Molecular Biology of Streptococci Chapter Abstracts
Chapter 1: Genetic Transformation in Streptococcus pneumoniae: Strategies for Genome Modification
Donald A. Morrison
Abstract
Competence for genetic transformation in Streptococcus pneumoniae depends on a developmental switch that is coordinated by quorum sensing via a peptide pheromone signal (CSP). Synthetic pheromone peptide can bypass requirements for endogenous pheromone production or response, eliciting competence in many media and strains, through a signal transduction pathway that includes a two-component signaling system and an alternative sigma factor. Donor DNA is processed through a single-strand intermediate and formation of heteroduplexes. Recovered recombination products depend on the topology of the donor molecule, especially if the donor is chimeric, and on the length of homologous segments presented by the donor. In nature, DNA may be obtained by cell lysis promoted by specialized proteins of the competence regulon, but in the laboratory, chimeric donor DNA can be designed according to experimental needs and constructed in vitro or in heterologous hosts. A variety of genetic tools has been developed for genome modification that utilize these pathways of homologous recombination, based on plasmid vectors, on circular integration of non-replicative donors, and on linear cassettes designed for insertion or gene replacement.
Chapter 2: Biologically Active Peptides in Streptococci
Ola Johnsborg, Trinelise Blomqvist, Mogens Kilian and Leiv Sigve Håvarstein
Abstract
A multitude of antimicrobial peptides termed bacteriocins have been purified from different species of Gram-positive bacteria during recent decades. A relatively small fraction of these have been isolated from members of the genus Streptococcus. The best-studied streptococcal bacteriocins are the posttranslationally modified lantibiotics (class I), which are characterized by the presence of lanthionine and methyllanthionine residues. However, streptococcal genomes also contain a large number of genes which seem to encode different unmodified class II bacteriocins. Consequently, streptococci appear to constitute a rich source of antimicrobial peptides that so far has been poorly exploited. Cell-cell communication by peptide pheromones regulates several processes in the life-cycles of streptococci that strongly contibute to their survival and proliferation in the host and their competitiveness towards other bacteria sharing the same niche. Two types of peptide pheromones, belonging to families 2 and 3, have so far been discovered in the genus Streptococcus. Family 2 pheromones are posttranslationally modified lantibiotic bacteriocins that regulate their own production, whereas family 3 pheromones are unmodified and regulate production of class II bacteriocins, competence for natural genetic transformation, biofilm formation and virulence.
Chapter 3: Competence in Streptococcus pneumoniae: What is it For?
Sébastien Guiral, Miriam Moscoso, Adilia Dagkessamanskaia, and Jean-Pierre Claverys
Abstract
Natural competence for genetic transformation is the best-characterized feature of the human pathogen Streptococcus pneumoniae. Competence has long been considered mainly as favoring long-term genetic plasticity through transformation-mediated exchanges. In this chapter, we discuss two sets of recent observations which change our views on the role of the competence regulon in this species. First, evidence that this regulon comprises a number of genes much larger than required solely for uptake and processing of transforming DNA into recombinants. Second, evidence that competent cells are endowed with the capability to promote lysis of noncompetent cells. We discuss how the latter could lead to transient (non-heritable) diversity and propose that competence be now viewed as a global adaptive response of S. pneumoniae allowing both short- and long-term variability.
Chapter 4: Structure and Biosynthesis of the Pneumococcal Cell Wall
Waldemar Vollmer
Abstract
The pneumococcal cell wall contains a multi-layered murein (peptidoglycan) with a covalently attached wall teichoic acid, and a membrane-bound lipoteichoic acid. The peptides in the murein are cross-linked either directly or via an interpeptide bridge. The repeating units of wall teichoic acid and of lipoteichoic acid have the same complex structure and are decorated with phosphorylcholine residues, that have a variety of physiological functions. The enlargement of the cell wall during growth and division occurs by incorporation of murein and teichoic acid at a central growth zone. Different cell wall hydrolases are required for the regulation of cell wall growth, separation of the daughter cells during division, autolysis, modification of the murein glycan strands to increase lysozyme resistance, and modulation of the number of choline residues on the surface.
