Staphylococcus aureus is a normal inhabitant of various parts of the human body, residing on the skin and in hair follicles, in the throat, and within nasal membranes of healthy individuals. However, it is also an opportunistic disease-causing organism responsible for a variety of diseases, including impetigo, cellulitis, food poisoning, toxic-shock syndrome, necrotizing pneumonia, endocarditis and sepsis (7, 10). Beta-lactam antimicrobial agents are the preferred drugs for serious S. aureus infections. However, since the introduction of methicillin into clinical use, methicillin-resistant S. aureus (MRSA) strains `have emerged worldwide as important nosocomial pathogens, and the prevalence of these strains in the community is now increasing substantially (5,8). Methicillin-resistant Staphylococcus aureus (MRSA) was first isolated in England in 1961 shortly after the development of methicillin, the first penicillinase-resistant semisynthetic penicillin (8). Since then, MRSA has become the most prevalent pathogen causing hospital infection throughout the world, and MRSA incidence is still increasing in many countries (2). MRSA is resistant to practically all b-lactam antibiotics, a class of antibiotics represented by penicillins and cephalosporins(4).
In order to control this infection, there is a need to develop novel agents with greater inhibitory activity against MRSA, searches for substances with antimicrobial activity are frequent, and medicinal natural products have been considered interesting by some researchers since they are frequently used in popular medicine as remedies for many infectious diseases. Some natural product extracts are known as medicinal because they contain active substances that cause certain reactions, from relenting to the cure of diseases, on the human organism (13).
In fact, natural products have always played an important role in medicine and, in particular, traditional medicinal plants have increasingly become major players in recent drug discovery. In this study, Calligonum comosum will used as a natural plant extract to develop a combination therapy. Calligonum comosum is a desert shrub distributed and growing throughout sandy desert of Saudi Arabia belongs to family Polygonaceae. It has been used by the healers in the treatment of stomach ailments. The stems and leaves of C. comosum are chewed for curing toothache (7). Also, its root decocting is used for gum sores (15).Some studies were demonstrated that the high levels of crude protein, potassium and calcium in C. comosum vegetative parts and Its flower can be eaten as it is high in sugar and nitrogenous components (10,13).
Adab is a gene encodes protein which is involved in the synthesis of methylated DNA-protein cysteine methyltransferase. The enzyme is produced by this gene play an important role in the DNA repair of Staphylococcus aureus cell. Therefore, Adab gene in staphylococcus aureus isolates will be used as a target protein to study the effect of natural plant (Calligonum comosum) extract and antibiotic on its sequence, in addition, the aim of this study is to develop a combination therapy for MRSA infection by using naturally derived extract and antibiotic formulation targeting selected infective protein.
To develop a combination therapy for MRSA infection by using naturally derived extract and antibiotic formulation targeting selected infective protein.
To investigate the effect of natural extracts on MRSA & MSSA:
- Based on growth inhibition of Staphylococcus aureus strains through disc diffusion and MIC assays.
- Based on sequencing analysis of adaB gene
To investigate the synergistic and antagonistic effects between natural extracts and antibiotics in combination on growth of MRSA & MSSA.
MATERIALS AND METHODS:
Strains of S. aureus will be obtained from the Medical Microbiology Research laboratory/ Faculty of Medicine and Health Sciences/ UPM and reference strains will be used as standards.
The strains will be inoculated into Mannitol Salt Agar and incubated for 36-48 h. Biochemical identification will be performed by using standard microbiological protocol (12). Colonies from pure culture will be subjected to Gram staining reaction, and coagulase test. Phenotypically confirmed isolates will be reconfirmed as S.aureus by species specific Sa442. This gene will be identified by using PCR technique as described by (Martineau et al., 1998)
Antimicrobial Susceptibility Testing
Antibiograms will be determined by disk diffusion on Mueller-Hinton agar according to the National Committee for Clinical Laboratory Standards (12). The following antibacterial agents will be included:
Calligonum comosum, documented (7) to possess bioactivity properties will be used. The extracts will be prepared using methanol extraction method.
antibacterial susceptibility tests:
Extract obtained will be tested for inhibitory activity against MRSA, MSSA and control. (both gram negative and gram positive isolates). Inhibitory extracts will be determined using disc diffusion and dilution tube assay according to established protocol (12).
