Date of Award

2009

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Biological Sciences

First Advisor

Dr. Jim O'Mahony

Abstract

Nontuberculosis Mycobacteria (NTM) are now considered a major source of opportunistic infection in immunocompromised patients. Reliable and rapid identification of NTM species is of the upmost importance for patient treatment regimes. Firstly, this study looked at the epidemiological trends of a small number of NTM isolates (n=50) from a large teaching hospital in Southern Ireland Cork University Hospital (C.U.H.). We found that 85% of clinical isolates were sampled from the respiratory tract, which is not unusual as the majority of samples analysed in the hospital lab originated from sputum samples. Also, the majority of patients were 59 years of age or older (62%). 42% of isolates donated were found to be MAC {Mycobacterium avium complex), which is regularly the most commonly extracted NTM strain in clinical environments. In addition to the Cork study, 50 MAC isolates were donated by St James' Hospital Dublin. No patient information was given with these isolates; although from the incidence data provided it was clear that MAC isolates were in general the most frequently identified NTM, with 86 / 259 isolates recovered over 7 years.

We identified the combined 100 NTM to a species and sub-species level, using several molecular methods. These results were then compared to biochemical analysis and identification methods carried-out by both hospitals. The first molecular method employed was 16S rRNA gene typing, which was compared to the genomic databases (BLAST and RIDOM). Both data bases gave identical species results for all 100 isolates, indicating the utility of these on-line systems. After 16S typing, 3 of the Cork isolates (6%) were shown to be different to the species typed, which was assigned following the biochemical analysis at C.U.H. There was 100% agreement between the species identification obtained from St James' Hospital laboratories and this study, although some of the MAC isolates were reclassified by the hospital after we received them. 16S typing on our part confirmed the validity of the re-classification.

One drawback of 16S typing is the poor discrimination at the sub-species level between closely related mycobacteria such as MAC. Therefore it was necessary to apply additional molecular profiling techniques for the identification of NTM at a sub-species level. IS (Insertion Sequence) elements (IS 1900, IS901, IS1311 and IS 1245) and sequevar analysis were used to identify 66 MAC isolates to a sub-species level. IS elements rapidly identify mycobacteria without the need for sequencing, and virulence potential can also be established using this system. It was determined using IS profiling that 94% of MAC isolates from Cork

and Dublin were virulent M avium subsp hominissuis. The remaining 6%, were positive for 1S1311, and were negative for all other IS elements (IS 1245, IS900 and IS901). This result in conjunction with 16S typing results identified these isolates as M. intracellulare.

The final molecular tool used in this study of 66 clinical MAC isolates was hsp65 sequevar profiling to confirm the sub-species identity. After PCR analysis and sequencing, each MAC isolate was aligned with the MAA104 reference genome to quickly identify strategic SNPs and assign a sequevar code. In total, 68% of the MAC isolates were represented by code 1, while 23% were identified as code 2. 3% were confirmed as code 3. All 3 sequevar codes represent M. avium subsp hominissuis which correlated with the IS profiling. Finally, 4 isolates (6%) of the cohort which were originally identified as MAC by St James' were re-classified as M intracellulare code 10 following sequevar analyses and 16S typing.

In tandem with this clinical analysis, an environmental study was also undertaken to establish the diversity of NTM from a variety of water sources. Two decontamination methods were chosen for this study (CPC and NaOH). It was necessary to optimise both of these methods prior to screening the environmental samples. In total 70 water samples were collected from the greater Cork region and processed using both decontamination procedures. From the analysis 5 isolates (7%) were positive for NTM and identified as M alvei, M. tokaiense, M.gordonae and M. murale. Only one of these isolates {M.gordonae) is commonly isolated from clinical environments as an opportunistic pathogen in immunocompromised individuals.

Access Level

info:eu-repo/semantics/openAccess

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