Chapter 5: Pneumococcal Capsular Polysaccharides: Biosynthesis and Regulation
James C. Paton and Judy K. Morona
Abstract
The serotype-determining capsular polysaccharide (CPS) is a crucial virulence determinant of Streptococcus pneumoniae, and is the target of existing pneumococcal vaccines. The complex genetic loci encoding CPS biosynthesis have been the subject of intensive study over many years, and complete nucleotide sequence data are now available for all 90 known S. pneumoniae serotypes. Almost all capsule loci comprise up to 20 or more closely-linked genes that appear to form a single transcriptional unit. They share a common organization. The 5' region comprises 4 common genes, which is followed by a type-specific region encoding glycosyl transferases, a polysaccharide polymerase and a repeat unit transporter. The 3' region encodes enzymes for synthesis of activated sugar precursors. Current studies are aimed at gaining a complete understanding of the molecular events involved in CPS biosynthesis and the mechanism whereby its expression is regulated. This will undoubtedly provide new insights into the mechanisms of pathogenesis of invasive pneumococcal disease, and may result in development of alternative antimicrobial or vaccination strategies targeted against components of the CPS biosynthetic machinery.
Chapter 6: Pneumococcal Virulence Factors and Adhesion Proteins Targeting the Host
Sven Hammerschmidt
Abstract
The pneumococcus is carried asymptomatically in the upper and lower respiratory tract in up to 60% of the population. However, the pneumococcus is also a common aetiologic agent of respiratory tract diseases and life-threatening invasive diseases. A prerequisite for the invasiveness of pneumococci is the pathogen's ability to breach epithelial as well as endothelial tissue barriers in order to gain access to the submucosa and the blood. The mechanisms promoting invasiveness are associated with the expression of virulence factors. Proteins, enzymes, toxins, or carbohydrate structures, which are secreted or deposited on the bacterial cell-outer surface, are import virulence determinants. These bacterial components, which most likely do not affect vitality of the microorganism, facilitate immune evasion as well as adherence and invasion into specific host cells. Here, the impact of pnneumococcal carbohydrate structures and proteins targeting specifically proteins or cellular receptors of the host and the underlying molecular events will be discussed. In addition, the bioactivities of some pneumococcal virulence determinants contributing to pneumonia and/or inflammation during sepsis and meningitis will be evaluated.
Chapter 7: Genetic Regulation of Virulence in Streptococcus pneumoniae
Tim J. Mitchell and Gavin K. Paterson
Abstract
Understanding how the pneumococcus perceives and adapts to its environment in the host offers insight into this bacterium and the potential opportunity for improved interventions. Molecular advances in the form of genome sequencing, signature-tagged mutagenesis, differential fluorescence induction and microarray analysis have yielded considerable progress in this area and will continue to do so. Recent advances in the genetic regulation of pneumococcal virulence are discussed here focusing on the regulation of the virulence phenotype rather than the detailed molecular mechanisms of the regulation.
Chapter 8: Genome-wide Screening for Essential Genes of Streptococcus pneumoniae: Current Approaches and Future Opportunities
Peter Burghout, Hester J. Bootsma, and Peter W.M. Hermans
Abstract
Prevention and treatment of pneumococcal disease is hampered by the limited strain coverage of today's vaccines and the emergence of drug-resistant strains. Therefore, the discovery of novel broad-range targets for therapy and prevention of Streptococcus pneumoniae infections is of utmost importance. This chapter describes and discusses the screening methods that have been used to uncover the molecular principles of the pneumococcal life cycle and pathogenesis. Most of these tools, such as signature tagged mutagenesis and differential fluorescence induction, exploit the genetic accessibility of this bacterium to study the characteristics of individual mutants in large mutant libraries. Others, like two-dimensional gel electrophoresis, examine alterations in protein expression profiles. Recently, the availability of genome sequences has facilitated the development of novel genome-wide screening technologies resulting in a considerable increase in experimental throughput. Most prominent amongst these tools are high-density DNA microarrays, which are especially useful for transcriptome analysis and comparative genomic hybridization. In addition, in silico approaches (functional and comparative genomics) have been used for more comprehensive predictions of gene function. It is expected that in the near future technological innovations will enable researchers to assign the contribution of individual pneumococcal genes during different stages of infection on a genome-wide scale.
