Introduction to antibiotics, marine, process

1. Marine MicrobialAntibiotics
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2. • Substances produced by living organisms found in nature have
played a critical role in the development of Antibiotics for life-
threatening conditions.
• Antibiotics were first produced in 1939. The term antibiotics
was introduced by S. A Waksman in 1942.
• Marine microbes including microalgae, bacteria, archaea,
protozoa, fungi, and viuses.
• Antibiotics are used to treat infections caused by organisms
that are sensitive to them, usually bacteria or fungi.
• AAntibiotics used to treat bacterial infections.
• Antibiotics are medicine that kills bacteria or inhibit the
growth of bacteria. There are used to cure diseases.
• Originally, an antibiotic was a substance produced by one
microorganism that selectively inhibits the growth of another.
INTRODUCTION
2
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3. Marine
• Marine drugs are compounds obtained from marine plant,
animal and microorganisms about 70% of earth surface covered
with water.
•Marine environment rich source of both biological chemical
diversity.
• Several of the compounds isolated from marine source exhibit
biological activity. The ocean is considered to be a source of
potential drugs. Marine organisms not only elaborate
pharmaceutically useful compounds but also produce toxic
substances.
• Several of these organisms are actively adapted to marine
environments (salt, pressure, etc)Serious problem is the presence
of numerous terrestrial microbes thriving in marine environment.
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4. Marine Organisms
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5. • Definition of marine bacteria is based on their ability to grow
only at certain sea water concentrations.
• Many marine bacteria requires sodium, potassium and
magnesium ions. Some of also require chloride ions and
ferric ions.
• Near coastal waters 95% of the bacterial population is salt
tolerant forms. Only 5% is true marine forms.
• In the open ocean and in deep sea the true marine forms
dominate.
• Significant portion the worlds bacteria live in the water
column of the oceans.
Marine Bacterial Diversity
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6. Cont....
• Bacteria are single-celled organisms without cell nuclei. They are
found in all portions of the water column, the sediment surface,
and the sediments themselves.
• Some are aerobic (requiring oxygen), whereas others are
anaerobic (not requiring oxygen).
• Most bacteria are free-living, but some live as partners
(symbionts) within other organisms.
• For instance, many deep-sea fish harbor symbiotic bacteria that
emit light, which the fish use to signal other members of their
species.
• The bacteria's ability to emit light is called bioluminescence.
Bioluminescence causes water to glow, a phenomenon most
noticeable at the surface but present at all depths.
•The largest known species of bacteria was found in 1999 in the
ocean sediments of Namibia, a country in southern Africa.
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7. Cont...
•The golden spheres inside the cell are accumulations of the
element sulfur (S) from the surrounding environment. The shiny
appearance and tendency of cells to group together in long strings
inspired this species' name: Thiomargarita namibiensis
• Name a chemical element or compound of elements, and there
are bacteria are using it, releasing it, recycling it, and
transforming it. This movement of elements through living
matter, the atmosphere, oceans, and sediments is referred to as
cycling.
• The carbon and nitrogen cycles are two of the most important
global cycles, and bacteria play a key role every step of the
way. Without bacteria, these cycles would not occur and life
could not exist.
• Bacterial decomposition is an important part of nutrient cycling,
and bacteria can decompose dead organisms.
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8. Cont...
•Bacterial decomposition is an important part of nutrient cycling,
and bacteria can decompose dead organisms as big as whales or as
small as other microbes.
• They break down organic molecules formed by biological
processes, making the nutrients in these molecules available for use
by other organisms, such as plants.
• The microbes themselves can also serve as food for larger
organisms. Some elements, such as iron (Fe) are rare but important
in the ocean, making it important to recycle them.
• When an organism dies or releases an iron-containing
compound, microbes "catch" that and get all the iron out of it
before it can sink too deep.
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9. Cyanobacteria
• One group of bacteria called cyanobacteria carry out
photosynthesis. They take carbon dioxide (CO2) from the air, turn
it into organic parts of their own cells, and release oxygen (O2)
just like plants. Half of the primary production, done on Earth is
in the ocean, and half of that is done by bacteria.
• In addition, bacteria are terrific chemists. Almost any chemical
reaction can be done by at least one group of bacteria.
• Some can take nitrogen gas (N2) from the air and turn it into
organic parts of their own cells—they can do the reverse too.
• Others play a key role in the sulfur (S) cycle, which is linked to
cloud formation, so even though they are tiny bacteria affect the
weather of our planet.
