Indian Patents. 269659:“A COMPOSITION FOR SELECTIVELY ELIMINATING A MICROORGANISM OR A GROUP OF MICROORGANISMS FROM BIOLOGICAL SAMPLE”

Title of Invention	“A COMPOSITION FOR SELECTIVELY ELIMINATING A MICROORGANISM OR A GROUP OF MICROORGANISMS FROM BIOLOGICAL SAMPLE”
Abstract	The present invention relates to a composition and method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest. Basically, the invention relates to a composition and method for controlling overgrowth of normal flora in a biological sample in liquid media.

Full Text	Field of the invention: The invention relates to a composition and method for controlling overgrowth of normal flora in biological samples in liquid media. Primarily, the Invention relates to a composition which selectively eliminates a microorganism or a group of microorganisms from a biological sample. The invention further relates to a method for selectively eliminating a microorganism or a group of microorganisms from a biological sample. Background of the invention: Microbial diseases caused by various microbial flora are major health concerns now-a-days. Rapid isolation and identification of causative microbe is one of the keys to develop an effective treatment for the disease. One such microbial disease tuberculosis caused by Mycobacterium tuberculosis remains a major health problem. Rapid diagnosis of tuberculosis is essential to break the chain of transmission especially in persons infected with multi drug resistant (MDR) tuberculosis and population dually infected with M. tuberculosis and HIV (HIV-TB). Delay in diagnosis of active pulmonary tuberculosis has been implicated with significant nosocomial outbreak (Greenaway et al., 2002) and increased mortality (Counsell et al., 1989) creating an urgency for developing new diagnostic tools that allow more rapid identification of M. tuberculosis and characterization of drug susceptibility patterns for the better management of TB. Growing M. tuberculosis in liquid medium is much faster when compared to growing on solid medium like Lowenstein Jensen medium. The Centers for the Disease Control and Prevention (CDC), USA has also recommended that the use of one liquid medium is mandatory for the primary isolation of mycobacteria (Kent and Kubica, 1985). The major problem while using liquid media is the over growth of normal flora, which compete for nutrients and mask the growth of tubercle bacilli (Vanaja Kumar et al., 2007). Antibiotic supplement in liquid media used for primary isolation of M. tuberculosis is either PANTA or PACT. Nalidixic acid in PANTA and carbenicillin in PACT are reported to affect the growth M. tuberculosis to some extent (Conville et al., 1995; Salfinger et al., 1988). It is also recommended that exposure of primary isolates to these selective antibiotics should be limited as it affects the growth of tubercle bacilli in liquid media. Therefore, there remains a need in the art to develop an effective method and composition for controlling overgrowth of normal flora in biological samples and for isolation of the microorganism of interest. The present team of inventors has provided a solution to this problem and has come out with a novel composition and method for controlling overgrowth of normal flora and for isolation of microorganism of interest. Objective of the invention: The objective of the invention is to derive a composition which selectively eliminates a microorganism or a group of microorganisms from a biological sample leaving behind the microorganism of interest. Another objective of the invention is to provide a method for controlling the overgrowth of normal flora and a method for isolation of microorganism of interest from a biological sample. Statement of the invention: Accordingly, the invention provides a composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest, said composition comprising phagebiotics supplemented with phage lysin, wherein the titer of phagebiotics in the composition ranges from ~ lxl 07 pfu/ml to~lx1011pfu/ml. Description of figures: Figure 1: Effect of lysin on the growth of Mycobacterium tuberculosis by LRP assay. Figure 2A: Growth of normal flora on blood agar. Figure 2B: Effect of phagebiotics, lysin and lysostaphin on sputum deposits Detailed description of the invention: The invention is about a composition for selectively eliminating a microorganism or a group of microorganisms and isolation of microorganism of interest from a biological sample, wherein said composition comprises phage lysin supplemented phagebiotics. The invention also provides a method for selectively eliminating a microorganism or a group of microorganisms and isolation of microorganism of interest from a biological sample. basically, the invention is about controlling the overgrowth of normal flora in biological sample such as pulmonary samples or extra pulmonary samples grown in liquid media using phage lysin added to phagebiotics. Processed sputum samples are most commonly used samples for isolation of Mycobacterium tuberculosis. Sputum specimens form the exclusive clinical samples for primary isolation and identification of Mycobacteria in pulmonary tuberculosis patients. The presence of normal flora and opportunistic microorganisms in sputum samples necessitate the decontaminating procedure as these organisms overgrow and mask the growth of tubercle bacilli otherwise. Sputum specimens have mucous strands, enzymes and other inhibitory factors that hamper the detection