I= PRODUCTION, PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN-LIKE INHIBITORY SUBSTANCES (BLIS) FROM INDIGENOUS ENTEROCOCCI
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Title of Thesis
PRODUCTION, PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN-LIKE INHIBITORY SUBSTANCES (BLIS) FROM INDIGENOUS ENTEROCOCCI

Author(s)
Samia Ahmed
Institute/University/Department Details
Department of Microbiology/ University of Karachi
Session
2007
Subject
Microbiology
Number of Pages
264
Keywords (Extracted from title, table of contents and abstract of thesis)
bacteriocin-like inhibitory substances, blis, enterococci, prokaryotic organisms, toxins, antibiotics, bacteriolytic enzymes, metabolic pathways, bacteriocins, bacteriocin, enterococcal isolates, ampicillin, penicillin g, cephazolin, cefuroxime, ceftazidime, vancomycin, teicoplanin, fosfomycin, gentamicin, streptomycin, chloramphenicol, tetracycline, norfloxacin, ciprofloxacin, enterococcus faeclum aar20, enterococcus faecalis aar71, enterococcus casseliflauus aar 102, enterococcus avium aar 124, enterococcus avium aar136, enterococcus faeclum aar170, enterocin aar20, enterocin aar71, enterocin aari02, enterocin aar124, enterocin aar136, enterocin aar170, bacteriocins

Abstract
Most prokaryotic organisms produce a heterogeneous array of molecules during their growth which are antagonistic to other organisms e.g. toxins, antibiotics, bacteriolytic enzymes, by-products of metabolic pathways, bacteriocins and bacteriocin-like substances. Among them bacteriocins comprise of most abundant diverse group responsible of bacterial defense systems, naturally produced by certain strains of gram positive and gram-negative bacteria. They are capable of providing natural support to protect people from undesirable bacterial infections. In this study a total of three hundred enterococcal isolates were collected from different pathological laboratories and hospitals of Karachi. Identification of their strains was done using both conventional and kits. method (RapID STR kit). Percentages of enterococci found in blood were 11 %, body effusions 6%, high vaginal swabs 6%, pus 18%, rectal swabs 11 % and urine 48%. As regard their strains, E. faecalis were found 69.3%; E. faecium, 16.6%; E. raffinosus, 4.3%; E. cassefilauus, 4%; E. gallinarum 3% and E. avium 2.6%.

They all were screened for antibiotic resistance against fourteen different drugs i.e. ampicillin, penicillin G, cephazolin, cefuroxime, ceftazidime, vancomycin, teicoplanin, fosfomycin, gentamicin, streptomycin, chloramphenicol, tetracycline, norfloxacin and ciprofloxacin by disc diffusion method. Antibiograms were drawn and resistance was interpreted according to the breakpoint values for enterococci as given in National Committee for Clinical Laboratory Standards (NCCLS). Antibiotic resistance pattern showed that teicoplanin, ampicillin, fosfomycin, vancomycin and penicillin G were significally effective on the enterococcal isolates showing 80%, 78.3%, 76.6%, 74% and 73.3% sensitivity respectively. Chloramphenicol occupied middle position showing 54% sensitivity. Rests of the antibiotics tested were towards lower side showing gentamicin 41%; norfloxacin 40.6%; ciprofloxacin 30.6%; cefuroxime 26.6%; tetracycline 21.3%; ceftazidime 8.3%; cephazolin 5.3% and streptomycin 4.3% sensitivity. MIC sensitivity profile was determined by plate dilution method against eight different antibiotics. Since 38.6% and 58.3% strains of enterococci showed resistance to gentamicin and streptomycin respectively up to a concentration of 500ug/ mL, it appears that enterococci have developed high-level aminoglycoside resistance (HLAR).

Enterococcal strains were screened for determining bacteriocinogenic potential by three conventional methods i.e. cross-streak, stab-overlay and agar-well diffusion assays. Percentage of the enterocin producers on solid media screened by cross-streak and stab-overlay methods remained same (i.e. 70%) while in liquid media screened by agar-well diffusion method it was found 51.6%. Since the inhibitory zone produced were not due to organic acids, hydrogen peroxide or bacteriophages, these substances can be classified as bacteriocins. Frequency of enterocin production was found high in E. faecalis strains and low in E. raffinosus strains in both the solid and liquid medium.

Initial characterization (both the physico-chemical) was performed in six strains namely Enterococcus faeclum AAR20, Enterococcus faecalis AAR71, Enterococcus casseliflauus AAR 102, Enterococcus avium AAR 124, Enterococcus avium AAR136 and Enterococcus faeclum AAR170. Their bacteriocins were named as enterocin AAR20, enterocin AAR71, enterocin AARI02, enterocin AAR124, enterocin AAR136 and enterocin AAR170 respectively. The inhibitory substances produced by these isolates showed narrow or broad spectrum of activity, essential protein moiety, bacteriolytic or bactericidal mode of action, attachment to cell receptors on susceptible cells and plasmid-encoded function which is in agreement with the criteria to call them as bacteriocins.

Partial purification was done using enterocins AAR71 and AAR170. Both of them were found inhibitory to gram-positive as well as gram-negative bacteria. Among gram-positive bacteria they were inhibitory to Enterococcus, Streptococcus and Staphylococcus species. They were also found inhibitory against vancomycin-resistant (VRE), multi-drug resistant (MDR) enterococci and methicillin-resistant Staphylococcus aureus (M RSA) , food spoilage bacteria like S. aureus and L. monocytogenes. Where as among gram-negative bacteria, they were found inhibitory against N. meningitidis and phytopathogens e.g. Xanthomonas species.

Proteolytic enzymes were observed rapidly inactivating the bacteriocin activity in above-mentioned enterocins whereas glycolytic and lipolytic enzymes had no effect. They remained stable in the presence of organic solvents, metal ions, protease inhibitors, at low temperatures (-20°C, O°C and 4°C for 6 months), at high temperatures (80°C, 100°C for 60min) and at 121°C (15 psi) for 15 min. As regard pH the enterocin AAR71 maintained its activity within pH range 2-14 and enterocin AAR170 within 2-12. The former was bacteriolytic while later was bactericidal in mode of action. Plasmid curing studies by ethidium bromide suggested correlation of bacteriocin production to plasmid borne genes. The electron micrographs of native negatively stained enterocin AAR170 revealed defective bacteriophage like structure Le. empty head with short tail. Both the enterocins were purified. to homogeneity by ammonium sulfate precipitation, ultra filtration and size exclusion chromatography. Purification of AAR71 and AAR170 resulted in 16.19 and 15.4 folds purification. Their electrophorogram as indicated by activity detection after SDS-PAGE revealed a single band of molecular mass of approximately 3.5KDa for enterocin AAR71 and 4KDa for enterocin AAR170 respectively. Their properties of thermo-tolerance, retention of activity after cold temperature and wide range of pH, antagonistic activity towards many pathogens give them desired characteristics for possible use as an additive in food preservatives as well as chemotherapeutic agents.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
687.96 KB
2 1 Introduction And Literature Review 1
1250.72 KB
3 2 Materials And Methods 52
949.06 KB
4 3 Tables 95
360.97 KB
5 4 Figures 114
733.1 KB
6 5 Graphs 136
886 KB
7 6 Results And Discussion 168
1243.62 KB
8 7 Conclusion 216
92.25 KB
9 8 References 220
936.05 KB