Gram-Positive Pathogens, Second Edition

GROUP A STREPTOCOCCI

• Chapter 3 : Intracellular Invasion by Streptococcus pyogenes: Invasins, Host Receptors, and Relevance to Human Disease

  Authors: Beinan Wang, David Cue, P. Patrick Cleary
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    This chapter presents a review of the invasins and pathways used by Streptococcus pyogenes to reach the intracellular state, and discusses the relationship between intracellular invasion and human disease. Intracellular invasion depends on at least two classes of surface proteins, the M proteins and fibronectin (Fn)-binding proteins. The function of these proteins in the context of intracellular invasion is described in the chapter. The best and most direct evidence that intracellular bacteria are an important source for dissemination of streptococci and the cause of recurrent tonsillitis is based on microscopic studies of surgically excised tonsils. Invasins are a subclass of bacterial adhesin molecules required for ingestion by host cells. Typically, invasions are proteins expressed on the surfaces of bacterial cells that directly or indirectly recognize specific host cell receptors. The internalization by a zipper mechanism is mediated by interactions between surface invasins, ligands, and host cell receptors. Studies of streptococcal-induced signal-transduction pathways have focused on two major invasins and Fn-binding proteins, SfbI/PrtF1 and M1 protein. The spectrum of target host cells, invasion efficiency, and requirement for serum agonists is determined by the extracellular matrix-binding proteins displayed on the bacterial surface. No single invasin or surface protein accounts for high-efficiency invasion of epithelial cells by all strains of streptococci. These findings suggest that intracellular invasion may be triggered by different agonists in different tissues or at different stages of infection.

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• Chapter 4 : Capsular Polysaccharide of Group A Streptococci

  Author: Michael R. Wessels
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    Careful observations by microbiologists and clinicians in the 1920s and 1930s pointed to a link between the hyaluronic acid capsule and group A streptococci (GAS) disease pathogenesis. More recent studies have defined the genetic locus that directs hyaluronic acid biosynthesis and have characterized the molecular mechanisms through which the capsule enhances GAS virulence. It is now appreciated that the has capsule synthesis operon is both highly conserved and widely distributed among GAS strains, attesting to the adaptive role served by the hyaluronic acid capsule in the coevolution of GAS with the human host. As a poorly immunogenic “self” antigen, the capsular polysaccharide appears to have persisted in an invariant form in GAS. Studies of acapsular mutant strains have demonstrated that the capsule protects GAS from complement-mediated phagocytic killing and is essential for full virulence in a variety of experimental infection models. The GAS capsule also influences attachment of the bacteria to human epithelial cells, both by modulating interaction of M protein and other potential adhesins and by itself serving as a ligand for attachment of GAS to CD44 on epithelial cells. The successful adaptation of GAS to survival in the human host involves regulation of capsule expression that is dependent on the fine structure of the has operon promoter, on the CsrRS two-component regulatory system, and, likely, on additional mechanisms yet to be uncovered.

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• Chapter 5 : Toxins and Superantigens of Group A Streptococci

  Authors: John K. McCormick, Marnie L. Peterson, Patrick M. Schlievert
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    Streptococcus pyogenes (group A streptococcus) is a remarkable and versatile human bacterial pathogen that is capable of producing an impressive arsenal of both surface-expressed and secreted virulence factors. This pathogen continues to generate significant morbidity by causing a variety of uncomplicated human diseases such as pharyngitis and skin infections, and more serious diseases such as acute rheumatic and scarlet fevers. In addition, group A streptococci cause some of the most devastating bacterial diseases known, such as necrotizing fasciitis/myositis and the streptococcal toxic shock syndrome (STSS). This chapter focuses on the true exotoxins of group A streptococci with regard to their structure, function, and genetics, as well as their roles in the pathogenesis of human disease. The streptococcal superantigens belong to a larger group of structurally conserved exotoxins that are also produced by coagulase-positive staphylococci. Some group C and group G beta-hemolytic streptococci also produce these toxins. The exotoxins discussed in the chapter are streptococcal pyrogenic toxin type B (SpeB, or cysteine protease), streptococcal pyrogenic toxin type F (SpeF, or mitogenic factor), streptococcal cytolytic toxins, streptokinase, and streptococcal inhibitor of complement (Sic). It has been reported that uncharacterized Spes exist, and they have also been detected in certain group B, C, F, and G streptococcal strains that may mimic the role of the group A streptococcal Spes.