Chapter 9: Streptococcus pneumoniae: From Colonization and Infection Towards Prevention Strategies
D. Bogaert, R. de Groot, and P.W.M. Hermans
Abstract
Streptococcus pneumoniae is an important pathogen causing life-threatening infections such as sepsis, meningitis, and pneumonia. The burden of disease is highest in the youngest and oldest age-groups in both developed and developing countries. Pneumococcal disease is preceded by asymptomatic colonisation, which is also particularly high in children. The treatment of pneumococcal infections is complicated by the worldwide emergence of multidrug-resistance among pneumococcal strains. Although the current seven-valent conjugate vaccine is highly effective against invasive disease caused by the vaccine-type strains, the vaccine coverage is limited, and replacement by non-vaccine serotypes resulting in disease is a serious threat for the near future. Therefore, other preventive strategies need to be considered. To illustrate the importance of pneumococcal colonisation in relation to pneumococcal disease and prevention of disease, we discuss the mechanism and epidemiology of colonisation, the complexity of interactions within and between species, and the consequences of the different preventive strategies for pneumococcal colonisation.
Chapter 10: Molecular Epidemiology and Mechanisms for Antibiotic Resistance in Streptococcus pneumoniae
Birgitta Henriques Normark
Abstract
Streptococcus pneumoniae is a major cause of morbidity and mortality world-wide. It causes a variety of infections ranging from milder respiratory tract infections to severe invasive diseases, sometimes with a lethal outcome. Resistance to several groups of antibiotics has emerged among clinical isolates of Streptococcus pneumoniae. Antibiotic resistant clones (closely related bacteria) are spreading causing treatment problems and multi-resistance is increasing. The success of clones in spreading is dependent on several factors, including bacterial factors such as the capsular type and genotype, deciding virulence properties such as capabilities of colonization, transmission, and invasiveness. However, host responses including herd immunity, are also of great importance, as are environmental factors such as antibiotic usage and vaccination. The available vaccines today are directed towards the capsular polysaccharide, the major virulence factor of pneumococci. Vaccination and reduction of antibiotic usage have been suggested to be tools in the battle against resistant pneumococci.
Chapter 11: Mechanisms of Penicillin Resistance in Streptococcus pneumoniae: Targets, Gene Transfer and Mutations
Dalia Denapaite, Fang Chi, Patrick Maurer, Oliver Nolte, and Regine Hakenbeck
Abstract
Penicillin-binding proteins, PBPs, are crucial enzymes important for the biosynthesis of murein, the typical procaryotic macromolecule. They represent the targets for beta lactam antibiotics and are thus involved in the evolution of penicillin resistance in Streptococcus pneumoniae. Whereas distinct point mutations in individual pbp genes occur in laboratory mutants selected for resistance, gene transfer events play an additional role for the emergence and spread in clinical isolates. Moreover, apparent PBP independent resistance mechanisms have been described. In this chapter, alterations in PBPs of resistant strains on the molecular level will be discussed and the different pathways now identified during the development of resistance to different beta lactams will be summarized.
