University of Lagos Department of Microbiology - PowerPoint Presentation

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University of Lagos

Department of Microbiology

1

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BIOPROSPECTING FOR ANTIMICROBIAL AND ANTICANCER COMPOUNDS FROM ACTINOMYCETES FROM LAGOS LAGOON SEDIMENTS.

Professor

I.A.

Adeleye

Dr. M.O. Akinleye

2

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BACKGROUND OF STUDY

About 23,000 bioactive secondary metabolites produced by microorganism have been reported and over 10,000 of these compounds are produced by actinomycetes, representing 45% of all bioactive microbial metabolites discovered.

Among

actinomycetes, around 7,600 compounds are produced by Streptomyces spp. (

Berdy

, 2005).

3

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Many of these secondary metabolites are potent antibiotics and clinically useful antitumor drugs

. However, the search for novel drugs is still a priority goal, due to the issue of drug resistance to multiple chemotherapeutics.In addition, the high toxicity usually associated with cancer chemotherapy drugs and their undesirable side effects has increased the demand for novel antitumor drugs which are active against recalcitrant tumors, with fewer side effects and/or with greater therapeutic efficiency (Demain and Sanchez, 2009). 4

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It is therefore imperative to continue the search for novel microbial natural products from underexplored habitats because researchers have hypothesized that since these microorganisms could thrive in the marine environment, fierce competition among the species encourage them to produce

novel bioactive compounds to antagonize other competitors to aid their survival. 5

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STATEMENT OF THE PROBLEM

Antibiotic resistance in clinical isolates continues to pose formidable challenges in medicine worldwide. Viral infections exact a great toll on human lives especially in resource limited countries of Africa including Nigeria which has the second largest AIDS burden in Africa with an estimate of 3.8% of her population of 160 million infected with the deadly virus.There has been an increase in the public health concern about cancer in low-income countries in Africa including Nigeria.6

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AIM OF STUDY

This study aimed at isolation, purification and identification of antimicrobial and antitumor agents from actinomycetes in Lagos estuarine sediments and provide information of the actinobacteria community profile and their diversity in these sediments.7

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OBJECTIVES OF THE STUDY

Isolate actinomycetes from Lagos marine sediments, characterize the isolates phenotypically and genotypically by sequencing pertinent strainsDetermine the actinobacterial community structure using culture-independent method (DGGE)Determine the antibacterial, antifungal and antiviral activity of metabolites obtained from the isolated actinomycetes8

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SIGNIFICANCE OF THE STUDY

Determine the antitumor activities of metabolites obtained from the isolated actinomycetes.Isolate and purify the bioactive metabolites using chromatographic toolsIdentify the purified bioactive compounds through structural elucidation using a combination of spectroscopic methods9

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MATERIALS AND METHODS10

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SAMPLES COLLECTION11

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Fig 1. Locations of sediments obtained from Lagos lagoon, Nigeria

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Isolation and identification of actinomycetes from Lagos lagoon sediment Isolation of actinomycetes was done using spread plate method using starch casein, Kuster’s, Gauze 1 and 2, Marine and Actinomycete isolation agar. Plates were incubated at 29°C for 1 to 5weeksIdentification of the isolates was done with API kits and through amplification of 16S rRNA gene and sequencing using actinobacterial-specific primers. (approximately 640 bp of 16S r DNA)Sequences were identified by NCBI BLAST and submitted to GenBank.

Phylogenetic tree construction. (Stach et al., 2003)13

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Culture-independent molecular analysis of actinobacterial community structure in Lagos Lagoon sediment samplesTotal actinobacterial community DNA was extracted from 12 sediment samples using QIAamp DNA kit (Qiagen, Germany) by enzymatic method (Muyzer et al., 1993)Nested PCR PCR products after analysis by agarose gel were separated on 8 % denaturing gradients polyacrylamide gel with a 50 % - 65 % Urea and Formamide denaturants at 140V for 5 hours (Adewumi et al., 2012).The major DGGE bands of interest were excised from the gels, eluted in 50μl sterile deionized water, amplified and sequenced

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Production and bioactivity screening of metabolitesProduction of metabolitesStandard ATCC Strains E.coli, P. aeruginosa, C. albicans, S. flexneri, S. epidermidis, S.

typhi, E. faecalis were first used to screen the actinomycetes for bioactivity potentials using cross streak methodPure cultures were fermented in 10ml broth for 3 days at pH7, 28OC and 180rpm and transferred to 200ml broth under same conditionsFermentation broth was scaled up by transferring to flasks containing 1000 mL sterile culture broth and shaken at 180 rpm for 10 days (

Janardhan

et al., 2014).

