Antimicrobial activity of Myxobacteria isolated from soil.pdf

MINISTRY OF EDUCATION AND TRAINING NGUYEN TAT THANH UNIVERSITY 0O0 FINAL REPORT TOPICS STUDENT SCIENCE RESEARCH PROGRAM YEAR 2020 Dissertation’s name: ANTIMICROBIAL ACTIVITY OF MYXOBACTERIA ISOLATED FROM SOIL Topic code: Topic manager: NGUYEN PHUONG CHAM Supervisor: M.S Pharm NGUYEN THI NGOC YEN Industry: Pharmacology The members participate' No Student's full name Student ID Class Nguyen Phuong Cham 1511540569 15DDS8A Ho Chi Minh City, 10/2020 i Nguyen Phuong Cham CONTENTS CONTENTS ii LIST OF ACRONYMS iv LIST OF TABLES V LIST OF FIGURES vi ABTRACT vii CHAPTER LITERATURE REVIEW CHAPTER SUBJECTS - RESEARCH METHOD 2.1 SUBJECTS 2.1.1 Soil samples 2.1.2 Test microorganisms 2.2 Media and chemicals 2.2.1 Media 2.2.2 Chemicals 2.2.3 Instruments 2.3 RESEARCH METHOD 2.3.1 Collection of samples 2.3.2 Myxobacterial isolation 2.3.3 Purification methods 2.3.4 Myxobacterial identification 2.3.5 Storage 2.3.6 Screening antimicrobial activities .10 CHAPTER RESULTS AND DISCUSSION 17 3.1 RESULTS 17 3.1.1 Collection of the soil samples 17 3.1.2 Isolation of myxobacterial isolates 18 3.1.3 Purification and identification of myxobacterial isolates 20 ii Nguyen Phuong Cham 3.1.4 The antimicrobial activities of myxobacterial strains 26 3.2 DISCUSSION 32 3.2.1 Isolation of myxobacterial isolates 32 3.2.2 Purification and identification of myxobacterial isolates 32 3.2.3 The antimicrobial activities of myxobacterial strains 33 CHAPTER CONCLUSION .35 REFERENCES 36 Hi Nguyen Phuong Cham LIST OF ACRONYMS An Aspergillus niger Ca Candida albicans CFU Colony-forming unit CMC Carboxyl methyl cellulose DMSO Dimethyl sulfoxide Ec E coll GC Growth control GMB medium MHA with 2% glucose and pg/mL methylene blue LB broth Luria Bertani Broth MHA Muller-Hinton agar MHB Muller-Hinton broth MHB+2% Muller-Hinton broth + 2% glucose glucose MIC Minimum Inhibitory Concentration MRSA Methicillin resistant Staphylococcus aureus MSSA Methicillin susceptible Staphylococcus aureus Mu Mucor sp P-medium Peptone medium Pa Pseudomonas aeruginosa PDA Potato dextrose agar Pe Penicillium Rh Rhizopus sp SC Sterility control Sf Streptococcus faecalis TSA Tryptic soy agar wcx Water agar with cycloheximide 100 pg/mL iv Nguyen Phuong Cham LIST OF TABLES Table 2.1 Test microbial strains Table 2.2 List of laboratory instruments used in this research Table 2.3 Preparartion of inoculum in agar well diffusion method 13 Table 2.4 Preparartion of inoculum in broth microdilution 14 Table 3.1 Total soil samples were collected 17 Table 3.2 The number of identified myxobacterial strains 20 Table 3.3 The number of identified strains from provinces/cities 20 Table 3.4 Morphological characteristics of some isolates 21 Table 3.5 Morphology of vegetative cell, fruiting body, myxospore, and colony 22 Table 3.6 Identification of new isolates based on 16S rDNA analysis 23 Table 3.7 Antimicrobial activities of myxobacterial extracts 26 Table 3.8 The number of myxobacterial strains exhibit inhibition on test microorganisms 28 Table 3.9 Distribution of antibacterial activity of the BDI 2.2 strains 30 Table 3.10 Distribution of antifungal activity of the BDI 2.2 strains 30 Table 3.11 96-well microtitre plates on MIC test 31 V Nguyen Phuong Cham LIST OF FIGURES Figure 2.1 The procedure of this study Figure 2.2 Different isolation methods Figure 2.3 Check the purity of the isolated strains by CEHmedium Figure 2.4 The procedure of myxobacterial extract preparation 10 Figure 2.5 The 96 well microtiter plate 15 Figure 3.1 Soil sampling locations in Vietnam 18 Figure 3.2 Fruiting bodies in wild