Chapter 12: Virulence Factors and Their Regulation in Group A Streptococcus
Kadaba S. Sriprakash and David J. McMillan
Abstract
Streptococcus pyogenes is a pathogen that colonises the skin and throat, survives intracellularly and also disseminates to sterile tissue sites within the host. The versatility of this pathogen is largely due to the timely expression of virulence factors that are involved in adhesion, protect from host innate and adaptive immune responses, promote bacterial dissemination, modulate the activity of other virulence factors which have toxic or cytotoxic activity. The co-ordinated expression of these factors is achieved through a network of regulatory molecules. Chief among them are two component and 'stand alone' response regulators that exert their influence at a global level, as well as through control of specific virulence loci. Additional control of other loci is provided through regulatory RNA molecules, post-translational modification and phase variation. The complexity of the regulatory network underscores the importance of co-ordinating the expression of specific virulence factor repertoires to specific host niches and successful host-pathogen interactions.
Chapter 13: Streptococcal Peptide Transmembrane Transport: Description of Transport Systems and Biological Consequences
Michael G. Caparon, Bert Poolman, and Andreas Podbielski
Abstract
Streptococci are auxotrophic for several amino acids and often satisfy their need by absorbing peptides. In turn, streptococci use peptide for signalling and for attacking each other or a potential host. In all cases transmembrane peptide transport systems are involved in these features. This chapter describes structural details of the streptococcal, lactococcal, and enterococcal machineries for exporting and importing peptides and proteins. In addition, functional consequences of peptide transport with respect to metabolism, growth kinetics, and virulence traits are discussed. Precisely, details about the Sec and cytolysin-mediated translocation pathways for peptide export as well as the ExPortal and the impact of double-glycine leader motifs are presented. With respect to import systems, ABC transporters for oligo- and dipeptides and ion-linked transporters are introduced.
Chapter 14: Gene Expression and Tagging of Streptococcal Proteins
Jason N. Cole, Martina L. Sanderson-Smith, Amanda J. Cork, Anna Henningham, Feildhlim Conlan, Marie Ranson, Jason D. McArthur and Mark J. Walker
Abstract
The Gram-positive bacterial pathogen Streptococcus pyogenes (Group A streptococcus; GAS) is responsible for a wide variety of human diseases ranging from mild infections of the skin and throat, to life-threatening infections such as "flesh-eating" disease and a toxic-shock-like syndrome. This chapter describes three different strategies for the cloning, expression and purification of recombinant histidine-tagged GAS proteins from Escherichia coli. These strategies include the traditional restriction enzyme cloning approach (pQE-9), topoisomerase cloning (pET101/D-TOPO(r)) and the use of GST fusion protein technology (pGEX-2T). A discussion on the advantages and limitations of each cloning system is presented.
Chapter 15: Molecular Pathogenesis of Group B Streptococcal Infections
Amanda L. Jones and Craig E. Rubens
Abstract
Early onset disease (EOD) caused by group B streptococcus (GBS) occurs in neonates during the first week of life. In mothers colonized with GBS in the genital tract, neonates are exposed to the organism in utero following an ascending infection or during passage through the birth canal at delivery. The clinical manifestations of EOD can include fulminant pneumonia, septicemia and meningitis. In recent years, there have been significant advances in our understanding of the pathogenesis of GBS infection and a substantial reduction in the burden of neonatal EOD GBS disease. This progress has taken two forms which include the identification and characterization of the virulence mechanisms used by GBS to cause disease and the prevention of infections by identifying infants at risk for EOD. However, GBS remains a significant neonatal pathogen. The significant virulence potential of this organism coupled with the relative immaturity of the neonatal immune system can tip the host:pathogen balance in favor of the pathogen. The outcome for the neonate can be potentially devastating. This chapter will review the pathogenic mechanisms relevant to EOD focusing on recent advances in the field. Discussed are strategies used by GBS for adherence to host surfaces, penetration of epithelial and endothelial cell barriers, adaptation to the host environment, evasion of host immune responses and regulation of gene expression.
