Academic literature on the topic 'PFGE typing'
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Journal articles on the topic "PFGE typing"
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Chen, Kuo-Wei, Hsiu-Jung Lo, Yu-Hui Lin, and Shu-Ying Li. "Comparison of four molecular typing methods to assess genetic relatedness of Candida albicans clinical isolates in Taiwan." Journal of Medical Microbiology 54, no. 3 (2005): 249–58. http://dx.doi.org/10.1099/jmm.0.45829-0.
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This report describes the investigation of the genetic profiles of 53 Candida albicans isolates collected from 18 hospitals in Taiwan using three PFGE-based typing methods (PFGE karyotyping, and PFGE of SfiI and BssHII restriction fragments) and one repetitive-sequence-PCR (rep-PCR) method. All four methods were able to identify clonal related isolates from the same patients. PFGE-BssHII exhibited the highest discriminatory power by discriminating 40 genotypes, followed by PFGE-SfiI (35 genotypes) and then by rep-PCR (31 genotypes), while PFGE karyotyping exhibited the lowest discriminatory power (19 genotypes). High discriminatory power can also be achieved by combining typing methods with different typing mechanisms, such as rep-PCR and PFGE-based typing methods. The results also showed that the genotype of each isolate was patient-specific and not associated with the source of the isolation, geographic origin or antifungal resistance.
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Doran, Geraldine, Dearbhaile Morris, Colette O'Hare, et al. "Cost-Effective Application of Pulsed-Field Gel Electrophoresis to Typing of Salmonella enterica Serovar Typhimurium." Applied and Environmental Microbiology 71, no. 12 (2005): 8236–40. http://dx.doi.org/10.1128/aem.71.12.8236-8240.2005.
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ABSTRACT Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.
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LOUIE, M., S. READ, L. LOUIE, et al. "Molecular typing methods to investigate transmission of Escherichia coli O157[ratio ]H7 from cattle to humans." Epidemiology and Infection 123, no. 1 (1999): 17–24. http://dx.doi.org/10.1017/s0950268899002551.
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The utility of phage typing, pulsed-field gel electrophoresis (PFGE), and plasmid profile analysis was compared, to differentiate between Canadian Escherichia coli O157[ratio ]H7 strains of human (n = 27) and cattle (n = 24) origin. The diversity indices for phage typing, plasmid analysis and PFGE were 0·85, 0·69 and 0·93, respectively. PFGE and phage typing were also applied to study the role of direct transmission of E. coli O157[ratio ]H7 from cattle to humans on isolates collected from two separate farm outbreaks. PFGE showed that more than one E. coli O157[ratio ]H7 strain with varying PFGE DNA subtype profiles, may be responsible for an outbreak, and that more than one E. coli O157[ratio ]H7 subtype may be circulating on a particular farm at any one time. To our knowledge, this is one of the first reports where PFGE typing was used to verify the direct transmission of E. coli O157[ratio ]H7 from cattle to humans.
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D'Agata, Erika M. C., Monique M. Gerrits, Yi-Wei Tang, M. Samore, and Johannes G. Kusters. "Comparison of Pulsed-Field Gel Electrophoresis and Amplified Fragment-Length Polymorphism for Epidemiological Investigations of Common Nosocomial Pathogens." Infection Control & Hospital Epidemiology 22, no. 9 (2001): 550–54. http://dx.doi.org/10.1086/501950.
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AbstractObjective:To compare molecular typing by amplified fragment-length polymorphism (AFLP) analysis with pulsed-field gel electrophoresis (PFGE) with respect to the ability to differentiate between epidemiologically related and unrelated isolates of common nosocomial pathogens recovered during a period of endemicity.Design:Retrospective laboratory analysis.Setting:Tertiary-care institution.Methods:17 isolates ofAcinetobacter baumannii,22 isolates ofPseudomonas aeruginosa,and 22 vancomycin-resistantEnterococcus faecium(VRE) were typed by both methods.Results:AFLP generated comparable results to PFGE forA baumanniiandP aeruginosaisolates; both methods identified epidemiologically related and unrelated isolates. However, strain typing of VRE isolates produced discordant results between the two methods. PFGE identified 10 different strain types and differentiated between all epidemiologically related and unrelated isolates. In contrast, AFLP generated only five different strain types, three of which contained both epidemiologically related and unrelated isolates.Conclusion:Molecular typing by AFLP is comparable to PFGE forA baumanniiandP aeruginosaisolates. For VRE isolates, however, PFGE remains the method of choice.