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• Chapter 6 : Genetics of Group A Streptococci

  Authors: Kyu Hong Cho, Michael Caparon
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    Rapid progress has been made in recent years in the development of sophisticated techniques for genetic analysis in Streptococcus pyogenes (the group A streptococcus). Much of this effort has been directed at the development of methods for the mutagenesis of known genes. A key element of any genetic system involves some system of genetic exchange between different bacterial hosts which allows the construction of an altered genome in the target host which can then be subjected to an analysis of its virulence phenotypes. Conjugative DNA transfer does occur in group A streptococci; however, this is restricted to the transfer of conjugative plasmids and conjugative transposons, and there is no evidence for mobilization of chromosomal markers. Much progress has also been made in the development of strategies for the identification of novel genes. It is likely that the widespread application of these techniques to the virulence properties of S. pyogenes will enrich our understanding of streptococcal pathogenesis with insight at the molecular level and will help to establish and clarify the contributions of specific genes. Additional use and development of methods for analysis of gene expression and heterologous expression will continue and allow analyses of virulence factors at much higher levels of resolution than previously possible.

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• Chapter 7 : Cross-Reactive Antigens of Group A Streptococci

  Author: Madeleine W. Cunningham
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    Cross-reactive antigens are molecules on the group A streptococcus that mimic host molecules and during infection induce an immune response against host tissues. The identification of cross-reactive antigens in group A streptococci is important in the understanding of the pathogenesis of autoimmune sequelae, such as rheumatic fever and glomerulonephritis, which may occur following group A streptococcal infection. Monoclonal antibodies (MAbs) cross-reactive with group A streptococci and human heart tissues were produced from mice immunized with streptococcal cell wall and membrane components and from rheumatic carditis patients. The cross-reactive antibodies were divided into three major subsets based on their cross-reactivity with (i) myosin and other alpha-helical molecules, (ii) DNA, or (iii) N-acetylglucosamine. All three subsets were identified among MAbs from mice immunized with group A streptococcal components, but in the human, the predominant subset reacted with the N-acetyl-glucosamine epitope and myosin and related molecules. This chapter provides more evidence about the identification and analysis of the cross-reactive antigens of the group A streptococcus. It also discusses immune responses to N-acetyl-β-d-glucosamine, dominant epitope of group a polysaccharide, in the pathogenesis of rheumatic heart disease and sydenham chorea in acute rheumatic fever.

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• Chapter 8 : Extracellular Matrix Interactions with Gram-Positive Pathogens

  Authors: Gursharan S. Chhatwal, Klaus T. Preissner
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    This chapter discusses the interaction of extracellular matrix (ECM) components with gram-positive pathogens. It first describes the structure and function of ECM, and then deals with the interactions of gram-positive pathogens with ECM components and their biological consequences. The chapter explains binding of collagen, laminin, elastin, fibronectin, vitronectin, fibrinogen, and thrombospondin, to gram-positive bacteria in detail. Adherence and invasion are the important disease-causing mechanisms of gram-positive pathogens. As interaction of these pathogens with the components of host ECM is involved both in adherence and in invasion, the underlying mechanisms of this interaction are of utmost importance for designing novel therapeutic strategies. A classic example of the potential use of bacterial cell surface components interacting with ECM is documented with fibronectin-binding protein SfbI from Streptococcus pyogenes. This and other examples clearly demonstrate the potential use of components involved in the interaction of ECM and gram-positive pathogens and justify further research in this field.