Genomic DNA will be extracted from staphylococcal positive cultures by using commercially DNA extraction kit according to manufacture's instructions.
Gene cloning will be performed by using commercially cloning kit according to manufacture's instructions.
Gene expression will be performed by using commercially gene expression kit according to manufacture's instructions.
Significant difference in growth inhibition by various extracts and antibiotic extract combination will be determined by statistical analysis.
Bacterial ID confirmation
Natural product preparation
- SFE extraction.
Antibacterial assays of :
- Natural Product Extract (NPE).
- Antibiotic (Antb).
- Combination of NPE and Antb.
- Disc diffusion plate.
- Tube dilution or MIC Assay.
- PCR of adab gene.
- Nucleotide sequence analyses of adaB gene.
- RT.PCR analysis.
- Cloning of treated and untreated adaB gene from MRSA & MSSA.
- Gene expressing of adaB gene with and without alterative nucleotide sequence.
- Inhibitory activities of NPE, Antb and NPE + Antb based on zone of inhibition and MIC.
- Synergistic effect of NPE + Antb. Based on largest zone of inhibition and lowest MIC.
- Nucleotide sequence changes in treated adaB gene.
- No express of altered adaB nucleotide sequence.
- Ayliffe, G. A. 1997. The progressive intercontinental spread of methicillin resistant Staphylococcus aureus. Clin. Infect. Dis. 24(Suppl. 1):74-79.
- Chambers, H. F., and H. C. Neu. 1995. Penicillins, p. 233-246. In G. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases. Churchill Livingstone, New York, N.Y.
- Chambers, H. F. 2001. The changing epidemiology of Staphylococcus aureus? Emerg. Infect. Dis. 7:178-182.
- Diekema, D.J., Pfaller, M.A., Schmitz, F.J., Smayevsky, J., Bell, J., Jones, R.N., and Beach, M. (2001) Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997-99. Clin Infect Dis 32 (Suppl. 2): S114- S132.
- Hiramatsu, K., K. Okuma, X. X. Ma, M. Yamamoto, S. Hori, and M. Kapi. 2002. New trends in Staphylococcus aureus infections: glycopeptide resistance in hospital and methicillin resistance in the community. Curr. Opin. Infect. Dis. 15:407-413.
- Jevons, M. P. 1961. "Celbenin"-resistant staphylococci. Br. Med. J. 1:124-125.
- Liu, X.M.; M.N.M. Zakaria; M.W. Islam; R. Radhakrishnan; A. Ismail; H.B. Chen; K. Chan and A. Al-Attas 2001. Antiinflamatory and anti-ulcer activity of Calligonum comosum in rats. Fitoterapia, 72: 487-491.
- Lowy, F.D. (1998) Staphylococcus aureus infections. N Engl J Med 339: 520-532
- Martineau, F., Picard, F. J., Roy, P. H., Ouellette, M. & Bergeron, M. G. 1998. Species-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus. J Clin Microbiol, 36:618-623.
- Munton,P. 1988. Vegetation and Forage Availability in the Sands. J. Oman Studies 3 (3): 241-250.
- National Committee for Clinical Laboratory Standards. 2000. Performance standards for antimicrobial disk susceptibility tests, 7th ed. Approved standard. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- Silva Junior AA, Vizotto VJ, Giorgi E, Macedo SG, Marques LF 1994. Plantas medicinais, caracterizao e cultivo. EPAGRI. Bol TcnicoFlorianpolis68: 1-71.
- Survival - Appendix B, 2002.Edible and medicinal Plants.http:www.aircav.com/survival/appb/asappb01.html.
- Yamada Y, Azuma K. "Evaluation of the in vitro antifungal activity of allicin." Antimicrob Agents Chemother 1977 Apr;11 (4):743-9
- Zoghet, M.F. and A. Al-sheikh, 1999. Wild plants in the Region of Riyadh. King Saud University. Academic Publishing and Press, pp: 194-195.