, an assay was carried out to measure superoxide anions after exposure of cells to silver nanoparticles. 1 ml lysate of bacterial cells exposed to silver nanoparticles was added to Nitro Blue Tetrazolium (156mM) & NADH (468mM). Presence of 15 superoxide anions was checked by measuring absorbance at 560 nm after 30 min. Higher levels of the anions were observed for both biogenic nanoparticles and the FP1 capped nanoparticles in comparison to the unexposed control cells. The results signify that superoxide anions generated by silver nanoparticles might be responsible for plasmid removal. Another mechanism could probably involve the inhibition of replication due to binding of positively charged silver ions to the plasmid DNA. During replication of the parent culture, the plasmids get unequally distributed in the daughter cells i.e. the dilution of plasmid from the parent culture occurs eventually leading to loss of plasmid. The silver nanoparticles could induce the formation of constrictions along the breadth of the cell. The cell wall is probably weakened in that region leading to leakage of small molecules such as plasmids that may be attached at the inner membrane near the constricted area eventually leading to their removal. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence. For replication of a plasmid uncoiling is required. Silver nanoparticles due to physical binding at the "origin of replication" probably keep plasmid DNA in the supercoiled form thus preventing its replication. During replication silver nanoparticles could interact with SL I and SLIII of 16 RNA giving a complex which can no longer bind plasmid DNA to continue replication. Due to this further process of replication probably stops. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. The invention will now be described with reference to the following non-limiting examples. Example 1: Preparation of plasmid bearing strains: For curing experiments, plasmid pUC19 (2386 bp) with ampicillin resistance marker was obtained from Bangalore Genei Pvt. Ltd., India. Competent cells of E. coli Nova Blue strain were prepared according to standard protocols using calcium chloride. Plasmid pUC19 was mixed with competent cells and transformation experiment was carried out by heat shock method. The mixtures were plated on Luria agar (LA) plates and LA + ampicillin (100 |ig/ml) plates for scoring the transformants. The transformation frequency was found to be > 90%. Five colonies selected randomly from the ampicillin plates were checked for the presence of plasmid. Plasmid isolation was carried out using rapid isolation method and its presence was checked by performing agarose gel electrophoresis at 50 V for 3 h. Subsequently presence/absence of plasmid DNA was checked by fluorescence in presence of ethidium bromide. It was found that all the 17 five preparations showed plasmid bands corresponding to standard pUC19band. In order to determine the dose required for the curing experiments, it was necessary to first determine the minimum inhibitory concentration (MIC) of silver nanoparticles. One transformant showing presence of pUC19 plasmid band was used for this experiment. The MIC was determined by double dilution technique performed in a 96-well microtitre plate. An 8 h old culture (lml, 104 cells/ml) was added to 200 ml Luria broth (LB) in each well. Silver nanoparticles of average size 10 nm were obtained by various methods including extracellular synthesis using yeast MKY (Issatchenkia orientalis) and reducing silver nitrate using borohydride followed by stabilization with biopolymers extracted from a fungus Itajahia sp. The typical concentration of the silver particles suspension was 100 mg/1. The suspension of silver particles was added to different wells in the 96 well microtitre plate in such quantity that the final silver concentration in each well was sub-MIC, typically ranging between 0.1-6.12 mg/1. The pH of the reaction mixture was 7.2. The plates were incubated for 24 h at 30°C. A control was kept without addition of silver nanoparticles. Ionic silver (as silver nitrate) and metallic silver (as fine silver powder, 0.1 g) were also used as controls. In order to determine whether the capping agents and cell-free lysate of MKY 3 culture had any inhibitory effect, 18 these were also added to the culture separately in amounts equivalent to those used for capping the nanoparticles. As seen in Table below, the MIC of silver nanoparticles varied with the method of preparation. The MIC of biogenic