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Title of Thesis

Sadia Saeed
Institute/University/Department Details
Department of Microbiology/ University of Karachi
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
staphylococcin 188, staphylococcus aureus, antibiotics, antimicrobial peptides, wound pus, s aureus, bacteriocin, lantibiotics, nisin, cell free neutralized supernatant

The emergence of antibiotic-resistant bacteria is of worldwide concern. Antibiotics have been indiscriminately used and this has contributed to the rise in antibiotic resistance in a wide range of bacteria, using a variety of resistance mechanisms. Antimicrobial peptides provide a new structural class of highly active antimicrobial agents and offer a new resource for the development of novel antimicrobial agents. They are found in all living species and can have antiviral, antibacterial, antifungal or anti protozoal activity in vitro. Bacteriocins are generally able to kill specific bacterial competitors while causing little or no harm to the host bacterium, due- to posttranscriptional modification and/or specific immunity mechanisms. These proteins and peptides are generally active against species closely related to the producer of very proteins or peptides. However, this restriction is very much pulled over by the reports of new bacteriocins and advent of new techniques to assay previously reported bacteriocin. They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria. In a classical sense, although bacteriocin activity is considered to be species specific, bacteriocins produced by Gram-positive bacteria have proved to have greater spectra of activity and thus possibly have a broader application.