rabbit dung of the rabbit dung method 19 Figure 3.3 Fruiting bodies of some isolates in filter paper/ST21 medium 19 Figure 3.4 Fruiting bodies of some isolates in wcx medium 19 Figure 3.5 The percentage of myxobacteria isolated by three methods 20 Figure 3.6 Gram stain of myxobacterial vegetative cells 23 Figure 3.7 Phylogenetic tree of some strains based on 16S rRNA gene sequences25 Figure 3.8 The number of extracts from p and VY/3 media exhibits antibacterial properties on each test organism 28 Figure 3.9 The antibacterial activities of the crude extracts 29 Figure 3.10 The number of extracts from p and VY/3 media exhibit antifungal properties on each test organism 29 Figure 3.11 The antifungal inhibition zones of myxobacterial extracts 29 vi Nguyen Phuong Cham ABTRACT DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE PHARMACIST - ACADEMIC YEAR 2020 ANTIMICROBIAL ACTIVITY OF MYXOBACTERIA ISOLATED FROM SOIL Nguyen Phuong Cham Instructor: M.Sc Pharm Nguyen Thi Ngoc Yen Introduction: Myxobacteria are Gram-negative bacteria commonly found in soil, tree bark, and decay wood These bacteria have unique social behaviors by forming fruiting bodies, moving by gliding motility, and preying on other microorganisms The purpose of the research is to explore new strains capable of producing secondary metabolites with antimicrobial activity that opened promising treatment direction on infectious disease in the future Materials and methods: Myxobacteria were isolated using three conventional methods (WCX with E coll bait, filter paper, and rabbit dung) Characterization of myxobacteria was performed with Gram staining, observation on the morphology of vegetative cells, fruiting bodies, and myxospores Molecular identification was conducted based on 16S rRNA gene sequence analysis Antimicrobial activities were evaluated by (i) Agar well method for screening inhibition and (ii) Broth microdilution using 96-well microtitre plates for MIC determination Results: From 110 soil samples collected at 19 cities and provinces, 43 identified strains /68 isolates with high similarity (from 91.1-100%) were distributed in six genera, including Corallococcus, Myxococcus, Cystobacter, Archagium, Angiococcus, and Chondromyces Most of myxobacterial strains exhibited the resistance to most of test microorganism, especially on yeast and fungi MIC values of BDI 2.2 raw extract on P-medium were 1024, 16, 1024, 256, 128, 256, and 1024 pg/mL for MSSA, MRSA, p aeruginosa, c albicans, Mucor sp., Rhizopus sp., and A niger, respectively MIC values of VY/3 extracts were 1024, 16, 256, 512, 32, 256, 256, and pg/mL for MSSA, MRSA, s faecalis, p aeruginosa, c albicans, Mucor sp., Rhizopus sp., and A niger, respectively Conclusion: Bioactive evaluation showed that most strains had antimicrobial capabilities, but BDI 2.2 strains exhibit the impressive inhibition of MRSA (16 pg/mL) and A niger (4 pg/mL) vii Nguyen Phuong Cham Final dissertation Introduction CHAPTER LITERATURE REVIEW Antimicrobial resistance is rising to dangerously high levels in all parts of the world because of greater access to antibiotic drugs in developing countries Each year, millions of people die as a result of treating the transmissible disease unsuccessfully It poses challenges to researchers and the whole community in research and development of new active ingredients that help treat such infections Currently, the research for active substances has expanded in various directions, from medicinal plant extracts, chemical synthesis to secondary metabolites from microorganisms In particular, research orientation on microorganisms has brought many