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Tang, Yi-Wei, Michael G. Waddington, Douglas H. Smith, et al. "Comparison of Protein A Gene Sequencing with Pulsed-Field Gel Electrophoresis and Epidemiologic Data for Molecular Typing of Methicillin-Resistant Staphylococcus aureus." Journal of Clinical Microbiology 38, no. 4 (2000): 1347–51. http://dx.doi.org/10.1128/jcm.38.4.1347-1351.2000.
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The epidemiologic relatedness of methicillin-resistantStaphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.
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Shaaly, Aishath, Marit Gjerde Tellevik, Nina Langeland, E. Arne Høiby, and Roland Jureen. "Comparison of serotyping, pulsed field gel electrophoresis and amplified fragment length polymorphism for typing of Streptococcus pneumoniae." Journal of Medical Microbiology 54, no. 5 (2005): 467–72. http://dx.doi.org/10.1099/jmm.0.45912-0.
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The aim of the present study was to compare serotyping, PFGE and AFLP for typing of Streptococcus pneumoniae with regard to discriminatory power, typeability and typing system concordance. Thirty-four isolates from cerobrospinal fluid and 34 time-matched blood culture isolates collected from in-patients at two hospitals in western Norway during the period from January 1994 to May 2002 were included in the study. The discriminatory powers of serotyping, PFGE and AFLP were 0.93, 0.99 and 0.95, respectively. The typeabilities for serotyping, PFGE and AFLP were 1, 1 and 0.99, respectively. A good concordance was shown between all the typing methods. Serotyping would most probably have a higher discriminatory power if further subtyping had been performed. PFGE was more discriminatory than AFLP, and AFLP grouped more-distantly related isolates together. The two typing methods thus provided different information, and therefore both could be useful adjuncts to serotyping for the characterization of S. pneumoniae.
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Boekhout, Teun, Marga Kamp, and Eveline Guého. "Molecular typing ofMalasseziaspecies with PFGE and RAPD." Medical Mycology 36, no. 6 (1998): 365–72. http://dx.doi.org/10.1080/02681219880000581.
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de Boer, Paulo, Birgitta Duim, Alan Rigter, Jan van der Plas, Wilma F. Jacobs-Reitsma, and Jaap A. Wagenaar. "Computer-Assisted Analysis and Epidemiological Value of Genotyping Methods for Campylobacter jejuni andCampylobacter coli." Journal of Clinical Microbiology 38, no. 5 (2000): 1940–46. http://dx.doi.org/10.1128/jcm.38.5.1940-1946.2000.
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For epidemiological tracing of the thermotolerantCampylobacter species C. jejuni and C. coli, reliable and highly discriminatory typing techniques are necessary. In this study the genotyping techniques of flagellin typing (flaA typing), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and amplified fragment length polymorphism (AFLP) fingerprinting were compared. The following aspects were compared: computer-assisted analysis, discriminatory power, and use for epidemiological typing of campylobacters. A set of 50 campylobacter poultry isolates from The Netherlands and neighboring countries was analyzed. Computer-assisted analysis made cluster analysis possible and eased the designation of different genotypes. AFLP fingerprinting was the most discriminatory technique, identifying 41 distinct genotypes, while PFGE identified 38 different types, flaA typing discriminated 31 different types, and ribotyping discriminated 26 different types. Furthermore, AFLP analysis was the most suitable method for computer-assisted data analysis. In some cases combining the results of AFLP fingerprinting, PFGE, and flaA typing increased our ability to differentiate strains that appeared genetically related. We conclude that AFLP is a highly discriminatory typing method and well suited for computer-assisted data analysis; however, for optimal typing of campylobacters, a combination of multiple typing methods is needed.