of viable tubercle bacilli in rapid broth based detection systems. Addition of the composition comprising phage lysin and phagebiotics is useful to control the overgrowth of normal flora and to allow the normal growth of M. tuberculosis. A composition comprising lysin and phagebiotics effective against the normal flora surviving the action of 4% NaOH, form a novel, suitable alternative to antimicrobials in minimizing the overgrowth of normal flora without affecting the growth of mycobacteria. Accordingly, the invention provides a composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest, said composition comprising phagebiotics supplemented with phage lysin, wherein the titer of phagebiotics in the composition ranges from ~1x10 pfu/ml to~lxl011pfu/ml. The lysin to be used in the composition is obtained from phages at a titer of ~ 1x10 pfu/ml to -lxl011 pfu/ml. The phage lysin is preferably obtained from the phages infecting Bacillus sp. more preferably, the phage lysin is obtained from phages Chedec 11, Chedec 20 and Chedec 21 (Chedec - Chennai Decontaminating) infecting Bacillus sp. Preferably, the lysin used for the purpose of the invention is pooled lysin obtained from three phages Chedec 11, Chedec 20 and Chedec 21. other bacteriophages have been used for control of Salmonella enterica, Listeria monocytogenes, Pseudomonas plecoglossida for similar purpose of invention in food preparations. In an embodiment of the invention the microorganism of interest is Mycobacterium tuberculosis. The biological sample employed in the invention is preferably pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens. The invention further provides a method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest using the composition comprising phagebiotics and phage lysin, said method comprising the steps of: i. obtaining the phagebiotics and phage lysin; ii. mixing phagebiotics and phage lysin to obtain composition with desired concentration; iii. mixing the composition in the medium containing the biological sample; iv. incubating the medium; v. obtaining a culture with inhibited growth of normal flora and isolating the microorganism of interest. The biological sample for the method is selected from the group comprising pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens. The concentration of lysin in the composition to be used in the method is preferably neat, 1/10 or 1/100. More preferably the concentration is 1/10. The incubation is done at a temperature of about 30°C to 40°C for a period of 18-24 hrs. Preferably, the incubation temperature is 37°C. The invention also provides phage lysin obtained from Chedec 11, Chedec 20 and Chedec 21, wherein the phage lysin is useful for preparing composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest. This is the first time, when a team of researchers have isolated lysin from Chedec 11, Chedec 20 and Chedec 21 and has incorporated the same in composition and method for controlling overgrowth of normal flora in biological samples in liquid media. The invention is illustrated further by the following examples which are only meant to illustrate the invention and not act as limitations. The embodiments which may be apparent to a person skilled in the art are deemed to fall within the scope of the present invention. Example 1- Preparation of phage lysin: Following method is used for preparing phage lysin: A. Bacterial cell preparation: i. Inoculating one loopful of Bacillus sp grown on nutrient agar medium in to nutrient broth and incubating at 37°C for 16 hours; ii. After incubation, adding the culture to 500 ml flask containing 300 ml of the same medium at a concentration of 5% (v/v); iii. Incubating the flask at 37°C in a water bath for 3-4 hours to reach the turbidity of 0.8 OD equivalent to ~1 x 108 cfu/ml; B. Lysin preparation: i. Adding the phage at MOI 2-6 to Bacillus sp grown in nutrient broth and incubating at 37°C until lysis (approximately 1 hour) and chilling the flask in an ice bath; ii. Adding dithiothreitol (DTT) (final concentration 0.001M) and neutralized EDTA (final concentration 0.005M) and storing the flasks at 4°C until further processed; iii. Processing further by the adding 5% (vol/vol) of 0.05M DTT and mixing thoroughly; iv. Centrifuging the suspension at 20,000 RPM for 1.5 hours at 4°C. (This step clarifies lysin solution and facilitates the precipitation of lysin by ammonium sulfate); v. Precipitating lysin by adding solid ammonium sulfate at 4°C with constant stirring to a final concentration of 50% saturation; vi. Allowing the precipitation to proceed at 4°C for 24-72 hours and separating the lysin from the fluid by centrifuging at 20, 000 RPM for 1.5 hours at 4°C; vii. Recovering the lysin from the walls of the beaker in which it precipitates and dissolving the precipitate in a small amount of 0.02M phosphate buffer (pH 6. 