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• Chapter 9 : Streptococcus-Mediated Host Cell Signaling

  Author: Vijay Pancholi
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    This chapter provides information on host cell-signaling events induced by streptococci. With the availability of complete genome sequence analyses of five group A Streptococcus (GAS) strains, including M1, M3 (two strains), M18, and M6, it is easy to predict the number of surface proteins, which may serve as potential adhesins during initial interactions with host cells. Pathogenic gram-positive cocci through their surface proteins interact with specific receptors on the target cell and induce a series of biochemical signals. These signals, which are characterized by the induction of phosphokinase enzymes and phosphorylation of several intracellular proteins, ultimately target the nucleus and lead to either generalized or specific gene activation. Activation of some of these genes may result in the modulation of interleukin or cytokine expression, which may then initiate a proinflammatory response. These induced signals, and subsequent products, could have several effects on the invasion of bacteria. For example, these induced signals may modulate cytoskeletal structure and/or specific host cell receptor expression or may destroy adjoining cells and disrupt natural protective barriers in autocrine or paracrine modes. This, in turn, could facilitate bacterial entry.

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• Chapter 10 : Vaccine Approaches To Protect against Group A Streptococcal Pharyngitis

  Author: Vincent A. Fischetti
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    Streptococcus pyogenes (Lancefield group A) is a human pathogen responsible for a wide range of diseases, the most common of which are nasopharyngeal infections and impetigo. About 3% of individuals with untreated or inadequately treated streptococcal pharyngitis develop rheumatic fever and rheumatic heart disease, a sequela of the streptococcal infection resulting in cardiac damage, particularly to the mitral valve. This chapter concentrates on the progress to date toward the development of a vaccine to protect against streptococcal nasopharyngeal infection. It was shown more than 50 years ago that the surface M protein would be a prime candidate for a vaccine to protect against streptococcal infection. The chapter describes type-specific protection, multivalent type-specific vaccine, mucosal vaccine for non-type-specific protection, passive protection, active immunization with conserved-region peptides, and the use of gram-positive commensals as vaccine vectors. In addition, it discusses non-M-protein approaches to protect against streptococcal infection. Perhaps a combination vaccine incorporating the serum opsonic power of the polyvalent type-specific approach combined with the mucosal protection offered by a mucosal vaccine will ultimately be the best way to control all pathogenic aspects of a streptococcus.

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• Chapter 11 : The Bacteriophages of Group A Streptococci

  Author: W. Michael McShan
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    This chapter focuses on the influence that the bacteriophages of Streptococcus pyogenes, both lytic and lysogenic or temperate, have on the biology and dissemination of virulence factors of this important gram-positive pathogen. The lytic bacteriophages infect their specific host bacterium, replicate their genome and assemble new virions, and then rupture the host to release the newly formed phages. Lytic phages can play important roles in the shaping of the biology of their GAS hosts, through elimination of the phagesusceptible members of a population consisting of more than one strain, selection for the rare phage-resistant variants in a mostly homogenous population, or by being the vectors of genetic exchange through generalized transduction. Although the role played by lytic streptococcal phages in pathogenesis may be indirect, acting as vehicles of genetic exchange through generalized transduction, lysogeny by GAS bacteriophages can directly enhance the pathogenic potential of the host streptococcus through toxigenic conversion. The allelic variation is more than would be expected to result from accumulated random mutations between genetically isolated individuals. Because transduction is the only known natural means of genetic exchange, it is likely that this mechanism and its associated bacteriophages play an important role in the genetic shifts seen in GAS. A better understanding of streptococcal transduction may prove key to understanding the flow of genetic information in natural populations of GAS and the horizontal transfer of information from other genera.

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• Chapter 12 : Molecular Epidemiology, Ecology, and Evolution of Group A Streptococci

  Authors: Debra E. Bessen, Susan K. Hollingshead
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    Within a bacterial species, there are often strains that differ from another in important biological properties. This is certainly the case for Streptococcus pyogenes (i.e., group A streptococci; GAS), whose members can cause a wide variety of human diseases, yet there does not appear to exist a single omnipotent strain. To better understand strain differences and their relevance to human disease, stable markers have been identified within organisms of this species. Epidemiological markers are useful for investigating outbreaks of disease, and they can also provide a reference point for deciphering the genetic organization of a bacterial population. The epidemiology of a microbial disease is often, in large part, a reflection of the ecology and evolution of the causative agent. For a bacterial species whose world is largely confined to the human population, the selective pressures that most profoundly shape its genetic structure are intrinsic to the human condition. Local microenvironmental conditions can shift during the course of infection within a single host. Also, ecological conditions can vary widely from host to host, and not all exposed hosts are susceptible to infection by a given strain. From the evolutionary standpoint, invasive disease is a dead end for the infecting GAS organism because the severely ill patient becomes immobilized; thus, opportunities for its transmission to new hosts are diminished.

