This document discusses the isolation of bacteria from various fly species and their antibiotic properties. Bacteria including Bacillus circulans, Staphylococcus aureus, and Pseudomonas aeruginosa were isolated. B. circulans strain 88T produced a potent metabolite that was purified and found to have antibiotic effects against various pathogens, including S. aureus and Salmonella, as determined by minimum inhibitory concentration testing. Mass spectrometry identified the metabolite's molecular weight as approximately 26kDa.

1. – –)

3. C30H37N4SO9
5 ug/ml

8. - -

11. -
- -

16. –

17. –

18. –
 Nutrient agar emended with 1% yeast extract.
 Nutrient agar emended with 5% sheep blood
 MaConkey's agar
 Starch nitrate agar
 Tryptose blood agar
 Staphylococcus media

(CFU
1944 Holt et al., 2004 Honda
et al

19. –
–

20. –
Bioautographic
technique pH
Thin layer and column chromatography
Spectroscopy
(UV) Spectrophotometer
(IR) Mass spectral Data
Hp mudel MS 5988

21. –
Agar Diffusion Method

25. Table (1): The viable plate count of bacterial flora (CFU/ml) isolated
from wings of P. papatasi, M. stabulans, M. domestica and C.
pipiens
Medium P. papatasi M. stabulans M. domestica C. pipiens
.
Right Left Right Left Right Left Right Left
wing wing wing wing wing wing wing wing
Nutrient agar 5 x 102 2 x 102 2.9 x 3.4 x 5.1 x 5.1 x Nil Nil
with y. extract 102 102 103 103
Nutrient blood 6 x 102 1 x 102 6.7 x 5.9 x Nil 4.3 x 3 x 102 Nil
103 103 103
MacConkey Nil Nil 3.9 x 3.9 x Nil Nil Nil Nil
103 103
Starch nitrate 1.7 x 102 Nil 5 x 102 4.8 x Nil Nil Nil Nil
102
Tryptose blood 1 x 102 Nil 3.1 x 2.7 x 3.3 x 3.5 x 1 x 102 1.4 x
103 103 103 103 102
Staphylococcus Nil Nil Nil Nil Nil Nil Nil Nil

26. Table (2): Organisms isolated from wings of the sandfly, the false stable
fly, the house fly and the mosquito.
Symbole Organism
175b Salmonella arizona
157y Erwina herbicola
68S Yeast
165y Bacillus subtilis
181y Yeast
191T Actinomycete
88T Bacillus circulans
132T Staphylococcus aureus
127T Lactobacillus animalis
98y Bacillus mycoides
113M Pseudomonas aeruginosa
201T Lactobacillus gasseri

27. Table (3): Antagonistic action of bacterial species between each other grown on nutrient broth
amended with yeast extract.
Organism S. arizona E. B. B. S. L. B. mycoides P. aeruginosa L. gasseri
175b herbicola subtilis circulans aureus animalis 98y 113M 201T
157y 165y 88T 132T 127T
S. arizona x –ve +ve +ve +ve 2+ve –ve +ve +ve
175b
E. herbicola –ve x +ve –ve –ve –ve –ve –ve –ve
157y
B. subtilis –ve +ve x +ve 2+ve 3+ve +ve –ve +ve
165y
B. circulans –ve –ve –ve x +ve 2+ve –ve +ve –ve
88T
S. aureus –ve –ve +ve +ve x 3+ve –ve +ve –ve
132T
L. animalis –ve –ve –ve +ve –ve x –ve –ve –ve
127T
B. mycoides –ve –ve +ve –ve –ve –ve x +ve –ve
98y
P. –ve –ve –ve –ve –ve –ve –ve x –ve
aeruginosa
113M
L. gasseri +ve –ve +ve +ve +ve +ve +ve –ve x
201T
–ve = no inhibition zone, + = weak inhibition zone, 2+ = moderate inhibition zone, 3+ = good inhibition
zone.