1) containing, final concentrations of 0.001M DTT and 0.005 M EDTA; viii. Adding about 6 ml of buffer to take up the lysin from 400 ml of crude lysate; ix. Centrifuging the concentrated lysin at 12, 000 RPM for 45 minutes at 4°C; x. Discarding the pellet, containing insoluble material including a small amount of phages and obtaining the clear supernatant fluid with its active lysin and freezing at -65°C. Example 2- Preparation of phagebiotics, phagebiotics supplemented with phage lysin and phagebiotics supplemented with lysostaphin: Phagebiotics is prepared by mixing equal volume of high titer (~lxl08 pfu/ml) lysate of phages Chedec 11, Chedec 20 and Chedec 21 harvested in nutrient broth prior to treating the biological sample. Phagebiotics supplemented with lysin is prepared by mixing 900µl of phagebiotics with l00µl of pooled lysin (neat) to reach a final concentration of 1/10 (N1). Similarly, phagebiotics supplemented with lysostaphin is prepared by mixing 900ul of phagebiotics with l00µl of lysostaphin (100µg/ml) to obtain a final concentration of lysostaphin of l0µg/ml. Example 3- Host range of phage lysin: Suspension of all selected 25 normal flora (Table 1) obtained from processed sputum samples is prepared in 1 ml of nutrient broth equivalent to # 2 McFarland's standard and the lawns of all normal flora on nutrient agar are prepared using 0.6-0.8% agar. Table 1: Staining properties and colony morphology of selected colonizing normal flora (CF) (Table Removed) a. Phagebiotics is incapable of controlling these organisms in processed sputum samples. Results shown in table 1 indicate that phagebiotics alone is capable of controlling the growth of 12 organisms. Example 4- Activity of phage lysin on normal flora at different concentrations Neat (N), decimal dilutions 1/10 (N1) and 1/100 (N2) of lysin from Chedec 11, Chedec 20 and Chedec 21 are prepared and about 3 µl of the same is dropped onto the lawns of organisms listed in Table 1. Cocktail of pooled lysins from Chedec 11, Chedec 20 and Chedec 21 is also prepared and is used in the same concentrations as Neat (N), decimal dilutions 1/10 (N1) and 1/100 (N2). The same are incubated at 37°C for 18-24 hours. The organisms are also subjected to Lysostaphin (Sigma), a commercial preparation of lysin from hages of Staphylococcus saprophyticus and are included as control. The concentration of lysostaphin used for the experiment is 10 µg/ml and 100 ug/ml. The results are shown below in table 2: Table 2: concentration of phage lysin active against normal flora (Table Removed) Compared to individual lysin, pooled lysins of Chedec 11, Chedec 20 and Chedec 21 show better activity in term of controlling the growth of many strains of normal flora. Pooled lysin at concentrations neat and 1/10 inhibits the growth of 24/25 normal flora but 1/100 concentration inhibits only 9/25 strains. Lysin of Chedec 20 is found to inhibit more number of normal flora when compared to that of Chedec 11 and Chedec 21. Lysostaphin is found to inhibit only staphylococci (7 strains of normal flora) at both concentrations. The only flora surviving the action of neat and 1/10 (N1) concentration of pooled lysin is Bacillus sp. Pooled lysin prepared from Chedec 11, Chedec 20 and Chedec 21 is found to inhibit 24/25 normal flora that survived the action of sputum processing as neat, 1/2,1/3, 1/4,1/5,1/6,1/7,1/8,1/9 and 1/10 dilutions. The surviving normal flora are found belonging to Bacillus sp, Micrococcus sp, Staphylococcus sp, Candida sp and coccobacillary forms. Dilutions ranging from neat to 1/50 are able to inhibit all organisms belonging to these genera. Dilutions ranging from 1/50 to 1/100 are able to inhibit all organisms of these genera excepting Staphylococcus sp. Example 5- Effect of phagebiotics, lysin and lysostaphin on the growth of M tuberculosis by LRP assay: Suspension of standard strain M. tuberculosis H37R.V and five clinical isolates of M. tuberculosis equivalent to # 2 McFarland's standard is prepared in 1 ml of 7H9 media supplemented with 5% glycerol and 10% albumin dextrose complex (G7H9) and treated as control. The same suspension is also prepared individually in G7H9 supplemented with 10% (v/v) of phagebiotics harvested in G7H9, lysin (final concentration N1) and lysostaphin (final concentration 10 µg/ml). Similarly, 0.02M phosphate buffer (pH 6. 1) containing final concentrations of 0.001M DTT and 0.005 M EDTA is also evaluated as the buffer is used to dissolve lysin in the final stage of lysin preparation. The reaction is allowed at 37°C for 24 hours. After incubation, luciferase reporter phage phAE129 is added at multiplicity of infection (MOI) 10 to all the reaction vials and incubated at 37°C. After 4 hours, relative light units (RLU) is measured in luminometer (Monolight) by mixing 100 ul of the sample with 100 µl of D- luciferin (R&D Systems, Minneapolis) at final concentration 0.33 mM in 05M sodium citrate buffer at pH 4.5. RLU of each sample is taken in duplicates and the mean value is used for interpretation of the results. Figure 1 indicates the results of the LRP assay conducted to study the effect of phagebiotics, lysin and lysostaphin on the growth of M. tuberculosis. The relative light units obtained as shown in the figure clearly indicate that no activity is shown by phagebiotics, lysin and lysostaphin against M. tuberculosis at concentrations tested. Example 6- Effect of phagebiotics, lysin and lysostaphin on sputum samples: 120 sputum samples are processed by modified Petroff s method and deposits are obtained. The deposits are washed again with distilled water and centrifuged. A loopful from each of the deposits is streaked on blood agar plates which are incubated at 37°C for 18-24 hrs (Stage I). The deposits are then aliquoted in to four portions each containing l00µl of the sample. To the first aliquot 200µl of nutrient broth is added (Stage II). This is to be used as control. To the second aliquot 200µl of phagebiotics is added (Stage III). To the third aliquot 200(4.1 of phagebiotics supplemented with lysostaphin is added (Stage