Detection, identification and genotyping of Pseudomonas aeruginosa isolated from clinical and environmental samples

Anuj, Snehal Neeraj (2012). Detection, identification and genotyping of Pseudomonas aeruginosa isolated from clinical and environmental samples PhD Thesis, School of Medicine, The University of Queensland.

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Author Anuj, Snehal Neeraj
Thesis Title Detection, identification and genotyping of Pseudomonas aeruginosa isolated from clinical and environmental samples
Formatted title
Detection, Identification and Genotyping of Pseudomonas aeruginosa isolated from clinical and environmental samples
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Claire Wainwright
Michael Nissen
David Whiley
Total pages 208
Total colour pages 23
Total black and white pages 185
Language eng
Subjects 110801 Medical Bacteriology
Formatted abstract
Cystic fibrosis (CF) is a genetic disorder affecting a number of organs in the body including the respiratory system. The lungs are unable to clear the mucus formed thus creating an environment suitable for pathogens and making patients more prone to respiratory infections. A number of pathogens have been identified to cause respiratory infections with Pseudomonas aeruginosa (P. aeruginosa) being considered as a major respiratory pathogen. Initial strains of P. aeruginosa isolated from the respiratory samples of the CF patients are usually non-mucoid, sensitive to a number of antibiotics and have a unique genotype. This supports the hypothesis that the initial P. aeruginosa infection may be acquired from the environment. However, other sources such as patient-to-patient transmission of P. aeruginosa strains have also been identified. Dominant and genetically related P. aeruginosa strains have been identified between patients in a number of CF centres (clonal strains) and have been associated with increased virulence when compared with genotypes only found in a single patient (unique strains). Studies have shown that infection with P. aeruginosa is a marker of poor prognosis and chronic infection is associated with increased morbidity and mortality. Therefore, timely and correct administration of anti-pseudomonal therapy will not only aid to eradicate the early infection but also prevent recurrent and chronic infection with P. aeruginosa. It is essential to have an identification and a genotyping assay for P. aeruginosa that can be readily transferred across laboratories, easily reproducible, is cheap and result interpretation is objective. Aims:
   - To investigate the home environment of patients with CF to find a possible source
     of P. aeruginosa that may be the cause of the initial or re-infection.
   - To identify and genotype P. aeruginosa isolates from the samples collected.
   - To identify if the most common clonal Australian epidemic strains (AES) 1, AES 2
      and pulsotype (P) 42 are prevalent in the home environment.
   - To design a real-time identification assay that is robust and reproducible.
   - To design a real-time PCR genotyping assay using high-resolution melting curve
     analysis and targeting single nucleotide polymorphisms.
   - The environment of twenty homes of families with CF patients’ were tested. Twenty
     homes of families that did not have any patients’ with CF were also tested as
     control homes.
   - A duplex real-time PCR identification assay targeting the ecfX and gyrB gene was
     designed with increased specificity and sensitivity for the identification of P.
     aeruginosa isolates isolated from clinical and environmental samples.
   - A 10 single nucleotide polymorphism (SNP) based genotyping assay using high
     resolution melting curve analysis (HRM) and targeting the SNPs found in the
     house-keeping genes of P. aeruginosa was successfully designed. This assay can
     be used to identify the three most common clonal strains AES1, AES 2 and P42
     isolated in patients attending the Brisbane clinics.
Results and Conclusion:
As part of this PhD study, problems using existing identification and genotyping methods were recognised and discussed. New assays for identification and genotyping were successfully designed and implemented for use. I have shown that these assays can be successfully used for P. aeruginosa strains isolated from both clinical and environmental samples. These assays have faster turnaround times with result interpretation not being subjective. An important finding as part of the environmental testing was that aerosols containing P. aeruginosa were generated in the home environment and they could potentially be a source of the initial or re-infection in patients with CF. This is the first time that aerosol testing has been performed in the home environment to study the role of contaminated aerosols in causing the initial or re-infection. No major common clonal strains were isolated from the home environment. Implementation of the identification and genotyping assays as designed as part of this PhD study, will make a significant contribution to the laboratory by providing a robust, re-producible and considerably cheaper methods than the current methods in use. Enabling correct and rapid identification of P. aeruginosa from the respiratory samples will not only aid in the implementation of correct antibiotic regime but will also help in maintaining correct segregation policies thus making a greater contribution in patient care and patient management.
Keyword Cystic fibrosis (CF)
Pseudomonas aeruginosa

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Created: Thu, 02 May 2013, 21:23:06 EST by Snehal Anuj on behalf of Scholarly Communication and Digitisation Service