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1
Al, Qurashi Yasir Mohammed A. "Molecular typing of adenoviruses." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.506268.
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Patel, Sushil. "Molecular typing and identification of mycobacteria." Thesis, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300635.
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3
Desai, Meeta. "Molecular epidemiological typing of Streptococcus pyogenes." Thesis, Open University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299021.
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4
Ngan, Chi-shing, and 顏志成. "Rapid typing of mycobacterium tuberculosis in respiratory specimens using PCR-based mycobacterial interspersed repetitive units (MIRU)typing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B4378334X.
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Matsheka, Maitshwarelo Ignatius. "Molecular identification and typing of campylobacter concisus." Doctoral thesis, University of Cape Town, 2000. http://hdl.handle.net/11427/2713.
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Hoff, Justin Wallace. "Molecular typing of wine yeasts : evaluation of typing techniques and establishment of a database." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/19942.
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Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: The yeast species, Saccharomyces cerevisiae and S. bayanus are well known for the key role
they play during alcoholic fermentation in both wine and beer industries. These yeasts are
available in pure active dried form and can be used to produce different wine styles and to
manage quality. There are more than 200 commercial wine yeast strains on the market and
include naturally isolated strains and hybrids. With all these commercial yeasts available, strain
authenticity is very important to the manufacturer of active dried wine yeasts (ADWY) because it
can prevent commercial losses and maintain market credibility. It is as important to the
winemaker as it may impact wine quality. Various traditional and molecular techniques have
been successfully applied to perform quality control of wine yeast strains.
The aims of this study were to evaluate electrophoretic karyotyping (CHEF) and PCRbased
methods to distinguish between Saccharomyces wine yeast strains and to establish a
database containing molecular profiles of commercial strains. CHEF karyotyping was chosen
because it is generally used in the wine industry to distinguish between wine yeast strains, but
can be time-consuming. Alternatively, PCR-based methods are considered to be reliable and
fast. These PCR methods included the evaluation of interdelta regions, multiplex-PCR of miniand
microsatellites, MET2 gene RFLP analysis and the use of several species-specific primers.
In this study, 62 commercial wine yeast strains, were randomly selected from various
manufacturers of ADWY, and two reference strains, S. bayanus CBS 380 and S. cerevisiae
CBS 1171, were evaluated. CHEF karyotyping could successfully differentiate between all 64
yeast strains. The two primer sets used for interdelta amplifications, delta1-2 and delta12-21,
yielded 59 and 62 profiles, respectively. Yeast strains considered to be similar or identical
according to interdelta amplification results, were resolved with CHEF karyotyping. CHEF
karyotyping was proven to be more accurate than interdelta amplifications in distinguishing
between commercial wine yeast strains. However, the results of interdelta amplifications were
very useful and less time-consuming. The multiplex-PCR of mini- and microsatellite primers only
succeeded in identifying a specific band within 55 of the 64 yeast strains including the S.
cerevisiae reference strain, a possible indication of species specificity. However, oenological
designation using MET2 gene RFLP analysis and species-specific primers indicated that all the
commercial strains in this study had a S. cerevisiae ancestry. Restriction analysis of the MET2
gene with EcoRI also successfully identified AWRI Fusion and Zymaflore X5 as hybrid yeast
strains. A wine yeast database was created and contains three libraries, i.e. CHEF karyotypes,
delta1-2 and delta12-21 electrophoretic profiles. The database was proven to be functional and
showed great accuracy in grouping and identifying test strains. The database has many
possible applications, but there is still some optimisation and refinement needed.<br>AFRIKAANSE OPSOMMING: Die Saccharomyces sensu stricto kompleks, is bekend vir die belangrike rol wat hierdie giste
speel tydens alkoholiese fermentasie in biede wyn en bier industrieë. Dit is om hierdie rede dat
kelders rein aktief gedroogte wyngis gebruik vir die produksie van spesifieke wynstyle, asook
kwaliteit. Daar is meer as 200 kommersiële wyngiste op die mark beskikbaar en dit sluit
natuurlike isolate en hibriede in. Daarom is gisras verifikasie baie belangrik vir die vervaardiger
van aktief gedroogde wyngiste asook die wynmaker om finansiële verliese te voorkom en mark
vertrouenswaardigheid te handhaaf. Verskeie tradisionele en molekulêre metodes word
suksesvol toegepas vir gehalte beheer van die gisrasse.
Die doel van hierdie studie was om elektroforetiese kariotipering (CHEF) en PKR
gebaseerde tegnieke se vermoë om tussen Saccharomyces wyngiste te onderskei, te
ondersoek. Ook deel van die doelwitte was om ‘n databasis te skep wat die verskillende
elektroforetiese profiele van die kommersiële gisrasse bevat. Tydens hierdie studie is 62
kommersiële gisrasse van verskeie vervaardigers ewekansig geselekteer. Saccharomyces
bayanus CBS 380 en S. cerevisiae CBS 1171 is as verwysingsrasse gebruik. Elektroforetiese
kariotipering (CHEF) is gekies omdat dit een van die mees algemeenste tegnieke is wat gebruik
word om tussen wyngiste te onderskei, maar dit word as tydrowend en arbeidsintensief beskou.
As ‘n alternatief is daar na PKR gebaseerde tegnieke gekyk. Hierdie tegnieke word as
betroubaar en vinnig beskou. Verskeie PKR gebaseerde tegnieke is ondersoek, naamlik PKR
van interdelta areas, multipleks-PKR van mini- en mikrosatelliete, MET2 geen RFLP analise en
die gebruik van spesie-spesifieke inleiers. Interdelta amplifikasies en mini- en makrosatelliet
inleiers is geselekteer as gevolg van hul vermoë om Saccharomyces wyngiste tot op spesie en
ras vlak te onderskei. Die MET2 geen en spesie-spesifieke inleiers is geselekteer om die
kommersiele wyngis as S. cerevisiae, S. bayanus of as hibriede te klassifiseer.
CHEF kariotipering kon tussen al 64 giste onderskeid tref. Die twee stelle inleiers wat vir
interdelta amplifikasie gebruik was, delta1-2 en delta12-21, het onderskeidelik 59 en 62 profiele
gelewer. Gis rasse wat identiese profiele met die delta inleiers gelewer het, kon egter met CHEF
kariotipering onderskei word. Die resultate het getoon dat CHEF kariotipering beter tussen die
kommersiële wyngiste kon onderskei as die interdelta amplifikasies, maar dat die interdelta
amplifikasies nogsteeds goeie onderskeiding toon en dat dit minder tydrowend is. Die
multipleks-PKR van mini- en mikrosatelliete kon slegs ‘n enkele band in 55 van die 64 giste uit
lig. ‘n Moontlike aanduiding van spesie spesifiekheid. Die oenologiese groepering volgens
MET2 geen analise en spesies-spesifieke inleiers dui aan dat al die kommersiele wyngiste wat
in hierdie studie gebruik is, moontlik van S. cerevisiae afkomstig is. Restriksie analise van die
MET2 geen met EcoRI het ook AWRI Fusion en Zymaflore X5 as hibriede geïdentifiseer. Die
CHEF kariotipes en interdelta elektroforetiese profiele is gebruik om ‘n databasis van die
kommersiële Saccharomyces wyngiste op te stel. Die databasis is funksioneel en het die toets rasse akkuraat geïdentifiseer en korrek gegroepeer. Die databasis moet egter nog verdere
optimisering en verfyning ondergaan.
7
Ng, Yi-ting, and 吳依婷. "Multilocus sequence typing for streptococcus agalactiae." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46699569.
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Ngan, Chi-shing. "Rapid typing of mycobacterium tuberculosis in respiratory specimens using PCR-based mycobacterial interspersed repetitive units (MIRU) typing." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B4378334X.
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9
Hielm, Sebastian. "Molecular detection, typing and epidemiology og Clostridium botulinum." Helsinki : University of Helsinki, 1999. http://ethesis.helsinki.fi/julkaisut/ela/elint/vk/hielm/.
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10
Hu, Honghua. "Molecular typing and evolution of Salmonella enterica serovar Typhimurium." Thesis, The University of Sydney, 2005. http://hdl.handle.net/2123/704.
Full textAbstract:
Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular 'phage' typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.