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Torpdahl, M., G. Sørensen, S. Ethelberg, G. Sandø, K. Gammelgard, and L. Jannok Porsbo. "A regional outbreak of S. Typhimurium in Denmark and identification of the source using MLVA typing." Eurosurveillance 11, no. 5 (2006): 5–6. http://dx.doi.org/10.2807/esm.11.05.00621-en.
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In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all S. Typhimurium isolates are currently sub-typed using phage typing, antibiogram typing, and pulsed-field gel electrophoresis (PFGE). However, the discriminatory ability of PFGE is not always high enough to discriminate within certain phage types, and it is not always possible to separate unrelated and related isolates. We have therefore applied multiple locus variable number of tandem repeats analysis (MLVA) for surveillance typing of S. Typhimurium since 2004. In May and June 2005, an outbreak with 26 cases of S. Typhimurium infection was identified by MLVA. The isolates were fully sensitive and had one of the most frequently occurring Danish phage types (DT12) and PFGE types. S. Typhimurium DT12 isolates from routine surveillance of animals and food were typed using MLVA and PFGE for comparison with the human isolates. The typing results revealed that an isolate from a pig herd and its corresponding slaughterhouse located in the same geographic region as the outbreak had the same PFGE and MLVA type as the human isolates. In contrast, all other DT12 isolates investigated, which had the same PFGE profile, had different MLVA types. The conclusion that the pig herd was the source of the human infections was supported by patient information, and pork from the herd stopped entering the market on 29 June. MLVA may contribute significantly to both surveillance and outbreak investigations of S. Typhimurium, as without MLVA typing this outbreak would not have been found nor its origin traced.
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Garaizar, Javier, Nuria L�pez-Molina, Idoia Laconcha, et al. "Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of Salmonella enterica Serovar Enteritidis." Applied and Environmental Microbiology 66, no. 12 (2000): 5273–81. http://dx.doi.org/10.1128/aem.66.12.5273-5281.2000.
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ABSTRACT Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D= 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI,BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance ofSalmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.
Dissertations / Theses on the topic "PFGE typing"
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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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Costa, Natália Silva da. "Análise do polimorfismo numérico de sequências repetitivas em múltiplos loci (MLVA) como instrumentos de avaliação da diversidade genética de Streptococcus pneumoniae do sorotipo 14." Universidade do Estado do Rio de Janeiro, 2012. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=6065.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>Streptococcus pneumoniae é um importante agente etiológico de infecções invasivas e não invasivas, incluindo meningite, pneumonia e otite média. A cápsula polissacarídica é o principal fator de virulência desse microrganismo, sendo também considerada um importante marcador em estudos epidemiológicos. Dentre os mais de 90 tipos capsulares conhecidos, o sorotipo 14 se destaca pela prevalência elevada em várias regiões, inclusive no Brasil. A avaliação da diversidade genética desse microrganismo também inclui a aplicação de métodos moleculares, como PFGE e MLST. Entretanto, essas metodologias são relativamente onerosas, consomem muito tempo e os resultados obtidos com a técnica de PFGE são de difícil comparação entre diferentes laboratórios. A técnica de análise do polimorfismo numérico de segmentos repetitivos em múltiplos loci [MLVA, do inglês Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis] se apresenta como uma alternativa, embora ainda necessite de padronização e avaliação mais ampla para a espécie em questão. No presente estudo, 60 amostras de Streptococcus pneumoniae pertencentes ao sorotipo 14, isoladas de diversas fontes clínicas, em diferentes locais e períodos de tempo, foram caracterizadas pelas técnicas