28. Table (4): Antagonistic action of most potent bacterial species
grown on peptone water during log phase
Organism S. aureus P. aeruginosa B. circulans L. animalis B. subtilis
132T 113M 88T 127T 165y
S. aureus x + 4+ + 4+
132T
P. aeruginosa –ve x –ve –ve –ve
113M
B. circulans ve + x 3+ +
88T
L. animalis –ve + 2+ x 2+
127T
B. subtilis + + 4+ 2+ x
165y
–ve = no inhibition zone, ve = doubolful inhibition zone, + = weak inhibition
zone, 2+ = moderate inhibition zone, 3+ = good inhibition zone, 4+ = very good

29. Table (5): Bioautography and migration (Rf) of the active
metabolite 88T with various developing solvents
Developing solvent system Rf value
. 0.00
Petroleum ether
Benzene (saturated with water) 0.00
Chloroform (saturated with water) 1.00
Carbon tetrachloride (saturated with water) 0.75
Methanol 0.85
N–Butanol (saturated with water) 0.80
Acetone 0.45
Diethyl ether 0.55
Ethyl acetate 0.50
Amyl acetate 0.00
3% ammonium chloride 0.10
N–Butanol : pyridine : water (1 : 0.6 : 1) 0.00
N–Butanol : Acetic acid : water (2 : 1 : 1) 0.00
Distilled water 0.20

30. Table (5): Bioautography and migration (Rf) of the active
metabolite 88T with various developing solvents
Methylene chloride (1 : 1)
Developing solvent system Rf
valu
e
Petroleum ether 0.00
Benzene (saturated with water) 0.00
Chloroform (saturated with water) 1.00
Carbon tetrachloride (saturated with water) 0.75
Methanol 0.85
N–Butanol (saturated with water) 0.80
Acetone 0.45
Diethyl ether 0.55
Ethyl acetate 0.50
Amyl acetate 0.00
3% ammonium chloride 0.10
N–Butanol : pyridine : water (1 : 0.6 : 1) 0.00
N–Butanol : Acetic acid : water (2 : 1 : 1) 0.00
Distilled water 0.20

31. Table (6): The MIC of active metabolite 88T
Test organism MIC ( g/ml)
Reference strains:
Bacillus subtilis NCTC 8236 <5
Bacillus pumilus NCTC 8241 <5
Micrococcus luteus ATCC 9341 12
Staphylococcus aureus NCTC 7447 12
E. coli BPP01 16
Pseudomonas aeruginosa ATCC 10145 83
Klebsiella pneumonia NCIB 9111 18
Candida albicans IMRU 3669 94
Saccharomyces cerevisiae CBS 1171 94

32. Table (6): The MIC of active metabolite 88T
Aspergillus niger LTU 131 >100
Local isolates:
Bacillus subtilis 165y <5
Bacillus mycoides 98y <5
Staphylococcus aureus 132T <5
Lactobacillus animalis 127T 32
Lactobacillus gasseri 201T 40
Salmonella arizona 175b <5
Erwina herbicola 157y >100
Pseudomonas aeruginosa 113M >100
Yeast 181y >100
Yeast 68y >100

33.  -B.subtilis cause ocular infection

 - B. pumilus
 - B. anthracis

 - Vibro cholerae
 - Salmonella
 - B. dysentri, Amoebic dysentri
 -
 - Poliomylitis
 - (HAV) A
 - Yaws
 - Infentile diarrhea

34.  Pseudomonas aeryginosa


 Staphylococcus aureus
 Furuncles (boils)
 Carbuncles
 Impetigo
 Cystitis

35.  Enterocolitis
 Osteomylitis
Bacteremia.
 Cerebritis.
 Abscesses of muscle, skin.
 Urinogenital tract.
 Central Nervous system.
 Various intraabdominal organs.
 Food poisoning.

36. (Spore forming) Lactobacillus

Lactoperoxidase system

 Lactobacilli
(Vincent, Veomell and Riley 1959)
(Gram-ve bacteria)

37.  (Spore forming) Bacillius

(The prokaryotes vol. II)
 proteolysis
power
 Bacilli
 polymyxin, gramicidin, Bactiracin

 Several proteinases, amylase, subtilysin
 B. circulus
 2000

38.  Ampusa muscae
(Gram-ve,+ve)


(Gram-ve,+ve)


39.  Sarcophaga
 Aspergillus sp. B.
circulus (1997)

 Ml.Wt
(2002) 26kDa.



40. Fig. (1): A simplified scheme for the extraction, isolation and purification of the active
metabolite 88T biosynthesized by Bacillus circulans 88T.

41. Fig. (2): A simplified scheme for the extraction, isolation and purification
of the active metabolite 88T biosynthesized by Bacillus circulans 88T

42. Fig. (3): Mass spectrum of the
active metabolite 88T

43. Fig. (4): IR spectrum of the active metabolite 88T .

44. Fig. (5): 1H–NMR spectrum of the active metabolite 88T

45. Fig. (6): The time killing curves of the active metabolite 88T using Bacillus mycoides
98y, Bacillus subtilis 165y, Staphylococcus aureus 132T and Salmonella arizona
175b.

47. 
B.circulans


48. 

49. 


50. 5 mg/ml

51. 

52. 

...