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Investigation of phage based approaches for the detection and drug susceptibility testing of mycobacteriaAl-Suwaidi, Zubaida Daham F. January 2002 (has links)
No description available.
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Isolation and identification of environmental mycobacteria and associated temperate phagesLukusa, Kambulu 21 October 2009 (has links)
The Mycobacteria are a genus of bacteria which are acid-fast, non-motile,
grampositive rods. The genus comprises several species classified into three main
groups. Firstly, the major group of these organisms, which poses the biggest threat, is
the M. tuberculosis complex which can cause tuberculosis-like disease. These include
M. bovis, M. africanum and M. microti. Members of the M. tuberculosis complex are
not found in the environment. The second group is M. leprae which is the causative
agent of leprosy. The last group constitutes the nontuberculous mycobacteria (NTM),
which are all the environmental mycobacteria that can cause various diseases
resembling tuberculosis. Due to the importance of environmental mycobacteria, 15
mycobacteria isolates were isolated from environmental samples such as soil, water
and drinking water biofilms. After PCR amplification of the hsp65 gene using genus
specific primers hsp65, the isolates revealed sequences similarities when compared
with the well characterized mycobacteria in the GenBank. Alignment of the
nucleotide sequences and homology analysis were done with Clustall. It has been
suggested that mycobacteria-associated phages (mycobacteriophages) may make an
important contribution to the evolution of pathogenic mycobacteria. Spontaneous
induction of phage associated with mycobacteria isolates using overlay and indicator
plate methods was not successful to detect the presence of any inducible phage. A
phage was isolated from soil samples that was designated the name A22. After
purification and characterization. A22 phage was compared morphologically to well
characterized L5 phage using electron microscopy. Morphological studies revealed
that A22 mycobacteriophage had a non-contractile tail approximately 150 nm long
with an isometric head approximately 60 nm, the phage could be assigned to the
family Siphoviridae, According to these criteria, both of the phages (A22 and L5)
belong to the order Caudovirales (tailed bacteriophages). Based on PCR amplification
of A22 phage DNA using L5 gp71 specific primers and the infection of M. smegmatis
L5 lysogen, we believe that this novel A22 phage differs from L5 phage.
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Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillusSilva, Joás Lucas da 28 November 2011 (has links)
O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula. / Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.
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Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillusJoás Lucas da Silva 28 November 2011 (has links)
O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula. / Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.
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Investigating Impact of Mycobacterial Physiology on Mycobacteriophage Life Cycles by Mass SpectrometryYi Li (5929964) 17 January 2019 (has links)
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<p>Mycobacteriophages are the viruses that infect mycobacteria. Due to the high
death rate and antibiotic-resistant strains, phage therapy is considered to be a promising treatment of tuberculosis. Current understanding of phage-bacteria interaction is
abstracted as phage lytic and lysogenic life cycles. However, bacterial physiology may
impact phage life cycles and bacterial cells with different physiology may have different
responses to phage infection. In order to improve the understanding of phage-bacteria
interaction and update phage therapy strategy, the impact of mycobacterial physiology on mycobacteriophage life cycles was studied in this research. In this research,
a mass spectrometry-based method was first developed to study phage proteins in
phage-bacteria mixture. Then five mycobacteriophages isolated at Purdue University were selected to infect exponential and stationary <i>Mycobacterium smegmatis</i> (<i>M.
smegmatis</i>) cell cultures. Growth curves of the <i>M. smegmatis</i> cell cultures infected
by the five phages were determined. Proteomics and lipidomics of the <i>M. smegmatis</i>
cells cultures infected by phages FrenchFry and MrGordo were analyzed by mass spectrometry. The correlations between individual proteins/lipids and the experimental
factors (bacterial growth phases, phages and phage infection time) were studied by
developing linear regression models using SAS. The mass spectrometry-based method
was proved to be able to detect phage proteins other than the structural proteins.
It also verified the phage protein annotation that had been accomplished <i>in silico</i>.
X! Tandem and a database consisting of six frame translation of the phage genome
and the annotated proteins of <i>M. smegmatis</i> were the optimal option for analyzing mass spectra data of phage-bacteria mixture. The growth curves of the <i>M. smegmatis</i>
infected by the phages displayed that growth of exponential <i>M. smegmatis</i> cell cultures were depressed by phages (except FrenchFry) and stationary <i>M. smegmatis</i> cell
cultures were not actively lysed by any of the phages. The proteomics results showed
that MrGrodo infection impacted more proteins than other factors did. Exponential
phase up-regulated proteins involved in cell division. Stationary phase up-regulated
proteins that may change cell surface properties. FrenchFry up-regulated LuxR protein. Infection time up-regulated the proteins associated with mycobacterial virulence. The lipidomics results indicated that growth phases impacted the most lipids.
Phage infection time increased the amount of the lipids related to mycobacterial virulence. In summary, the mass spectrometry-based method developed in this research
can be employed to study phage proteins in phage-bacteria mixture and verify phage
genome annotation. Mycobacterial physiology alters mycobacteriophage life cycles.
Phage-bacteria interaction is the interaction between the two populations instead of
between an individual phage particle and an individual bacterial cell. Virulence of
<i>M. smegmatis</i> improves as a response to phage infection.</p></div></div></div>
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The development of a novel fluorescentmarker phage technology system for the early diagnosis of tuberculosis diseaseVan der Merwe, Ruben Gerhard 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and
morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread
and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have
emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly
enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics
are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role.
Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost
and skill requirements. Novel TB diagnostics are thus required that meet these requirements.
Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective
alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is
described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior
to current existing mycobacteriophage-based TB diagnostics. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van
mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n
aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige
tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika
behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika
weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word
benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans
nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm
instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB
diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat
mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir
vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende
rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese
toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde
TB-diagnostiese toetse.
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