The cells were separated from broth by centrifugation at 6000 rpm and

10

O

C

for 30

minutes and metabolites extracted

by liquid-liquid extraction method

in ratio 1:1

ethylacetate

and

cultre

broth (

Janardhan

et al

., 2014

)

Antibacterial and antifungal assay of the metabolites was determined using agar well diffusion method.

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Bioactivity screening of metabolitesMIC, MBC and MFC of the crude extracts were determined by broth dilution method (Ramalivhana et al., 2014)Antiviral activity of Influenza virus strain X-31 (H3N2)

using Madin–Darby canine kidney (MDCK) cells using plaque reduction assay technique (Shimizu et al., 2008).Antitumor activities were determined against five cell lines - K562 (Human acute myelocytic leukemia), HeLa (cervical carcinoma), AGS (Human gastric celline

), MCF-7 (breast adenocarcinoma), HL-60 (Human acute

promyelocytic

leukaemia) cell-lines using CCK8 assay (Ravikumar

et al., 2012)

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Isolation and identification of bioactive metabolites

The actinomycete strain with the best overall antimicrobial activity was selected for the large-scale fermentation by scaling up from 1250mL to 10L fermentationExtraction of metabolites was achieved using Amberlite XAD7 and Amberlite XAD 16, eluted with acetone and concentrated to dryness yielding 8 grams of crude extract (Kwon et al., 2009)The crude extract was separated by partitioning Kwon et al. (2009) using the solvents Dichloromethane, water and 10% Dichloromethane in 2-propanol at ratio 1:1:1 (Kwon et al., 2009) 17

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The fraction with highest bioactivity were fractionated further using flash column chromatography using n-hexane/ethylacetate (0-100 % and changed to ethylacetate/methanol (60 % : 40 %) and monitored at UV 254 nm Structural elucidation was done using IR, ESI-MS and NMR Spectra (1H, 13C, COSY, HMBC) Isolation and identification of bioactive metabolites (contd.)Statistical analysisStatistical analysis was done using Microsoft Excel 2013 and GraphPad Prism software (GraphPad Software Inc., San Diego, CA). Log IC50 calculations were done using algorithms

for dose-response curve with variable slope18

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RESULTS19

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1. ISOLATION AND IDENTIFICATION OF ACTINOMYCETES FROM LAGOS LAGOON SEDIMENT 20

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1. IDENTIFICATION OF ISOLATED ACTINOBACTERIAL STRAINS21

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Table 1. Physiological characteristics of isolated actinobacterial strains from Lagos Lagoon

Key: IND: Indole, URE: Urease, GLU: Glucose, MAN: Mannitol, LAC: Lactose, SAC: Saccharose, MAL: Maltose, SAL: Salicin, XYL: Xylose, ARA:Arabinose

, GEL:

Gelatin

, ESC: Esculin, GLY: Glycerol, CEL:

Cellobiose

, MNE: Mannose, MLZ:

Melezitose

, RAF:

Raffinose

, SOR: Sorbitol,

RHA:Rhamnose

, TRE:

Trehalose

, CAT: Catalase, SPO-Spores, GRA: Gram reaction, STA: Starch hydrolysis, CAS: Casein hydrolysis, +: positive, -: negative,

+

/-: variable

 

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Table 2. Identification of actinomycetes isolated from Lagos Lagoon based on 16Sr RNA gene sequencesStrainsSampling Location

16 Sr RNA gene of closest known relative% Similarity

Accession Numbers

ULMa27

Imope, Ikosi

Micromonospora aurantica

97%

KX352083

ULG2.23

Imope, Ikosi,

Micromonospora sp.