11
Hu, Honghua. "Molecular typing and evolution of Salmonella enterica serovar Typhimurium." University of Sydney. School of Molecular and Microbial Biosciences, 2005. http://hdl.handle.net/2123/704.
Full textAbstract:
Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular �phage� typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.
12
Robinson, Sally Rae. "Isolation, characterisation and molecular typing of feline mycoplasma species." Connect to thesis, 2009. http://repository.unimelb.edu.au/10187/5512.
Full textAbstract:
The exact role of mycoplasma in feline ocular and respiratory disease is not yet understood. The results of previous studies are contradictory in this regard. There is some evidence to suggest that M. felis has a pathogenic role in such diseases, but it is inconclusive.<br>The aim of this study was to investigate the prevalence and anatomical distribution of mycoplasmas in a population of shelter cats, to determine which species were present, and establish the association of their presence with ocular or respiratory disease.<br>The prevalence of mycoplasma in the 110 cats examined was 71.8%, as determined by in vitro culture. Mycoplasma was most commonly isolated from the pharynx, followed by the bronchus and conjunctiva. In infected cats, mycoplasmas were likely to be isolated from multiple anatomical sites.<br>The polymerase chain reaction (PCR) was used to amplify part of the 16S rRNA gene, and the mutation scanning technique non-isotopic single-strand conformation polymorphism (SSCP) was utilised to delineate mycoplasma isolates based on nucleotide sequence variation. PCR-SSCP proved to be a useful method to screen large numbers of samples for variation and to group them according to species.<br>The species of mycoplasma identified by nucleotide sequencing were M. felis and M. gateae. It was not determined whether it was possible to differentiate between M. gateae and M. arginini based on SSCP profile results with the target DNA region used due to their almost identical nucleotide sequence. This group of M. gateae/M. arginini served as a useful non-pathogenic comparison group to M. felis.<br>There was no statistically significant difference between M. felis and the M. gateae/M. arginini group with respect to prevalence or anatomic distribution. There was no evidence of any association of mycoplasma with disease linked to any of the anatomic locations studied.<br>Mycoplasmas were isolated from the lower respiratory tract in 42.7% of cats. The isolation of mycoplasmas from the lower respiratory tract of healthy cats has been reported once, but this is the first report of M. felis being isolated from this location in healthy cats. This finding indicates that the isolation of mycoplasmas from the lower respiratory tract is not sufficient evidence to implicate a role in respiratory disease.<br>Mycoplasmas were not significantly involved in ocular or respiratory disease in the population of cats studied. More likely, they are commensal organisms in the conjunctiva, pharynx and bronchus. Whether they are capable of playing an opportunistic role in disease, or what conditions may facilitate such a role remains to be determined.
13
Gracias, Kiev S. "Molecular typing of virulence genes in enterotoxigenic Bacillus cereus." Virtual Press, 2007. http://liblink.bsu.edu/uhtbin/catkey/1378147.
Full textAbstract:
Bacillus cereus causes emesis and/or diarrhea following ingestion of contaminated food due to the production of emetic toxins and enterotoxins. SYBR Green I is used as an intercalating dye and its florescence increases as a result of DNA amplification during real-time PCR. A second-derivative plot is obtained at the end of the PCR run, where amplicons are differentiated based on their DNA melting temperature (Tm). DNA was extracted from Tryptic Soy Broth (TSB) and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus at cell densities of 10',106,105,104,0,102, and 101 cfu/ml. In order to detect the multiple virulence determinants in pathogenic B. cereus, specific primers were used to target three enterotoxigenic genes (hblC, nheA, and hblA), followed by melt-curve analysis to confirm identity. Conditions used for this experiment allowed for the reproducible distinction of melt curves (characteristic Tm) for each amplicon (hblC = 74.5°C in TSB and 75°C in NFDM; nheA = 78°C; and hblA = 85.5°C in TSB and 84°C in NFDM) with an assay sensitivity of 106 CFU/ml in TSB and 10' CFU/ml in NFDM. B. cereus, nheA expression was examined in cells grown in TSB using transcript-specific, real-time nucleic acid sequence-based amplification (NASBA) with SYBR Green II. NASBA was applied to ascertain relative levels of nheA expression, when cells were subjected to subinhibitory levels of chloramphenicol as a stressor. B. cereus demonstrated consistently high levels of nheA expression at 15 hours when grown in TSB containing subinhibitory concentration (SIC) chloramphenicol (15.625 mg/ml). Relative levels of nheA expression differed in stressed B. cereus cells grown during the 30 hours incubation.<br>Department of Biology
14
Sharma, Naresh Kumar. "Development of novel molecular typing methods for Staphylococcus aureus." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361470.
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Abdullah, Saleh Mohammed S. "Blood group typing by molecular techniques : application to immunohaematology." Thesis, University of Aberdeen, 2004. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU486962.
Full textAbstract:
The initial aim was to establish conventional DNA typing technology for blood group typing using cell derived genomic DNA material from donors. Accordingly, blood samples were collected from 200 healthy, randomly selected, Aberdeen Regional blood donors. The DNA extracted from these samples was used to develop and validate PCR assays for RHD, RHDPsi, RHC/c, KEL1 and KEL2, Fya and Fyb and ABO blood group alleles. All samples were also phenotyped serologically for RH, KEL, Duffy and ABO blood groups. Complete concordance was observed between phenotype and genotype. The developed RHD, RHDPsi and Fya and Fyb genotyping assays were then applied to study the molecular basis of blood groups in a cohort of 170 native Saudi donors. This study demonstrated there was no significant difference between the Saudi and Scottish (Caucasian) populations with respect to the RHD or RHDPsi blood groups. However, a significant difference was recorded with respect to the molecular basis of the FY blood group in which 30.6% of the Saudi donors were typed as Fy/Fy. The Fyx allele was also observed in this cohort. Further study of the Scottish blood donor population was performed to define the molecular basis of Weak D in 58 SNBTS (Scottish National Blood Transfusion Services) blood donors. Weak D types 1 and 2 represented more than 95% of all Weak D types tested in this study. Of the rest, nine samples were typed as DVI type II, one sample typed as DVII, one sample typed as DVa type II and a new partial D was identified in one sample to be lodged with the Genbank DNA database. In the second part of the project, the overall aim was to develop fetal DNA typing from maternal plasma.
16
Zhao, Shuai. "Studies on molecular typing and pathogenicity of Xanthomonas oryzae." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20043/document.