silver nanoparticles (those synthesized extracellularly by yeast, Isatchenkia orientalis (MKY 3) and those stabilized by polymer from fungus, Itajaia sp. [fungal polymer (FP1)], was 3.06 mg/1 for E. coli Nova Blue. Uncapped nanoparticles showed a high MIC of 50 mg/1. Ionic silver had very low MIC value of 0.04 mg/1 for E. coli. Metallic silver showed no inhibitory effect. The capping agent fungal polymer (FP1) and the lysate from MKY 3 culture were found not to exert any inhibitory effect on the cells. For curing experiments the culture (10 ml) of pUC19 transformed E. coli Nova Blue (O.D60o =0.2) was inoculated into a series of tubes containing lml LB. Cells were exposed to sub-inhibitory concentrations (Table 1) of silver nanoparticles for a period of 24 h at 37°C. Appropriate dilution (10'6) of the exposed cells were plated on LA plates and incubated at 37°C overnight. The colonies appearing on the plates were transferred to LA+ampicillin plates using sterile toothpicks. After overnight incubation at 37°C, the plates were checked for cells on LA that failed to grow on LA+ampicillin (considered as cured derivatives). Based on these data, the curing efficiency was determined for the different types of nanoparticles. The biogenic nanoparticles and FP1 capped nanoparticles showed curing efficiencies of 81% and 75% respectively for E. coli Nova blue strain. No curing was observed with uncapped or PE1 capped nanoparticles. Cured 19 derivatives picked up randomly from LA plates were inoculated into 200ml LB in a microtiter plate. After incubation at 37°C overnight, these cultures were used for plasmid isolation by rapid isolation method (Birnboim, H.C. and Doly, J. (1979), Nucleic Acid Res., 7:1513-1523). The plasmid preparations were subsequently loaded on agarose gel and electrophoresed at 50 V. It was observed that plasmid band corresponding to pUC19 was not present in case of cells exposed to biogenic and FP1 capped silver nanoparticles (Figure 1). For the uncapped and PE1 capped nanoparticles the plasmid band was present. Table 1various Nova B : MIC and Plasmid curing using silver nanoparticles E. coli lue(pUC19) MIC (mg/L) Plasmid curing (%) Biogenic 1.53 81 Uncapped 25 0 PE1 25 0 FP1 1.53 75 AgNO3 0.02 0 The Figure 1 of the accompanying drawings is a photograph of agarose gel electrophoresis showing plasmid bands wherein Lane lis for standard molecular weight DNA ladder; Lane 2 is for E. coli Nova Blue transformant (with plasmid PUC19); Lane 3 is transformant (with plasmid PUC19) treated with uncapped silver particles; Lane 4 is for transformant (with plasmid PUC19) treated with PE1 capped silver particles; Lane 5 is for transformant (with plasmid PUC19) treated with FP1 20 capped silver particles and Lane 6 is for transformant (with plasmid PUC19) treated with biogenic ( MKY3) silver particles. It is to be noted that Lane 5 and Lane 6 do not show any plasmid band indicating removal of plasmid. The plasmid-free bacteria were removed from growth medium silver particles mixture in the 96-well plates by centrifugation at 8000 rpm in a microcentrifuge. Examples 2 to 7 The method was repeated by changing the particle size of silver to an average size of 25 nm, 50 nm, and 80 nm (examples 2,3 and 4) and rest of the method was carried out as per Example 1. The data obtained were very similar to that of Example 1. The method was repeated by changing the temperature of the reaction to 20, 25, and 40 C (examples 5,6 and 7) and rest of the method was carried out as per Example 1. The data obtained were very similar to that of Example 1. Example 8: Standard E. coli NovaBlue strain transformed with drug resistance plasmid eg. pUC 18 conferring ampicillin resistance was used. Transformants showed beta-galactosidase due to a-complementation (pink colonies on MacConkey's Agar medium). The E. coli-pUC18 transformant was then 21 plated onto the MacConkey agar plates containing a gradient of concentration of silver nanoparticles. If the silver nanoparticles are an inhibitor of plasmid replication, then as the concentration of the silver nanoparticles is increased the number of pink colonies would decrease. In actual assay, silver nanoparticles in suspension were incorporated at concentration equal to four times MIC (4X MIC) and gradient was prepared using 12.5 ml medium in an 8.5 cm diameter culture dish. The other part of the plate was filled with another 12.5 ml medium. Upon solidification the plasmid bearing E. coli strain was spread (0.1 ml/plate, cell density 1 X 105 CFU/ml) and