100%

KX352058

ULMa40

Imope, Ikosi, Egbin

Micromonospora sediminicola

99%

KX352076

ULMa33

Okobaba, Offin, Iddo, Ikosi, Egbin

Micromonospora humi

99%

KX352075

ULMa30

Imope, Ikosi, Egbin

Micromonospora sp.

100%

KX352073

ULG1.08

Ejirin, Imoru

Micromonospora sp.

100%

KX352080

ULMa32

Ejirin, Imoru, Itokin

Agromyces sp.

94%

KX352074

ULK2

Folawiyo, Ejirin, Imoru

Streptomyces albus

100%

KX352059

ULK3

Okobaba, Offin, Folawiyo, Iddo, Ejirin, Imoru, Imope, Ikosi, Egbin, Ijede, Bayeku

Streptomyces avermitilis

99%

KX352077

ULS7

Okobaba, Offin, Folawiyo, Iddo, Ejirin, Imoru, Imope, Ikosi, Egbin, Ijede

Streptomyces coelicolor

100%

KX352086

ULS14

Folawiyo, Iddo

Streptomyces bingchenggensis

98%

KX352065

ULS13

OffinStreptomyces fulvissimus100%KX352087ULK11OffinStreptomyces albus99%KX352062ULK7Ikosi, EgbinStreptomyces pratensis98%KX352060ULG2.17Imope, Ikosi, BayekuStreptomyces albus100%KX352081UL28fImope, Ikosi,Streptomyces albus99%KX352082UL28a Ikosi, EgbinStreptomyces albus99%KX352079ULT1Okobaba, EgbinStreptomyces albus100%KX352088ULMa36Imope, Ikosi, EgbinMicromonospora sp.100%KX352085UL7BOkobaba, Offin, Folawiyo, Iddo, Ejirin, Imoru, Imope, Ikosi, Egbin, Ijede, BayekuStreptomyces albus99%KX352066UL28dEjirin, Imoru, ItokinStreptomyces pratensis98%KX352068UL31bFolawiyo, Ejirin, ImoruStreptomyces albus99%KX352070ULA9 Ikosi, EgbinStreptomyces avermitilis99%KX352071ULK10 FolawiyoStreptomyces sp.98%KX352061UL23aImope, IkosiStreptomyces albus99%KX352057UL19bOffinStreptomyces fulvissimus100%KX352067UL030OffinMicromonospora sp.99%KX352063ULAct2Ikosi, EgbinStreptomyces pratensis98%KX352072UL6aImope, IkosiStreptomyces albus100%KX352078ULS12Imope, Ikosi,Streptomyces sp.100%KX352064UL31a Ikosi, EgbinStreptomyces albus99%KX352069ULMa26Okobaba, EgbinStreptomyces albus99%KX352084

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Figure 2. Phylogenetic tree showing multiple sequence alignment of 16S rRNA gene sequences of actinomycetes Actinomycetes isolated in Lagos LagoonAgromyces sp. 24

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Figure 3. Phylogenetic tree showing multiple sequence alignment of 16S rRNA gene sequences of actinomycetes isolated in Lagos Lagoon and other marine environments worldwide and some other known strains. Pairwise phylogenetic distances were calculated based on 16S rRNA gene.25

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Figure 4. Scanning electron micrograph of Streptomyces bingchenggensis ULS14. a) The strain growing on starch casein agar at 100x magnification. b) Strain morphology at 10.00KX magnification26

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2. IDENTIFICATION OF MAJOR ACTINOBACTERIAL PCR-DGGE BANDS27

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Figure 5. DGGE profiles of PCR-amplified 16S rRNA gene fragments of 12 sampling sites of Lagos Lagoon showing actinobacterial diversityLegend: ID Act- Already Identified actinomycetes

strains, ID Micro- Already identified Micromonospora strains. All soil- Combined PCR products for all the soil from sample sites, OK- Okobaba, OFF- Offin, FOL-Folawiyo, IDD-Iddo

, ST1-Ejirin, ST2-Imoru, ST3-Itokin, ST4-Imope, ST5-Ikosi, ST6-Egbin, ST7-Ijede, STA 8-

Bayeku

.Bands a.

Micromonospora

aurantica

;

b.

Micromonospora

sp.;

c.

Micromonospora

sediminicola

;

d.

Micromonospora

humi

;

e.

Micrococcus

luteus

;

f.