Chapter 16: Oral Streptococci: Commensals and Opportunistic Pathogens
Charles F. Schachtele, Angela Nobbs, Yongshu Zhang, Massimo Costalonga, and Mark C. Herzberg
Abstract
The oral streptococci are intriguing to microbiologists because they can quickly change from friendly commensals to harmful pathogens. Under the proper conditions, indigenous oral streptococci can initiate diseases ranging from dental caries to endocarditis. Twenty-five species of streptococci call the human oral cavity their home and the taxonomy of these bacteria is complex. The various species have developed unique mechanisms for colonizing the different oral surfaces and their existence in biofilms on both hard tissue and soft tissue allows them to survive in a constantly changing environment. Oral streptococci have to fight off competing bacteria, biological and mechanical shocks, and activation of innate and adaptive host immunity. Using the latest molecular technologies, pathogenic mechanisms used by the oral streptococci are becoming uncovered. Likewise, genomic and proteomic investigations on streptococcal biofilms may suggest means to prevent the conversion of oral streptococci from commensals to opportunistic pathogens.
Chapter 17: Virulence Mechanisms of Streptococcus suis
Laurentiu Benga and Peter Valentin-Weigand
Abstract
Since its identification almost two decades ago Streptococcus suis has received increasing scientific interest as an important porcine pathogen. In addition, S. suis can cause diseases in humans, and, thus, represents an animal pathogenic streptococcus, of which some strains might have adapted to humans. This chapter focuses on the virulence mechanisms of S. suis, many of which have been discovered very recently. Since S. suis is a relatively "young" pathogen, the chapter starts with an introduction on its biology and clinical significance. Following, major putative virulence factors identified over the last years are presented, and current concepts of pathogenesis are described, which have been proposed based on these factors and on experimental infections. Taken together, it appears that S. suis shares a number of virulence mechanisms as well as its high diversity with other pathogenic streptococcal species, in particular group B streptococci and pneumococci, but also exhibits specific features. Nevertheless, the molecular mechanisms of the entry, survival and spread of S. suis within its host(s) still remain a mystery and are a challenge for future work on pathogenesis of this zoonotic pathogen.
Chapter 18: Adhesion and Invasion of Streptococci in Eukaryotic Cells
D. Patric Nitsche-Schmitz, Manfred Rohde, and Gursharan S. Chhatwal
Abstract
Streptococcal adhesion and invasion of eukaryotic cells has a variety of implications in the infection pathogenesis. Cell adhesion establishes the initial host contact. Host cells are exploited by adhering bacteria, for their own benefit. Internalisation into the host cell contributes to bacterial survival and dissemination, thus playing a key role in the course of infection. This chapter summarizes the current knowledge about the mechanisms of streptococcal adhesion to and invasion into different eukaryotic cells. It sheds light on the benefits of these processes for the bacteria and on the consequences for the pathogenesis of streptococcal infections.
Chapter 19: In Vitro Mariner Mutagenesis of Streptococcus pneumoniae: Tools and Traps
Marc Prudhomme, Andrew Camilli, and Jean-Pierre Claverys
Abstract
There is no abstract for this chapter. Therefore the first paragraph of the introduction is provided instead. Since the original description of GAMBIT ('Genomic Analysis and Mapping By In vitro Transposition' (Akerley et al., 1998), in vitro transposon mutagenesis with mariner-derived minitransposons has proved particularly useful for the study of Streptococcus pneumoniae. It was successfully used both to create a bank of transposon mutations spanning the entire genome (Martin et al., 2000) and for targeted mutagenesis of many genes [endA, comEA/C, comFA/C, comGA/B and dprA (Bergé et al., 2002); srtBC and rrgABC (Hava and Camilli, 2002); lytA (Moscoso and Claverys, 2004); cbpD, cibABC, comE, lytA and ply (Guiral et al., 2005)]. A comprehensive description of GAMBIT by (Akerley and Lampe, 2002) incorporated details of the steps leading to the creation of banks of mariner transposon mutations. This section is therefore not intended as a practical guide to the use of mariner mutagenesis in general, but rather as a source of informations on its use with S. pneumoniae, based on our experience. We include a description of tools available (see "Donor Plasmids") and discuss possible traps identified in the course of our experiments (see "Transposase Purification", "In Vitro Transposition And Repair Of Gaps" and "Transformation And Phenotypic Expression").
