I also authorize Chattogram Veterinary and Animal Sciences University (CVASU) to give this thesis to other institutions or individuals for the purpose of scientific research. I would like to express my thanks and grateful feelings to all those who have helped and supported me in various ways during my graduation of Master of Science in Biochemistry under Department of Physiology, Biochemistry and Pharmacology, Chattogram Veterinary and Animal Sciences University. CVASU Chittagong Veterinary and Animal Sciences University et al Et alii/ et aliae/ et alia.
Introduction
However, traditional plants have been shown to be a better source of antimicrobial drugs (Hammer et al., 1999). In addition, plants are considered as one of the most important sources of secondary metabolites and essential oils (Singh et al., 2010). Much of the problem has been shown to be due to the presence of transmissible plasmids encoding multidrug resistance and their spread among different enterobacterial species (Blake et al., 2003).
Review of Literature
Tulsi
- Pharmacological Properties of Ocimum sanctum
- Anti-microbial activity
- Anti-fungal activity
- Antioxidant effect
- Antidiabetic effect
- Wound healing effect
- Hypolipidemic activity
- Anti-carcinogenic property
- Antigenotoxic effect
Phytochemical investigations of stem and leaves of Ocimum sanctum revealed constituents such as saponins, flavonoids, triterpenoids and tannins (Pattanayak et al., 2010). Phytochemical investigations of Ocimum sanctum leaf extract show phenols (eugenol, cirsilineol, isotimucin, apigenin and vosamarinic acid) and flavonoids (orientin and vicenin). Banerjee et al. reported the anticancer activity of Ocimum sanctum against several carcinogens (Banerjee et al., 1996).
Swertia chirayita (Chirota)
- Medicinal Uses
- Phytochemistry of Swertia chirayita
- Pharmacological Activity of Swertia chirayita
Herbal formulations such as Ayush-64, Diabecon, Mensturyl syrup and Melicon V ointment (Edwin and Chungath, 1988; Mitra et al., 1996) contain S. Mangiferin is also reported to have anti-diabetic, anti-atherosclerotic properties (Pardo-Andreu et al., 2008), anticancer, anti-HIV (Guha et al., 1996), antiparkinsonian (Kavitha et al., 2013) and chemopreventive (Yoshimi et al., 2001) activities. Swerchirin at different concentrations (1, 10 and 100μM) significantly increased glucose-stimulated insulin release from isolated islets (Saxena et al., 1993).
Plasmid
Accessory genes may also be present and often confer clinically relevant traits such as virulence and antibiotic resistance (Thomas and Summers, 2008). In particular, during conjugation, a plasmid promotes its transfer (and that of a co-resident plasmid) from one bacterial cell to another (Norman et al., 2009). Classification of plasmids according to a typing scheme provides useful insights into the epidemiology of plasmid-mediated antibiotic resistance: for example, examining the composition of plasmid types can indicate whether an antibiotic resistance epidemic is driven by different plasmids or a dominant plasmid type (Valverde et al., 2009).
In addition, hypotheses of resistance transmission during outbreaks can be refined according to the relatedness of resistance plasmids harbored by clinical strains (Pecora et al., 2015). The main plasmid classification schemes are replicon based backbone loci encoding plasmid replication and mobility functions (Carattoli et al., 2005; Garcillán-Barcia et al., 2009). Although these single-locus typing schemes have been widely and successfully used, they provide limited resolution (Fricke et al., 2009), limiting epidemiological inference: in an outbreak setting, if two patients are infected by unrelated strains harboring resistance plasmids of the same type, this increases the possibility of plasmid transmission, but plasmid transmission cannot be definitively verified using single-locus plasmid typing alone; further studies at higher resolution would be required (Foxman et al., 2005).
Plasmid type can provide a cornerstone for higher resolution analyses; the identification of shared ("core") genes between related plasmids can inform the development of plasmid multilocus sequence typing (pMLST) schemes (García-Fernández et al., 2011) or allow phylogenetic relationships to be reconstructed based on in single nucleotide polymorphism of basic genes. (SNPs) (de Been et al., 2014). Unfortunately, determining plasmid relationships at high resolution is challenging: the tendency of plasmids to gain, lose and rearrange genetic content means that groups of plasmids – even if they are of the same type – will tend to share some genes essentially phylogenetically compatible (Fondi et al., 2010; Tazzyman and Bonhoeffer, 2014), hindering subtyping and phylogenetic analysis (Maiden, 2006). Even backbone genes may not be well conserved across all plasmids of the same strain (Lanza et al., 2014), and sometimes show mosaic phylogenetic origins (Sen et al., 2013).
However, recent analyzes suggest that plasmids can be frequently transferred between strains, even over short periods of time (Conlan et al., 2014; Sheppard et al., 2016).
Polymerase chain reaction (PCR)
Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermal cyclers without a heated lid require a layer of oil on top of the reaction mixture or a bead of wax inside the tube. PCR can be used for the diagnosis of many human diseases, a wide range of experiments and analysis (Wang et al., 2012).
PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which are currently the most developed in cancer research and are already routinely used. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity at least 10,000 times higher than that of other methods (Rahman et al., 2013). PCR also allows identification of non-cultivable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria or viruses from tissue culture tests and animal models.
The basis for diagnostic applications of PCR in microbiology is the detection of infectious agents and the differentiation of non-pathogenic from pathogenic strains based on specific genes. In contrast to living organisms, the PCR process can copy only short fragments of DNA; usually up to 10 kb (kb means kilogram of base pairs). Certain methods can copy fragments as small as 40 kb, which is still much smaller than the chromosomal DNA of a eukaryotic cell—a human cell, for example, contains about three billion base pairs.