plates were incubated at 37°C for 24 - 48 h until visible colonies were obtained. It was observed that growth (pink colored colonies) was obtained in areas containing low amounts of silver, indicating inhibition of culture by presence of silver. White colonies were very few. Example 9: In another experiment, MIC for silver nanoparticles (biostabilized silver nanoparticles) for E. coli cells containing plasmid pBR322 was determined using standard double dilution method. A silver nanoparticles free medium served as inoculated control. The microtitre plate was incubated at 37°C for 18 h. On day 1, results on MIC were recorded and the growth in the well corresponding to sub-MIC (here 12 mg/1) was used as inoculum for subsequent day and again exposed to various concentrations of silver nanoparticles (various concentrations achieved according to double dilution 22 scheme). Simultaneously, an aliquot of cells from each well was suitably diluted and spread-plated on Luria Agar+ Ampicillin containing plates. Plates and freshly inoculated microtitre plate was incubated for 24 h at 37°C. Table 2 : Effect of exposure to silver nanoparticles average size 30 nm Silver concentrations tested mg/1 MIC result after 18 h Average CFU obtained on LA + ampicillin (50mg/ml) agar Average CFU obtained on LA 12 + No growth No growth 6 + 26X106 23 X 106 3 + 57X106 76 X 106 1.5 + 119X106 161 X106 0.8 + 61 X 106 90 X 106 0.4 + Not determined Not determined Positive control + 61 X 106 96X106 Negative control - 0 0 Table 3 : Effect of re-exposure to silver nanoparticles Silverconcentrations tested mg/1 MIC result after 18 h Average CFU obtainedonLA +ampicillin (50mg/ml)agar Average CFU obtained on LA 12 - No growth No growth 6 - No growth No growth 3 - No growth No growth 1.5 + No growth No growth 0.8 + 1X106 1X106 0.4 + 82 X 106 106 X106 Positive control + 123 X 106 169 X106 Negative control - 0 0 Data in Table 2 & 3 indicate that survivor population of cells exposed to a particular concentration of silver nanoparticles, exhibit greater sensitivity on 23 re-exposure (i.e. are killed at 4-fold lower dosages). Reduction in cell count on exposure to ampicillin containing plates demonstrates sensitivity to ampicillin which might be due to loss of plasmid. Example 10: In another experiment, loss of plasmid encoded antibiotic resistance characteristic was assessed. In this experiment the time and dose dependent loss of antibiotic resistance was monitored. The experiment was initiated by inoculating the plasmid bearing E. coli strain to determine the MIC against silver nanoparticles preparation using the standard double dilution technique. A positive control containing cells not exposed to silver nanoparticles was maintained. The experimental microtitre plate was incubated at 37°C for 18 h. After this an aliquot of cells from each well was removed, culture diluted 1000 times in sterile saline and 10 fil was added to another set for determination of MIC keeping the silver concentrations same. This ensured use of pre-exposed cells as inoculum for the subsequent MIC testing. The fresh plate was incubated as described above and entire process was followed up to 50 h. At the end of 18, 36 and 50 h exposure aliquots of cells (suitably diluted) were used to determine the TVC on Luria Agar plates and Luria Agar + ampicillin plates. Plates were incubated at 37C for 18 h and results were recorded. For E. coli MTCC 393 (pKT231) the MIC for silver nanoparticles preparation was 12 ug/ml, culture resistant to kanamycin (plasmid borne 24 resistance) and streptomycin (non-plasmid encoded). It was observed that (Table 4 ) at -50 generations there was no curing of kanamycin resistance character for control, whereas 30-45% curing was observed in concentration range 1.5-6 ug/ml. Spontaneous curing occurred (13%) at 5Oh and during this period cells exposed to 1.5-6 ug/ml were completely killed. At -50 h, even 0.8 ug/ml silver nanoparticles were sufficient to inhibit surviving cells. Table 4: Cell count of E. coli MTCC 393 (pKT231) at various Ag concentrations CFU/ml 0.8 mg/ml0.125XMIC 1.5 mg/ml0.25XMIC 3.0 mg/ml 0.5X MIC 6.0 mg/ml IX MIC 0 mg/ml Control 18 h (-50 generations) MIC 6 mg/ml On Luria agar 391 X 106 266 X106 >300 X 106 203 X 106 196 X106 On Luria agar + kanamycin (50 mg/ml) 325 X 106 145 X 106 199 X106 130 X106 246 X 106 % curing of KanR 27 46 33 36 0 36 h (-100 generations) MIC 1.5 mg/ml On Luria agar 47X106 0 0 0 130 X106 On Luria agar + kanamycin (50 mg/ml) 35X106 0 0 0 114X106 % curing of KanR 26 13 50 h (-150 generations) MIC 0.8 ug/ml 25 For E. coli MTCC 398 (pRK 2013) the MIC for silver nanoparticles preparation was 12 ug/ml, culture resistant to kanamycin (plasmid borne resistance) and ampicillin. It was observed that (Table 5) at -50 generations there was only -10% curing of kanamycin resistance character for control and, whereas 13 and 42% curing was observed at concentrations 3.0 and 6 ug/ml. At -36 h, 70-80% curing was observed at 0.8 and 1.5 ug/ml respectively and cells exposed to 3 and 6 ug/ml were completely killed. At 50 h 1.5 ug/ml silver nanoparticles were sufficient to inhibit surviving cells. Table 5 : Cell count of E. coli MTCC 398 (pKT231) at various Ag concentrations average size of particles 20 nm CFU/ml 0.8 mg/ml0.125XMIC 1.5 mg/ml0.25XMIC 3.0 mg/ml 0.5X MIC 6.0 mg/ml IX MIC 0 mg/ml Control 18 h ( generations) MIC 12 ug/ml On Luria agar 99X106 52X106 42 X 106 164 X106 186 X106 On Luria agar + kanamycin (50 mg/ml) 102 X106 70X106 32X106 96X106 166 X106 % curing of KanR 0 0 13 42 11 36 h ( generations) MIC 3 mg/ml On Luria agar 300 X107 10X106 NIL NIL >300 X 106 On Luria agar + kanamycin (50 mg/ml) 840 X106 2X106 NIL NIL > 300 X106 % curing ofKanR 72 80 0 50 h( generations) MIC 1.5 ug/ml 26 We Claim: 1. A culture independent method of removal of plasmids from live and multiplying plasmid containing bacteria comprising the following steps:--Preparing an aqueous first suspension of submicronic silver particles; - estimating the MIC of the silver particles for the bacteria to determine the inhibitory concentration of the particles suspension for the bacteria; - Adding in a reaction vessel, the first suspension and growth medium of the bacteria to obtain a second suspension containing sub-MIC concentration of silver particles; - Introducing the bacteria in the reaction vessel under conditions favouring the multiplication of the bacteria, for 12 to 48 hrs, to obtain subsequent generations of the bacteria; - testing the bacterial generations for absence of plasmids to obtain a generation of plasmid free bacteria, and - separating the plasmid free bacteria from the growth media/silver particles suspension mixture by a known method. 2. A method as claimed in claim 1 wherein the particle size of silver is in the range of 1 to 999nm. 3. A method as claimed in claim 1 wherein the particle size of silver is in the range of 5 to 80nm. 4. A method as claimed in claim 1 wherein the concentration of silver particles in the first suspension is in the range of about 50 mg/L to about 500 mg/L. 27 4. A method as claimed in claim 1 wherein the concentration of silver particles in the first suspension is in the range of about 50 mg/L to about 500 mg/L. 5. A method as claimed in claim 1 wherein the concentration of silver particles in the second suspension is in the range of about 0.1 mg/L to about 50 mg/L. 6. A method as claimed in claim 1 wherein the concentration of silver particles in the second suspension is in the range of about 25 to 50 % of the MIC value for the bacteria. 7. A method as claimed in claim 1 wherein the reaction vessel is shaken during multiplication of the bacteria. 8. A method as claimed in claim 1 wherein the MIC for the bacteria is estimated by double dilution method. 9. A method as claimed in claim 1 wherein the testing of the generation is done by gel electrophoresis. 10. A method as claimed in claim 1 wherein the bacteria are Gram negative bacteria. 11. A method as claimed in claim 1 wherein the testing of the generation is done by using at least one antibiotic as a marker. 28 12. A method as claimed in claim 1 wherein the first suspension is prepared by a method selected from a group of methods consisting of physical, chemical, biological methods and combinations thereof. 13. A method as claimed in claim 1 wherein the first suspension contains biostabilized silver submicronic particles. 14. A method as claimed in claim 1 wherein the bacteria are treated by the second suspension at temperatures ranging from 5 to 90 C, preferably 20 to 45 C. 15.A method as claimed in claim 1 wherein the bacteria are treated by the second suspension at pH ranging from 1 to 13, preferably 6 to 8. 16.A method as described herein with reference to the accompanying examples. Dated this 2nd day of August, 2007. 29 ABSTRACT A method of removal of plasmids from live, multiplying plasmid containing bacteria is disclosed. The method comprises treating the bacteria with silver submicronic particles of a predetermined size and concentration under specific conditions.