de MLVA (baseada na análise de 18 loci distintos), MLST, PFGE e tipagem do gene pspA. O gene pspA2 predominou entre as amostras analisadas, seguido pelo gene pspA1. Os tipos de MLVA, perfis de PFGE, e STs encontrados apresentaram resultados, em geral, concordantes, indicando o elevado poder discriminatório da versão da técnica de MLVA empregada. Cinco complexos clonais (CC) de MLVA e cinco singletons puderam ser definidos. O CC de MLVA denominado de L7 foi o predominante, compreendendo 36,7% da amostragem estudada. O CC L7 mostrou-se relacionado com genes pspA da família 2, com o CC1 de MLST, com o CC Pen14-H de PFGE, e com a não susceptibilidade à penicilina, Entre os complexos clonais de MLST, o CC1 foi o prevalente e incluiu predominantemente o ST156, pertencente ao clone internacional Spain9V-3. O CC L3 e o singleton L17 de MLVA apresentaram-se associados ao CC de PFGE Eri14-A, a família 1 de PspA e ao CC2 de MLST, que por sua vez também estava relacionado com o clone internacional England14-9. O CC L15 de MLVA esteve associado ao CC de PFGE Pen14-A, ao gene pspA2, aos CC3 e CC4 de MLST e ao clone internacional do PMEN Tennessee14-18. A técnica de MLVA revelou-se significativamente mais discriminatória que as técnicas de PFGE e MLST, conforme exemplificado pela detecção de 21 perfis de MLVA, 13 perfis de PFGE e cinco STs, entre as 22 amostras pertencentes ao CC de MLVA L7. Uma versão de MLVA, compreendendo um painel com os oito loci de maior poder discriminatório, pôde ser proposta a partir da análise dos resultados obtidos. Estes aspectos, aliados ao menor tempo e custo de execução, indicam que a técnica de MLVA constitui uma alternativa importante e satisfatória para uso em estudos sobre a diversidade genética de S. pneumoniae.<br>Streptococcus pneumoniae is a major pathogen causing invasive and non-invasive diseases in humans, including meningitis, pneumonia and otitis media. The polysaccharide capsule of this microorganism is considered a major virulence factor and an important marker for epidemiological studies. More than 90 pneumococcal capsular serotypes are recognized, and serotype 14 is highly prevalent in many regions, including Brazil. Genotyping methods, such as PFGE and MLST, are essential to evaluate genetic diversity of this bacterium. However, these methods are expensive, time-consuming and results from different laboratories are difficult to compare. Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis (MLVA) appears as an alternative, despite the fact that standardization and wide evaluation for application to this species is still required. In the present study, a total of 60 S. pneumoniae isolates belonging to serotype 14, isolated from different sources, regions and periods of time, were analyzed by MLVA (based on the analysis of 18 distinct loci), MLST, PFGE and pspA typing methods. Gene pspA2 was the predominant, followed by pspA1. Overall, the results of PFGE, MLST and MLVA typing were congruent, and indicated the discriminatory power of the MLVA method used. Five clonal complexes (CC) and five singletons were identified by MLVA. CC L7 was the predominant MLVA CC, comprising 36.7% of all the isolates. L7 was associated with pspA2 gene and non-susceptibility to penicillin, and it was related to MLST CC1, and to PFGE Pen14-H. CC1 was the prevalent MLST CC and included mostly ST156 that belongs to international clone (IC) Spain9V-3. Another MLVA CC, named L3, and the singleton L17 were related to PFGE CC Eri14-A, MLST CC2, and IC England14-9. MLVA CC L15 was related to PFGE Pen14-A, MLST CC3 and CC4, and IC Tennessee14-18. MLVA was found to be more discriminatory than PFGE and MLST, as exemplified by detection of 21 MLVA types, 13 PFGE profiles and 5 STs among the 22 strains belonging to L7, the predominant MLVA CC. A modified version of the MLVA method, based on the analysis of 8 loci only, is proposed. This aspect, in conjunction with reducing time and costs, indicate that MLVA represents an important and satisfactory alternative method to evaluate the genetic diversity of S. pneumoniae.
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Patel, Nehal Jitendralal. "Comparison of Antibiotic Sensitivity Profiles, Molecular Typing Patterns, and Attribution of Salmonella Enterica Serotype Newport in the U.S., 2003-2006." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/iph_theses/12.