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10. Cyanobacteria (“Blue-GreenAlgae”)
Photosynthetic Bacteria
Oldest Organisms on the Earth
(Fossil Record - 3.5 Billions Years!)
Marine, Freshwater and Terrestrial
Symbiosis - e.g. Lichen
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11. •Cyanobacteria, a type of bacteria, played an important role in
the history of Earth and in ocean processes.
•cyanobacteria produced oxygen during the process of
photosynthesis, which generated the oxygen in the Earth's
atmosphere that many living beings require
•cyanobacteria also are called "blue-green algae," it is important
to remember that cyanobacteria are relatives of bacteria and not
algae.
• They are, however, related to the chloroplasts within algae;
the chloroplasts used by some plants to produce food are
actually cyanobacteria living within plants' cell walls.
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12. List of Marine Bacteria
•Bacillus cirroflagellosus
•Bacillus epiphytus
•Bacillus filicolonicus
•Bacillus licheniformis
•Bacillus subtilis
•Brevibacterium stationis
• Corynebacterium stationis
•Brevibacterium vitarumen
•Corynebacterium vitaeruminis
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13. • True marine fungi are defined on the basis of their ability to
grow and sporulate in sea water.
• Where as faculative marine fungi are from land, which are
capable of grow in sea water
• The first faculative marine fungus described was
phaeosphaeria typharum and the first obligate marine fungus
isolated from rhizome of a seagrass was posidonia oceanica.
• Marine fungi are species of fungi that live in marine
environments.
• Obligate marine fungi grow exclusively in the marine habitat
sporadically submerged in sea water.
• Facultative marine fungi normally occupy freshwater habitats
but are capable of living or even sporulating in a marine
habitat.
Marine Fungal Diversity
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14. Marine Toxins
• The majority of marine toxins are produced by microalgae,
especially dinoflagellates. Some of the toxins are also
produced by bacteria and a few by macroalgae.
Brevetoxins:
• The dinoflagellate Gymnodinium breve has yielded several
toxins named brevetoxins. Chemically they are highly
oxygenated polyethers of the isolated toxins, brevetoxin-A is
most potent toxin.
• breve isolated during an outbreak at Florid were grown in an
artificial sea-water medium. The medium containing the cells
were acidified to pH 5.5 and extracted with diethyl ether to
give 90 mg of crude brevetoxins.
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15. compounds from
marine derived fungi
• Aspergillus flavus- Desmethylnomifensine- Antibacterial
• Penicillium viridicatum-Fumigaclavine-Antibacterial Antifungal
• Penicillium griseofulvum Y19-07 - 4-Hydroxyphenethyl methyl
succinate 4-Hydroxy-phenethyl 2-4- hydroxyphenyl acetate –
Cytotoxicity
•Penicillium expansum Expansol A Expansol B Cytotoxicity
• Aspergillus fumigatus-Bismethylthiogliotoxin 6-
methoxyspirotryprostatin B –Antiparasitic
•Fusarium sp-Fusaquinon A Fusaquinon B Fusaquinon C-
Cytotoxicity
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16. • The serially diluted inoculum of 0.1 ml will be spread
uniformly with sterile glass spreader on the Nutrient agar
medium.
• These materials will be incubated at room temperature and
observed from 1-5 days.
• Colonies will be counted and viable count will be made as
No. of colonies appered on the plates, mulitiplied by dilution
factor.
Isolation Method
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17. • For identification, a series of biochemical, physiological and
serological tests have to be performed or using prescribed
test kits,the identification will be confirmed.
Antibiotics Assay :
• This can be used to identify strains with in a given species.
• Which is especially important in epidemiological studies.
• Different tests are available, the disc method is probably the
simplest to perform and interpret.
Identification Method
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18. Cross Streak Method :
• The strains were cross streaked against the pathogens an MHA
plates.
• The strains was streaked horizontally on the MHA and the
Pathogens were streaked vertically towards the strain till a
marginal line.
• The pathogen crossing the marginal line is taken as positive
result.
Screening for Anti-Microbial Activity
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19. • Agar well Diffusion Method :
• 5 test tubes were prepared each containing 2 mi of LB.
• After autoclaving the pathogenic strains were inoculated in it
kept in shaker at 180 rpm at 37 C.
• The autoclaved buds were dipped in the broth and swabbed on
the MHA plates.
• The 4 test bacterial strains were inoculated in 5 ml of NB after
autoclaving. It was kept on shanker for 48 hours.
• The cultures of the strains were poured on to the wells. The
cultures were then centrifuged at 6000 rpm for 10 minutes.