Full textAbstract:
La bactériose vasculaire du riz (BLB) et bactériose non-vasculaire du riz (BLS), causées respectivement par Xanthomonas oryzae pv. oryzae (Xoo) et X. oryzae pv. oryzicola (Xoc), sont les deux plus importantes maladies bactériennes du riz. Ces maladies limitent le rendement de la production de riz dans les zones rizicoles en Asie et dans certaines régions d'Afrique. L'infection et la multiplication bactérienne dans les tissus de l'hôte dépendent souvent des facteurs de virulence de ces bactéries dont le type le système de sécrétion de type III (T3SS) et ses substrats. Dans cette thèse, nous avons identifié neuf effecteurs non-TAL (transcription Transcription activator-like) sécrétés par des effecteurs de type III de la souche chinoise 13751 de Xoo en utilisant le domaine d'induction HR de la protéine avirulente AvrBs1 comme gène reporter. Parmi eux, XopAE13751 a été expérimentalement confirmé pour la première fois comme étant un effecteur non-TAL. Ensuite, par l'analyse mutationnelle de ces gènes effecteurs identifiés dans Xoo, nous avons constaté que l'effecteur non-TAL XopR13751 était nécessaire pour la virulence de la souche chinoise de Xoo sur le riz hybride Teyou63. En parallèle, nous avons démontré que le gène rsmA (repressor of secondary metabolism) - comme le gène rsmAXoo de l'espèce chinoise Xoo 13751- régule positivement l'expression des gènes associés aux facteurs de virulence, tels que le système de sécrétion de type III, les enzymes extracellulaires et le DSF (diffusible signal factor). De plus, le gène effecteur non-TAL xopO s'est avéré être peu répandu chez les Xanthomonas puisqu'il est présent uniquement chez X. euvesicatoria (Xe) et Xoc mais est absent chez Xoo. En considérant les deux pathovars de X. oryzae, avec deux modes d'infection différents, xopO a été examiné comme un facteur de la spécificité du tissu par l'inactivation mutationnelle du gène dans Xoc et par l'expression du gène dans Xoo. Les résultats ont montré que xopO n'est pas la cause déterminante de la spécificité de tissu chez Xoc. Enfin, nous avons étudié les VNTRs (Variable Number of Tandem Repeats) comme outil de typage moléculaire rapide, fiable et rentable, pour améliorer le contrôle des épidémies et pour évaluer la structure de population des souches de Xoc. 28 loci candidats VNTR ont été prédits par le criblage de trois génomes de Xoc (souche philippine BLS256, souche chinoise GX01 et souche malienne MAI10). Des paires d'amorces pour l'amplification de PCR de chacun des 28 loci ont été conçues et testées à un pannel de 20 souches de Xoc provenant de l'Asie et de l'Afrique. Le séquençage des amplicons de PCR a confirmé 25 loci VNTR robustes et polymorphes communs entre les souches Xoc asiatiques et africaines. Un dendrogramme, construit à partir de la combinaison des 25 loci de VNTR (MLVA-25), a montré que la plupart des souches asiatiques sont clairement distinguables des souches africaines. Cependant, en accord avec de précédents rapports, une souche Malienne se distingue et semble être liée aux souches asiatiques, suggérant une introduction possible de souches sur le continent africain. Ce nouvel outil de typage basé sur les VNTR sera utile pour l'étude de structures de populations et pour la surveillance épidémiologique de Xoc<br>Bacterial leaf blight (BLB) and Bacterial leaf streak (BLS), caused respectively by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), are two most important bacterial diseases on rice, constraining severely the rice yield in the rice-growing areas in Asia and in parts of Africa. Successful infection and bacterial multiplication in host tissue often depend on virulence factors from these bacteria including type Ⅲ secretion system (T3SS) and its substrates. In this thesis, we identified nine type Ⅲ secreted non-TAL (Transcription activator-like) effectors of Xoo Chinese strain 13751 using HR-inducing domain of avirulence protein AvrBs1 as the reporter, among them, XopAE13751 was first found experimentally to be non-TAL effector. Subsequently, through mutational analysis of these identified effector genes in Xoo, we showed non-TAL effector XopR13751 was found to be required for full virulence of Xoo Chinese strain in hybrid rice Teyou63. In parallel, we demonstrated that rsmA (repressor of secondary metabolism)-like gene rsmAXoo of Xoo Chinese strain 13751 positively regulated the expression of genes associated with virulence factors such as type Ⅲ secretion system, extracellular enzymes and diffusible signal factor (DSF). Furthermore, non-TAL effector gene xopO was found to be narrowly distributed in Xanthomonas, which was only present in X. euvesicatoria (Xe) and Xoc, but not in Xoo. Based on the consideration of two X. oryzae pathovars carrying two different infection ways, xopO was tested in host and tissue specificity by analysis of mutational analysis of the gene in Xoc and expression of the gene in Xoo. The results showed that xopO of Xoc did not function as a determinant in host and tissue specificity. Finally, we explored Variable Number of Tandem Repeats (VNTRs) as a fast, reliable and cost-effective molecular typing tool, to better monitoring epidemics and assess the population structure of Xoc strains. 28 candidate VNTR loci were predicted by screening of three Xoc genome sequences (Philippine strain BLS256, Chinese strain GX01 and Malian strain MAI10). Primer pairs for PCR amplification of all 28 loci were designed and applied to a panel of 20 Xoc strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci which are shared among Asian and African strains of Xoc. A dendrogram was constructed from 25 VNTR loci-combinating data (MLVA-25), indicating that most Asian strains were clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali appeared to be related to Asian strains, pointing to a possible introduction of strains to the African continent. A detailed analysis of the evolutionary relationships among a larger set of Xoc strains from China will be presented, considering different spatial scales. In conclusion, a new VNTR-based tool useful for studies of population structures and epidemiological monitoring of Xoc was successfully established
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Hadi, Syed Sibte. "A molecular study of Pakistani populations using short tandem repeat markers." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341966.
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Kan, Man-yee Elsie. "Application of neisseria gonorrhoeae molecular typing techniques in forensic medicine." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31971854.
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Octavia, Sophie Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Molecular typing and evolutionary relationships of Salmonella enterica serovar Typhi." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/43115.
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The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.
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Kan, Man-yee Elsie, and 簡文意. "Application of neisseria gonorrhoeae molecular typing techniques in forensic medicine." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31971854.
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Thomas, Prasad [Verfasser]. "Genome sequencing and molecular typing of Clostridium chauvoei / Prasad Thomas." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1153769794/34.
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Sims, Catriona Margaret. "Molecular typing of bacteria from poultry and poultry processing environments." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319674.
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Anthony, Richard Michael. "Molecular typing methods applied to yeasts from the genus Malassezia." Thesis, King's College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338779.
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Magalhães, Bárbara Gomes. "Evaluation of a new molecular typing strategy of Pseudomonas aeruginosa." Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/13861.
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Mestrado em Microbiologia<br>Pseudomonas aeruginosa is the third leading cause of hospital acquired infection in intensive care unit (ICU) patients. This microorganism holds responsibility in a high number of nosocomial infections and their severity. Because it is ubiquitous in the environment and also constitutes the endogenous microbiota of hospitalized patients, there is a need to use powerful molecular typing methods to establish clonal relationships between individual isolates. Double Locus Sequence Typing (DLST) has recently been used in the analysis of P. aeruginosa strains relatedness, proving to be efficient, easy, and also reducing the working time and costs of analysis. Another typing technique called Double Digest Sequence Label (DDSL) had also been reported in the molecular study of this microorganism. A higher discriminatory power makes DDSL a putative typing complement to resolve DLST clusters in specific situations.
From 2010 to 2012, an increase in P. aeruginosa infections incidence was observed in the ICUs of the Lausanne University Hospital, Switzerland. During this period, 689 isolates were retrieved from 254 patients. All isolates were analyzed with DLST and grouped in 46 DLST clusters, from which 4 clusters were further investigated in this study (cluster 1_18, 1_21, 6_7 and 28_77). These 4 clusters were retrospectively typed with the DDSL method to verify if an improved discrimination of isolates could be achieved. To do so, a first DDSL optimization step was performed, which resulted in good quality fingerprinting profiles. However, a quantitative analysis of the results using BioNumerics software was not possible. Visual comparison of DDSL fingerprinting patterns within each cluster allowed the formation of different DDSL types, but not the determination of bands differences between them.
Epidemiological data showed that contamination of humid environments probably played an important role in the dissemination of P. aeruginosa strains in this outbreak. Comparison of epidemiological and molecular information showed that most of undistinguishable DDSL types were epidemiologically linked, leading to the assumption that patient-to-patient transmission should be highly suspected, as seen for cluster 1_18. Nevertheless, strain evolution should be considered when studying a long period outbreak.
In conclusion, this new typing strategy of P. aeruginosa allowed the acquisition of a general picture about this outbreak’s epidemiology. Nevertheless, the DDSL is a technically complex, time consuming and subjective technique, not efficient to be use for epidemiological investigation purposes.<br>Pseudomonas aeruginosa é a terceira causa de infeção adquirida em hospitais, em pacientes hospitalizados em unidades de cuidado intensivo (UCIs). Este microrganismo é responsável por um elevado número de doenças nosocomiais, e pelo sua gravidade. Uma vez que é ubíquo no ambiente e também constitiu a microbita endógena de pacientes hospitalizados, existe a necessidade de utilizar métodos de tipagem molecular eficientes no estabelecimento de relações clonais entre isolados. Double Locus Sequence Typing (DLST) tem sido usado recentemente na análise de relações clonais entre estirpes de P. aeruginosa, provando ser eficaz, fácil, e reduzindo também o tempo de manipulação e custos de análise. Outra técnica de tipagem chamada Double Digest Sequence Label (DDSL) foi também descrita no estudo molecular deste microrganismo. Um elevado poder discriminatório torna DDSL num complemento putativo à tipagem para resolver clusters de DLST em situações específicas.