Materials and Methods
- Experimental Design
- Plants used in this study
- Collection of plant materials
- Drying and grinding
- Preparation of ethanolic extracts
- Collection of E. coli, Salmonella spp. and Staphylococcus aureus
- Preparation of Stock
- Phytochemical activity test
- Detection of Alkaloids
- Detection of Flavonoids
- Detection of Saponins
- Detection of Tannins
- Detection of Phenolic Compound
- Detection of Glycosides
- Detection of Carbohydrates
- Detection of Reducing sugar
- Detection of Protein and Amino Acid
- Detection of Acidic Compound
- Detection of Phytosterol
- Detection of Terpenoids
- Antimicrobial discs
- Culture and sensitivity test
- McFarland Standard preparation
- Preparation and components of Agar
- Antimicrobial Activity test of plant extracts
- Plasmid Isolation
- PCR
- Phytochemical
- Antimicrobial Sensitivity
- Sensitivity Pattern
- Plasmid Identification
- PCR for detection plasmid resistant bacteria
A few mg of the test residue was mixed with 2 drops of naphthol in 100 ml of 95% alcohol. No reddish-purple ring at the junction of the bilayers appeared in the presence of sugars. 5 ml of Benedict's solution was added to the test tube, boiled for 5 minutes and in the test tube.
The absence of yellow color indicated increased deficiency on the side of the test tube. Each of the discs was cut from filter paper No. 1 of Whatman with an approximate diameter of 6 mm using a drill. The prepared disc with filter paper was sterilized by autoclaving at 121°C for 15 minutes and inoculated with 30 μl of the extract of the experimental plant.
McFarland Standards were used as a reference to adjust the turbidity of the liquid/bacterial suspension in the vial or tube in the microbiology laboratory. The McFarland standard can be prepared with varying concentration ranging from 0.5 to 4 and depending on the concentration the cell number density varies. In good light, visually compare the opacity of the test suspension to that of the McFarland standard by comparing the brightness of the lines on the Wickerham chart.
Then the filter paper disks were placed on the surface of the agar plate equidistant from each other and 15 mm from the edge of the plate as described by Clutter Buck et al. The time for this step depends both on the DNA Polymerase itself and on the length of the DNA fragment to be amplified. The size of the PCR product can be determined by comparing it to a DNA ladder, which contains DNA fragments of known size, also in the gel (Rahman et al., 2013).
Discussion
The herbs are cheap, available in large quantities around us and they pose no danger to the living organisms, the environment and the consumers and therefore very useful for living organisms (Raynor et al., 2011). In addition, the number of antimicrobials used also increases when targeting specific diseases or to prevent the spread of a particular disease, or in times of stress (Studdert et al., 1998). Antimicrobial screening of Ocimum sanctum showed the highest zone of inhibition in 14 days (S1) and 21 days (S2) with 0.2 mg/μL concentration than other doses.
Here they also achieved a higher zone of inhibition in lower concentrations compared to the present study. These variations may occur due to the difference in the quality of Ocimum sanctum leaves or extraction efficiency. The present study showed that some bacterial isolates showed 100% resistant, which is similar to the Cardoso et al., 2006 that found 100%.
Variations in the pattern of antimicrobial resistance may be due to differences in bacterial isolates and types of antimicrobial use, as well as geographic location of farms. This result suggests that the extent of antibiotic resistance is related to the extent of antibiotic use (Bebora et al., 1997; Mastour et al., 1999). We randomly analyzed fourteen isolates each for the presence of plasmid DNA and among these four isolates of each bacterium, 28% of all samples were found to contain a high molecular weight plasmid (size greater than 10 kbp) with a high degree of resistance as reported by John et al.
Our study concludes that multiple resistant E. coli isolates and plasmids containing multidrug resistance genes are present in poultry farms, and poultry may act as a possible source of transmission of these highly resistant pathogens and their genes to humans, as previously reported. Wooley et al. 1999), we agree with calls to ban the use of antibiotics for growth promotion and treatment in the poultry sectors.
Conclusion and Recommendation
Evaluation of anti-inflammatory effects of Swertia chirata in acute and chronic experimental models in male albino rats. Evaluation of antimicrobial activity of Ocimum sanctum (Tulsi) extract against Streptococcus mutans and Lactobacillus acidophilus - an in vitro study. Inhibition of lipid peroxidation by botanical extracts of Ocimum sanctum: in vivo and in vitro studies.
Evaluation of alpha-amylase inhibitory activity of Nepalese medicinal plants used in the treatment of diabetes mellitus. Evaluation of the in-vivo activity and toxicity of amarogentin, an antileishmanial agent, in both liposomal and niosomal forms. Study of antibacterial activity of Ocimum sanctum extract against gram positive and gram negative bacteria.
Phenolic biochemical rationale for the antidiabetic properties of Swertia chirayita (Roxb. ex Flem.) Karst. Therapeutic use of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: a brief review. Effect of different fractions of Swertia chirayita on the blood sugar level of albino rats.
Evaluation of antioxidant and wound healing effects of alcoholic and aqueous extract of Ocimum sanctum Linn in rats. Evaluation of anti-inflammatory potential of fixed oil of Ocimum sanctum (Holy basil) and its possible mechanism of action. Comparison of hepatoprotective activity of Swertia chirayita and Andrographis paniculata plant of Northeast India against CCl4-induced hepatotoxic rats.