results Myxobacteria is also one of the most promising strains since they were functionally characterized to assess their ability to produce antibacterial and anticancer metabolites Most compounds from myxobacteria were not found in other organisms and are unique to the bacterium The secondary metabolites have antimicrobial and antifungal activities that can mention as Nannochelin, Pyrrolnitrin, Ripostatin, e.g Not only that, secondary metabolites from myxobacteria often target cellular structures, and many of the isolated substances showed cytotoxic activity against the human cancer cell lines [4] There have been many studies around the world aiming to isolate and survey the biological activities of myxobacterial strains However, in our country, research topics relating to myxobacteria have not been paid much attention The dissertation named “Antimicrobial activity of myxobacteria isolated from the soir was carried out for isolation, identification, and screening the active secondary metabolites of myxobacteria strains in Vietnam, which can be further applied in therapeutic purposes in the future: - Isolation of myxobacteria three conventional three methods: (1) Using wild rabbit dung, (2) Using E coli baiting on wcx medium, and (3) Isolation of cellulolytic myxobacteria using ST21 medium with filter paper as a carbon source; - Purification of myxobacteria isolates; - Myxobacterial identification using morphological characteristics and analysis of 16S ribosomal RNA sequencing databases; Nguyen Phuong Cham Final dissertation Introduction - Preparation of crude extracts from isolated myxobacterial strains and screening antibacterial and antifungal activities using agar well diffusion and MIC determination by microdilution method in 96-plate microtitre plates Nguyen Phuong Cham Final dissertation Subjects - Research methods CHAPTER SUBJECTS - RESEARCH METHOD 2.1 SUBJECTS 2.1.1 Soil samples Soil samples were collected from some provinces in Vietnam: Ho Chi Minh City, Vinh Long, Hau Giang, Binh Duong, Thanh Hoa, Binh Phuoc, Binh Thuan, Ninh Binh, Long An, Tien Giang, e.g Soil samples were collected aseptically from a depth of 10-15 cm using clean, dry, and sterile polythene bags The samples were immediately air-dried at room temperature as soon as possible to reduce the development of mold and worms before being used for direct isolation of myxobacteria [29] Six strains of myxobacteria ( VL4.5, VL4.7, HG2.8, HG2.12, HG2.23, HG2.25) were obtained from the results of the thesis “Isolation of Myxobacteria with biological activity from the soil” of Ms Nguyen Le Bao Ngoc in 2019 2.1.2 Test microorganisms Table 2.1 Test microbial strains Strains Code Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 Methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 25923 Streptococcus faecalis ATCC 29212 Pseudomonas aeruginosa ATCC 27853 Escherichia coll ATCC 25922 Candida albicans ATCC 10231 Aspergillus niger ATCC 16404 Penicillium sp - Mucor sp - Rhizopus sp - Nguyen Phuong Cham ... isolation of myxobacteria [29] Six strains of myxobacteria ( VL4.5, VL4.7, HG2.8, HG2.12, HG2.23, HG2.25) were obtained from the results of the thesis “Isolation of Myxobacteria with biological activity. .. activities of myxobacterial strains However, in our country, research topics relating to myxobacteria have not been paid much attention The dissertation named ? ?Antimicrobial activity of myxobacteria isolated. .. ABTRACT DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE PHARMACIST - ACADEMIC YEAR 2020 ANTIMICROBIAL ACTIVITY OF MYXOBACTERIA ISOLATED FROM SOIL Nguyen Phuong Cham Instructor: M.Sc