De 2010 a 2012 observou-se um aumento da incidência de infeções por P. aeruginosa nas UCIs do Hospital Universitário de Lausana, na Suíça. Durante este período, 689 isolados foram recolhidos de 254 pacientes. Todos os isolados foram analisados com DLST e agrupados em 46 DLST clusters, dos quais 4 clusters foram posteriormente investigados neste estudo (cluster 1_18, 1_21, 6_7 e 28_77). Estes 4 clusters foram retrospectivamente tipados com o método DDSL para verificar se se poderia alcançar uma melhor discriminação dos isolados. Para isso, um primeiro passo de optimização de DDSL foi realizado, o qual resultou em perfis de fingerprinting de boa qualidade. Contudo, a análise quantitativa dos resultados usando o software BioNumerics não foi possível. A comparação visual dos perfis de fingerprinting de DDSL para cada cluster permitiu a formação de diferentes tipos de DDSL, mas não a determinação de bandas diferentes entre os mesmos.
Os dados epidemiológicos mostraram que a contaminação de ambientes húmidos provavelmente desempenhou um papel importante na disseminação de estirpes de P. aeruginosa neste surto. Comparação de informação epidemiológica e molecular mostrou que a maioria dos tipos de DDSL não distinguíveis estavam epidemiologicamente ligados, levando à suposição de que a transmissão paciente-para-paciente deveria ser altamente considerada, como visto para o cluster 1_18. No entanto, a evolução da estirpe deve ser considerada aquando do estudo de um surto de longa duração.
Concluindo, esta nova estratégia de tipagem de P. aeruginosa permitiu obter uma imagem geral acerca da epidemiologia deste surto. Todavia, DDSL é um método tecnicamente complexo, demorado e subjectivo, não eficiente para ser usado para fins de investigação epidemiológica.
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Ip, Ka-fai, and 葉嘉輝. "Molecular epidemiological study of mycobacterium tuberculosis using IS6110-RFLP and MIRU typing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45010092.
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Klint, Markus. "Chlamydia trachomatis: Development of molecular typing methods and applications in epidemiology." Doctoral thesis, Uppsala universitet, Klinisk bakteriologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-108930.
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A general aim was to combine molecular typing methods with clinical background information to increase epidemiological knowledge about Chlamydia trachomatis infections. An outbreak of Lymfogranuloma venereum (LGV), caused by a more invasive variant of C. trachomatis, was reported from the Netherlands in 2003 among men who have sex with men (MSM). All Chlamydia positive specimens from a venereal disease clinic for MSM in Stockholm during one year were genotyped. No spread of LGV was found, apart from three symptomatic cases. The same ompA genotypes were found among MSM in Melbourne, but the genotype distribution was different compared to findings among the heterosexual population in Sweden. The standard method for genotyping of Chlamydia is ompA-sequencing, but it has low resolution because one genotype predominates. A multilocus sequence typing (MLST) system based on five targets was developed. In a sample of 47 specimens, 32 variants were found with MLST, but only 12 variants with ompA-sequencing. The polymorphisms in the hctB gene, one MLST target, are caused by an element of 108 bp that is present in two to four repetitions and in different variants. Although the DNA-binding function of Hc2 that is encoded by hctB has been studied, our findings of a considerable size variation show that new studies are needed. In 2006, specimens with a 377 bp deletion in the cryptic plasmid covering the target region for diagnostic test systems from Abbott and Roche were discovered in Sweden. Applying MLST to these specimens indicated that there was a single clone, denoted nvCT. The proportion of nvCT in all detected Chlamydia cases was higher (20% to 65%) in counties using Abbott/Roche compared to counties using the BectonDickinson test system (7% to 20%). The proportions of nvCT converge in counties with high or low levels when detection systems were adjusted to detect nvCT.
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de, Jong Anton. "Detection and molecular typing of Cryptosporidium in South African wastewater plants." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-338855.
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Cryptosporidium is a protozoan parasite infecting the intestines of its hosts, leading to acute diarrheal disease. Out of 26 recognized species, 14 are known to infect humans. Of most importance, from a human perspective are Cryptosporidium parvum and Cryptosporidium hominis, of which the former is known to have zoonotic potential. Globally, cryptosporidiosis affect people with lowered immune status particularly hard; among children under five it is the most important parasitic cause of gastroenteritis. In the region of KwaZulu-Natal, on the east coast of South Africa, Cryptosporidium is considered endemic. Drinking water is frequently collected from river systems and as Cryptosporidium spp. can be transmitted via contaminated water, this may be one source of infection. Research on the species distribution is important for outbreak investigations and prevention efforts. In water and wastewater such speciation is commonly performed using immunomagnetic separation, an antibody dependent method. There is however a suspicion that these antibodies have less affinity to some species and hence contorts the detected species distribution. An alternative approach is therefore of interest. In the present study, Cryptosporidium diversity in wastewater collected from four different wastewater treatment plants in KwaZulu-Natal, is evaluated with an optimized antibody-free workflow and a single cell platform. It was shown that the workflow is suitable for complex samples, such as wastewater. Furthermore, diversity was assessed with amplicon sequencing, revealing four different species and genotypes. Further modifications of the methods used could benefit the field of Cryptosporidium research, along with improving global health and preventing disease outbreaks.
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Owen, Jonathan Paul. "Molecular strain typing and environmental persistence of ruminant transmissible spongiform encephalopathies." Thesis, University of Leicester, 2012. http://hdl.handle.net/2381/10186.
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A novel thermolysin digestion method for the molecular strain-typing of ruminant TSEs has been developed which resulted in the clearance of cellular prion protein (PrPc) from healthy sheep or cattle brain homogenates, while digestion of scrapie or BSE infected samples resulted in the generation of the full-length disease-related isoform, PrPSc. Using antibodies against the amino terminal region of PrP it was possible to distinguish ovine scrapie from ovine BSE, permitting the potential identification of BSE infected sheep within the UK flock. The identification of a disease-associated, endogenously-generated fragment of PrP (termed C2) in scrapie infected sheep is also described. Absent in healthy brain homogenates, the neuroanatomical distribution of both C2 fragments and thermolysin-resistant PrPSc permitted the classification of four groups within a sample of natural scrapie cases which may be representative of scrapie strain heterogeneity in the UK. The retention of ovine scrapie and bovine BSE prion protein during incubation with six UK lowland soils in soil-packed columns over a period of 18 months was also monitored. Data was collected on the persistence, the vertical migration, and the distribution of PrPSc in soil component fractions. PrPSc bound to all six soils, with elution being dependent on soil type. The majority of PrPSc bound irreversibly, and no migration of PrPSc was observed. Differences in PrPSc persistence were dependent on soil type and prion strain. PrPSc persistence on a single soil at defined pH, temperature, or moisture contents indicated the initial deposition of PrPSc within the column was dependent on soil pH and persistence was inversely correlated to temperature. Reduced soil moisture content resulted in increased elution and persistence of PrPSc. Data suggests that the interaction of PrPSc with soil is a complex phenomenon dependent not only upon soil type and TSE strain but also environmental factors such as soil temperature, pH, and moisture content.
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Baquar, Namoos. "Molecular epidemiological typing of Salmonella with the DNA insertion sequence IS200." Thesis, Open University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262529.
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Sommerhalter, Kristin McCarthy. "Use of molecular typing methods in characterizing MRSA infections in Ohio." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1440090373.
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Schulte, Kathleen Q. "Mutagenized HLA DNA Constructs: Tools for Validating Molecular HLA Typing Methodologies." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc500888/.
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This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.
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Abreu, Juliana Aizawa Porto de. "Estudo da diversidade molecular de linhagens de Staphylococcus aureus de isolados de mastite bovina." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-17042017-172505/.