Micromonospora

sp

.;

g.

Micromonospora

sp

.; .

h.

Streptomyces

albus

;

i

.

Streptomyces

avermitilis

;

j.

Streptomyces

coelicolor

;

k.

Streptomyces

bingchenggensis

;

l.

Arthrobacter

phenanthrenivorans

;

m. Arthrobacter sp.; n. Arthrobacter chlorophenolicus28

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3. DETERMINATION OF ANTIBACTERIAL, ANTIFUNGAL AND ANTIVIRAL ACTIVITY OF METABOLITES OBTAINED FROM THE ISOLATED ACTINOMYCETES29

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FractionsE.coliPseudomonas

aeruginosaShigellaflexneri

Staphylococcus

e

pidermidis

Salmonella typhi

Enterococcus faecalis

UL7B

-

-

-

5mm

-

 7mm

ULS7

-

-

-

5mm

-

5mm

ULK2

-

-

-

-

6mm

5mm

ULS12

-

-

-

6mm

-

-

ULS13

4mm

-

4mm

7mm

-

-

ULK3

-

-

10mm

-

-

-

ULK10

-

-

5mm

-

-

7mm

ULK11

-

-

5mm7mm--ULMA40--10mm7mm-5mmULG2.17---4mm--UL28f----5mm-UL28a---7mm--UL23a---3mm--ULMa275mm----4mmULS1415mm2mm15mm27mm25mm10mmULT1---7mm--ULMa36--8mm--6mmUL28d---5mm--ULG1.08--5mm---UL31c---5mm--UL19b- -5mm--Table 4. Antibacterial activity of bioactive actinomycetes strains (cross-streaking assay)30

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Table 5. Antifungal activity of bioactive crude extracts of actinomycetes strains (Kirby-Bauer assay)

Test OrganismsTrichophyton mentagrophytes A1Trichophyton mentagrophytes

A2

Trichophyton

rubrum

Candida

a

lbicans

UL7B

33mm

33mm

29mm

16mm

ULS7

32mm

34mm

24mm

20mm

ULK2

28mm

30mm

26mm

20mm

ULS12

25mm

26mm

23mm

18mm

ULK7

32mm

27mm

21mm

-

ULK3

24mm

29mm

22mm

23mm

ULK10

-

-

-

15mm

ULK11

26mm

29mm

26mm

12mm

ULMA40

28mm

29mm

21mm

4mm

ULMa27

29mm

32mm

27mm

-

ULS14

27mm28mm24mm20mmULS13---5mm31

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Table 6. Minimum Inhibitory Concentration (MIC) (mg/ml) of bioactive crude extracts against bacterial pathogens  

  Test OrganismsGram Reaction

UL7B

ULS7

ULS12

ULS13

ULK3

ULK10

ULK11

ULMA40

UL

MA27

ULS14

ULK2

E. coli

-

-

-

-

-

-

 

-

-

-

10

5

-

Pseudomonas

a

eruginosa

-

-

-

-

-

-

-

-

-

-

-

-

Shigella

f

lexneri

-

-

-

-

-

5

-

-

2.5

-

2.5

-

Staphylococcus

epidermidis +10101010--1010-0.078-Salmonella typhi----------0.15610Enterococcus faecalis+1010---10--1051032

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Table 7. Minimum Inhibitory Concentration (MIC) (mg/ml) of bioactive crude extracts against fungal pathogensTest OrganismsUL7B

ULS7ULK7

ULS12

ULS13

ULK3

ULK10

ULK11

ULMA40

UL

MA27

ULS14

ULK2

Candida

a

lbicans

5

1.25

0.625

1.25

0.156

0.156

2.5

2.5

-

-

0.625

1.25

Trichophyton mentagrophytes

A1

0.156

0.156

0.156

0.156

-

0.156

-

0.156

0.156

0.156

0.156

0.156

Trichophyton mentagrophytes

A2

0.156

0.156

0.156

0.156

-

0.313

-

0.625

0.156

0.156

0.156

0.313

Trichophyton

rubrum

0.156

0.156

-0.156-0.313-0.1560.1560.1560.1560.31333

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Table 8. Minimum Bactericidal Concentration (MBC) (mg/ml) of bioactive crude extracts of actinomycetesTest Organisms

