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Procedure and Results on the Identification of Staphylococcus aureus

Discuss About The Discovery Of An Archetypal Protein Transport.

The Staphylococcus aureus is one of the many normal florae of the body that is mostly found in the respiratory tract, skin as well as the skin. In our case, the patient has a diabetic foot hence laboratory samples will be taken from the foot, skin and nose swabs for microbiological tests (Bakker, Apelqvist, Schaper & International Working Group on the Diabetic Foot Editorial Board, 2012). This is because, in diabetic patients, the foot ulcers can have a lifelong incidence rate and become infected especially with Staphylococcus aureus (Bakker, Apeldvist & Schaper, 2012). The infection can spread to the soft tissue and bone hence early detection and identification experiments have to be carried out as soon as possible.

  • To determine whether the recurrence of Staphylococcus aureus is due to a new infection or the same bacteria, the samples will be obtained from the wound mechanically through debris obtained using a scalpel or a sterile curette and cleaning the wound with a gauze that has been soaked in sterile physiological saline.
  • The samples will be obtained by scraping the wound base, aspiration of the purulent secretions by use of a needle via the healthy skin or biopsy.
  • Sampling will be repeated a couple of times and taken for microbiology laboratory for DNA micro-assay and multiple real-time PCR to analyze the virulence of the Staphylococcus aureus and the potential resistance of the microorganism.

The results obtained will show the presence of surface polymers and proteins that have DNA characteristics similar to those of the previous tests. This will prove that the infection is the recurrence of the previous infection due to the identifications of various genes involved in virulence as well as antibiotic resistance factors. These results reveal that staphylococcus aureus is a very resistant to antimicrobial agents hence causes recurrent infection.

Research Plan To Identify The Molecular Mechanisms Used By Staphylococcus Aureus Isolate For Its Colonization

To determine the molecular mechanisms that are used by Staphylococcus aureus isolate for its colonization one has to explore the virulence factors that enable S. aureus to survive the extreme conditions within the human host. These experiments should include polymerase chain reaction (PCR) and serological tests to determine the virulence factors that lead to S. aureus colonization.

  • We will use the direct plate count method
  • We will then prepare the staphylococcus samples to be used in PCR.
  • Synthetic oligonucleotide primers of 24 and 21 bases will be used so as to amplify a series of the agr gene that usually encodes for the nuclease of S. aureus.
  • A DNA fragment of S. aureus will be amplified and analyzed using agarose gel electrophoresis and the southern blot.
  • The PCR products will be obtained by using vitro-cultured S. aureus to prepare specimens of blood, urine and CSF.
  • We will then isolate and enumerate the staphylococcus aureus.
  • Finally, we will count and record colonies.

The results will show mucoid colonies with plasmids and virulence genes. These colonial morphology enables the staphylococcus aureus able to cause humoral responses that are induced by s. aureus colonization. The genes detected lead to S. aureus colonization and virulence and are the accessory gene regulator agr which controls much virulence and colonizing factors (DeLeo, Otto, Kreiswirth & Chambers, 2010). Therefore, the PCR amplification of the agr gene has the potential to determine colonization and diagnosis of the staphylococcus aureus infection by the direct testing of the clinical specimen which includes specimen obtained from patients ongoing antimicrobial therapy.


Research Plan To Identify The Molecular Mechanisms For Staphylococcus Aureus Persistence And The Recurrent Cause Of DFI Disease

The recurrent Diabetic Foot Infection leads to ulcers that result in open wounds which are sites for Staphylococcus aureus entry to the body through the bloodstream. The molecular mechanisms for the identification of DFI (Diabetic Foot Infection) involve culture mechanisms.

Research Plan to Identify the Molecular Mechanisms Used by Staphylococcus Aureus

Procedure

  • We will use culture based mechanisms for the identification process of DFI disease
  • We will carry out wound biopsy culture of the 16S pyrosequencing (Boulton, 2010).
  • The two microorganisms will be cultured and genes that cause DFI determine through DNA assays.
  • Finally, we will observe clinical features such as discolored granulation tissue, odor, secretions, delayed healing, and necrosis.  