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Salmonella causes gastrointestinal illness in humans. The purpose of the study was to determine the relative contribution of different food commodities to sporadic cases of salmonellosis (attribution analysis) caused by Salmonella Newport (SN) using Pulsed-Field Gel Electrophoresis (PFGE) patterns and antimicrobial sensitivity (AST) data submitted by public health laboratories and regulatory agencies from 2003 to 2006. The genetic relationship between isolates from non-human (348) and human (10,848) sources was studied by two unique clustering methods: UPGMA and Ward. Results show poultry was the highest contributor of human SN infections, followed by tomatoes and beef. Beef was the largest contributing food commodity of multi-drug resistant (MDR)-AmpC infection patterns. Results from this pilot study show that PFGE and AST can be useful tools in performing attribution analysis at the national level and that SN MDR-AmpC patterns are decreasing and seem to be restricted to isolates from animal sources.
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Sauerwald, Claudia. "Die subklinische Staphylococcus aureus-Mastitis - Sanierung eines hessischen Milcherzeugerbetriebes und epidemiologische Untersuchungen mittels Staphylokokken-Protein A (spa)-Typisierung." Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-127925.
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In der vorliegenden Studie wurde die Sanierung einer durch S. aureus verursachten Eutergesundheitsstörung in einem hessischen Milchviehbestand mittels klassischer Sanierungsmaßnahmen über einen Zeitraum von 18 Monaten begleitet.
Durch konsequente Einhaltung der Sanierungsmaßnahmen nach dem Fünf-Punkte-Plan und die räumliche Trennung der Herde in eine S. aureus-positive und -negative Gruppe sank die S. aureus-Prävalenz im Betrieb innerhalb von 15 Monaten von 30% auf <1%. Nach 18 Monaten waren erstmalig keine Neuinfektionen mehr mit S. aureus zu verzeichnen.
Zusätzlich zu den im Abstand von drei Monaten durchgeführten Viertelanfangsgemelk (VAG)- Gesamtbestandsuntersuchungen wurden Umweltproben bakteriologisch auf das Vorhandensein von S. aureus untersucht. Diese Untersuchungen verliefen ausschließlich mit negativem Ergebnis.
Die im Untersuchungszeitraum asservierten 218 S. aureus-Isolate aus diesem Betrieb wurden genotypisch mittels PCR untersucht. Thermonuklease-Gen, Protein A-Gen (IgG-bindende Region) und Polymorphismen bei Protein A-Gen (Xr-Region) sowie Koagulase-Gen ermöglichten die Unterscheidung in sechs Typen.
Zusätzlich wurden 80 dieser Isolate mittels Pulsfeldgelelektrophorese (Pfge, Gold Standard) typisiert. Es konnten zwei Pfge-Typen gefunden werden: Pfge-Typ I mit insgesamt 10 Subtypen (bei 78 Isolaten) und Pfge-Typ II (bei zwei Isolaten). Der Pfge-Typ II wurde ausschließlich bei einem Zukaufstier nachgewiesen, das vor der Einstallung in diesen Betrieb nicht zytobakteriologisch untersucht worden war.
Die 12 unterschiedlichen Pfge-Typen bzw. -Subtypen wurden zusätzlich mittels Staphylokokken-Protein A- (spa)-Typisierung untersucht. Dabei zeigte sich, dass alle Subtypen des Pfge-Typs I dem spa-Typ t2067 zugeordnet werden konnten und der Pfge-Typ II dem spa-Typ t2112 entsprach.
92 weitere bovine S. aureus-Mastitisisolate wurden möglichst randomisiert über das Landesgebiet Hessens entnommen und mittels dieser Typisierungsmethode untersucht. Die Isolate stammten ebenfalls aus S. aureus-Problembetrieben und wurden aus VAG von (sub-) klinischen Mastitiden in Reinkultur isoliert. Insgesamt konnten 28 spa-Typen unterschieden werden. Durch den Algorithmus BURP wurden 57 dieser Isolate dem Clonal Complex CC543 zugeordnet und waren demnach genetisch eng miteinander verwandt. Der in dem Sanierungsbetrieb vorherrschende spa-Typ t2067 war dem CC543 nicht zuzuordnen.
Die Anwendung der spa-Typisierung in der Routinediagnostik und damit die Möglichkeit der laborübergreifenden, möglichst zentralisierten Datendokumentation der Ergebnisse könnten zukünftig die Identifizierung besonders häufig vorkommender und damit hochkontagiöser S. aureus-Stämme ermöglichen.