IV). And to the fourth aliquot 200ul of phagebiotics supplemented with lysin is added (Stage V). All these samples are incubated at 37°C for 18-24 hrs. A loopful from stages II-V are streaked on blood agar plates and incubated at 37°C for 18-24 hrs. The results obtained are summarized in table 3: Table 3: Effect of phagebiotics, lysin and lysostaphin on sputum deposits (Table Removed) Stage I: Soon after processing of sputum samples Stage II: Grown overnight in nutrient broth (control) Stage III: Grown overnight in phagebiotics Stage IV: Grown overnight in phagebiotics supplemented with lysostaphin Stage V: Grown overnight in phagebiotics supplemented with lysin Minimal growth of normal flora in low numbers of samples (26/120) is observed soon after processing the sputum samples (Stage I). All the surviving organisms are identified as Staphylococcus sp. Confluent and mixed growth of normal flora is observed in 105/120 of the samples when incubated overnight with nutrient broth (Stage II-control). And pure growth of Staphylococcus sp is observed only in 3 of the samples (Stage II-control). In the samples treated overnight with phagebiotics (Stage III) growth of normal flora is observed in 50/120 samples, 24 being Bacillus sp and 21 belonging to Staphylococcus sp while 5 samples shows confluent growth of normal flora consisting of both hemolytic and non-hemolytic organisms. Samples treated by phagebiotics supplemented with lysostaphin (Stage IV) controls the growth of normal flora in 81/120 samples. Growth of Bacillus sp and Staphylococcus sp are observed in 23 and 13 samples respectively while confluent mixed growth is seen in 3 samples. Phagebiotics supplemented with phage lysin controls the growth of normal flora in 112/120 samples during overnight incubation (Stage V). The remaining 8 samples shows minimal growth of normal flora consisting of Bacillus sp in 3 samples and Staphylococcus sp in 5 samples (Figure 2). Lysostaphin is found to inhibit 9 out of 22 staphylococcal strains which survived the action of phagebiotics while lysin inhibits 17 of them. Among 23 samples resulting in the growth of Bacillus sp, lysin is found to inhibit 20 while lysostaphin inhibits none (Table 3). Stage V i.e. phagebiotics supplemented with lysin gives the best results as no microbial growth is observed in 112 samples. Only Staphylococcus sp. growth is observed in 5 samples and only Bacillus sp. growth is observed in 3 samples. Advantages: 1. The composition and method of the invention provides a non-chemical and eco-friendly substitute to antibiotics for controlling the growth of normal flora in biological samples; 2. Selectively kills normal flora without affecting the growth of microorganism of interest; 3. Provides flexibility of usage in liquid media for rapid diagnosis of tuberculosis by any method. WE CLAIM: 1. A composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest, said composition comprising phagebiotics supplemented with phage lysin, wherein the titer of phagebiotics in the composition ranges from ~ 1x107 pfu/ml to ~ 1x1011 pfu/ml. 2. A composition as claimed in claim 1 wherein the lysin is obtained from phages at a titer of- lxl07 pfu/ml to ~ lxl011 pfu/ml. 3. A composition as claimed in claim 1, wherein the microorganism of interest is Mycobacterium tuberculosis. 4. A composition as claimed in claim 1, wherein the biological sample is selected from the group comprising pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens. 5. A composition as claimed in claim 1 or 2, wherein the phage lysin is preferably obtained from the phages infecting Bacillus sp. 6. A composition as claimed in any of the claims 1, 2 or 5, wherein the phages are preferably Chedec 11, Chedec 20 and Chedec 21. 7. A composition as claimed in any of the claims 1, 2, 5 or 6, wherein the lysin is preferably pooled lysin obtained from phages Chedec 11, Chedec 20 and Chedec 21. 8. A method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest using the composition as claimed in claim 1, said method comprising the steps of: i. obtaining the phagebiotics and phage lysin; ii. mixing phagebiotics and lysin to obtain composition with desired concentration; iii. mixing the composition in the medium containing the biological sample; iv. incubating the medium; v. obtaining a culture with inhibited growth of normal flora and isolating the microorganism of interest. A method as claimed in claim 8, wherein the biological sample is selected from the group comprising pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens. 10. A method as claimed in claim 8 wherein the concentration of lysin in the composition is preferably neat, 1/10 or 1/100. 11. A method as claimed in claims 8 and 10, wherein more preferably the concentration is 1/10. 12. A method as claimed in claim 8, wherein the incubation is done at a temperature of about 30°C to 40°C for a period of 18-24 hrs. 13. A method as claimed in claims 8 and 12, wherein preferably the incubation is done at a temperature of 37°C. 14. Phage lysin obtained from Chedec 11, Chedec 20 and Chedec 21, wherein the phage lysin is useful for preparing composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest. 15. A composition and a method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest and a phage lysin obtained from Chedec 11, Chedec 20 and Chedec 21 substantially such as herein described with reference to the accompanying drawings and as illustrated in the foregoing examples.