Chapter 20: There Will be a Light: The Use of luc Transcriptional Fusions in Living Pneumococcal Cells
Marc Prudhomme and Jean-Pierre Claverys
Abstract
There is no abstract for this chapter. Therefore the first paragraph of the introduction is provided instead. The firefly luciferase gene, luc, was first used in Streptococcus pneumoniae (pneumococcus) by Stieger and coworkers (1999) to demonstrate the usefulness of a tetracycline-regulated gene-expression system. The luc gene then served to report the induction of competence in pneumococcal cultures, through the use of com::luc transcriptional fusions (i.e. fusions with a competence-induced gene, either comC or ssbB; Chastanet et al., 2001; Bergé et al., 2002; Dagkessamanskaia et al., 2004; Moscoso and Claverys, 2004). Unlike other reporter genes (e.g. lacZ or cat), measurement of luciferase activity does not rely on the preparation of cell extracts and can be carried out in vivo, with actively growing cells. We discuss below observations that led us to consider the luc gene as the best tool available for the analysis of gene expression in living pneumococccal cells, including the intermediate stability of luciferase, which allows the detection of successive waves of expression during a growth cycle, and the remarkable sensitivity of the assay, which allows detection of luciferase from a single-copy fusion with as few as ~150-200 cfu (colony-forming-unit, each unit corresponding to ~2 cells).
Chapter 21: Protocol for Conjugal Transfer of Genetic Elements in Streptococcus pneumoniae
Francesco Iannelli, Catiuscia Orienti, and Gianni Pozzi
Abstract
Conjugation is a common mechanism for horizontal gene transfer in bacteria. We described a protocol for in vitro conjugal transfer of genetic elements in Streptococcus pneumoniae. Conjugal transfer is performed on plate and selection of transconjugants is carried out with a multilayer plating. This procedure tipically allows the transfer of large genetic elements (longer than 50-kb) with a frequency of 1x10-3 transconjugants/donor cells.
Chapter 22: Preparation and Analysis of Pneumococcal Murein (Peptidoglycan)
Waldemar Vollmer
Abstract
There is no abstract for this chapter. Therefore the first paragraph of the introduction is provided instead. Pneumococci contain a complex cell wall composed of murein (peptidoglycan) with covalently bound wall teichoic acid (WTA) (see chapter 4, this book). The high-resolution HPLC technique for the quantitative analysis of the muropeptide (disaccharide-peptide) composition was first established for the murein from E. coli and allowed the identification and quantification of more than 50 different subunits (Glauner, 1988; Glauner et al., 1988). Alexander Tomasz and co-workers developed the methods for the isolation of cell wall and murein from pneumococci and the HPLC technique for their quantitative analysis (Garcia-Bustos et al., 1988; Severin and Tomasz, 1996).
Chapter 23: Detection of Penicillin-Binding Proteins
Jens Rutschmann, Patrick Maurer, and Regine Hakenbeck
Abstract
There is no abstract for this chapter. Therefore the first paragraph of the introduction is provided instead. Penicillin-resistance in Streptococcus pneumoniae and related viridans streptococci is mediated by alterations in penicillin-binding proteins. These proteins bind beta-lactams covalently via the active site serine, and this covalent penicilloyl (cephalosporoyl) -complex is enzymatically inactive. In resistant strains, altered PBPs have a decreased affinity to beta-lactam antibiotics; hence higher concentrations of the drug are required for inhibition of the enzymatic function of the PBP. PBP profile analysis is a rapid way for describing resistant strains since it represents a clone specific fingerprint in most cases (Hakenbeck et al., 1991a; Jabes et al., 1989), and affinity changes can also be estimated by this analysis.