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Roberts, Jill Carolyne. "Characterization of Community-acquired Methicillin-resistant Staphylococcus aureus by Pulsed-field Gel Electrophoresis, Multilocus Sequence Typing, and Staphylococcal Protein A Sequencing: Establishing a Strain Typing Database." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001489.
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Wang, Qinning. "Erysipelothrix rhusiopathiae : epidemiology, virulence factors and neuraminidase studies." University of Western Australia. Microbiology Discipline Group, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0043.
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Erysipelothrix rhusiopathiae, a Gram-positive bacillus, has long been an important pathogen in veterinary medicine as well as a cause of serious disease in humans. Infections caused by this organism have economic impact on animal industries, causing erysipelas in swine and morbidities in other farmed animals. Human infections are commonly erysipeloid (skin cellulitis) and occasionally septicaemia or endocarditis. Little is known of the diagnosis, epidemiology and pathogenesis of such infections in Western Australia. The aims of this thesis were to establish new diagnostic techniques for the detection and recovery of E. rhusiopathiae, to describe the epidemiology of Erysipelothrix infection in Western Australia in humans and animals, and to characterize virulence-associated characteristics, especially focusing on the neuraminidase produced by the organism. A protocol using 48 h Brain Heart Infusion enrichment followed by subculture to selective agar containing antibiotics achieved the highest recovery rate of 37% in a seafood survey. Twentyone isolates of Erysipelothrix spp., of which 19 were identified as E. rhusiopathiae, were obtained. Two published PCR assays for differentiating E. rhusiopathiae and other Erysipelothrix species were evaluated and the best PCR detection rate achieved was 67% following selective enrichment. The PCR method was 50% more sensitive than the culture method. Epidemiological surveys using the above methods showed that E. rhusiopathiae infection is present in farmed animals in Western Australia. The PCR positive frequencies (3.3-3.7%) and isolate recovery rate (2.8-3.3%) in samples from pig and sheep abattoirs and carcass washings indicate a potential threat to the economy of the farmed animal industry as well as a public health concern with the occurrence of E. rhusiopathiae in meat for consumption. Positive PCR results (1.1%) from human skin swabs of patients with cellulitis and wounds may suggest the existence of Erysipelothrix colonization in the general population. Genetic relatedness of 92 isolates of Erysipelothrix species from various sources was analyzed and a total of 64 distinct PFGE patterns identified. Isolates were further classified into 20 clonal groups based on pattern similarities, and most E. rhusiopathiae were clustered into six groups. A few patterns of other Erysipelothrix species were clustered into separate groups from E. rhusiopathiae but shared greater than 70% similarity with E. rhusiopathiae. The genetic relatedness of colonial variants was well demonstrated using this method. PFGE typing promises to be a useful tool for epidemiological and taxonomic studies of Erysipelothrix. Several virulence-associated factors were characterized in 86 isolates of Erysipelothrix spp. A rapid and sensitive peanut lectin hemagglutination assay for neuraminidase was developed and the influence of media, incubation conditions and pH on the production of the enzyme was investigated. All 61 isolates of E. rhusiopathiae produced neuraminidase in cooked meat broth with titres between 1:10 and 1:320, with no significant difference in titre among isolates from different sources. The enzyme activity was not detected in non-pathogenic Erysipelothrix spp. Capsule was produced by 78.7% of isolates of E. rhusiopathiae but not by other species, while both hyaluronidase and haemolysin were produced by non-pathogenic Erysipelothrix spp. It was concluded that neuraminidase and capsule are most likely to be virulence factors of E. rhusiopathiae. The gene encoding neuraminidase was cloned from the type strain E. rhusiopathiae ATCC 19414. The cloned fragment was a functional partial nanH gene with a mol% G+C of 39.7. The predicted amino acid sequence displayed homology with many microbial neuraminidases and contained conserved sequences found in most bacterial neuraminidases. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial genomic DNA. A neuraminidasenegative mutant vector was constructed by insertional inactivation using a tetM cassette. This has provided starting material for developing a neuraminidase-deficient E. rhusiopathiae mutant, which will permit the study of the role of neuraminidase in pathogenesis. Based on the cloned sequence, a sensitive neuraminidase-specific nested PCR technique was designed and optimized. The specificity was tested in 61 isolates of E. rhusiopathiae, 25 Erysipelothrix species, and 62 other species of neuraminidaseproducing and non-producing bacteria. All isolates of E. rhusiopathiae were PCR positive and all other bacteria were negative; thus this PCR is a highly specific method suitable for application in clinical investigations of Erysipelothrix infection. In conclusion, the present study has contributed new knowledge of the biology of Erysipelothrix spp. and current occurrence of Erysipelothrix infections in Western Australia, as well as to the understanding of pathogenesis of E. rhusiopathiae. Development of several new cultural and molecular approaches in combination with other established techniques will facilitate future studies of the epidemiology, taxonomy and pathogenesis of this bacterial species.