The test on DFI helps one to distinguish S. aureus into two populations; the toxinogenic Staphylococcus aureus strains and the non-toxinogenic strains (Lipsky et al. 2012). Then DNA assays should be carried out on the toxinogenic s. aureus to determine the encoding genes for TSST and genes that harbor exfoliatin which include PVL and EDIN. Testing these genes provides information on virulence profile of the bacteria which is a very effective way to regulate and forecast the conduct of the Staphylococcus aureus in long-lasting lesions like in our case study. However, since microorganisms usually colonize all chronic and recurrent lesions, the analysis of DFI done was based on both the bacteriological examination of the wound culture as well as medical results. The classical signs of DFI are discolored granulation tissue, odor, secretions, delayed healing, and necrosis. 

How And Why The Bioinformatics Analysis Led To The Conclude That The Protein Is A Type V(a) Autotransporter Protein, And Why It May Contribute To Virulence.

The bioinformatics analysis had led to the conclusion that the protein present is a type V(a) autotransporter protein since the pathogenic E. coli possess an external membrane protein of type V autotransporter group of proteins that contributes to its pathogenicity and virulence (Selkrig et al. 2012). This protein has a wide range of functions that include giving the pathogenic strain the ability to modulate apoptosis and condense the host cells hence leading to pathogenicity and virulence. The non-pathogenic E. coli do not possess this protein hence it does not cause pathogenicity (Clermont,  Bonacorsi & Bingen, 2010). Therefore, the DNA sequence analysis derived genetic and molecular investigations of E. coli done in this experiment show that the pathogenic E.coli differs from the non-pathogenic E.coli due to the V autotransporter protein that determines virulence in the form of mobility mediators, enzymes, toxins, and adhesins. The pathogenic E.coli secretes these proteins to increase their survival abilities and multiplication in the host (Clermont et al.  2010).

Series Of Experiments To Determine If The Protein Is Actually Located In The Outer Membrane Of The Pathogenic E. Coli Strains, and/or If Is Secreted.

So as to determine that the protein detected by bioinformatics is located in the surface membrane of the pathogenic E. coli the series of experiments to be carried out that include DNA techniques, PCR, electron microscopy and the separation of the outer and inner membrane fractions.

Procedure

  1.    We will apply the DNA techniques to encode the target proteins that were amplified from the E. Coli by the use of PCR through;
  • Protein denaturation of the membrane.
  • Annealing of the pathogenic E. coli.
  • Extension and Elongation of the Escherichia coli outer membrane.

The results that are obtained from the DNA analysis then should be run against the known DNA of the autotransporter protein to positively identify it.

  1.    We will use the Electric microscopy to be able to visualize and identify the protein present in the outer membrane as V(a) autotransporter protein through its characteristic morphological features.
  2.   We will separate the inner and outer membrane fractions- Through the separation of the inner and outer membranes, we will be able to determine that the autotransporter protein is in the outer membrane through the use of an electric microscope and that is secreted.

Research Plan to Identify the Molecular Mechanisms for Staphylococcus Aureus Persistence and the Recurrent Cause of DFI Disease

The above procedures in PCR, electric microscopy and DNA analysis show the presence of  a protein that has been identified as an autotransporter protein that is located in the outer membrane of the pathogenic E.coli and is secreted since it produces exotoxins. This protein is involved in virulence and pathogenicity. The results also show that the V(a) autotransporter protein that is located in the outer membrane of the pathogenic E. coli is secreted as it produces toxins that spread to various parts of the host to cause disease. The location of this protein that enhance virulence and pathogenicity of the pathogenic E. coli in the outer membrane and the production/secretion of the protein as an exotoxin helps the microorganism in causing disease in the host.