Gram ReactionULS13ULK3

ULMA40

UL

MA27

ULS14

Escherichia coli

-

-

-

-

10

10

Pseudomonas

a

eruginosa

-

-

-

-

-

-

Shigella

f

lexneri

-

-

10

10

-

5

Staphylococcus

e

pidermidis

+

10

-

10

-

0.313

Salmonella typhi

-

-

-

-

-

0.625

Enterococcus faecalis

+

-

-

-

10

10

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Test OrganismsUL7B

ULS7ULK7ULS12

ULS13

ULK3

ULK10

ULK11

ULMA40

UL

MA27

ULS14

ULK2

Trichophyton mentagrophytes

A1

0.156

0.156

0.156

0.156

-

0.156

-

0.156

0.156

0.156

0.156

0.156

Trichophyton mentagrophytes

A2

0.156

0.156

0.156

0.156

-

0.313

-

0.625

0.156

0.156

0.156

0.313

Trichophyton

rubrum

0.156

0.156

-

0.156

-

0.313

-

0.156

0.156

0.156

0.156

0.313

Candida

a

lbicans

-

10

-

2.510-105--0.6252.5Table 9. Minimum Fungicidal Concentration (MFC) (mg/mL) of bioactive crude extracts of actinomycetes35

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Figure 6. Antiviral activity of crude extracts of actinomyetes against H3N2 influenza virus Legend: C= control (cells) without virus, C+V= control (cells) infected with virus, K2 (0.8-0.3) = concentrations of crude extracts of ULK2, K3 (1-0.3) = concentrations of crude extracts of ULK3 in mg/mL, K10 (1-0.4) = concentrations of crude extracts of ULK10 in mg/mL, K11 (1-0.4) = concentrations of crude extracts of ULK11 in mg/mL36

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4. DETERMINATION OF THE ANTITUMOR ACTIVITIES OF METABOLITES OF METABOLITES OBTAINED FROM THE ISOLATED ACTINOMYCETES37

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Table 10. Antitumor effect of actinomycete crude extracts on cellines (mg/mL)38

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5. ISOLATION AND PURIFICATION OF BIOACTIVE METABOLITES USING CHROMATOGRAPHIC TOOLS39

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Figure 7: Isolation of compounds from the crude extract of actinomycete using flash chromatography 40

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Table 11. Minimum Inhibitory Concentration (MIC) (µg/mL) of purified bioactive compounds from crude extract of Streptomyces bingchengensis ULS14Compounds

Staphylococcus epidermidisCandida albicans

ULDF4

5

5

ULDF5

-

5

Chloramphenicol

3

na

Pimaricin

na

5

Legend:

na

-

not

applicable

41

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Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (µg/mL) of purified bioactive compounds from crude extract of Streptomyces bingchengensis ULS14

42

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(IC50= 0.075µg/mL)

Log10 Concentration (µg/mL)Figure

8.

Inhibition concentration (IC

50

) of compound ULDF4 against

HeLa

cell line

43

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(IC50= 0.034µg/mL)

Log10 Concentration (µg/mL)Figure 9. Inhibition concentration (IC50

) of compound ULDF5 against

HeLa

cell line

44

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6. IDENTIFICATION OF PURIFIED BIOACTIVE COMPOUNDS THROUGH STRUCTURAL ELUCIDATION USING A COMBINATION OF SPECTROSCOPIC METHODS45

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Figure 10. Mass Spectroscopy (ESI-MS) of compound ULDF4C34H35NO1346

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Figure 11. Infrared spectroscopy (IR) of compound ULDF447

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Figure 12. Proton NMR (1H-NMR) of compound ULDF448

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Figure 13. Carbon NMR (13C-NMR) of compound ULDF449

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Figure 14. COSY of compound ULDF450

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Figure 15. HMBC of compound ULDF451

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Figure 16. Proposed structure of compound ULDF4

 521

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Figure 17. Mass Spectroscopy (ESI-MS) of compound ULDF5C28H26N4O3

53

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Figure 18. Infrared spectroscopy (IR) of compound ULDF554

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Figure 19. Proton NMR (1H-NMR) of compound ULDF555

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Figure 20. Carbon NMR (13C-NMR) of compound ULDF556

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Figure 21. COSY of compound ULDF557

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Figure 22. HMBC of compound ULDF558

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Figure 23. Proposed structures of compound ULDF5  59

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SUMMARY OF FINDINGS60

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Specific Objectives

Findings1.Isolate actinomycetes

from Lagos lagoon sediments, characterize the isolates phenotypically and

genotypically

• A total of 32

actinomycetes

isolates were obtained from the Lagos lagoon sediment.