Chapter 24: Approaches to Elucidate Pneumococci-host Interactions
Sven Hammerschmidt
Abstract
There is no abstract for this chapter. Therefore the first paragraph of the introduction is provided instead. The impact of pneumococcal components on pathogenesis can be investigated in cell culture infection experiments using host-specific human derived epithelial and endothelial cells. Human lung alveolar carcinoma epithelial cell line A549 (type II pneumocyte; ATCC CCL-185), HEp-2 larynx carcinoma cell line (ATCC CCL-23), Detroit 562 (human pharynx carcinoma; ATCC CCL 138), and Calu-3 cells (human lung epithelium; ATCC HTB-55) are most commonly used as epithelial cell lines. A549 and HEp-2 are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin G (100 IU/ml), and streptomycin (100 μg/ml). The medium for Calu-3 cells is further supplemented with 1 mM sodium pyruvate and 0.1 mM non-essential amino acids. Detroit 562 are grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, and 1 mM sodium pyruvate. The human brain-derived microvascular endothelial cells HBMEC (Stins et al., 1997), representing a model for the blood-brain barrier, and human umbilical vein-derived endothelial cells (HUVEC) are used as endothelial cell lines. HBMEC are cultured in RPMI 1640 based medium supplemented with 10% FCS, 10% Nu-Serum IV (Becton Dickinson), 1% non-essential amino acids, 1% MEM vitamins (Invitrogen), 1 mM sodium pyruvate, 2 mM glutamine, penicillin (100 Units/ml) and streptomycin (0.1 mg/ml) and HUVEC are grown in endothelial cell growth medium 2 (PromoCell, Heidelberg, Germany) in the presence of the SupplementMix (PromoCell). Out of these cells, Detroit 562 and Calu-3 express the human polymeric Ig receptor which interacts with the major pneumococcal adhesin. All cells were cultured at 37ºC under 5 % CO2.
Chapter 25: Mouse Models of Pneumococcal Infection
Tim J. Mitchell and Gavin K. Paterson
Abstract
There is no abstract for this chapter. Therefore the first paragraph of the introduction is provided instead. Animal models of pneumococcal infection have provided a valuable research tool to understand this bacterium and to develop therapeutic measures. Despite the obvious disadvantage that experimental animals are not humans these models have nonetheless allowed helpful insight beyond that provided by in vitro studies alone. Animal systems provide the ability to investigate pneumococcal virulence factors, the immune response to the pneumococcus and the ability of new vaccine candidates to protect against disease in a physiologically intact setting. A number of animal species have been used to study pneumococcal infection but the mouse has been the animal most used because it is small and easy to house, there is a large body of data related to immune function in this species and there is the ability to manipulate this host by transgenic technology. The complete genome sequence of this species is now also available.
Chapter 26: Protocol for Pulsed-field Gel Electrophoresis for Streptococcus pneumoniae
Raquel Sá-Leão and Hermonia de Lencastre
Abstract
There is no abstract for this chapter. Therefore the first paragraph is presented instead. In the last fifteen years pulsed-field gel electrophoresis (PFGE) become one of the most widely used techniques for molecular typing of bacteria. In fact, until the development of multilocus sequence typing (Maiden et al., 1998), it was considered as the "gold standard" for typing StreptococcuS. pneumoniae (and other bacterial genera and species) since it has optimal typeability, reproducibility, and resolving power (McGee et al., 2001; Tenover et al., 1995). In addition, the typing costs associated with reagents and disposable material is low (less than 3 euros per strain) although the initial investment in equipment can be high (around 20,000 euros). The technique is easy to perform but may require initial training with an experienced technician. PFGE is very useful for local epidemiology and it can also be applied for global epidemiology once standardized (Sá-Leão et al., 2000).
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