Full Text

Field of the invention:
The invention relates to a composition and method for controlling overgrowth of normal flora in biological samples in liquid media. Primarily, the Invention relates to a composition which selectively eliminates a microorganism or a group of microorganisms from a biological sample. The invention further relates to a method for selectively eliminating a microorganism or a group of microorganisms from a biological sample.
Background of the invention:
Microbial diseases caused by various microbial flora are major health concerns now-a-days. Rapid isolation and identification of causative microbe is one of the keys to develop an effective treatment for the disease. One such microbial disease tuberculosis caused by Mycobacterium tuberculosis remains a major health problem. Rapid diagnosis of tuberculosis is essential to break the chain of transmission especially in persons infected with multi drug resistant (MDR) tuberculosis and population dually infected with M. tuberculosis and HIV (HIV-TB). Delay in diagnosis of active pulmonary tuberculosis has been implicated with significant nosocomial outbreak (Greenaway et al., 2002) and increased mortality (Counsell et al., 1989) creating an urgency for developing new diagnostic tools that allow more rapid identification of M. tuberculosis and characterization of drug susceptibility patterns for the better management of TB.
Growing M. tuberculosis in liquid medium is much faster when compared to growing on solid medium like Lowenstein Jensen medium. The Centers for the Disease Control and Prevention (CDC), USA has also recommended that the use of one liquid medium is mandatory for the primary isolation of mycobacteria (Kent and Kubica, 1985). The major problem while using liquid media is the over growth of normal flora, which compete for nutrients and mask the growth of tubercle bacilli (Vanaja Kumar et al., 2007). Antibiotic supplement in liquid media used for primary isolation of M. tuberculosis is either PANTA or PACT. Nalidixic acid in PANTA and carbenicillin in PACT are reported to affect the growth M. tuberculosis to some extent (Conville et al., 1995; Salfinger et al., 1988). It is also recommended that exposure of primary isolates to these selective antibiotics should be limited as it affects the growth of tubercle bacilli in liquid media.
Therefore, there remains a need in the art to develop an effective method and composition for controlling overgrowth of normal flora in biological samples and for isolation of the

microorganism of interest. The present team of inventors has provided a solution to this problem and has come out with a novel composition and method for controlling overgrowth of normal flora and for isolation of microorganism of interest.
Objective of the invention:
The objective of the invention is to derive a composition which selectively eliminates a microorganism or a group of microorganisms from a biological sample leaving behind the microorganism of interest.
Another objective of the invention is to provide a method for controlling the overgrowth of normal flora and a method for isolation of microorganism of interest from a biological sample.
Statement of the invention:
Accordingly, the invention provides a composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest, said composition comprising phagebiotics supplemented with phage lysin, wherein the titer of phagebiotics in the composition ranges from ~ lxl 07 pfu/ml to~lx1011pfu/ml.
Description of figures:
Figure 1: Effect of lysin on the growth of Mycobacterium tuberculosis by LRP assay.
Figure 2A: Growth of normal flora on blood agar.
Figure 2B: Effect of phagebiotics, lysin and lysostaphin on sputum deposits
Detailed description of the invention:
The invention is about a composition for selectively eliminating a microorganism or a group of microorganisms and isolation of microorganism of interest from a biological sample, wherein said composition comprises phage lysin supplemented phagebiotics. The invention also provides a method for selectively eliminating a microorganism or a group of microorganisms and isolation of microorganism of interest from a biological sample.

basically, the invention is about controlling the overgrowth of normal flora in biological sample such as pulmonary samples or extra pulmonary samples grown in liquid media using phage lysin added to phagebiotics.
Processed sputum samples are most commonly used samples for isolation of Mycobacterium tuberculosis. Sputum specimens form the exclusive clinical samples for primary isolation and identification of Mycobacteria in pulmonary tuberculosis patients. The presence of normal flora and opportunistic microorganisms in sputum samples necessitate the decontaminating procedure as these organisms overgrow and mask the growth of tubercle bacilli otherwise. Sputum specimens have mucous strands, enzymes and other inhibitory factors that hamper the detection of viable tubercle bacilli in rapid broth based detection systems. Addition of the composition comprising phage lysin and phagebiotics is useful to control the overgrowth of normal flora and to allow the normal growth of M. tuberculosis.
A composition comprising lysin and phagebiotics effective against the normal flora surviving the action of 4% NaOH, form a novel, suitable alternative to antimicrobials in minimizing the overgrowth of normal flora without affecting the growth of mycobacteria.
Accordingly, the invention provides a composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest, said composition comprising phagebiotics supplemented with phage lysin, wherein the titer of phagebiotics in the composition ranges from ~1x10 pfu/ml to~lxl011pfu/ml.
The lysin to be used in the composition is obtained from phages at a titer of ~ 1x10 pfu/ml to -lxl011 pfu/ml.
The phage lysin is preferably obtained from the phages infecting Bacillus sp. more preferably, the phage lysin is obtained from phages Chedec 11, Chedec 20 and Chedec 21 (Chedec - Chennai Decontaminating) infecting Bacillus sp.
Preferably, the lysin used for the purpose of the invention is pooled lysin obtained from three phages Chedec 11, Chedec 20 and Chedec 21.