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GROUP B STREPTOCOCCI

• Chapter 13 : Pathogenic Mechanisms and Virulence Factors of Group B Streptococci

  Authors: Victor Nizet, Craig E. Rubens
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    Although group B streptococci (GBS) commonly colonize the lower gastrointestinal tract and vaginal epithelium of healthy adults, they remain a potentially devastating pathogen to susceptible infants. As the newborn is quantitatively and qualitatively deficient in host defenses, including phagocytes, complement, and specific antibody, an environment exists in which a variety of potential GBS virulence factors are unveiled. The complex interactions between the bacterium and the newborn host that lead to disease manifestation can be divided into several important categories. This chapter reviews GBS pathogenic mechanisms involved in adherence to epithelial surfaces, cellular invasion of epithelial and endothelial barriers, direct injury to host tissues, avoidance of immunologic clearance, and induction of the sepsis syndrome. Special attention is focused on recent molecular genetic discoveries, including the sequencing of three complete GBS genomes, which have led to the identification of specific virulence determinants implicated in the pathogenesis of newborn infection.

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• Chapter 14 : Surface Structures of Group B Streptococci Important in Human Immunity

  Authors: Lawrence C. Madoff, Lawrence C. Paoletti, Dennis L. Kasper
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    The seminal findings by Rebecca Lancefield and coworkers on the role of carbohydrate and protein antigens in group B streptococcus (GBS) immunity have led a generation of researchers not only to a better understanding of these antigens in immunity but also toward the development of effective vaccine. This chapter highlights critical advances in our understanding of the role(s) of GBS surface antigens (namely, the group B carbohydrate, the type-specific capsular polysaccharides [CPSs], and proteins) in immunity and their application as components of experimental vaccines. With few exceptions, all strains of GBS isolated from humans are encapsulated and can be classified on the basis of serology and CPS structure. Nine distinct GBS serotypes have thus far been identified: Ia, Ib, II, III, IV, V, VI, VII, and VIII. In the past, serotypes Ia, Ib, II, and III were equally prevalent in normal vaginal carriage and early-onset sepsis. The protein antigens of GBS discussed are alphalike proteins, beta C protein, and other surface proteins.

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• Chapter 15 : Epidemiology of Group B Streptococcal Infections

  Authors: Anne Schuchat, Sharon Balter
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    Although group B streptococcus (GBS) is commonly thought of as a cause of disease in neonates and pregnant women, it causes substantial morbidity and mortality among nonpregnant adults, and appears to be increasing in incidence in that population. The epidemiology of GBS endocarditis has undergone a shift since the preantibiotic era. Previously, endocarditis was one of the most common syndromes caused by GBS infection, but GBS endocarditis is much rarer today. Many pregnant women are colonized with GBS but are asymptomatic. However, GBS colonization in pregnant women is important because of the risk for transmission to their newborns. Infections in newborns commonly present as bacteremia, meningitis, or pneumonia. There are two distinct syndromes: early-onset disease (EOD), which appears in the first week of life, usually within the first 24 h, and late-onset disease (LOD), which occurs on or after 7 days of age. Risk factors for late-onset GBS disease have recently been characterized, though they are less well understood than those for EOD. A recent hospital matched case-control study of risk factors for LOD conducted in Houston identified prematurity as the major risk factor for late-onset GBS disease, with maternal GBS colonization and black race as other independent factors associated with higher risk of LOD. Development of a vaccine against GBS may provide a better long-term solution than chemoprophylaxis. A vaccine could be given to women during pregnancy or to adolescent girls; transfer of antibodies across the placenta late in pregnancy would confer protection to the newborn.