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Hernandez, Charles Andrew. "Molecular Typing and Antimicrobial Resistance of Campylobacter Isolated During Commercial Broiler Production." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8643.
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Campylobacter jejuni is a commensal microorganism of the poultry
gastrointestinal tract. Broilers, layers, ducks, turkeys, and quails can be colonized by
Campylobacter without illness occurring. The vast majority of human Campylobacter
infections are recognized as being foodborne. For 2008, preliminary FoodNet data
showed that the Campylobacter incidence of infection, 12.68 per 100,000 of the U.S.
population, is the second highest, only behind Salmonella at 16.20 per 100,000. To
further understand Campylobacter’s role as a foodborne pathogen, analysis at the
molecular level is needed.
Microbial molecular typing allows for identification and differentiation of
bacterial strains beneath the species level. In this study, the “gold standard” method for
molecular subtyping, Pulsed Field Gel Electrophoresis (PFGE), along with Diversilab®
repetitive element Polymerase Chain Reaction (rep-PCR) and 16S-23S Internal Spacer
Region Denaturing Gradient Gel Electrophoresis (ISR DGGE) were used for the
molecular typing of Campylobacter jejuni isolates obtained during different stages of
commercial broiler production and processing. In addition, the C. jejuni isolates were tested for resistance to antimicrobials commonly used in both veterinary and human
medicine. Antimicrobial resistance testing was carried out using a broth dilution system.
The majority of recovered isolates came from post-harvest carcass rinsates. Carcass
rinses were obtained at post-evisceration, post-chill stages. All isolates (n = 46) were
identified by the Polymerase Chain Reaction as Campylobacter jejuni. Three genotypes
(n = 44, n = 1, n = 1) were identified by PFGE. The 46 rep-PCR products grouped into
seven clusters and two outliers. Clustering of rep-PCR products by sample source was
not observed. No relatedness trends were observed for isolates recovered from the same
source. The combination of PFGE and Diversilab rep-PCR methods provides highly
discriminatory molecular typing results.
These results provide practical epidemiological information that shows postevisceration
and post-chill stages are still important targets for intervention studies. The
very high occurrence of C. jejuni isolates exhibiting genotype A suggests it may
differentially express certain gene(s) that enable this strain to more favorably survive
under the different harsh environmental conditions encountered during production and
processing.
In addition, phenotypic testing revealed all of the isolates were not resistant to
the antimicrobials azithromycin, ciprofloxacin, erythromycin, gentamycin, tetracycline,
florfenicol, nalidixic acid, telithromycin, and clindamycin at any of the concentrations
tested. All the C. jejuni isolates exhibited an indistinguishable two-band 16S-23S ISR
DGGE profile. Overall, these C. jejuni commercial broiler pre- and post-harvest isolates
exhibited an extremely low degree of molecular and phenotypic variability.
8
Oosthuysen, Wilhelm Frederick. "Molecular characterisation of methicillin-resistant Staphylococcus aureus (MRSA) from South Africa." Thesis, 2008. http://hdl.handle.net/10539/4919.
