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1. Isolation, molecular identification and application of probiotic Lactobacillus against biofilm forming Klebsiella pneumoniae Submitted by Sneha K P222030 Under the guidance of Dr. Rishad K S UniBiosys Biotech Research Lab, Kochi

2. INTRODUCTION The genus Lactobacillus encompasses a diverse assemblage of Gram-positive, catalase-negative, non-sporing rod-shaped organisms. Over 50 species are currently recognized. Although members of the group have been extensively studied, it is widely acknowledged that the taxonomy of the genus is unsatisfactory. The genus Lactobacillus as constituted in Bergey's Manual of Systematic Bacteriology is phenotypically very heterogeneous. Probiotic bacteria, particularly Lactobacillus species, have gained significant attention for their potential health-promoting properties and antimicrobial activities. In this study, we aimed to identify a probiotic Lactobacillus strain and investigate its antibiofilm activity against Pseudomonas sp ., a common pathogenic bacterium associated with various infections. To achieve this, a Lactobacillus strain was isolated from a commercially available probiotic product and characterized through molecular identification using 16S rRNA gene sequencing. The most widely used techniques for bacterial identification and quantification are based on the 16S rRNA gene. Previous studies have indicated that the 16S rRNA gene is present in multiple copies in most bacterial genomes and the copy numbers vary from one to as many as 15. The 16S rRNA gene copy number is useful for 16S rRNA gene-based quantification technique.

3. MATERIALS AND METHOD 1. SAMPLE COLLECTION AND ISOLATION OF BACTERIA (Lactobacillus) Collect 3 different curd samples. Serial dilution of the collected samples from 10 -1 to 10 -5 . Spread plate method : Prepare 120 ml of MRS Agar media. After sterilization pour the media into four different petri plates. Mark one plate as control others as 10 -2 ,10 -3 and 10 -4 respectively. Take 0.1ml of serially diluted samples from 10 -2 ,10 -3 and 10 -4 test tubes and pour into respective plates and then spread plate it. After that keep the plates in incubator for 24 hours. And count the colonies after 24 hours of incubation. Quadrant streaking: Prepare MRS Agar plate. Take a loopful of inoculum from the 10 -4 spread plate and quadrant streak it. And observe after 24 hours of incubation. Nutrient Broth inoculation : Prepare 5 ml of nutrient broth. Then take a loopful of bacterial colony from quadrant streak plate and inoculate it into the nutrient broth. Incubate it for 24 hours.

4. 2.MORPHOLOGICAL ANALYSIS Gram staining Materials required: Glass slide, inoculation loop, crystal violet, gram’s iodine, 95% ethanol, safranin, bacterial culture   Procedure: Air dry or heat fix the bacteria smear Flood the slide with crystal violet staining reagent for one minute Wash the smear in a gentle and direct stream of tap water for one minute Flood the slide with gram’s iodine solution for one minute Wash the slide and decolourize with 95%ethanol for a few seconds Flood the slide with water and then immerse the smear for one minute in counter stain called safranin Wash the slide, dry and observe under microscope (10X /40X)  

5. 3.LACTIC ACID PRODUCION AND PROPERTIES LACTIC ACID ESTIMATION TEST: Make 5 ml of 0.2% FeCl3 solution in a beaker. Take 2 test tubes, mark one as control and other one as test. Add 2 ml FeCl3 in to each test tube. Then add 50 µl bacterial culture into the test tube which mark as test. And measure the absorbance with a spectrophotometer at 390nm. BILE TOLERENCE TEST: T ake 3 test tubes and add 1 ml of inoculated nutrient broth into each test tubes. Then mark one as control and others as test 1 and test 2. Add 0.1 ml of 0.3% bile solution into test 1 and add 0.1 ml of 0.1% bile solution into test 2 and mix well. After 3 hours of incubation measure the absorbance with a spectrophotometer at 600nm.  ACID TOLERENCE TEST: Take 3 test tubes and add 1 ml of inoculated nutrient broth into each test tubes. Mark one tube as control and others as test 1and test 2. Check pH of the control and make the pH of test 1 into 2 and pH of test 2 into 3 by using 5 N HCl or 0.1% NaOH. After 3 hours of incubation measure the absorbance with a spectrophotometer at 600nm.

6. RESULTS .   SAMPLE NUMBER OF COLONIES 10 -2 spread plate 10 -3 spread plate 10 -4 spread plate Curd sample 1 Numerous Single colony Single colony Curd sample 2 Numerous 149 132 Curd sample 3 Numerous 190 41 Isolation of probiotic lactobacillus on MRS agar Fig.1: Spread plate of Lactobacillus sp . Table 1: number of colonies counted on spread plate

7. Fig.2: Gram stain of Lactobacillus sp . Table2: Morphological and microscopical characteristics Morphological and microscopical characteristics Result Colony morphology Creamy, little sticks and smooth round colonies Gram staining Gram positive, rod Motility Non-motile Growth in MRS broth uniform turbidity

8. LACTIC ACID PRODUCTION AND PROPERTIES   Graph 1: lactic acid estimation standard graph Table 3: lactic acid estimation test result   OD at390 nm Conc. Of lactic acid(µg/ml) Curd sample 1 0.183 0.0853 Curd sample 2 0.317 0.208 Both curd samples follow the lactic acid estimation standard graph.