1. Discovery of A Novel Source of Chloramphenicol Production by Endophytic Streptomyces SUK 25 sp., Isolated from Zingiber spectabile Against Methicillin Resistance Staphylococcus aureus (MRSA)
Muhanna M Alshaibani¹. Juriyati Jalil2. Nik M. Sidik3. Noraziah Mohamad Zin¹*
1. Faculty of Health Sciences,Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur, Malysia.
*E.Mail :
INTRODUCTION
RESULTS and DISCUSSION
Actinobacteria are prokaryotes of Gram-positive bacteria but are well-known from other bacteria by their morphology. Streptomyces produce many important bioactive substances that have high commercial value; more than 70% of naturally occurring antibiotics have been isolated from different genus of actinobacteria.
Molecular identification results using 16S rRNA gene sequencing showed that SUK 25 has 95 % sequence similarity to Streptomyces omiyaensis NBRC 13449T which was produced chloramphenicol ( Cm).
(Cm) obtained originally from the bacterium Streptomyces venezuelae. It is a broad-spectrum bacteriostatic antibiotic against various bacteria and it is also bactericidal for some other bacteria. Due to potential side effects in humans, the drug is not recommended for the treatment of minor diseases, but is reserved for the treatment of serious infection.
4- NMR: (1D & 2 D)- 1H proton . 13 C. of Peak No3.and standard Chloramphenicol
OBJECTIVE
C13- of standard Chloramphenicol
Isolation, extraction, and identification of active new source of secondary metabolites production by endophytic Streptomyces
SUK 25 sp. isolated from Zingiber spectabile against Methicillin Resistance Staphylococcus aureus (MRSA).
Fermentation in Thronton's broth media for 7 days, Temp280C, at PH 7.
Extraction by 1/2 V of EA.
Evaporation of EA by rotary evaporator.
(Crude extract 700mg)
TLC , C.C
Bioautography using
(Disc diffusion assay).
HPLC , NMR , LC- MS .
MIC and MBC against MRSA
METHODOLOGY
Figure 4 : 1D- NMR spectra of compound and standard chloramphenicol were obtained on Bruker NMR 600 MHz Cryo-Probe instrument. Chemical shifts were calibrated internally against the residual signal of the solvent in which the sample was dissolved in (CD MeOD, δH at and δ C13 at 47.7). Results Indicated 12 proton and 11 Carbon for both compound from SUK 25 and standard chloramphenicol. 1 2
RESULTS and DISCUSSION
1- Primary, secondary screening and Bioautography of SUK25 against MRSA. 4 3
Figure 1: Primary, secondary screening of SUK25 against MRSA, and Bioautography. The MIC of chloramphenicol against MRSA = 8 µg/ ml and BMC= 32 µg/ ml.
Figure5 : The 2D-HSQC, HMBC and COSY NMR spectra of
Cm are shown in the Fig. 1,2, 3 and 4, respectively, Molecular Formula C11H12Cl2N2O5.
2- Isolation , purification of compound by Analytical and preparative HPLC Agilent 1200.
Fourier Transform Infra-Red (FT- IR) spectrum showed a strong and sharp vibrational band at cm-1 indicated the presence of (N-O) stretch(ArNo2)& C–C stretching vibrations and cm-1 indicated the presence of O-H bending. .
An. HPLC. Single peak
Pr. HPLC
Figure2 : Isolation and purification of F.no.7.using RP-HPLC Agilent 1200 using gradient ACN : H2O for 20 mins . Results shows max. peaks at R.T ( 11.6, 14.5mins). Confirmation purity by analytical HPLC for each peak. Single peak confirm the purity of this compound.
CONCLUSION
The observations from this study confirmed that Streptomyces SUK 25 isolated from Zingiber spectabile in the novel antibiotic research laboratory may be used for extracting chloramphenicol as a new source of natural product for treating bacterial infection.
3- Liquid Chromatography-Mass Spectroscopy (LC-MS)
ACKNOWLEDGEMENT:
The researchers are grateful to the MOHE for financially supported the research grant
code FRGS/1/2011/ST/UKM/02/1
REFERENCES:
1- Ehrlich, J., Q. R. Bartz, R. M. Smith, D. A. Joslyn & P. R. Burkholder Chloromycetin, a new antibiotic from a soil actinomycete. Science (New York, NY) 106(2757):
2- Zin, N. M., Sarmin, N. I. M., Ghadin, N., Basri, D. F., Sidik, N. M., Hess, W. & Strobel, G. A Bioactive endophytic streptomycetes from the Malay Peninsula. FEMS microbiology letters, 274,
3- Hopwood, D. A Streptomyces in nature and medicine: the antibiotic makers Ed.: Oxford University Press New York.
Figure 3:The molecular weight of Cm was determined by LC- MS. The (m/z) of this active compound was ( Mwt + H).