Full textAbstract:
ABSTRACT
Few antibiotics are left that are effective against methicillin-resistant Staphylococcus
aureus (MRSA) and even strains resistant to these agents have been isolated. Previous
studies have identified five distinct MRSA clonotypes, which are present globally. No
comprehensive national study has previously been undertaken to investigate the MRSA
types in South Africa, and this study was aimed at elucidating the genotypic population
structure of South African MRSA isolates. SmaI digested genomic DNA, separated by
pulsed-field gel electrophoresis, was used to characterise 349 S. aureus isolates, obtained
from various state and private diagnostic laboratories. PFGE results were complemented
with those of spa typing and staphylococcal cassette chromosome mec (SCCmec) typing
results. Two-hundred-and-five different PFGE patterns were identified, which were
grouped into twenty-four clusters. Three were major lineages, containing more than 20%
of the isolates with a similarity cut-off of 70%. Only thirty-seven spa types were identified
(fourteen novel spa types), which clustered into six spa-Clonal Complexes after BURP
analysis. SCCmec types I-IV were identified, including variants of each type. Data
suggest that the Archaic clone (RSA05), oldest of the epidemic clones, represents one of
the major clones in South Africa. Strains that were part of this complex (n=98 (28.2%);
t064; SCCmec type I-pls) clustered together with strain E2125/ATCC BAA-38 (t051;
SCCmec type I). Another major complex, RSA16 (n=90 (25.7%); t012; SCCmec type
II/IIB) possessed a single-locus variant (SLV) spa type and the same or a SLV SCCmec
types as EMRSA-16 (t018; SCCmec type II). The third major complex, RSA03 (n=74
(21.2%); t037; SCCmec type III/IIIE), had similar spa and SCCmec types to control strainANS46 (t037; SCCmec type III). One MRSA and twelve MSSA isolates were also
identified as carrying genes for the toxin Panton-Valentine leukocidin, which was
confirmed by DNA nucleotide sequencing.
Book chapters on the topic "PFGE typing"
1
Ayling, R. D., L. E. Johnson, S. Evans, and D. G. Newell. "PCR/RFLP and PFGE Sub-Typing of Thermophilic Campylobacter Isolates from Poultry Epidemiological Investigations." In Campylobacters, Helicobacters, and Related Organisms. Springer US, 1996. http://dx.doi.org/10.1007/978-1-4757-9558-5_34.
Full text
2
Struelens, Marc J., Raf De Ryck, and Ariane Deplano. "Analysis of Microbial Genomic Macrorestriction Patterns by Pulsed-Field Gel Electrophoresis (PFGE) Typing." In New Approaches for the Generation and Analysis of Microbial Typing Data. Elsevier, 2001. http://dx.doi.org/10.1016/b978-044450740-2/50008-3.
Full text
3
Rebić, Velma, Mufida Aljičević, Sajra Vinčević-Smajlović, and Damir Rebić. "Distribution and Molecular Detection of Methicilin-Resistant Staphylococcus aureus." In Infectious Diseases and Sepsis [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.98655.
Full textAbstract:
Isolation of Staphylococcus aureus is quite common in both the general population and hospital environment. The heterogeneity of the disease and the unique ability of S. aureus to develop resistance to the most recently discovered antibacterial drugs points to its ability to adapt and survive in different conditions. CA-MRSA is different from hospital strains of MRSA by its epidemiological, phenotypic and genotypic characteristics. The emergence of MRSA in the community suggests the need for a new approach to managing the indications and the certification of staphylococcal infections, with special emphasis on the selection of empiric antibiotic therapy. In the study, we analised of MRSA from 4341 samples taken from patients from the general population of Sarajevo Canton in the six-month period of follow-up processed at the Public Health Institute of Sarajevo Canton. We determined the epidemiological characteristics of the isolated strains. Methicillin resistance was determined by phenotypic methods. The following molecular methods were used for the confirmation of methicillin resistance: determination of the mecA gene, PFGE profile, genetic type of MRSA being determined by spa typing, the distribution of SCCmec types being examined, and the detected gene for PVL. The study stresses the need for national monitoring of spreading of the existing epidemic strains, as well as the monitoring of emergence of new strains which would enable the inclusion of our country in the international network of monitoring bacterial resistance.