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A mastite bovina é considerada a enfermidade de maior impacto na pecuária leiteira mundial. Apesar da adoção de práticas de manejo para minimizar a ocorrência desta, os resultados não são satisfatórios para o controle de Staphylococcus aureus, o principal agente causador. Além da importância veterinária, possui implicações na saúde humana devido ao uso extensivo de antibióticos no tratamento e controle da doença e devido ao potencial de disseminação zoonótica de S. aureus. A principal preocupação em relação à resistência aos antimicrobianos é referente à meticilina, mediada pelo gene mecA. Estudos moleculares são importantes para o conhecimento da diversidade genética dos agentes causadores de mastite. A tipagem por sequenciamento de um único locus, do gene da região X da proteína A de S. aureus (spa Typing), tem sido utilizada com sucesso para investigar estrutura populacional, sendo adequada para estudos epidemiológicos. O presente projeto teve como objetivo pesquisar o perfil de resistência aos antimicrobianos e a presença de genes de resistência à meticilina e de S. aureus isolados de mastite bovina, além de compreender a diversidade genética, identificar possíveis novos perfis e conhecer a distribuição destes isolados. Os resultados encontrados foram a ausência de resistência à meticilina no antibiograma, confirmada pela ausência do gene mecA, e a baixa prevalência de multirresistência aos antimicrobianos testados. Apesar de mais de 58,74% dos isolados (242/412) serem resistentes a pelo menos um antibiótico, 53,64% (221/412) correspondem a resistência à ampicilina. Foram determinados 44 spa tipos diferentes, sendo os três mais frequentes t605 (57,45%), t002 (8,4%) e t127 (8,13%). Não houve associação entre a distribuição dos spa tipos e as variáveis analisadas de ano, estado, município e fazenda, e perfil de resitência aos antibicrobianos. Este é o primeiro estudo que utiliza o spa typing para avaliar uma coleção temporalmente diversa e, portanto, representativa da região estudada ao longo de 22 anos. Logo, espera-se que a descrição dos tipos de S. aureus obtidos contribua para a compreensão dos aspectos epidemiológicos envolvidos na transmissão do patógeno e do papel dos hospedeiros como reservatórios para linhagens resistentes. Tal compreensão é imprescindível para a prevenção, controle e tratamento da mastite bovina.<br>Bovine mastitis is considered the disease of greatest impact in global dairy industry. Despite the adoption of management practices to minimize its occurrence, the results are not satisfactory for the control of Staphylococcus aureus, the major causative agent. Besides veterinary importance, there are also implications for human health due to the extensive use of antibiotics in treatment and control of the disease and due to the potential for zoonotic spread of S. aureus. The main concern regarding antimicrobial resistance is methicillin resistance mediated by the mecA gene. Molecular epidemiology studies are important for the understanding of the genetic diversity of the causative agents of mastitis. Typing by sequencing of a single locus of the X region gene of the S. aureus protein A (spa Typing) has been successfully used to investigate population structure. This project aims to investigate the antimicrobial susceptibility profile and the presence of methicillin resistance genes in S. aureus isolated from bovine mastitis, in addition to understanding the genetic diversity, identifying possible new profiles and knowing the distribution of these isolates. The results obtained to the present were the absence of methicillin resistance in the antibiogram, confirmed by the absence of mecA gene, and the low prevalence of multidrug resistance to antimicrobials. Even though resistance to at least one antibiotic was present in more than 58,74% of the isolates (242/412), ampicillin resistance corresponds to 53.64% (221/412). Different spa types were determined, the three most frequent being t605 (57.45%), t002 (8.4%) and t127 (8.13%). There was no association between the spa type distribution and the variables of year, state, municipality, farm, and antibiotic resistance profile analyzed. This is the first study that uses spa Typing to evaluate a collection as temporarily diverse and representative of the region studied in 22 years. Therefore, it is expected that the results of this study, by allowing comparison of the profile of S. aureus from different sources (animals and property), will contribute to the understanding of the epidemiological aspects involved in the transmission of the pathogen and the role of hosts as reservoirs for pathogenic strains. Such understanding is essential for the prevention, control and treatment of bovine mastitis
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Ip, Ka-fai. "Molecular epidemiological study of mycobacterium tuberculosis using IS6110-RFLP and MIRU typing /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31495473.
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Cebeci, Aydin Aysun. "Molecular Identification And Typing Of Lactobacillus Delbrueckii Subspecies Bulgaricus And Streptococcus Thermophilus." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12609333/index.pdf.
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Lactic acid bacteria are associated with preservation of foods, including milk, meat and vegetables. Yoghurt is produced by the cooperative action of two starter bacteria<br>S. thermophilus and L. delbrueckii subsp. bulgaricus. In this study, identification and typing of yoghurt starter bacteria were aimed. Traditional home made yoghurts were collected from different areas of Turkey, identification of those isolates at species and subspecies level and typing at strain level were achieved using PCR based methods.
In our study, identification of yogurt starter bacteria was studied using species specific primers and ARDRA. These methods were inefficient in identification of yoghurt starter bacteria, at species and subspecies level. Consequently, a reliable and quick method for accurate identification of yoghurt starter bacteria was developed. The new method focuses on amplification of methionine biosynthesis genes, for selective identification of yoghurt starter bacteria together with some cheese starters. Further discrimination by ARDRA enabled differentiation of yoghurt starter bacteria from cheese starters. Confirmation of the proposed method has been accomplished by partial sequencing of the 16S rRNA gene.
After correct identification of starter bacteria had been achieved, the strains were typed at strain level using RAPD-PCR and MLST. RAPD-PCR with primer 1254 resulted better fingerprints, compared to primer M13 at strain level. Comparisons of the two typing methods showed that RAPD-PCR revealed strain diversity better than MLST, however MLST was a more robust and reliable method and resulted in clustering of the strains depending on the isolation source.
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Miah, Aklak. "Characterisation and molecular typing of clinical and environmental isolates of Vibrio parahaemolyticus." Thesis, University of Plymouth, 2009. http://hdl.handle.net/10026.1/1977.
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Vibrio parahaemolyticus is a natural inhabitant of coastal waters worldwide and is the leading cause of seafood-borne gastroenteritis. This study reports on the use of several molecular characterisation methods to screen clinical and environmental isolates of V parahaemolyticus to assess whether such techniques can be used to distinguish pathogenic isolates reliably. In a total of 86 isolates mainly of V parahaemolyticus but also including V cholerae, V vulnificus and several other species, serotypes of the more virulent clonal group 03:K6 were identified, but otherwise there appeared no association with serotype and phenotype. The tdh and trh genes encoding haemolysins that are typically associated with virulent isolates were found in a significantly large number of isolates; however, poor concordance between haemolytic activity and the presence of the gene tdh was found. In an effort to establish more accurate relationships amongst clinical and environmental isolates of V parahaemolyticus, four molecular typing systems were employed; namely pulsed-field gel electrophoresis (PFGE), intergenic transcribed spacer (ITS) analysis, tDNA intergenic length polymorphisms (tDNA-ILPs) and randomly amplified polymorphic DNA (RAPD). Typing patterns and clustering analysis using these methods differentiated V parahaemolyticus from other marine species as well as at the subspecies level. PFGE with NotI was shown to be the most discriminative but suffered from not being universally applicable. Both ITS and tDNA-ILP methods were sufficiently discriminatory with discrimination indices (DI) of between 0.568 and 0.724, depending on the primers employed. The discriminatory ability of RAPD was also affected by the primers used (DI= 0.959 - 0.965) but closely matched that of PFGE (DI = 0.976). Additionally, both RAPD methods were able to distinguish putative markers for the pandemic clonal group. Typing systems appeared largely stable in duplicate and triplicate analyses with multiple primer pairs with some obvious variability in the reproduction of faint amplicons. All methods except PFGE were simple to execute but none of the methods could distinguish V parahaemolyticus into obvious lineages based on the clinical or environmental source. With the recent implication of a type Ill secretion system {TTSS) involved in the pathogenicity of V parahaemolyticus, a multiplex PCR system using PCR primers that spanned both TTSSl and TTSS2 regions was developed. Dot-blot analysis confirmed TTSS2 genes in at least 30% of environmental isolates. Nucleotide sequence analysis revealed l00% sequence homology in three loci of TTSS2 putative structural genes. In comparison, a total of 34 single nucleotide polymorphisms (SNP) were identified in three TTSS1 regions. In two of the regions, the SNPs were synonymous, whereas a non-synonymous substitution in the structural gene vcrDI resulted in valine replacement with isoleucine. In addition, nucleotide deletions in TTSS1 with resultant frameshift mutations were identified. The finding that significant numbers of environmental isolates also possess TTSS2 genes is contrary to currently held opinion that TTSS2 is only present in clinical isolates. It is hypothesed that the high incidences of V parahaemolyticus infections may be related to active TTSS2 genes, whereas a high degree of polymorphisms in TTSS1 suggest it may be inactive.
