Detection of virulence genes and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis among raw vegetables isolates of Campylobacter jejuni

A total of 20 (n=20) raw vegetable isolates of Campylobacter jejuni were examined for their virulence genes and genetic diversity by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis. All raw vegetable isolates of C. jejuni were encoded cadF and ceuE genes. None of the C. jejuni isolates was cdtA positive. Whereas, 12 (12/20) isolates were positive for cdtB and 6 (6/20) were positive for cdtC. All the 20 isolates of C. jejuni were subtyped and they produced 18 ERIC-PCR profiles namely E1 to E18. Dendrogram performed from cluster analysis showed that the 20 isolates were grouped into 2 major clusters, cluster I and II indicating the high level of local geographical genetic variation. The detection of virulence genes among C. jejuni strains isolated from raw vegetables raised concern on the threat of campylobacteriosis

Orbiting Resonances and Bound States in Molecular Scattering

A family of orbiting resonances in molecular scattering is globally described by using a single pole moving in the complex angular momentum plane. The extrapolation of this pole at negative energies gives the location of the bound states. Then a single pole trajectory, that connects a rotational band of bound states and orbiting resonances, is obtained. These complex angular momentum singularities are derived through a geometrical theory of the orbiting. The downward crossing of the phase-shifts through pi/2, due to the repulsive region of the molecular potential, is estimated by using a simple hard-core model. Some remarks about the difference between diffracted rays and orbiting are also given.Comment: 18 pages, 3 figures, to appear in Physical Review

Restrição do 16S-23S DNAr intergênico para avaliação da diversidade de Azospirillum amazonense isolado de Brachiaria spp.

The aim of this work was to study the intra-specific diversity of Azospirillum amazonense isolates and to establish possible influences of different Brachiaria spp. and edaphoclimatic conditions. The characterization of the diversity among the isolates of A. amazonense studied was conducted using restriction analysis of the 16S-23S rDNA intergenic spacer region. The evaluated strains were separated in two groups, defined at 56% of similarity. Brachiaria spp. showed effects on strain diversity. Most part of the isolates from B. decumbens and B. brizantha are inserted in the first group, while B. humidicola isolates concentrate in the second group.O objetivo deste trabalho foi avaliar a diversidade intra-específica de isolados de Azospirillum amazonense e estabelecer a possível influência de diferentes espécies de Brachiaria ssp. e diferentes condições edafoclimáticas. A caracterização da diversidade desses isolados foi conduzida, utilizando-se a análise de restrição da região intergênica 16S-23S DNAr. As estirpes estudadas separaram-se em dois grupos, definidos a 56% de similaridade. As espécies de Brachiaria ssp. influenciaram a diversidade de estirpes. A maioria dos isolados oriundos de B. decumbens e B. brizantha está inserida no primeiro grupo, enquanto os oriundos de B. humidicola concentram-se no segundo grupo

Application of molecular techniques for identification and ennumeration of acetic acid bacteria

Application of molecular techniques for identification and enumeration of acetic acid bacteria:Los principales objetivos de la tesis son el desarrollo de técnicas de biología molecular rápidas y fiables para caracterizar bacterias acéticas.Las bacterias acéticas son las principales responsables del picado de los vinos y de la producción de vinagre. Sin embargo, existe un desconocimiento importante sobre su comportamiento y evolución. Las técnicas de enumeración y de identificación basadas en características fisico-químicas existentes son lentas y poco fiables mientras que las técnicas moleculares utilizadas son muy lentas y excesivamente caras y no son aplicables en un trabajo de rutina en el laboratorio ni adecuadas para una gran cantidad de muestras. En primer lugar se ha conseguido identificar a nivel de especie, mediante dos técnicas de biología molecular como son la PCR-RFLP del rDNA 16S y PCR-RFLP del rDNA 16S-23S ITS. Se lograron identificar todas las especies de bacterias acéticas existentes hasta el momento mediante la combinación de ambas técnicas. Estas técnicas se han utilizado también en estudios ecológicos.Se han desarrollado dos técnicas para la caracterización a nivel de cepa: ERIC- y REP-PCR. Estas técnicas han permitido la realización de estudios ecológicos en fermentaciones en distintas condiciones, permitiendo profundizar en el conocimiento de su comportamiento y desarrollo en fermentaciones vínicas. Se han desarrollado dos técnicas para la enumeración, real-time PCR y la detección nested-PCR respectivamente de bacterias acéticas. Estas técnicas presentan la ventaja de no tener que cultivar y por lo tanto de detectar la presencia de bacterias viables pero no cultivables, que han sido descritas dentro de las bacterias acéticas.Artículos en revistas internacionales:A González, N Hierro, M Poblet, N Rozès, A Mas, JM Guillamón: Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation Journal of Applied Microbiology, 96, 853-860, 2004A González, N Hierro, M Poblet, A Mas, JM Guillamón: Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production. International Journal of Food Microbiology (en prensa), 2005. A González, JM Guillamón, A Mas, M Poblet: Application of molecular methods for routine identification of acetic acid bacteria. International Journal of Systematic and Evolutionary Microbiology (enviado).A. González, Núria Hierro, Montserrat Poblet, Albert Mas and José Manuel Guillamón. Enumeration and detection of acetic acid bacteria by real-time PCR and nested-PCR. FEMS Microbial Letters. (enviado).JM Guillamón, Hierro, N., A González, A Mas: New PCR-based methods for yeast identification. Journal of Applied Microbiology, 97, 792-801; 2004.Núria Hierro, Ángel González, Albert Mas, José Manuel Guillamón.(2005) Diversity and evolution of non-Saccharomyces yeast populations during wine fermentations: Efecto of grape ripeness and cold maceration. FEMS yeast research. (enviado).Artículos en revistas nacionales:A Mas, A González, N Hierro, M Poblet, N Rozès, JM Guillamón: Bacterias acéticas durante la fermentación vínica: Interacciones con otros microorganismos. Tecnología del vino, 11: 27-30, 2003.A Mas, N Hierro, JM Guillamón, A González, M Poblet, N Rozès: Nuevas técnicas de identificación, detección y cuantificación de levaduras de importancia en Enología. Tecnología del vino, 15, 65-70, 2004. Y. Quintero, A González, M Poblet, JM Guillamón, A Mas: Importancia de las bacterias acéticas en el vino. Enorigen, 1, 6-12, 2004Congresos y capítulos:A. González, M. Poblet, N. Rozés, JM. Guillamón, A. Mas. Desarrollo de técnicas moleculares de análisis de bacterias acéticas. Valencia, España. Gienol, 2001.A. González, N. Hierro, M. Poblet, N. Rozés, A. Mas, JM. Guillamón. Development of molécular techniques for the análisis of acetic acid bacteria. París, Francia. Xth international congress of Bacteriology and applied microbiology. 2002.J.M. Guillamón, A. González, M. Poblet, A. Mas. Development of molecular techniques for the analysis of acetic acid bacteria in winemaking. En: Yeast-Bacteria Interactions. Lallemand Tecnical Meetings, 10. Lallemand, S.A., 45-49, 2002.N. Hierro, A. González, N. Rozès, A. Mas, J.M. Guillamón: Nuevos métodos de identificación de levaduras basados en PCR. VII Jornadas Grupos de Investigación Enológica. Logroño.195-197, 2003Gonzalez, A.; Hierro, N.; Chiva, R.; Poblet, M.; Rozès, N.; Mas, A.; Guillamón, JM.: Dinámica poblacional de las bacterias acéticas en fermentaciones de vino tinto. VII Jornadas Grupos de Investigación Enológica. Logroño. 204 - 206. 2003.A. González, N. Hierro, M. Poblet, N. Rozés, A. Mas, JM. Guillamón. Population dynamics of acetic acid bacteria during red wine fermentations. 1st FEMS congress of European microbiologists. Lubiana, Eslovenia, 2003.N. Hierro, A. González, A. Mas, JM Guillamón. New PCR-based methods for yeast identification. Ponencia. Budapest, Hungría. ISSY 2003J.M. Guillamón, A. González, N. Hierro, N. Rozès, A. Mas, M. Poblet: Técnicas de identificación de bacterias acéticas. En: Primeras Jornadas de I+D+i en la Elaboración de Vinagres de vino. URV-CeRTA. 9-16, 2003.A. González, N. Hierro, M. Poblet, A. Mas, N. Rozès, J.M. Guillamón Bacterias acéticas durante la fermentación vínica. En: Primeras Jornadas de I+D+i en la Elaboración de Vinagres de vino. URV-CeRTA. 25-30, 2003.JM Guillamón, A González, M Poblet, A Mas: I microorganismi dell'Aceto Balsamico. Modena. 2004A. González, N. Hierro, M. Poblet, JM. Guillamón, A. Mas. Quantification of acetic acid bacteria in wine and vinegar samples using rt-PCR. Badajoz, España. BioMicroWorld 2005.J.M. Guillamón, A. González, M. Poblet, A. Mas: Molecualr techniques of identification and quantification of acetic acid bacteria. Vinegars and Acetic Acid Bacteria International Symposium. Reggio Emilia, 2005.A. Mas, A. González, J.M. Guillamón, M. Poblet: Acetic acid bacteria population dynamics during wine fermentation. Vinegars and Acetic Acid Bacteria International Symposium. Reggio Emilia, 2005.F. Barja, A. González, M. Mesa, M. Macías, D. Cantero. Molecular and morphological characterization of acetic acid bacteria from industrial fermenters of wine vinegar production. Vinegars and Acetic Acid Bacteria International Symposium. Reggio Emilia, 2005.A. González, N. Hierro, M. Poblet, JM. Guillamón, A. Mas. Quantitative-PCR for rapid detection of acetic acid bacteria. Vinegars and Acetic Acid Bacteria International Symposium. Reggio Emilia, 2005.N. Hierro, A. González, N. Rozés, A. Mas, JM. Guillamón. PCR-cuantitativa para la detección y cuantificación de levaduras vínicas. Palencia, España. Gienol, 2005.A. González, N. Hierro, M. Poblet, JM. Guillamón, A. Mas. Evolución de la población de las cepas de bacterias acéticas durante la fermentación alcohólica. Palencia, España. Gienol, 2005.Application of molecular techniques for identification and enumeration of acetic acid bacteria:The main objectives of the present thesis is the development of fast and reliable molecular techniques for the identification and characterization of acetic acid bacteria.Acetic acid bacteria are the main responsible of the acetification of the wines and of the Viniegra production. However, there is an important lack of knowledge about their behaviour and evolution through the different processes they are involved. The techniques of enumeration and identification based on their chemo-taxonomic properties are tedious and not reliable meanwhile molecular techniques already used are slow, tedious, very expensive and not useful for routine analysis of big amounts of samples in the laboratory.We have been able to identify at species level, with two molecular techniques such as PCR-RFLP del rDNA 16S and PCR-RFLP del rDNA 16S-23S ITS. We were able to identify all the described acetic acid bacteria species with the combination of the two techniques. Those, have also been used in ecological studies in wine.We have developed two techniques for the characterization of aceic acid bacteria at strain level: ERIC- and REP-PCR. Those techniques have allowed us the development of ecological studies in wine fermentations in different condition, allowing us to improve our knowledge about these bacteria behaviour and development through the wine fermentations.We have developed two techniques for the enumeration, real-time PCR and for the detection, nested-PCR respectively of acetic acid bacteria. Those techniques allow us not to plate the samples before the analysis, therefore they allow the detection of the viable but non-culturable cells, already describes inside the acetic acid bacteria

Molecular and epidemiological studies of salmonella enterica serotype enteritidis in Hong Kong.

by Koo Ching Irene.Thesis (M.Phil.)--Chinese University of Hong Kong, 1997.Includes bibliographical references (leaves 105-118).Abstract also in Chinese.Acknowledgments --- p.iAbstract --- p.iiContent --- p.ivList of tables --- p.viiiList of figures --- p.xChapter Chapter 1: --- Introduction --- p.1Chapter A. --- Classification of salmonellae --- p.1Chapter B. --- Salmonellae enterica ser Enteritidis --- p.4Chapter C. --- Global increase in the prevalence of S. Enteritidis --- p.6Chapter D. --- Susceptibility of S. Enteritidis to antimicrobial agents --- p.13Chapter E. --- Methods for the epidemiological typing of S. Enteritidis --- p.17Chapter 1. --- Phenotypic methods --- p.17Chapter (1) --- Biotyping --- p.17Chapter (2) --- Antibiotic resistance pattern --- p.18Chapter (3) --- Phage typing --- p.18Chapter (4) --- Characterization of plasmids --- p.19Chapter a. --- Resistance plasmids --- p.20Chapter b. --- Transferability of plasmids --- p.20Chapter c. --- Incompatibility --- p.21Chapter 2. --- Molecular methods --- p.21Chapter (1) --- Plasmid analysis --- p.21Chapter a. --- Plasmid profile --- p.22Chapter b. --- Plasmid fingerprinting --- p.22Chapter (2) --- Chromosomal DNA fingerprinting --- p.24Chapter a. --- Restriction fragment length polymorphism (RFLP) of chromosomal DNA --- p.25Chapter b. --- Pulsed-field gel electrophoresis (PFGE) --- p.25Chapter c. --- Hybridization with specific gene probes --- p.26Chapter d. --- Ribotyping --- p.27Chapter e. --- Insertion sequence IS200 fingerprinting --- p.29Chapter (3) --- Polymerase chain reaction (PCR) --- p.30Chapter 3. --- Other --- p.32Chapter (1) --- Lipopoly saccharide (LPS) analysis --- p.32Chapter (2) --- "Whole cell protein profile analysis, fatty acid profile analysis, multilocus enzyme electrophoresis and Fourier-transform injfrared spectroscopy" --- p.33Chapter F. --- Epidemiology of S. Enteritidis in different parts of the world --- p.34Chapter G. --- Objectives --- p.35Chapter Chapter 2: --- Materials and Methods --- p.36Materials --- p.36Methods --- p.38Chapter A. --- Identification --- p.38Chapter 1. --- Biochemical tests --- p.38Chapter 2. --- Serotyping --- p.38Chapter B. --- Antimicrobial susceptibility testing --- p.39Chapter C. --- Characterization of β-lactamases --- p.39Chapter 1. --- Extraction of β-lactamases --- p.39Chapter 2. --- Determination of isoelectric points (pIs) --- p.41Chapter D. --- Characterization ofplasmids --- p.42Chapter 1. --- Genetic studies --- p.42Chapter (1) --- Transferability of resistance plasmids --- p.42Chapter (2) --- Mobilization of resistances --- p.43Chapter 2. --- Molecular studies --- p.44Chapter (1) --- Plasmid profile analysis --- p.44Chapter a. --- Plasmid extraction --- p.44Chapter b. --- Agarose gel electrophoresis --- p.45Chapter (2) --- Plasmid DNA fingerprinting --- p.45Chapter a. --- Preparation of pure plasmid DNA --- p.46Chapter b. --- Preparation of individual plasmid from strains harbouring more than one plasmid --- p.46Chapter c. --- Restriction endonuclease digestion of plasmid DNA --- p.47Chapter E. --- Total DNA fingerprinting --- p.47Chapter 1. --- Total DNA preparation --- p.48Chapter 2. --- Restriction endonuclease digestion of total DNA --- p.48Chapter 3. --- Ribotyping --- p.49Chapter (1) --- Restriction enzyme digestion of total DNA --- p.49Chapter (2) --- Transfer of DNA fragments to solid support --- p.49Chapter (3) --- Reverse transcription of rRNA into cDNA and labelling of cDNA --- p.50Chapter (4) --- Hybridization --- p.50Chapter (5) --- Detection of hybridized fragments --- p.51Chapter 4. --- AP-PCR (Arbitrary primed-PCR) --- p.51Chapter F. --- Experimental design --- p.52Chapter Chapter 3: --- Results --- p.54Chapter A. --- Prevalence of S. Enteritidis in the Prince of Wales Hospital --- p.54Chapter B. --- Antimicrobial susceptibilities --- p.58Chapter C. --- β-lactamases produced by β-lactam-resistant S. Enteritidis --- p.66Chapter D. --- Plasmid profile analysis --- p.66Chapter E. --- Characterization of resistance plasmids --- p.69Chapter F. --- Plasmid fingerprinting --- p.72Chapter G. --- Total DNA fingerprinting --- p.76Chapter H. --- Ribotyping --- p.82Chapter I. --- AP-PCR --- p.85Chapter J. --- "Correlation of plasmid analysis, total DNA fingerprinting, ribotyping and AP-PCR results" --- p.88Chapter Chapter 4: --- Discussion --- p.91Chapter A. --- Prevalence of S. Enteritidis --- p.91Chapter B. --- Susceptibilities of S. Enteritidis to antimicrobial agents --- p.92Chapter C. --- Evaluation of methods for epidemiological typing of S. Enteritidis --- p.94Chapter D. --- Molecular epidemiology of S. Enteritidis in Hong Kong --- p.99Chapter E. --- Areas for future research --- p.102References --- p.105Appendix --- p.11

The use of PCR-based methodologies to characterize salmonella serotypes of poultry origin

Three studies were conducted to investigate the use of molecular techniques to identify Salmonella serotypes in poultry. In the first experiment, two polymerase chain reaction (PCR)-based techniques: denaturing gradient gel electrophoresis (DGGE) and polyacrylamide gel electrophoresis (PAGE) were used to analyze Salmonella serotype isolates from two turkey processing plants (A and B). Genotypic patterns of each isolate were compared with those of known serotypes identified by traditional antibody precipitation methods. In Plant A, four different Salmonella serotypes were identified: Derby, Hadar, Montevideo, and Senftenberg. In plant B, ten serotypes were identified: Agona, Anatum, Brandenburg, Derby, Hadar, Meleagridis, Montevideo, Reading, Senftenberg, and Typhimurium. S. Derby was predominant in Plant A (83%) while S. Typhimurium was the most common serotype recovered in Plant B (39%). Overall, DGGE was more sensitive than PAGE. Isolates of the same serotypes were all grouped together by DGGE, while PAGE failed to group all like serotypes. Next, DGGE and REP-PCR were used as genotyping tools for identifying Salmonella. Fifty-four Salmonella isolates from two turkey processing plants (A and B) were evaluated. The isolates were comprised of the following serotypes: Brandenburg, Derby, Hadar, and Typhimurium (n = 6, 21, 12, and 15, respectively). Both methods were very sensitive and detected diverse fingerprint profiles among the isolates. The data suggested that REP-PCR and DGGE are useful tools for identifying Salmonella serotypes in research trials of this type. The final trial was carried out to track Salmonella serotypes throughout an integrated poultry operation using DGGE. Four flocks were sampled from grow-out through processing. The data showed that there was correlation between Salmonella serotypes found on processed carcasses and during grow-out. In addition, the isolates were compared against 15 known serotypes in our data base and only S. Hadar from the data base matched the unknown Salmonella isolates. Overall, these studies demonstrate that PCR-based methods could be considered as an alternative to conventional methods of antibody-based serotyping. Molecular methods were found to be reliable, sensitive, inexpensive, reproducible, and less labor intensive than conventional methods