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GROUP C AND G STREPTOCOCCI

• Chapter 16 : Genetics and Pathogenicity Factors of Group C and G Streptococci

  Author: Horst Malke
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    The application of recombinant DNA techniques has advanced one's understanding of group C streptococci (GCS) and group G streptococci (GGS) in diverse areas, and this chapter concentrates on the structure and function of pathogenetically relevant genes and proteins studied at the molecular level in recent years. On the basis of 16S rRNA comparative sequence analysis, GCS and GGS fall into two species groups, the pyogenic and the anginosus group; the latter is also known as “Streptococcus milleri” group. One hallmark of the gram-positive pathogens is the synthesis of specific cell wall-associated proteins that enable them to interact in various ways with proteins present in the body fluids or extracellular tissue matrix of their mammalian hosts. Such interactions may facilitate colonization, lead to molecular host mimicry, or interfere with various host defenses against invasion. The cell wall-associated proteins discussed are M and M-like proteins, immunoglobulin G (IgG)-binding proteins, fibronectin-binding proteins, and plasmin(ogen)-binding proteins. Other covered topics are cytoplasmic membrane-associated enzymes such as hyaluronan synthase and cytoplasmic membrane lipoprotein acid phosphatase, and extracellular proteins. The chapter ends with a discussion on the stringent and relaxed responses of Streptococcus dysgalactiae subsp. equisimilis.

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• Chapter 17 : Pathogenicity Factors in Group C and G Streptococci

  Authors: Gursharan S. Chhatwal, David J. McMillan, Susanne R. Talay
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    Group C and G streptococci constitute a heterogeneous complex of streptococcal species that reside as apathogenic commensals in humans and animals or act as causative agents of severe infection and organ damage associated with high mortality rates. This chapter gives a short overview of the various group C and group G streptococcal species, the diseases they cause, and the major pathogenicity factors that contribute to the virulence of these organisms. It discusses the major factors that enable group C and G streptococci to infect their hosts and cause disease. Such factors include adhesive structures that initiate the infection process, antiphagocytic factors that enable the bacterium to evade the host’s immune system, factors that are potentially involved in spreading in tissues, and factors that specifically bind, degrade, or damage host components. Virulence factors that enable these bacteria to colonize the host, avoid immune responses, and cause disease have been characterized in some detail in Streptococcus dysgalactiae and S. equi. Analysis of the bacterial factors that specifically interact with components of the host has allowed insight into the biochemical principles as well as some functional strategies of these streptococci; analysis has also revealed interesting evolutionary aspects, including convergent development, horizontal spread, and module shuffling. As group C and group G streptococci-associated diseases are coming under greater scrutiny it has become apparent that in many instances the virulence factors of these species have close homologues in S. pyogenes.

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• Chapter 18 : Group C and Group G Streptococcal Infections: Epidemiologic and Clinical Aspects

  Authors: Gio J. Baracco, Alan L. Bisno
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    Streptococci possessing Lancefield group C and G cell wall carbohydrates are heterogeneous in regard to biochemical reactions, hemolytic characteristics, predilection for host species, and clinical illnesses produced in humans and animals. These organisms are found as commensals in the throat, skin, and occasionally the female genitourinary tract, and their epidemiologic patterns and clinical manifestations reflect this distribution. This chapter focuses on the more common infections caused by Streptococcus dysgalactiae subsp. equisimilis, as well as on the few reported human cases caused by S. equi subsp. zooepidemicus, S. equi subsp. equi, S. dysgalactiae subsp. dysgalactiae, and S. canis. Strains of these streptococci have been associated with infections of many body sites. Treatment with penicillin is adequate under most circumstances.