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Fonseka, Thithalapitige Sunil Gamini. "Molecular typing of food poisoning bacteria isolated from farm shrimp and poultry." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316950.
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Herrmann, Daland C. "Molecular typing of Toxoplasma gondii isolates from cats and humans in Germany." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16606.
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Toxoplasma gondii weist eine weltweite Verbreitung auf und kann fast alle Wirbeltiere, vor allem Vögel und Menschen infizieren. Felide sind Endwirte von T. gondii, welche das infektiöse und umweltresistente Oozysten-Stadium ausscheiden können. T. gondii kann auch ohne sexuelle Vermehrung unterschiedlichste Zwischenwirte und Zwischenwirtspezies infizieren. Obwohl eine sexuelle Phase ein Teil des Lebenszyklus ist, werden rekombinierte Genotypen nur selten beobachtet. Dies ist ein Grund dafür, warum T. gondii eine klonale Populationsstruktur erkennen lässt. Während in Nordamerika und Europa drei klonale Genotypen (Typ I, II und III) dominieren, werden in Südamerika und Asien, neben den drei bekannten auch atypische und andere Genotypen beobachtet. Auch innerhalb der atypischen Genotypen lässt sich eine klonale Populationsstruktur erkennen. Die Linien I, II und III weisen Unterschiede in ihrer Virulenz für Mäuse auf. Typ-I-Stämme sind hochvirulent. Die Infektion mit nur einem Organismus führt bereits zum Tod. Klonale Typ-II- und Typ-III-Stämme sind avirulent für Mäuse. Nur Infektionen mit mehr als 103 Organismen führen zum Tod. In dieser Studie zeige ich, dass die Mehrzahl isolierter T. gondii-Oozysten dem Typ II zuzuordnen ist. Es wurde keine Typ-I-, dafür aber eine Typ-III-Infektion und vereinzelte Hinweise auf Mischinfektionen und nicht-kanonische T. gondii Genotypen beobachtet. Erstmalig kann gezeigt werden, dass aus einer Rekombination zwischen den Typen II und III genetisch unterschiedliche T. gondii in einer natürlich infizierten Katze entstanden sind. Die identifizierten nicht-kanonischen T. gondii Klone weisen unterschiedliche, meist hohe Virulenz im Mausmodell auf. Eine geringe Anzahl von T. gondii-DNA Proben von humanen Toxoplasmose-Fällen deutet auf eine Infektion mit T. gondii des Typs II hin. Ich zeige mit dieser Studie, dass sexuelle Rekombination von T. gondii in Deutschland möglich ist, und diese zur Entstehung von hochvirulenten T. gondii führen kann.<br>Toxoplasma gondii is a protozoan parasite that can infect almost all warm-blooded animals, including birds and humans. The definitive host is the cat which excretes the highly infectious and environmental resistant oocyst stage. Equally important for the spread of T. gondii is the transmission of T. gondii by ingestion of contaminated food (prey) between host species. Sexual recombination is a rare event observed in T. gondii. This is one of the reasons why a clonal population structure of T. gondii is observed. In Europe and North America the majority of genotypes (types) of T. gondii are members of only three clonal types, designated type I, type II and type III. In South America and Asia, T. gondii is shown to have an increased genetic diversity with a high prevalence of atypical genotypes. However, even within those atypical genotypes, a clonal population structure can be observed. More importantly, mouse virulence of types I, II and III differ markedly. While infection with T. gondii type I is always lethal in mice, infections with 104–106 parasites of type II or type III are needed to have the same effect in mice. This study shows that the majority of cats in Germany excrete T. gondii oocysts of type II. We have not observed any type I T. gondii, but show that type III and, importantly, mixed type infection as well as non-canonical T. gondii are present in Germany. For the first time we demonstrate that a sexual cross between T. gondii type II and type III in a single, naturally infected cat occurred in Germany resulting in excretion of many genetically different non-canonical T. gondii. Most of the identified non-canonical T. gondii show a high virulence in mice. The RFLP-typing analysis of a limited number of T. gondii-DNA isolated from human samples revealed only alleles of T. gondii type II. I show that genetic recombination of different T. gondii types in Germany can lead to a higher genetic diversity and generation of highly mouse-virulent T. gondii.
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Rettberg, Jill Walker. "The molecular characterisation and rapid detection of methicillin-resistant Staphylococcus aureus." Thesis, Manchester Metropolitan University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311205.
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Carvalho, Vanessa Rafaela de [UNESP]. "Determinação da diversidade bacteriana em culturas de caldo Macconkey de materiais clínicos de portadores de doença de Crohn e análise de propriedades de isolados de Escherichia coli identificados." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/143087.
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Previous issue date: 2016-08-09<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>Desequilíbrio na composição de espécies bacterianas (disbiose) do intestino é uma característica da doença de Crohn (DC), a mais grave das doenças inflamatórias intestinais (DII). Na disbiose da DII, há um aumento de certos grupos de bactérias, tais como as Proteobacteria, em alguns pacientes. Acredita-se que o aumento destas bactérias deve contribuir para as reações inflamatórias responsáveis pelas lesões observadas no intestino de portadores de DC e retocolite ulcerativa, ao estimular a produção de citocinas por células locais do sistema imune. Proteobacteria, um dos filos predominantes da microbiota intestinal compreende bacilos gram-negativos, dos quais Escherichia coli (E. coli) é o mais conhecido. Dada a importância de Proteobacteria como patógeno ou membro da microbiota intestinal residente humana, este trabalho teve dois objetivos: 1) Investigar a ocorrência de variação numérica na população de Proteobacteria na DC e 2) Caracterizar amostras de E. coli encontradas nesta população, em relação a propriedades de virulência. Visando o primeiro objetivo, DNA de culturas em caldo MacConkey de diferentes materiais clínicos (fezes e biópsias intestinais) de 8 controles e 9 pacientes com DC foram amplificados com primers para as regiões V6-V8 do gene que codifica a fração 16S do rRNA (16S V6-V8 rDNA) e, em seguida, sequências específicas (representando táxons bacterianos particulares) contidas nos amplicons foram separadas por temperature gradient gel electrophoresis (TGGE), definindo perfis de banda que refletem a diversidade bacteriana. O segundo objetivo compreendeu a tipagem dos isolados de E. coli destas culturas através da classificação em filogrupos (A, B1, B2 e D) da coleção de referência de E. coli (EcoR), a pesquisa de sorogrupos O25 e O83, determinação das propriedades de aderência e capacidade de produzir biofilme. Análise de 32 culturas de 9 portadores de DC e 24 culturas de 8 controles mostrou uma clara diferença no número de bandas nos perfis de TGGE dos amplicons de 16S V6-V8 rDNA. Amplicons de 8 culturas de 9 portadores de DC tinham no máximo 2 bandas, ao passo que o número de bandas em amplicons de controles tinham pelo menos 3 bandas. Uma das bandas dos casos em que havia duas bandas ou a banda única dos perfis de cultura de casos teve a mesma mobilidade do amplicon de E. coli, colocado no gel como referência da corrida. PCR para detecção de genes de O25 e O83 demonstraram que estes sorogrupos foram dominantes tanto na população de E. coli de portadores de DC como de controles (apenas um paciente controle deu resultado negativo na PCR para ambos sorogrupos). Também, a maioria das cepas de E. coli, tanto de casos como de controles pertenceu ao filogrupo B2. Resultados parciais de testes de adesão em células Hep-2 revelaram que todos os pacientes (controles ou não) apresentaram E. coli aderentes, expressando o padrão agregativo. Testes para avaliar a capacidade de formação de biofilme indicaram que isolados de E. coli das regiões proximais encontrados em controles produzem biofilme de forma mais intensa do que isolados equivalentes de portadores de DC. Como conclusão, os dados apresentados sugerem que a população intestinal de Proteobacteria em portadores de CD apresenta disbiose, associada com um aumento do número de E. coli, uma propriedade que foi determinada por TGGE e confirmada com seqüenciamento de amplicons da região V6, pela técnica Ion Torrent. Por fim, com base nos resultados de tipagem e caracterização bacteriana foi possível observar que amostras tanto de caso como de controles, não houve uma prevalência dos sorogrupos e com base nesse resultado podemos associar que ambos os grupos de estudos freqüentam de forma rotineira o mesmo ambiente hospitalar, podendo indicar um favorecimento na aquisição desses tipos bacterianos. Resultados semelhantes ocorreram com as análises dos sorogrupos, onde apenas o filogrupo D teve prevalência significativa em indivíduos controle. Em relação à produção de biofilme, as amostras encontradas na região proximal do intestino de pacientes controles produzem mais biofilmes do que amostras de portadores de DC da mesma região intestinal, indicando que a parede da mucosa intestinal serve como substrato pode influenciar na formação do biofilme.