• The isolates were found to be close relatives of

Micromonospora

sp.,

Streptomyces

sp. and

Agromyces

sp.

• The isolated strains include

Streptomyces

avermitilis

, S.

coelicolor

, S.

bingchenggensis

Streptomyces

fulvisssimus

, S.

pratensis

,

Micromonospora

aurantica

, M.

sedimennicola

and

M.

humi

.

2.

Determine the

actinobacterial

community structure of Lagos lagoon using culture-independent method

The identities of the sequenced major bands were found to belong to the genera

Micromonospora

,

Streptomyces,

Micromonospora

,

Arthrobacter

and

Micrococcus

sp.

Streptomyces

avertimitilis

and

S.

coelicolor

occurred most

frequently and were present in all the samples.61

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Specific Objectives

Findings3.Determine the antibacterial, antifungal and antiviral potential of metabolites derived from the isolated actinomycetes

The

actinomycete

strain,

Streptomyces

bingchenggensis

ULS14 had the highest antibacterial and antifungal activity against all the test pathogens.

The extract from

Streptomyces

bingchenggensis

ULS14 had the lowest inhibitory concentration at 0.078 mg/ml against

Staphylococcus

epidermidis

.

62

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Specific Objectives

Findings3.Determine the antibacterial, antifungal and antiviral potential of metabolites derived from the isolated actinomycetes

The crude extract from

Streptomyces

bingchenggensis

ULS14 had the highest bactericidal activity at concentration of 0.313mg/ml against

S.

epidermidis

while the MFC of

Streptomyces

bingchenggensis

ULS14,

Streptomyces

avermitilis

ULK3,

Streptomyces

albus

ULK2 and

Streptomyces

sp

.

ULK10 was 0.313 mg/ml against

Candida

albicans

,

Trichophyton

mentagrophytes

A1

,

Trichophyton

rubrum

.

Extracts from the

actinomycete

strain,

Streptomyces

avermitilis

ULK3 had the highest antiviral activity at 0.5mg/ml concentration against Influenza virus

X-31 (H3N2)

without having any cytotoxic effect on the MDCK cell line.

63

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Specific Objectives

Findings4.

5.

Determine the antitumor potential of metabolites derived from the isolated marine

actinomycetes

Isolate pure compounds and determine the bioactivity of the purified compounds

The

actinomycete

strain

Streptomyces

fulvissimus

ULS13 showed the highest antitumor activity with extracts from the strain having activity at concentration as low as 0.030mg/ml

The compound ULDF4 showed antimicrobial activity against both

Staphylococcus

epidermidis

and

Candida

albicans

with MIC at 5µg/ml against both pathogens. Compound ULDF5 showed the antitumor activity at 34ng/ml against

HeLa

cell line

64

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Specific Objectives

Findings6.

Identify the purified bioactive compounds through structural elucidation using a combination of spectroscopic methods

Two compounds were purified and identified based on ESI-MS, IR and NMR data obtained. The compounds were structurally related to

Kigamicin

and

Staurosporine

respectively

65

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CONTRIBUTIONS TO KNOWLEDGE66

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1. Combination of culture dependent and independent molecular techniques used in this study has revealed the diversity of actinomycetes

in Lagos Lagoon sediments.

2.

For the first time,

actinomycetes from the Lagos lagoon has been screened for production of antibacterial, antifungal and antiviral secondary metabolites

3.

Antimicrobial and anticancer compounds were purified from crude extracts of

actinomycetes

isolated from Lagos Lagoon which makes this report the first in West Africa.

67

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4. The antimicrobial and anticancer compounds, Kigamicin and

Staurosporine were obtained for the first time from Streptomyces binggchengensis 68

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THANK YOU ALL FOR LISTENING69