other bacteriophages have been used for control of Salmonella enterica, Listeria monocytogenes, Pseudomonas plecoglossida for similar purpose of invention in food preparations.
In an embodiment of the invention the microorganism of interest is Mycobacterium tuberculosis.
The biological sample employed in the invention is preferably pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens.
The invention further provides a method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest using the composition comprising phagebiotics and phage lysin, said method comprising the steps of:
i. obtaining the phagebiotics and phage lysin;
ii. mixing phagebiotics and phage lysin to obtain composition with desired concentration;
iii. mixing the composition in the medium containing the biological sample;
iv. incubating the medium;
v. obtaining a culture with inhibited growth of normal flora and isolating the microorganism of interest.
The biological sample for the method is selected from the group comprising pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens.
The concentration of lysin in the composition to be used in the method is preferably neat, 1/10 or 1/100. More preferably the concentration is 1/10.
The incubation is done at a temperature of about 30°C to 40°C for a period of 18-24 hrs. Preferably, the incubation temperature is 37°C.
The invention also provides phage lysin obtained from Chedec 11, Chedec 20 and Chedec 21, wherein the phage lysin is useful for preparing composition for selectively eliminating a

microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest. This is the first time, when a team of researchers have isolated lysin from Chedec 11, Chedec 20 and Chedec 21 and has incorporated the same in composition and method for controlling overgrowth of normal flora in biological samples in liquid media.
The invention is illustrated further by the following examples which are only meant to illustrate the invention and not act as limitations. The embodiments which may be apparent to a person skilled in the art are deemed to fall within the scope of the present invention.
Example 1- Preparation of phage lysin:
Following method is used for preparing phage lysin:
A. Bacterial cell preparation:
i. Inoculating one loopful of Bacillus sp grown on nutrient agar medium in to
nutrient broth and incubating at 37°C for 16 hours; ii. After incubation, adding the culture to 500 ml flask containing 300 ml of
the same medium at a concentration of 5% (v/v); iii. Incubating the flask at 37°C in a water bath for 3-4 hours to reach the
turbidity of 0.8 OD equivalent to ~1 x 108 cfu/ml;
B. Lysin preparation:
i. Adding the phage at MOI 2-6 to Bacillus sp grown in nutrient broth and
incubating at 37°C until lysis (approximately 1 hour) and chilling the flask
in an ice bath; ii. Adding dithiothreitol (DTT) (final concentration 0.001M) and neutralized
EDTA (final concentration 0.005M) and storing the flasks at 4°C until
further processed; iii. Processing further by the adding 5% (vol/vol) of 0.05M DTT and mixing
thoroughly; iv. Centrifuging the suspension at 20,000 RPM for 1.5 hours at 4°C. (This
step clarifies lysin solution and facilitates the precipitation of lysin by
ammonium sulfate); v. Precipitating lysin by adding solid ammonium sulfate at 4°C with constant
stirring to a final concentration of 50% saturation;

vi. Allowing the precipitation to proceed at 4°C for 24-72 hours and
separating the lysin from the fluid by centrifuging at 20, 000 RPM for 1.5
hours at 4°C; vii. Recovering the lysin from the walls of the beaker in which it precipitates
and dissolving the precipitate in a small amount of 0.02M phosphate buffer
(pH 6. 1) containing, final concentrations of 0.001M DTT and 0.005 M
EDTA; viii. Adding about 6 ml of buffer to take up the lysin from 400 ml of crude
lysate; ix. Centrifuging the concentrated lysin at 12, 000 RPM for 45 minutes at 4°C; x. Discarding the pellet, containing insoluble material including a small
amount of phages and obtaining the clear supernatant fluid with its active
lysin and freezing at -65°C.
Example 2- Preparation of phagebiotics, phagebiotics supplemented with phage lysin and phagebiotics supplemented with lysostaphin:
Phagebiotics is prepared by mixing equal volume of high titer (~lxl08 pfu/ml) lysate of phages Chedec 11, Chedec 20 and Chedec 21 harvested in nutrient broth prior to treating the biological sample. Phagebiotics supplemented with lysin is prepared by mixing 900µl of phagebiotics with l00µl of pooled lysin (neat) to reach a final concentration of 1/10 (N1). Similarly, phagebiotics supplemented with lysostaphin is prepared by mixing 900ul of phagebiotics with l00µl of lysostaphin (100µg/ml) to obtain a final concentration of lysostaphin of l0µg/ml.
Example 3- Host range of phage lysin:
Suspension of all selected 25 normal flora (Table 1) obtained from processed sputum samples is prepared in 1 ml of nutrient broth equivalent to # 2 McFarland's standard and the lawns of all normal flora on nutrient agar are prepared using 0.6-0.8% agar.
Table 1: Staining properties and colony morphology of selected colonizing normal flora
(CF)
(Table Removed)