Documents:

1477-MUM-2007-ABSTRACT(GRANTED)-(11-1-2012).pdf

1477-mum-2007-abstract.doc

1477-mum-2007-abstract.pdf

1477-MUM-2007-ASSIGNMENT(9-7-2009).pdf

1477-MUM-2007-CANCELLED PAGES(15-12-2011).pdf

1477-MUM-2007-CLAIMS(AMENDED)-(15-12-2011).pdf

1477-MUM-2007-CLAIMS(AMENDED)-(16-6-2011).pdf

1477-MUM-2007-CLAIMS(GRANTED)-(11-1-2012).pdf

1477-MUM-2007-CLAIMS(MARKED COPY)-(16-6-2011).pdf

1477-mum-2007-claims.doc

1477-mum-2007-claims.pdf

1477-MUM-2007-CORRESPONDENCE(11-11-2011).pdf

1477-MUM-2007-CORRESPONDENCE(16-4-2009).pdf

1477-MUM-2007-CORRESPONDENCE(21-9-2011).pdf

1477-MUM-2007-CORRESPONDENCE(29-6-2009).pdf

1477-MUM-2007-CORRESPONDENCE(9-7-2009).pdf

1477-MUM-2007-CORRESPONDENCE(IPO)-(11-1-2012).pdf

1477-mum-2007-correspondence-received.pdf

1477-mum-2007-description (complete).pdf

1477-MUM-2007-DESCRIPTION(GRANTED)-(11-1-2012).pdf

1477-MUM-2007-DRAWING(GRANTED)-(11-1-2012).pdf

1477-mum-2007-drawings.pdf

1477-MUM-2007-FORM 1(15-12-2011).pdf

1477-MUM-2007-FORM 1(16-6-2011).pdf

1477-MUM-2007-FORM 1(29-6-2009).pdf

1477-MUM-2007-FORM 18(16-4-2009).pdf

1477-MUM-2007-FORM 2(GRANTED)-(11-1-2012).pdf

1477-MUM-2007-FORM 2(TITLE PAGE)-(15-12-2011).pdf

1477-MUM-2007-FORM 2(TITLE PAGE)-(16-6-2011).pdf

1477-MUM-2007-FORM 2(TITLE PAGE)-(COMPLETE)-(2-8-2007).pdf

1477-MUM-2007-FORM 2(TITLE PAGE)-(GRANTED)-(11-1-2012).pdf

1477-MUM-2007-FORM 26(15-12-2011).pdf

1477-MUM-2007-FORM 26(16-6-2011).pdf

1477-MUM-2007-FORM 3(16-6-2011).pdf

1477-mum-2007-form 6(9-7-2009).pdf

1477-mum-2007-form-1.pdf

1477-mum-2007-form-2.doc

1477-mum-2007-form-2.pdf

1477-mum-2007-form-26.pdf

1477-mum-2007-form-3.pdf

1477-MUM-2007-MARKED COPY(15-12-2011).pdf

1477-MUM-2007-OTHER DOCUMENT(16-6-2011).pdf

1477-MUM-2007-PETITION UNDER RULE 137(16-6-2011).pdf

1477-MUM-2007-POWER OF AUTHORITY (15-12-2011).pdf

1477-MUM-2007-POWER OF AUTHORITY(15-12-2011).pdf

1477-MUM-2007-REPLY TO EXAMINATION REPORT(16-6-2011).pdf

1477-MUM-2007-REPLY TO HEARING(15-12-2011).pdf

1477-MUM-2007-SPECIFICATION(AMENDED)-(15-12-2011).pdf

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