Their supernatants were then poured in the wells. The plates
were than kept for 48 hours incubation. The inhibitiob zones
were observed.
Screening for Anti-Microbial Activity
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20. • Disc Diffusion Method :
• The selected strains were inoculted in 50 ml of Zorbell’s marin
broth and was kept for shanking in 160 rpm at 32 C for 48
hours.
• After 48 hours 5 ml of broth culture was centrifuged at 5000
rpm for 10 minutes at 4 C. 1 ml supernatant was collected and
added with 1 ml ethyl acetate.
• Then supernatant with ethyl acetate was shaken for 2 minutes
and crystal layer formed on the surface was pipetted out.
Crude extract was collected.
• The crude extract was then mixed with 2 ml of dmso and the
extract was made to dissolve in it.
Screening for Anti-Microbial Activity
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21. • Small disc was punched out from the filter paper and
autoclaved. The pathogenic stains were grown in LB. 100 µl of
the pathoenic strains were spread on MHA plates.
• The filtered was dippes in the crude extract solution mixed
with DMSO and then kept on the MHA plates.
• The plates were then kept for 48 hours incubation. The zones
were obsered around the filter paper.
Cont…
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22. • Life has originated from the oceans that cover over 70% of the
earth’s surface and contain highly ecological, chemical and
biological diversity starting from microorganisms to
vertebrates.
• The source of unique chemical compounds, which hold
tremendous pharmaceutical potential.
• Sources emphasize on investigation of the marine ecosystem
to explore numerous complex and novel chemical entities.
• The sources of new leads for treatment of many diseases such
as cancer, AIDS, inflammatory conditions, and a large variety
of viral, bacterial and fungal diseases.
Marine Organisms for Antibiotic Discovery
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23. • The discovery process begins with isolation of unique
microbes and generation of chemical extracts of biologically
active molecules produced by the microbes.
• Information on the microorganisms, their sources, culture and
extraction conditions, novel chemistries and biological
activities have been developed that allow for a concentrated
effort on the most productive organisms.
• Primary targets leads to identification of novel, biologically
active discovery leads in the selected therapeutic areas of
oncology and infectious diseases
Discovery Process and Productivity
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24. • Cancer
- Pancreatic Cancer
- Multi Antibiotic resistant cancers
- Cancer “Specific” Agents (NCDDG)
• Infectious Disease
- Antibiotic resistant staphylococcus aureus
- Anti-malarial-collaborative
Neurodegenerative Disease-collaborative
- Neuroprotection (Stroke)
• Inflammation-collaborative
Prospects for Success
Finding Antibiotic to Treat
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25. Antibiotics from microorganisms
• The first antibiotics from the marine bacteria to be isolated and
identified was 2-(3’,5’-dibromo-2’hydroxphenyl)
– 3,4,5-tribromopyrrole by burkholder.
• The marine bacterium pseudomonas bromoutilis was isoted by
Burkholder from Thalasia.
• This antibiotics highly active against gram positive bacterial
stains of Staphylococcus aureus, Diplococcus pnemoniae and
Staphylococcus pyogenes.
• A yellow marine pudomonas of altermona species produce
three antibiotics compounds 4-hydroxybenzaldehyde, 2n-pentyl-
4- guinoilinol and 2n-heptyl-4-guinolinol.
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26. Cont...
• The compound 2n-pentyl – 4 guinoline isolated from bacteria
pseudomonas aeroginosa.
• This antibiotics active against Staphylococcus aureus,
pseudomonas aeruginosa and vibrio harveyi.
• An actinomycete chainia purpurogana ss-228 later referred to
as chainia species was shown to produce antibiotics which
inhibit growth of gram negative bacteria.
• Penicillic acid were obtained from a marine derived fungus
aspergillus ostianus strain.
• Altermonas rubra is an organisms under current investigation
in the university of Hawii. It contains an antibiotics molecule
that is active against multi-drug resistant pathogenic bacteria
such as methicillin – resistant staphylococcus aureus, &
vancomycin resistant Enterococcus.
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27. some of Marine Products
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28. • We are dependent on antibiotics for the treatment of
infectious diseases and they are critical for the success of
advanced surgical procedures.
• There exists high-level resistance both to antibiotics that
have for decades served as gold standard treatments and
to those only recently approved for human use.
Conclusion
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29. Reference
• Villa FA, Gernwick L. Marine natural product drug
discover.
• Yasuhara-Bell J, Lu Y. Marine compounds and their
antiviral activities.
• Carte, B. K. Biomedical potential of marine natural
products.
• http://www.oceansonline.com
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