a. Phagebiotics is incapable of controlling these organisms in processed sputum samples. Results shown in table 1 indicate that phagebiotics alone is capable of controlling the growth of 12 organisms.
Example 4- Activity of phage lysin on normal flora at different concentrations Neat (N), decimal dilutions 1/10 (N1) and 1/100 (N2) of lysin from Chedec 11, Chedec 20 and Chedec 21 are prepared and about 3 µl of the same is dropped onto the lawns of organisms listed in Table 1. Cocktail of pooled lysins from Chedec 11, Chedec 20 and Chedec 21 is also prepared and is used in the same concentrations as Neat (N), decimal dilutions 1/10 (N1) and 1/100 (N2). The same are incubated at 37°C for 18-24 hours. The organisms are also subjected to Lysostaphin (Sigma), a commercial preparation of lysin from
hages of Staphylococcus saprophyticus and are included as control. The concentration of lysostaphin used for the experiment is 10 µg/ml and 100 ug/ml. The results are shown below in table 2:
Table 2: concentration of phage lysin active against normal flora
(Table Removed)
Compared to individual lysin, pooled lysins of Chedec 11, Chedec 20 and Chedec 21 show
better activity in term of controlling the growth of many strains of normal flora.
Pooled lysin at concentrations neat and 1/10 inhibits the growth of 24/25 normal flora but
1/100 concentration inhibits only 9/25 strains.
Lysin of Chedec 20 is found to inhibit more number of normal flora when compared to that
of Chedec 11 and Chedec 21.
Lysostaphin is found to inhibit only staphylococci (7 strains of normal flora) at both
concentrations.
The only flora surviving the action of neat and 1/10 (N1) concentration of pooled lysin is
Bacillus sp.
Pooled lysin prepared from Chedec 11, Chedec 20 and Chedec 21 is found to inhibit 24/25 normal flora that survived the action of sputum processing as neat, 1/2,1/3, 1/4,1/5,1/6,1/7,1/8,1/9 and 1/10 dilutions. The surviving normal flora are found belonging to Bacillus sp, Micrococcus sp, Staphylococcus sp, Candida sp and coccobacillary forms. Dilutions ranging from neat to 1/50 are able to inhibit all organisms belonging to these genera. Dilutions ranging from 1/50 to 1/100 are able to inhibit all organisms of these genera excepting Staphylococcus sp.
Example 5- Effect of phagebiotics, lysin and lysostaphin on the growth of M tuberculosis by LRP assay:
Suspension of standard strain M. tuberculosis H37R.V and five clinical isolates of M. tuberculosis equivalent to # 2 McFarland's standard is prepared in 1 ml of 7H9 media supplemented with 5% glycerol and 10% albumin dextrose complex (G7H9) and treated as control. The same suspension is also prepared individually in G7H9 supplemented with 10% (v/v) of phagebiotics harvested in G7H9, lysin (final concentration N1) and lysostaphin (final concentration 10 µg/ml). Similarly, 0.02M phosphate buffer (pH 6. 1) containing final concentrations of 0.001M DTT and 0.005 M EDTA is also evaluated as the buffer is used to dissolve lysin in the final stage of lysin preparation. The reaction is allowed at 37°C for 24 hours. After incubation, luciferase reporter phage phAE129 is added at multiplicity of infection (MOI) 10 to all the reaction vials and incubated at 37°C. After 4 hours, relative light units (RLU) is measured in luminometer (Monolight) by mixing 100 ul of the sample with 100 µl of D- luciferin (R&D Systems, Minneapolis) at final concentration 0.33 mM in
05M sodium citrate buffer at pH 4.5. RLU of each sample is taken in duplicates and the mean value is used for interpretation of the results.
Figure 1 indicates the results of the LRP assay conducted to study the effect of phagebiotics, lysin and lysostaphin on the growth of M. tuberculosis. The relative light units obtained as shown in the figure clearly indicate that no activity is shown by phagebiotics, lysin and lysostaphin against M. tuberculosis at concentrations tested.
Example 6- Effect of phagebiotics, lysin and lysostaphin on sputum samples: 120 sputum samples are processed by modified Petroff s method and deposits are obtained. The deposits are washed again with distilled water and centrifuged. A loopful from each of the deposits is streaked on blood agar plates which are incubated at 37°C for 18-24 hrs (Stage
I).
The deposits are then aliquoted in to four portions each containing l00µl of the sample.
To the first aliquot 200µl of nutrient broth is added (Stage II). This is to be used as control.
To the second aliquot 200µl of phagebiotics is added (Stage III).
To the third aliquot 200(4.1 of phagebiotics supplemented with lysostaphin is added (Stage IV).
And to the fourth aliquot 200ul of phagebiotics supplemented with lysin is added (Stage V).
All these samples are incubated at 37°C for 18-24 hrs. A loopful from stages II-V are
streaked on blood agar plates and incubated at 37°C for 18-24 hrs. The results obtained are
summarized in table 3:
Table 3: Effect of phagebiotics, lysin and lysostaphin on sputum deposits
(Table Removed)