<br>Imbalance in intestinal bacterial composition (dysbiosis) is a hallmark of Crohn’s disease (CD), the most severe of inflammatory bowel diseases (IBD). A characteristic of IBD dysbiosis is the elevation of certain groups of bacteria such as Proteobacteria in some patients. It is believed that the augmented bacteria may contribute for the inflammatory reactions underlying the typical lesions observed in the gut of CD and ulcerative colitis patients, by stimulating the production of cytokines by local cells of the immune system. Proteobacteria, one the most prevalent bacterial phyla of the gut microbiota are Gram negative rods of which Escherichia coli (E. coli) is the most well-known member. Given the significance of Proteobacteria as pathogens or member of resident gut microbiota, this work had two purposes: 1) to investigate the occurrence of numerical variation in the population of these bacteria in CD and 2) Characterizing E. coli isolates found in this population, in search for some virulence associated properties. For the first purpose, DNA from MacConkey broth cultures of distinct clinical materials (stools and gut mucosal biopsies) of 9 CD and 8 control patients were amplified with primers for the V6 and V8 regions of the 16S ribosomal RNA gene (16S V6-V8 rDNA) and distinct gene sequences (representing particular bacterial taxa) within the amplicons were resolved by temperature gradient gel electrophoresis (TGGE), defining band profiles, which reflect the bacterial diversity. The second objective consisted in phylotyping (determining A, B1, B2 and D phylogroups), serotyping (search for O25 and O83 genes), analyzing the E. coli isolates of the cultures for adherence properties and competence to produce biofilm. Analysis of 32 cultures from the 9 CD patients and 24 cultures from 8 control subjects showed a clear case-control difference in the number of bands in the TGGE profiles of the 16S V6-V8 rDNA amplicons. Cultures from 8 of 9 CD patients had at most 2 bands, while the number of bands in the cultures from 7 of the 8 controls subjects had at least 3 bands. One of the two bands found in the cultures from CD patients had the same mobility as E. coli, put in the gelas reference for the run. PCR screening for O25 and O83 demonstrated that these serogroups were dominant among the E. coli population from both controls and CD patients (only one control subject was negative for each of these O groups). Also, most of E. coli isolates from both control and CD patients belonged to the B2 phylogroup. Partial results of bacterial adherence to Hep-2 cells revealed that all patients (controls and CD) had aggregative adherent E. coli isolates. Tests to determine the ability to produce biofilm indicated that isolates from the proximal, but not from distal region of the gut or the stools, found in controls were stronger biofilm formers than isolates from equivalent sites of CD patients. In conclusion, the data presented here reveal that intestinal population of Proteobacteria of CD patients show dysbiosis associated with the elevation of Escherichia coli and that this variation can be assessed by TGGE and confirmed by sequencing of amplicons V6 region, the technique Ion Torrent. Finally, on the basis of the bacterial typing and characterization results, was observed that samples of both the case and controls, there was a prevalence of serogroups, based on this result we can associate both study groups frequent routinely the same hospital environment and may indicate a favoring the acquisition of these bacterial types. Similar results occurred with the analysis of serogroups, where only the filogrupo D had a significant prevalence in control subjects. For biofilm production, the samples found in the proximal region of the control patients intestine produce more biofilm than DC bearing samples of the same intestinal region, indicating that the intestinal mucosa serves as a substrate may influence the formation of biofilms.<br>FAPESP: 2013/04475-3
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Carvalho, Vanessa Rafaela de. "Determinação da diversidade bacteriana em culturas de caldo Macconkey de materiais clínicos de portadores de doença de Crohn e análise de propriedades de isolados de Escherichia coli identificados." Botucatu, 2016. http://hdl.handle.net/11449/143087.
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Orientador: Josias Rodrigues<br>Resumo: Desequilíbrio na composição de espécies bacterianas (disbiose) do intestino é uma característica da doença de Crohn (DC), a mais grave das doenças inflamatórias intestinais (DII). Na disbiose da DII, há um aumento de certos grupos de bactérias, tais como as Proteobacteria, em alguns pacientes. Acredita-se que o aumento destas bactérias deve contribuir para as reações inflamatórias responsáveis pelas lesões observadas no intestino de portadores de DC e retocolite ulcerativa, ao estimular a produção de citocinas por células locais do sistema imune. Proteobacteria, um dos filos predominantes da microbiota intestinal compreende bacilos gram-negativos, dos quais Escherichia coli (E. coli) é o mais conhecido. Dada a importância de Proteobacteria como patógeno ou membro da microbiota intestinal residente humana, este trabalho teve dois objetivos: 1) Investigar a ocorrência de variação numérica na população de Proteobacteria na DC e 2) Caracterizar amostras de E. coli encontradas nesta população, em relação a propriedades de virulência. Visando o primeiro objetivo, DNA de culturas em caldo MacConkey de diferentes materiais clínicos (fezes e biópsias intestinais) de 8 controles e 9 pacientes com DC foram amplificados com primers para as regiões V6-V8 do gene que codifica a fração 16S do rRNA (16S V6-V8 rDNA) e, em seguida, sequências específicas (representando táxons bacterianos particulares) contidas nos amplicons foram separadas por temperature gradient gel electrophores... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Imbalance in intestinal bacterial composition (dysbiosis) is a hallmark of Crohn’s disease (CD), the most severe of inflammatory bowel diseases (IBD). A characteristic of IBD dysbiosis is the elevation of certain groups of bacteria such as Proteobacteria in some patients. It is believed that the augmented bacteria may contribute for the inflammatory reactions underlying the typical lesions observed in the gut of CD and ulcerative colitis patients, by stimulating the production of cytokines by local cells of the immune system. Proteobacteria, one the most prevalent bacterial phyla of the gut microbiota are Gram negative rods of which Escherichia coli (E. coli) is the most well-known member. Given the significance of Proteobacteria as pathogens or member of resident gut microbiota, this work had two purposes: 1) to investigate the occurrence of numerical variation in the population of these bacteria in CD and 2) Characterizing E. coli isolates found in this population, in search for some virulence associated properties. For the first purpose, DNA from MacConkey broth cultures of distinct clinical materials (stools and gut mucosal biopsies) of 9 CD and 8 control patients were amplified with primers for the V6 and V8 regions of the 16S ribosomal RNA gene (16S V6-V8 rDNA) and distinct gene sequences (representing particular bacterial taxa) within the amplicons were resolved by temperature gradient gel electrophoresis (TGGE), defining band profiles, which reflect the bact... (Complete abstract click electronic access below)<br>Mestre
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Scott, Allison. "Sensitivity and specificity of spoligotyping and MIRU-VNTR typing in tuberculosis molecular epidemiology." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81436.
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The standard genotyping technique in tuberculosis molecular epidemiology is IS6110 Restriction Fragment Length Polymorphism (RFLP). Several public health organizations intend to switch from IS6110 RFLP to spoligotyping and Mycobacterium Interspersed Repetitive Units - Variable Number Tandem Repeats (MIRU-VNTR); however, the relevant test characteristics have not been studied. Objective. Estimate the sensitivities and specificities of spoligotyping and MIRU-VNTR using IS6110 RFLP as the reference standard.<br>Population. Residents of the island of Montreal diagnosed with active tuberculosis between 1996-1998, who were culture positive for Mycobacterium tuberculosis and had ≥6 copies of IS6110 upon RFLP. Outcomes. Sensitivity, specificity, percent transmission.<br>Estimated sensitivities. spoligotyping 83% (95% CI 63%-95%), MIRU-VNTR 52% (31%-72%), MIRU-VNTR and spoligotyping used in combination (clustered = both identical) 50% (29%-71%). Estimated specificities. spoligotyping 40% (35%-46%), MIRU-VNTR 56% (51%-62%), combination 70% (65%-76%). Percent transmission increased from 4% (IS6110 identical RFLP) to 23% (combination), 33% (MIRU-VNTR), and 53% (spoligotyping). There was evidence of misclassification.<br>The poor test characteristics of spoligotyping and MIRU-VNTR typing suggest that these methods are not suitable for population-based molecular epidemiology studies.
