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Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy. / NATALIA KAPEL

Swansea University Author: NATALIA KAPEL

Abstract

Infectious diseases remain a serious threat to human life and health as well as having important economical factor. One way of successful combating diseases is designing the most appropriate treatment plan following the correct diagnosis. Therefore, there is a need for a method combining reproducibi...

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Published: 2013
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa42219
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first_indexed 2018-08-02T18:54:10Z
last_indexed 2020-09-04T03:03:02Z
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spelling 2020-09-03T09:05:41.6301453 v2 42219 2018-08-02 Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy. b7123335713b17ea5461340903437229 NATALIA KAPEL NATALIA KAPEL true false 2018-08-02 Infectious diseases remain a serious threat to human life and health as well as having important economical factor. One way of successful combating diseases is designing the most appropriate treatment plan following the correct diagnosis. Therefore, there is a need for a method combining reproducibility, precision and speed. The aim of this work was to evaluate the potential of micro-Raman spectroscopy for identifying bacteria at different taxonomic levels, strains revealing different antibiotic resistance profiles, and for phylogenetic investigation. The project was based on a selection of bacteria: Staphylococcus aureus (6571, Cowanl), Staphylococcus epidermidis (1457, 9142), Escherichia coli including wild- types (strain B, K12, Top 10), transformants expressing ampicillin and kanamycin resistance (Top10Amp, Top10Kan) and clinical isolates expressing extended-spectrum beta- lactamases (ESBL). Following a precise and detailed protocol, Raman spectra were recorded from bacterial colonies grown overnight on a Colombia Blood Agar. In order to remove background fluorescence, rolling-circle filter procedure was applied. The most critical peaks for differentiation between organisms as well as for characterising each microorganism were determined. The spectral data were analyzed using principal component and cluster analysis techniques. As expected, the degree of separation decreased in the order genus&rarr;species&rarr;strain. It was determined that DNA/RNA, proteins and amino-acids are responsible for the differentiation between strains on a lower level of similarity with more influence of the constituents of the bacterial envelope between more closely related organisms. Raman spectroscopy was capable of differentiating between susceptible and resistant strains as well as monitoring whether the organism has been grown under antibiotic pressure. Based on triplex PCR, clinical isolates of ESBL strains were assigned to one of the phylogenetic group characterising Esherichia genus and it was revealed that within CTX- M TEM-1 there were two distinct clusters of D and B2 groups. Overall we have demonstrated that the combination of micro-Raman spectroscopy, microbiology and bioinformatics has the potential for the successful discrimination of bacteria species and strains, for the determination of antibiotic resistance profiles and investigating phylogenetic grouping in a clinical environment. E-Thesis Medicine.;Analytical chemistry. 31 12 2013 2013-12-31 COLLEGE NANME COLLEGE CODE Swansea University Doctoral Ph.D 2020-09-03T09:05:41.6301453 2018-08-02T16:24:28.4641899 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine NATALIA KAPEL 1 0042219-02082018162437.pdf 10797921.pdf 2018-08-02T16:24:37.4330000 Output 21029868 application/pdf E-Thesis true 2018-08-02T00:00:00.0000000 false
title Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
spellingShingle Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
NATALIA KAPEL
title_short Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
title_full Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
title_fullStr Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
title_full_unstemmed Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
title_sort Rapid species identification and antimicrobial susceptibility testing using Raman spectroscopy.
author_id_str_mv b7123335713b17ea5461340903437229
author_id_fullname_str_mv b7123335713b17ea5461340903437229_***_NATALIA KAPEL
author NATALIA KAPEL
author2 NATALIA KAPEL
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publishDate 2013
institution Swansea University
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hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description Infectious diseases remain a serious threat to human life and health as well as having important economical factor. One way of successful combating diseases is designing the most appropriate treatment plan following the correct diagnosis. Therefore, there is a need for a method combining reproducibility, precision and speed. The aim of this work was to evaluate the potential of micro-Raman spectroscopy for identifying bacteria at different taxonomic levels, strains revealing different antibiotic resistance profiles, and for phylogenetic investigation. The project was based on a selection of bacteria: Staphylococcus aureus (6571, Cowanl), Staphylococcus epidermidis (1457, 9142), Escherichia coli including wild- types (strain B, K12, Top 10), transformants expressing ampicillin and kanamycin resistance (Top10Amp, Top10Kan) and clinical isolates expressing extended-spectrum beta- lactamases (ESBL). Following a precise and detailed protocol, Raman spectra were recorded from bacterial colonies grown overnight on a Colombia Blood Agar. In order to remove background fluorescence, rolling-circle filter procedure was applied. The most critical peaks for differentiation between organisms as well as for characterising each microorganism were determined. The spectral data were analyzed using principal component and cluster analysis techniques. As expected, the degree of separation decreased in the order genus&rarr;species&rarr;strain. It was determined that DNA/RNA, proteins and amino-acids are responsible for the differentiation between strains on a lower level of similarity with more influence of the constituents of the bacterial envelope between more closely related organisms. Raman spectroscopy was capable of differentiating between susceptible and resistant strains as well as monitoring whether the organism has been grown under antibiotic pressure. Based on triplex PCR, clinical isolates of ESBL strains were assigned to one of the phylogenetic group characterising Esherichia genus and it was revealed that within CTX- M TEM-1 there were two distinct clusters of D and B2 groups. Overall we have demonstrated that the combination of micro-Raman spectroscopy, microbiology and bioinformatics has the potential for the successful discrimination of bacteria species and strains, for the determination of antibiotic resistance profiles and investigating phylogenetic grouping in a clinical environment.
published_date 2013-12-31T03:52:32Z
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