Staphylococcus aureus AB188 (a clinical isolate obtained from wound pus) has been found to produce bacteriocins or/and bacteriocin-like inhibitory substance tentatively termed as staphylococcin 188. The inhibitory activity could not be related to organic acids, bacteriophages and hydrogen peroxide. Staphylococcin 188 shows wide thermostability and could be stored at 4ºC for 6 months without loss of activity and remained stable at 60 ºC, 80 ºC, 100 ºC for 20 and 30 minutes and autoclaving temperature (121 ºC for 15 minutes). It remained stable at wide pH range (pH 2-14). It is also resistant to treatment with chloroform, catalase, lipase, lysozyme but sensitive to proteinase K, trypsin and protease suggesting its proteinaceous nature. It also remained stable in the presence of organic solvents and metal ions. Temperature mediated (44 ºC) plasmid curing studies suggested that the gene responsible for bacteriocin production is located on plasmid. The staphylococcin 188 was purified to homogeneity by 80% ammonium sulfate precipitation and conventional size exclusion gel chromatography using Sephadex G-75 column equilibrated and eluted with 50mM sodium phosphate buffer pH 7.0. This separation profile resulted in two major and well separated peaks designated as peak I and peak 11. Bacteriocin activity was trailed in peak 11 with minor activity in peak I. This purification step resulted in maximum specific activity of 5000A U/mg and a recovery' of 7.8% of the total activity. Staphylococcin 188 had an estimated molecular weight of 4kDa as indicated by activity detection after SDS-P AGE. Purified staphylococcin 188 was found to be thermostable, since full biological activity was retained after different treatments (i.e. pH, temperature and enzymes). Purified staphylococcin 188 has a broad activity spectrum against many Gram-positive (e.g. Corynebacterium diphtheriae, S. aureus, E. faecalis), . . Gram-negative bacteria (e.g. E. coli, S. typhi and S. dysenteriae), and dermatophytes including M canis, M gypseum, T. mentagrophytes, T. longi and T. rubrum (MIC against the sensitive isolates ranged from 10-18µg/mL). Interestingly, staphylococcin 188 also shows very potent activity against many clinical isolates of Mycobacterium tuberculosis (MIC against the sensitive isolates ranged from 3.12-100µg/mL). There was notable increase in the antimicrobial titre as compared to individual titre when combined effects of staphylococcin 188, Ent-71 and Ent-74 were studied. (bacteriocin like substances from Enterococcus faecalis SA-71 and Enterococ.cus faecalis SA-74 respectively). Staphylococcin 188 reveals bactericidal effect on S. aureus SS-1 sensitive strain (MIC = 2.2µg/mL) as well as E. coli and S. typhi, suggesting a similar mode of action on both Gram-negative and Gram-positive organisms. The activity unit of staphylococcin 188 against M /uteus SS-l00 and S. aureus SS-1 was 1280AU/mL and 80AU/mL respectively. Its production starts in early logarithmic phase and then remains constant throughout the incubation. It has bactericidal effect and causes rapid cessation of the growth of the sensitive strains. The acute and delayed toxicity studies with purified staphylococcin 188 did not show any toxic effects in the rabbits when injected with different regimen of doses (20, 40, 60 and 80µg/mL). Hematological studies with staphylococcin 188 showed that except for marginal increase in lymphocyte count in the test animal groups, no other significant effect was observed as compared to the control (rabbits injected with plain sterile saline). Further, different doses of staphylococcin 188 showed no significant biochemical changes in the normal profile of rabbit blood biochemistry. The in vivo anti-viral studies of purified staphylococcin 188 against animal viruses were performed by chick embryo technique using Newcastle disease virus (NCDV). Accordingly, the ELD50 (50% e.gg lethal dose) in case of virus injected system was found to be 100-9 while in the presence of staphylococcin 188; the ELD50 of viruses was dropped to 10-5. These observations indicate that in the absence of bacteriocin, the viruses had infected chick embryo at high titer (less viral load) while in the presence of bacteriocin, low titer of virus (high viral load) was required (ELD50) to infect chick embryo. Purified Staphylococcin 188 was checked for its in vitro anti-phage activity against bacterial virus (a coliphage HSA isolated from a raw sewage sample collected from a local sewage treatment plant of Karachi metropolitan city) by plaque assay. Accordingly, staphylococcin 188 possess demonstrable anti-phage activity witnessed as a reduction in PFU. In the control plates (plates without bacteriocin preparation) the PFU/mL was found to be 4.2 x 103 while in the presence of bacteriocin, the PFU/mL dropped up to 40. The" in vitro anti-viral studies of purified staphylococcin 188 against polio viruses (propagated on Human Rhabdosarcoma cells (RD) and L20B cell lines (Mouse L cells cloned by human poliovirus receptors) were performed by serial tube dilution and 96 well plate method. Staphylococcin 188 did not show anti-viral activity against this single strand RNA (polio) virus. The transmission electron microscopic (TEM) studies of staphylococcin 188 revealed numerous more or less circular bodies averaging from 20 to 40 nm in" diameter.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
553.01 KB
2 1 Introduction 1
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  1.1 General Introduction 1
  1.2 Bacterial Genera That Produce Bacteriocins 4
  1.3 Potential Uses Of Bacteriocins 5
  1.4 Microbiology Of Staphylococci 7
  1.5 Clinical Manifestations Of S Aureus 8
  1.6 Pathogenesis Of S. Aureus Infections 9
  1.7 Adherence Of S. Aureus To Host Tissues 10
  1.8 Bacteriocin Or Bacteriocin -Like Inhibitory 12
  1.9 Plan Of Work 17
3 2 Literature Review 19
711.61 KB
  2.1 Histological Background 19
  2.2 Microbiological Relevance To Bacteriocins 20
  2.3 Characterization And Synthesis 22
  2.4 Biochemical Nature Of Bacteriocins 23
  2.5 Inhibitory Spectrum 24
  2.6 Genetics And Regulation 25
  2.7 Determination Of Bacteriocin Activity 26
  2.8 Classification Of Bacteriocins 27
  2.9 Mode Of Action 28
  2.10 Immunity 30
  2.11 What Are Lantibiotics 31
  2.12 Activity Spectra And Properties Of Class I And Class 32
  2.13 What Is Nisin 34
4 3 Materials And Methods 45
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  3.1 Culture And Media 45
  3.2 Assays For Bacteriocin Activity 46
  3.3 Preparation Of Cell Free Neutralized Supernatant (CFNS ) 48
  3.4 Bacteriocin Activity Or Lytic Phages 48
  3.5 Physical Characterization 49
  3.6 Chemical Characterization 50
  3.7 Titration Of Bactericocins 54
  3.8 Lacunna Count 56
  3.9 Isolation Of Resistant Mutants 57
  3.10 Determination Of Location Bac + Markers/ Genes( Chromosomal Or Plasmid Borne) By Plasmid Curing 57
  3.11 Growth Curve With Simultaneous Measurement Of Staphylococcin 188 Production 58
  3.12 Combination Of Staphylococcin 188 With Other Bacteriocins 58
  3.13 Partial Purification Of Staphylococcin 188 59
  3.14 Mode Of Action Of Staphylococcin 188 On Susceptible Host Cell 63
  3.15 In Vitro Anti- Dermatophytic Activity Of Staphylococcin 188 64
  3.16 In Vitro Anti- Mycobacterial Activity Of Staphylococcin 188 65
  3.17 Hematological /Toxicological Studies Of Staphylococcin 188 66
  3.18 Anti-Viral Activity Of Staphylococcin 188 67
  3.19 Transmission Electron Microcopy (TEM) Of Staphylococcin 188 72
5 4 Results 73
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  4.1 Isolation, Identification And Characterization Of Bacteriocin Producing And The Sensitive Strains 73
  4.2 Production Of Bacteriocin Like Inhibitory Substances By Staphylococcus Aureus Ab 188 74
  4.3 Effect Of Different Treatments On Staphylococcin 188 Activity 75
  4.4 Effect Of Different Treatment On Staphylococcin 188 Production 76
  4.5 Titration Of Staphylococcin 188 77
  4.6 Lacuna Counts 78
  4.7 Isolation Of The Resistant Mutant Of Staphylococcus Aureus Ab 188 78
  4.8 Plasmid Curing 79
  4.9 Combination Of Bacteriocins 79
  4.10 Activity Directed Purification Of Staphylococcin 188 79
  4.11 Mode Of Action Of Staphylococcin 188 82
  4.12 Hematological And Biochemical Studies Of Staphylococcin 188 83
  4.13 Anti-Bacterial Activity Of Staphylococcin 188 84
  4.14 Anti-Dermatophytic Activity Of Staphylococcin 188 85
  4.15 Anti-Viral Studies Of Staphylococcin 188 86
  4.16 Transmission Electron Microscopy (Tem) Of Staphylococcin 188 87
6 5 List Of Tables 88
423.08 KB
7 6 List Of Figures 111
646.01 KB
8 7 Discussion 135
752.42 KB
9 8 Conclusion 164
115.92 KB
10 9 References 168
804.01 KB