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Webster, Carol Ann. "The use of molecular typing systems for studying the epidemiology of Acinetobacter infections." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388315.
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Fazaeli, Asghar. "Development of molecular markers for the typing and genetic analysis of Toxoplasma gondii." Thesis, University of Aberdeen, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367484.
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To develop robust and reproducible methods for molecular typing of <I>Toxoplasma </I>strains, the DNA regions of 5<I>S</I> rDNA, 28S-18S rDNA <I>IGS SAG2</I>, and <I>GRA6</I> loci were examined. The 5<I>S</I> sequences were identical among 24 different strains; sequencing of the <I>IGS</I> region showed a few polymorphisms (0.66%) distinguishing virulence types. The IGS PCR-RFLP methods were developed and used to examine 29 strains of different virulence types. Sequence analysis of the IGS 5'-end showed great diversity between <I>Neospora caninum </I>and <I>T. gondii. </I>The IGS-RFLPs also clearly distinguished between those two closely related species. Nucleotide sequencing of the <I>SAG2</I> locus (a surface antigen coding gene) showed 1.37% polymorphisms among 24 strains. Apart from a single nucleotide change at the 5'-flanking region, the type III and type I strains were identical. However, three new alleles of this locus were identified in minor variants of the strains. Analysis of the coding region of the <I>GRA6</I> locus (a dense granule antigen coding gene) revealed a great degree of polymorphisms (3.24%) among 33 strains. Nine different alleles, representing the three current types and the minor variants of strains were characterised at this locus. A PCR-RFLP based on <I>GRA6</I> polymorphisms was developed which could distinguish the three major types of <I>T. gondii</I>. This marker proved to be a suitable tool for a population study of the <I>Toxoplasma </I>parasite. The predominance of non-synonymous nucleotide substitutions in <I>SAG2</I> and <I>GRA6</I> genes confirmed positive selection in these loci, suggesting they play an important role in the parasite virulence. Phylogenetic analysis based on the multi-locus sequence alignment showed the existence of more than three lineages in <I>Toxoplasma </I>populations.
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Hamdi, Cassandra. "Clostridium difficile : Rapid typing Clostridium difficile using MALDI-TOF MS analysis." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17659.
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Olvera, van der Stoep Alexandre. "Application of molecular techniques to the diagnosis and epidemiology of Haemophilus parasuis." Doctoral thesis, Universitat Autònoma de Barcelona, 2007. http://hdl.handle.net/10803/3573.
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Haemophilus parasuis es l'agent etiológic de la malaltia de Glässer's, però aquesta bactèria pot causar altres manifestacions clíniques, a més a més de poder ser aïllat del tracte respiratori superior de porcs sans. Els aïllaments de H. parasuis poden presentar diferents fenotips (per exemple diferent perfil de proteïnes, morfologia de colònia o bé producció de càpsula) i diferent capacitat patogènica. Les diferencies entre soques també s'han demostrat a nivell genètic. S'han emprat varis mètodes de tipat per classificar soques de camp d' H. parasuis, però totes presentaven problemes de resolució o implementació. Per resoldre aquestes limitacions es van avaluar diferents tècniques basades en seqüenciació d'ADN. Conseqüentment l'objectiu d'aquest estudi fou millorar el tipat d' H. parasuis i examinar l'associació entre grups de soques i aparició de malaltia. <br/>En el primer capítol d'aquest treball s'estudià l'ús d'una seqüència parcial del gen "heat shock protein 60 KDa" (hsp60) com a marcador epidemiológic en un esquema de "single locus sequence typing" (SLST). Es compararen els resultats obtinguts emprant patrons de "enterobacterial repetitive intergenic consensus" (ERIC)-PCR, seqüències parcials de hsp60 i 16S rARN de 103 soques d' H. parasuis i altres espècies relacionades. En el segon capítol d'aquest treball es va desenvolupar un esquema de "multilocus sequence typing" (MLST) fent servir seqüències parcial del gens "house-keeping" mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB. Onze soques de referència i 120 soques de camp van ser incloses en aquest darrer estudi. <br/>Els nostres resultats mostren que la hsp60 es un marcador fiable per estudis epidemiológics d' H. parasuis, i que l'anàlisi d'aquesta seqüència es una aproximació millor que els mètodes basats en patrons de bandes. Sorprenentment els gen 16S rARN mostrà prou variabilitat com per ser emprat en el tipatge de H. parasuis enlloc de només en la identificació a nivell d'espècie. A més a més, l'anàlisi de les seqüències de hsp60 i 16S rARN revelaren l'existència de un llinatge divergent de soques associades a l'aparició de malaltia. Ambdós estudis, un amb SLST i l'altre amb MLST, indicaren l'existència de transferències laterals de gens entre soques de H. parasuis i Actinobacillus invalidant l'ús d'aproximacions basades en un sol gen en l'anàlisi filogenètic d'aquesta espècie. L'anàlisi amb MLST mostrà l'existència de 6 "clusters". Quan s'examina l'origen clínic dels aïllaments es veié que un dels "clusters" estava estadísticament associat amb l'aïllament nasal mentre que un altre era associat amb l'aïllament de lesions. El darrer "cluster" incloïa les mateixes soques que el llinatge associat amb l'aparició de malaltia prèviament descrit amb els gens hsp60 i 16S rARN. Finalment, tot i que H. parasuis presenta una estructura de població lliurement recombinant es van trobar dues branques divergents en construir un arbre "neighbour-joining" amb les seqüències del MLST concatenades. Aquesta troballa dona suport als resultats obtinguts amb el gen 16S rARN indicant que H. parasuis sembla tenir una especiació críptica enlloc de una estructura de població panmíctica.<br>Haemophilus parasuis is the etiological agent of Glässer's disease, but this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates of H. parasuis differ in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classify H. parasuis field strains, but they showed resolution or implementation problems. To overcome these limitations, different DNA sequence based techniques were evaluated. Consequently, the final goal of this study was to improve H. parasuis typing and examine the association of groups of strains with disease outcome. <br/>In the first chapter of this work, a partial sequence from the heat shock protein 60 KDa (hsp60) gene was assessed as epidemiological marker in a single locus sequence typing (SLST). We compared enterobacterial repetitive intergenic consensus (ERIC)-PCR patterns, partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species. In the second chapter of this work, we developed a multilocus sequence typing (MLST) system using partial sequences of the house-keeping genes mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB. Eleven reference strains and 120 field strains were included in this latter study. <br/>Our results showed that hsp60 is a reliable marker for epidemiological studies in H. parasuis, and the analysis of its sequence is a better approach than fingerprinting methods. Surprisingly, the 16S rRNA gene showed enough variability to be used, not only for species identification, but also for typing. Furthermore, the analysis of the hsp60 and 16S rRNA sequences revealed the presence of a separated lineage of disease-associated strains. Both SLST and MLST studies indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains invalidating the use of single gene approaches in the phylogenetic analysis of these species. MLST analysis revealed the existence of 6 clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation, while another cluster was associated with isolation from lesions. The latter cluster was the same disease-associated cluster identified by hsp60 and 16S rRNA gene analysis. Finally, although a freely recombining population structure was reported, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. The latter, supports the results obtained by 16S rRNA gene sequencing and indicate that H. parasuis is more likely to have a cryptic speciation than a true panmictic population structure.
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Caddick, James M. "Molecular typing of hospital-acquired, community-acquired and multidrug-resistant methicillin-resistant staphylococcus aureus." Thesis, Aston University, 2005. http://publications.aston.ac.uk/11028/.
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Abdel-Glil, Mostafa Y. [Verfasser]. "In silico genome analysis and molecular typing of Clostridium perfringens / Mostafa Y. Abdel-Glil." Berlin : Freie Universität Berlin, 2020. http://d-nb.info/1204432899/34.
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