Essay Example on Staphylococcus aureus infection is a severe pathological Condition

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Staphylococcus aureus infection is a severe pathological condition which not only affects the infected organ but extensive antimicrobial therapy used to treat such an infection also produces detrimental effects on overall health of the infected individual There are disparate pathophysiological mechanisms associated with acute and chronic S aureus infection The acute infection is associated with more aggressive and invasive phenotype whereas the chronic infection is associated with less aggressive and mild inflammatory phenotype characterised by small colony variants SCV There are several regulatory factors which play a crucial role in such a dynamic shift in S aureus phenotype from a greater inflammatory and shorter intracellular persistence associated acute phase to a less cytotoxic and longer intracellular persistence associated chronic phase The study aimed at finding the specific role of such essential factors i e agr sig B sarA in dynamic switching of bacterial phenotype in acute and chronic S aureus infection To identify the role of these regulating factors in acute and chronic infection the investigators designed different mutant models single double triple mutant and studied the virulence of wild and mutant strains in different in vivo and in vitro infection models Wild type and mutant strains of S aureus LS1 and SH1000 were used for all the experiments under the study Mutant strains of agr Sig B sarA were generated by transduction of antibiotic resistant cassettes i e RN6911 IK181 and ALC136 respe using different phages i e 11 80 85 respe Wild type and mutant strains were grown stepwise i e wild type and single mutants were grown in first step whereas double and triple mutants were grown in second step the protein fraction were extracted sequentially 



Proteomic analysis was performed using mass spectrometry LTQ Orbitrap Velos Mass Spectrometer and resulting data was analysed using appropriate software i e Rosetta Elucidator 3 3 01 for the differential expression of proteins As a part of in vitro studies primary human osteoblasts HUVECs Human umbilical venous endothelial cells and human polymorphonuclear cells PMNs were used as hosts to study the S aureus infection patterns In addition haemolytic effects of S aureus infection were studied using haemolysis assay i e incubating different mutant and wild type strains with extracted human red blood cells Cytokine fractions were analysed using cytokine release assay For in vivo studies wistar adult rats were used to develop chronic S aureus infection i e osteomyelitis Quantitative real time PCR was used to study the differential gene expression in bacterial strains Appropriate statistical tests were used to perform statistical analysis of the data i e one way ANOVA agr serA single mutant showed severe reduction in release of virulence factors and corresponding reduced inflammation and toxicity compared to wild type strains All mutants i e single double triple showed reduced inflammation and decreased cytotoxicity except sigB mutant which showed increased α toxin level and corresponding increased cytotoxic effects on PMNs Similar results were observed for HUVECs and osteoblasts in vitro assays where all double and triple mutants showed reduced cell activation and inflammation compared to wild type whereas sigB mutant showed increased cell activation and inflammation This showed that in acute phase agr and sarA are essential to develop an aggressive and inflammatory phenotype whereas sigB is associated with less aggressive phenotype To study S aureus infection pattern in chronic phase HUVECs and osteoblasts were treated with wild type and mutant bacterial strains and later checked for the presence of bacteria in intracellular compartment after 7 9 days Single agr serA double and triple mutants were able to localize in intracellular compartment for longer period but sigB mutants were totally cleared out from the host cells But when sigB mutants were complemented with complete sigB operon the effects were reversed i e complemented strains were localized intracellularly for longer time 



Moreover it has also been observed that sigB mutants were unable to show SCV a characteristic of chronic infection But reverse effects were observed when sigB mutants were complemented with complete sigB operon Gene expression analysis of wild type and mutant strain showed agr and sarA upregulation in acute phase and sigB upregulation in chronic phase This showed that agr and sarA downregulation and sigB upregulation is essential for long term intracellular persistence of S aureus in chronic phase agr serA mutants could not escape phagosomal lysis thus confirming the essential role of agr and sarA in phagosomal escape In vivo studies showed that the single double and triple mutant strains caused less aggressive phenotype and reduced toxicity than wild type strain Mutant strains either single double or triple could not induce severe and chronic infection compared to wild type Thus investigators hypothesized that the combined action of agr sarA and sigB is crucial for an acute chronic S aureus in vivo infection Thus by using different in vitro and in vivo models study demonstrated the importance of specific S aureus regulators in switching from acute to chronic phase infection i e it showed that active agr and sarA are crucial for acute toxicity and severe inflammation whereas sigB is essential for long term intracellular persistence Study also highlighted the dynamic interaction of important S aureus regulators agr sarA and sigB and the necessity of their combined action to induce severe inflammation and toxicity More importantly study also furnished a novel therapeutic marker target to treat severe chronic S aureus infection


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