Stage I: Soon after processing of sputum samples
Stage II: Grown overnight in nutrient broth (control)
Stage III: Grown overnight in phagebiotics
Stage IV: Grown overnight in phagebiotics supplemented with lysostaphin
Stage V: Grown overnight in phagebiotics supplemented with lysin
Minimal growth of normal flora in low numbers of samples (26/120) is observed soon after processing the sputum samples (Stage I). All the surviving organisms are identified as Staphylococcus sp. Confluent and mixed growth of normal flora is observed in 105/120 of the samples when incubated overnight with nutrient broth (Stage II-control). And pure growth of Staphylococcus sp is observed only in 3 of the samples (Stage II-control). In the samples treated overnight with phagebiotics (Stage III) growth of normal flora is observed in 50/120 samples, 24 being Bacillus sp and 21 belonging to Staphylococcus sp while 5 samples shows confluent growth of normal flora consisting of both hemolytic and non-hemolytic organisms. Samples treated by phagebiotics supplemented with lysostaphin (Stage IV) controls the growth of normal flora in 81/120 samples. Growth of Bacillus sp and Staphylococcus sp are observed in 23 and 13 samples respectively while confluent mixed growth is seen in 3 samples. Phagebiotics supplemented with phage lysin controls the growth of normal flora in 112/120 samples during overnight incubation (Stage V). The remaining 8 samples shows minimal growth of normal flora consisting of Bacillus sp in 3 samples and Staphylococcus sp in 5 samples (Figure 2).
Lysostaphin is found to inhibit 9 out of 22 staphylococcal strains which survived the action of phagebiotics while lysin inhibits 17 of them. Among 23 samples resulting in the growth of Bacillus sp, lysin is found to inhibit 20 while lysostaphin inhibits none (Table 3).
Stage V i.e. phagebiotics supplemented with lysin gives the best results as no microbial growth is observed in 112 samples. Only Staphylococcus sp. growth is observed in 5 samples and only Bacillus sp. growth is observed in 3 samples.

Advantages:
1. The composition and method of the invention provides a non-chemical and eco-friendly substitute to antibiotics for controlling the growth of normal flora in biological samples;
2. Selectively kills normal flora without affecting the growth of microorganism of interest;
3. Provides flexibility of usage in liquid media for rapid diagnosis of tuberculosis by any method.

WE CLAIM:
1. A composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest, said composition comprising phagebiotics supplemented with phage lysin, wherein
the titer of phagebiotics in the composition ranges from ~ 1x107 pfu/ml to ~ 1x1011 pfu/ml.
2. A composition as claimed in claim 1 wherein the lysin is obtained from phages at a titer of- lxl07 pfu/ml to ~ lxl011 pfu/ml.
3. A composition as claimed in claim 1, wherein the microorganism of interest is Mycobacterium tuberculosis.
4. A composition as claimed in claim 1, wherein the biological sample is selected from the group comprising pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens.
5. A composition as claimed in claim 1 or 2, wherein the phage lysin is preferably obtained from the phages infecting Bacillus sp.
6. A composition as claimed in any of the claims 1, 2 or 5, wherein the phages are preferably Chedec 11, Chedec 20 and Chedec 21.
7. A composition as claimed in any of the claims 1, 2, 5 or 6, wherein the lysin is preferably pooled lysin obtained from phages Chedec 11, Chedec 20 and Chedec 21.
8. A method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest using the composition as claimed in claim 1, said method comprising the steps of:
i. obtaining the phagebiotics and phage lysin;
ii. mixing phagebiotics and lysin to obtain composition with desired concentration; iii. mixing the composition in the medium containing the biological sample; iv. incubating the medium;
v. obtaining a culture with inhibited growth of normal flora and isolating the microorganism of interest.

A method as claimed in claim 8, wherein the biological sample is selected from the group comprising pulmonary samples such as sputum sample and extra pulmonary samples such as gastric lavage and pus specimens.
10. A method as claimed in claim 8 wherein the concentration of lysin in the composition is preferably neat, 1/10 or 1/100.
11. A method as claimed in claims 8 and 10, wherein more preferably the concentration is 1/10.
12. A method as claimed in claim 8, wherein the incubation is done at a temperature of about 30°C to 40°C for a period of 18-24 hrs.
13. A method as claimed in claims 8 and 12, wherein preferably the incubation is done at a temperature of 37°C.
14. Phage lysin obtained from Chedec 11, Chedec 20 and Chedec 21, wherein the phage lysin is useful for preparing composition for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest.
15. A composition and a method for selectively eliminating a microorganism or a group of microorganisms from a biological sample and isolating the microorganism of interest and a phage lysin obtained from Chedec 11, Chedec 20 and Chedec 21 substantially such as herein described with reference to the accompanying drawings
and as illustrated in the foregoing examples.

Documents:

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Patent Number

269659

Indian Patent Application Number

2819/DEL/2008

PG Journal Number

45/2015

Publication Date

06-Nov-2015

Grant Date

30-Oct-2015

Date of Filing

12-Dec-2008

Name of Patentee

INDIAN COUNCIL OF MEDICAL RESEARCH

Applicant Address

V. Ramalingaswami Bhawan Ansari Nagar Post Box 4911 New Delhi – 110 029 India

Inventors:

#	Inventor's Name	Inventor's Address
1	Vanaja Kumar	Tuberculosis Research Centre Chetput Chennai- 600 031 India

PCT International Classification Number

C12N 1/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA