Steps and Tools for PCR-Based Technique Design

The identity and clonal differences within bacterial populations have been broadly explored through PCR-based techniques. Thus, bacterial identification and elucidation of DNA fingerprinting have provided insights regarding their phenotypic and genotypic variations. Indeed, some diversity of rates may reflect changes among subpopulations that have their own ecological dynamic and individual traits on coexisting genotypes. Therefore, identification of polymorphic regions from nucleic acid sequences is based on the identification of both conserved and variable regions. Advantages of PCR-based methods are high sensitivity, specificity, speed, cost-effectiveness, and the opportunity for simultaneous detection of many microbial agents or variants. Fingerprint information might allow the tracking of certain outbreaks globally in several reference databases containing valuable genotyping information. In this chapter, we will review applications from Web resources and computational tools online for the designing of PCR-based methods to identify bacterial species. We will also focus on lab applications and key conditions for technique standardization

Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

<p>Abstract</p> <p>Background</p> <p>cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively.</p> <p>Results</p> <p>With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literatur and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and <it>Arabidopsis thaliana</it>. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set.</p> <p>Conclusion</p> <p>Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For <it>A. thaliana</it>, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.</p

An overview of molecular marker methods for plants

The development and use of molecular markers for the detection and exploitation of DNA polymorphism is one of the most significant developments in the field of molecular genetics. The presence of various types of molecular markers, and differences in their principles, methodologies, and applications require careful consideration in choosing one or more of such methods. No molecular markers are available yet that fulfill all requirements needed by researchers. According to the kind of study to be undertaken, one can choose among the variety of molecular techniques, each of which combines at least some desirable properties. This article provides detail review for 11 different molecular marker methods: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequencerepeats (ISSRs), sequence characterized regions (SCARs), sequence tag sites (STSs), cleaved amplified polymorphic sequences (CAPS), microsatellites or simple sequence repeats (SSRs), expressedsequence tags (ESTs), single nucleotide polymorphisms (SNPs), and diversity arrays technology (DArT)

Genetic diversity of predominant nosocomial meticillin-resistant Staphylococcus aureus lineages in the United Kingdom

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the major human pathogens and is a common cause of hospital-associated infection in immunocompromised patients and individuals with open wounds. However, infections affecting the healthy and young have increased in the past few decades and are a rising clinical concern. Understanding the genetic diversity of S.aureus can therefore result in a better understanding of the pathogenesis, spread and evolution of S.aureus, which in turn can lead to the development of effective infection control measures. In the present study, molecular methods were utilised to investigate differences in the distribution and variability of bacterial genes present between successful and unsuccessful lineages of MRSA. Lineages were assigned based on multilocus sequence typing (MLST) clonal complexes and successful lineages were defined as those lineages that have given rise to multiple epidemic MRSA done. In addition to well-established molecular methods such as MLST and staphylococcal cassette chromosome mec typing, a novel fluorescent amplified fragment length polymorphism assay was developed to identify regions of genetic heterogeneity between and within lineages. These differences were attributed to lineage-specific sequence variation and differences in the distribution of mobile genetic elements (MGEs) between lineages. These genetic differences were investigated in relation to functional differences within the bacterium, to explore the,role of cell functions encoded by these regions in the emergence and prevalence (or spread) of dominant genetic lineages. This study indicates a combination of factors play a role in the success of major MRSA lineages. The genetic diversity amongst core regions and MGEs appear to alter antimicrobial resistance and virulence factors that are vital to their success, as they enable the rapid adaption of lineages to their environment

Characterisation of the endophytic bacterial communities associated with South African sorghum plants: looking for potential plant growth-promoting endophytes

>Magister Scientiae - MScThe term endophyte is used to define all microorganisms that, during a part of their life cycle, colonize the internal tissues of a plant host. Many endophytes have been found to promote plant growth by acting either as biocontrol agents, biofertilizers or phytohormone producers. This study aimed to characterise the endophytic microbial community diversity associated with sorghum farmed in South Africa. Members of any common endophytic bacterial species identified during the study might in future studies be developed to improve sorghum production. Sorghum tissues (roots, shoots, stems) were sampled in three South African provinces (Free State, Limpopo and North West), each site being characterised by the use of different agricultural practices. Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses were used to characterise the endophytic bacterial communities. The analysis clearly demonstrated that the endophytic bacterial community structure in the three sorghum tissue types differed, suggesting that endophyte colonization is tissue-specific. The endophytic bacterial community structure is quite similar in each tissue when comparing the populations present in the sampling sites. In the sorghum endophytic microbial communities, common bacterial species were identified using molecular tools: The cyanobacterium Synechococcus and Staphylococcus saprophyticus were identified in the root samples. Pantoea sp., Erwinia sp., Enterobacter sp. and Klebsiella sp. were found in all shoot samples. Nocardia fluminea, Bacillus cereus and Microbacterium sp. were isolated as common shoot endophytic bacteria. This study defines, for the first time, the endophytic bacterial species associated with South African sorghum plants. These common endophytic bacterial species can be used to enhance the yield of sorghum crops

Whole genome sequence of mycoplasma mycoides subsp. Mycoides LC : application to the development of new diagnostic tools and heterologous gene expression

Les mycoplasmes sont des bactéries dépourvues de paroi qui dérivent de bactéries Gram plus. Il existe un groupe d'espèces, appelé le «Groupe mycoides» qui rassemble des espèces pathogènes pour les ruminants bien qu'il soit phylogénétiquement proche d'espèces pathogènes de plante. Leur génome est très réduit, environ un million de paires de bases et peu riche en G + C, environ 25 %. Parmi ce groupe, Mycoplasma mycoides subsp. mycoides LC (MmmLC) est un agent responsable d'agalaxie contagieuse chez les chèvres. Il est très proche de Mycoplasma mycoides subsp. mycoides SC (MmmSC) qui est l'agent responsable de la péripneumonie contagieuse bovine et dont le génome de la souche de référence était disponible. En raison de cette proximité nous avons décidé de séquencer complètement le génome d'une souche de MmmLC afin de pouvoir réaliser des études de génomique comparative. Préalablement au séquençage et à l'assemblage, nous avons évalué la taille du génome avec la technique d'électrophorèse en champs pulsé. Nous avons pu ensuite contrôler l'assemblage obtenu en comparant les données expérimentales «in-silico» avec les résultats d'électrophorèse. De plus nous avons réalisé des «Southern blots» afin de vérifier si les séquences dupliquées chez MmmSC l'étaient également chez MmmLC. La comparaison avec les génomes complets déjà disponibles pour des souches du «Groupe mycoides» a permis d'identifier un locus intéressant pour développer des PCR spécifiques. Ce locus comprend des gènes ou des fragments de gènes appartenant chez certaines bactéries à un opéron de «voie de déimination de l'arginine». Le nombre ainsi que l'agencement et la séquence de ces gènes varie d'une espèce à l'autre au sein du «Groupe mycoides». Il a ainsi été possible de développer une PCR spécifique pour Mycoplasma capricolum subsp. capripneumoniae, l'agent de la pleuropneumonie contagieuse caprine en amplifiant un fragment du gène arcD et une PCR spécifique de M. putrefaciens, un agent d'agalaxie contagieuse, en amplifiant un fragment du gène arcB. Pour la détection de l'ensemble des espèces du «Groupe mycoides» nous avons choisi un gène plus conservé, glk, situé en aval de l'opéron. L'annotation du génome de MmmLC a également permis d'identifier des séquences d'insertion. L'une d'entre elles, appartenant à la famille IS3, n'avait pas encore été décrite et a été appelée ISMmy2. Elle est présente chez certaines espèces du «Groupe mycoides» mais pas chez toutes les souches. Un variant de cette IS existe chez des espèces proches du «Groupe mycoides» et il en existe une copie non fonctionnelle chez MmmSC. Enfin nous avons voulu évaluer les capacités de MmmLC à exprimer des antigènes hétérologues dans le but ultime d'en faire un vecteur d'expression vaccinal. C'est pourquoi nous avons choisi un gène d'intérêt vétérinaire majeur, le gène H du virus de la peste des petits ruminants. ABSTRACT : Mycoplasmas are the smallest bacteria without a cell wall derived from Gram positive bacteria. A group of mycoplasma known as the “Mycoplasma mycoides cluster” is composed of five subspecies and an unassigned group of strains known for their pathogenicity in ruminant hosts. Phylogenetically, this cluster is found to be closely related to species of mycoplasma plant pathogens. Mycoplasmas have a reduced genome size of about 1 Mbp, characterized by a low GC content of about 25 %. Among members of the Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides large colony biotype (MmmLC) is one of the agents responsible for contagious agalactia in goats. This organism is closely related to Mycoplasma mycoides subsp. mycoides small colony biotype, the causative agent of contagious bovine pleuropneumonia (CBPP), for which the whole genome sequence is available. Because of the close relationship of these two species we have decided to sequence the genome of an MmmLC strain for comparative genomics. Before whole genome sequencing and assembly, we have estimated the genome size of MmmLC using pulse field gel electrophoresis (PFGE). Data generated from this initial study have permitted us to verify the genome assembly by comparing in-silico profiles. In addition the preliminary analysis included DNA hybridization tests to verify the presence of duplicated genes in MmmLC as that of the genome of MmmSC. Comparative genomics made from the available whole genome sequence data of species within the M. mycoides cluster has permitted the identification of target genes, which were used for the development of specific PCR tests. The target genes chosen included genes of the “arginine deiminase operon”, in most bacteria genes of this operon code for enzymes involved in the degradation of arginine to produce energy. The number of these genes as well as their organization within the operon found to vary between members of the M. mycoides cluster. From this operon arcD has been used to develop a specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP), and arcB has been used for the development of specific PCR for the identification of M. putrefaciens, another causative agent of the contagious agalactia syndrome. The glk gene, flanking the operon on the 3' end, was found to be highly conserved among all members of the M. mycoides cluster and was used for the design of specific primers able to detect all members of M. mycoides cluster. Furthermore, annotation of the genome sequence of MmmLC allowed the discovery of two new insertion sequence elements. One of these two insertion sequence elements was found in higher copy in the genome and belongs to the IS3 family. This insertion sequence was not described in any other mycoplasma species or bacteria, was given a new name: ISMmy2. It was also found in some species of the M. mycoides cluster, but not in all the strains under these species. Interestingly, a non-functional vestige of ISMmy2 was also found in the MmmSC genome. Copies of this ISMmy2 were also found in species closely related to the M. mycoides cluster. Finally, we have tried to evaluate the capacity of MmmLC to be transformed and to express a heterologous gene with the ultimate aim to create a multivalent vaccine. For this aim we have chosen the H-gene of peste des petits ruminant virus of veterinary health importanc

Evaluation of sensitivity an specificity of molecular diagnosis for staphylococcus aureus in milk of cows affected by mastitis

RESUMEN: Entre los agentes contagiosos que inducen mastitis severas en vacas, se encuentra el Staphylococcus aureus (S. aureus), de difícil curación clínica y alta resistencia a antimicrobianos. Debido a que el cultivo microbiológico de las muestras clínicas, solo es capaz de detectar alrededor del 50% de los casos positivos (Koskinen et al., 2009), el diagnóstico por reacción de la polimerasa en cadena (PCR) ofrece una posible alternativa. El objetivo de esta investigación fue contribuir a la estandarización de un diagnóstico rápido a partir de PCR para detectar S. aureus en leche de vacas afectadas por mastitis. Los resultados demostraron que con los siguientes fragmentos de nucleótidos empleados como cebadores: F 5' AGC TGT GGA TTG TCC TTT GG 3' y R 5' TCG CTC GCT CAC CTT AGA A 3', se obtenían secuencias amplificadas de 499 pb, que si bien no serían adecuadas para el diagnóstico de muestras clínicas por tener una especificidad del 63%, detectaron S. aureus en 12 de 13 muestras positivas por cultivo microbiológico, lo que indica una alta sensibilidad. En conclusión, se requiere buscar nuevos cebadores que amplifiquen regiones del S. aureus que no sean compartidas con otras bacterias, en especial aquellas que producen mastitis en vacas de leche.ABSTRACT: Staphylococcus aureus (S. aureus) is one of the infectious agents that induce severe mastitis in cows with a difficult bacteriological cure and high antimicrobial resistance. Because the microbiological culture of clinical samples only shows results in 50% of the cases (Koskinen et al., 2009), diagnostic through PCR is an alternative. The aim of this study was to prove if the primers described by Cremonesi et al. (2006) for the S. aureus diagnosis, with good sensitivity and specificity, could be used in clinical samples too. The results showed that the following nucleotide sequences can be used as primers: F 5’ AGC TGT GGA TTG TCC TTT GG 3’ and R 5’ TCG CTC GCT CAC CTT AGA A 3’ in order to obtain a 499 pb enlargemenr are not useful in clinical samples due their low specificity (62.95%). It is required to search new primers to amplify S. aureus regions not shared with other bacteria, especially those cauding mastitis in dairy cows

Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid

Bifidobacteria Ecology of non-Human Primates: Characterization of Novel Species with Unexpected Functionalities for Probiotic Applications and a Co-Evolutionary Host-Microbe Analysis

Bifidobacterium spp. are known as probiotic strains and recently new features emphasize their importance for human health, as EPSs and folate production. The relationship between Bifidobacterium spp. and their hosts is unknown, but probably links to peculiarities in the bifidobacterial cell-wall structures or to bifidobacterial ability to metabolize substrates from the host diet. Recently, a richness and diversity of bifidobacteria was observed in Callithrix jacchus and Saguinus midas, introducing the existence of a storehouse in primate guts. Several techniques were developed to deepen the microbial diversity, mainly based on the PCR. The RFLP-PCR of 16S rRNA gene represents a fast tool to distinguish human or animal origin bifidobacteria, useful in “Microbial Source Tracking” and probiotic selection. The project aim was the exploration of the bifidobacterial occurrence and diversity in evolutionary primate hosts to improve the knowledge about bifidobacteria distribution in non-human primates, and to identify bifidobacteria with new probiotic features (EPSs and folate production). 17 subjects from Strepsirrhini, Eulemur macaco, Eulemur rubriventer, Hapalemur alaotrensis and Lemur catta, and from Simiiformes, the New World Monkeys Callithrix jaccus, Pithecia pithecia, Saguinus oedipus and Saguinus imperator, and the Old World Monkeys, Chlorocebo aethiops and Macaca Sylvanus, were studied. Strains tested for probiotics traits, acid and bile tolerance, revealed B. aesculapii, B. myosotis and B. spp. MRM_8.19 strains as the most resistance. The folate production on strains from ring-tailed lemur and common marmoset revealed autotrophy only in strains from common marmoset. The distribution of microbial communities in non-human primates from 8 babies of common marmosets, golden faced saki and Barbary macaques and 11 adults of ring-tail lemurs, black lemurs, red-bellied lemur, Alaotran bamboo lemur, Barbary macaques, grivet, cotton top-tamarin and emperor tamarin, was carried out using ARDRA and rep-PCR. Results revealed a richness in both abundance and diversity of Bifidobacterium in primates