Experiments To Determine The Protein’s Intrinsic Virulence Factor Activity That May Contribute To Coli pathogenesis include;

We will determine Bacterial invasion using in vitro model systems- The ability of the bacteria to cause invasion through in vitro models show the intrinsic capabilities of the autotransporter to cause virulence that leads to pathogenesis.

Procedure

  1.    We will use the electron microscope examine the cytoskeleton rearrangement determined through pathogenetic analysis.
  2.    We will induce signal transduction.
  3.    We will culture the polarized epithelial cell.

The cytoskeleton arrangement that will be identified in the morphological form of the pathogenic e. coli indicate its ability to cause virulence through the presence of plasmids. The signal transduction pathways detected aid in the virulence and pathogenesis of E. coli by use of kinases of the autotransporter that control gene expression. Since the autotransporter is located in the outer membrane of the pathogenic E. coli, the culture of these epithelial cells  positively identify the virulence role played by the protein autotransporter through the actions of the protein’s lipids and polypeptides. (Maurelli, Fernandez,, Bloch, Rode & Fasano, 2008).

Experiments To Show That The Autotransporter Protein Is A Virulence Factor That Contributes Significantly To The Ability Of The Pathogenic E. Coli Strain To Cause Disease

As stated above, the autotransporter protein contributes to the pathogenic E. coli pathogenesis and virulence through mobility mediators, enzymes, toxins and adhesins hence giving it the ability to cause disease.

Procedure

  • We will determine the virulence and pathogenesis due to autotransporter protein through;
  • Bacterial genomes and gene annotations through Ribosomal RNA Analysis.

The results obtained will enable us to predicted proteome analysis and prevalence of genomic islands which contribute to virulence and pathogenesis (Peterson, Tian, Ieva, Dautin & Bernstein, 2010). Therefore, using these results we are able to confirm that the autotransporter protein is a virulence factor that contributes significantly to the ability of the pathogenic e. coli strain to cause disease. The V(a) autotransporter protein helps in the virulence and pathogenicity of the pathogenic E. coli through the production of mobility mediators, enzymes, toxins and adhesins which gives this microorganism the ability to cause disease.

References

Bakker, K., Apelqvist, J., Schaper, N. C., & International Working Group on the Diabetic Foot Editorial Board. (2012). Practical guidelines on the management and prevention of the diabetic foot 2011. Diabetes/metabolism research and reviews, 28, 225-231.

Boulton, A. J. (2010). Foot problems in patients with diabetes. Textbook of diabetes, 725-742.

Clermont, O., Bonacorsi, S., & Bingen, E. (2010). Rapid and simple determination of the Escherichia coli phylogenetic group. Applied and environmental microbiology, 66(10), 4555-4558.

DeLeo, F. R., Otto, M., Kreiswirth, B. N., & Chambers, H. F. (2010). Community-associated meticillin-resistant Staphylococcus aureus. The Lancet, 375(9725), 1557-1568.

Lipsky, B. A., Berendt, A. R., Cornia, P. B., Pile, J. C., Peters, E. J., Armstrong, D. G., ... & Pinzur, M. S. (2012). 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clinical infectious diseases, 54(12), e132-e173.

Maurelli, A. T., FERNAndez, R. E., Bloch, C. A., Rode, C. K., & Fasano, A. (2008). “Black holes” and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proceedings of the National Academy of Sciences, 95(7), 3943-3948.

Peterson, J. H., Tian, P., Ieva, R., Dautin, N., & Bernstein, H. D. (2010). Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment. Proceedings of the National Academy of Sciences, 107(41), 17739-17744.

Selkrig, J., Mosbahi, K., Webb, C. T., Belousoff, M. J., Perry, A. J., Wells, T. J., ... & Celik, N. (2012). Discovery of an archetypal protein transport system in bacterial outer membranes. Nature Structural and Molecular Biology, 19(5), 506.

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