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SREPTOCOCCUS PNEUMONIAE

• Chapter 19 : The Cell Wall of Streptococcus pneumoniae

  Authors: Alexander Tomasz, Werner Fischer
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    This chapter focuses on biochemical and genetic aspects of covalently linked components of the pneumococcal cell wall. Historically, studies of the pneumococcal cell wall were motivated by such unique features as the presence of choline in the teichoic acids (TAs), the pleiomorphic changes that accompany removal or alteration of choline residues, and structural changes in peptidoglycan that are associated with penicillin resistance. Most recent contributions to the field include immunofluorescence microscopic localization of cell wall synthetic enzymes at sites of wall synthesis, identification of genetic determinants and enzymes that are involved with the synthesis of muropeptide branches, sortase-dependent attachment of proteins, removal of N-acetyl groups from N-acetyl hexosamine residues in the cell wall glycan chains, and removal of phosphoryl choline residues from TAs. The chapter discusses functional anatomy of the Streptococcus pneumoniae cell wall. A considerable clarification concerning determinants of cell wall structure and its relationship to penicillin resistance was obtained by the recent identification of the murMN operon, which encodes enzymes involved in the synthesis of branched structured muropeptides in the pneumococcal peptidoglycan. Choline is an essential growth factor for all natural isolates of pneumococci, which have to import this nutrient from the growth medium. In this hypothesis, the nutritional requirement for choline resides in a recognition site of the transferase for phospho-amino-alcohols on TA, which may have been altered in the choline-independent strains.

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• Chapter 20 : Streptococcus pneumoniae Capsular Polysaccharide

  Authors: James C. Paton, Judy K. Morona
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    This chapter summarizes the current state of knowledge on the capsular polysaccharide (CPS) of Streptococcus pneumoniae, with particular reference to the genes encoding biosynthesis of this most important of all pneumococcal surface antigens. The functions of many of the individual genes in the cps loci await confirmation by conventional biochemical and genetic analysis. Nevertheless, access to the enormous body of information now available on sequence databases, combined with knowledge of the chemical structures for many of the CPS repeat units, has enabled accurate predictions of function for a significant proportion of these genes. It has also been possible to predict the mechanisms of CPS biosynthesis in pneumococci by analogy with those operating in gram-negative bacteria. The existence of two distinct mechanisms for CPS biosynthesis in S. pneumoniae has already been recognized. However, much remains to be learned about the precise molecular events involved in both of these processes, and about how CPS production in pneumococci is regulated. Further biochemical and mutational analyses are also required to elucidate the precise functions of the four genes at the 5' end of the cps loci, which clearly encode important common steps in polysaccharide biosynthesis in pneumococci, as well as in other gram-positive genera. Given the importance of capsules to the virulence of S. pneumoniae and several other gram-positive pathogens, such conserved components of the CPS biosynthesis machinery may prove to be useful targets for novel antimicrobial strategies.

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• Chapter 21 : Streptococcus pneumoniae: Invasion and Inflammation

  Authors: Carlos J. Orihuela, Elaine Tuomanen
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    Streptococcus pneumoniae (the pneumococcus) is the leading cause of otitis media (OM), community-acquired pneumonia, and bacterial meningitis. Pneumococcal models of invasive disease must account for the commensal nature of the bacteria, yet also take into account the wide spectrum of disease the pneumococcus is capable of causing. This chapter first reviews the molecular mechanisms that allow the pneumococcus to colonize and spread from one anatomical site to the next. Then, it discusses the mechanisms of inflammation and cytotoxicity during pneumococcal infection.

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• Chapter 22 : Phase Variation of Streptococcus pneumoniae

  Author: Jeffrey N. Weiser
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    Streptococcus pneumoniae undergoes spontaneous, reversible phenotypic variation, or phase variation, which is readily visualized as differences in colony morphology. This chapter describes phase variation in S. pneumoniae, the pneumococcus, and characterizes its relationship to colonization and the pathogenesis of infection. In particular, the focus is on the identification of variably expressed cell surface components as a means of gaining insight into the pathogenesis of pneumococcal disease at a molecular level. S. pneumoniae is highly proficient at colonization of its human host. The pneumococcus has the capacity to thrive in a number of diverse host environments, including the bloodstream and the mucosal surface of the nasopharynx. As is the case for other respiratory tract pathogens that frequently cause invasive infection, the ability of the pneumococcus to adapt to these varied environments requires changes in the expression of specific cell surface molecules.

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• Chapter 23 : Genetics of Streptococcus pneumoniae

  Authors: Janet Yother, Susan K. Hollingshead
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    The origins of genetics in Streptococcus pneumoniae can be traced to studies that began in the late 1800s with the isolation of nonencapsulated variants and ultimately led to the discovery of bacterial gene transfer by Griffith in 1928 and the identification of DNA as the genetic material by Avery, MacLeod, and McCarty in 1944. This chapter highlights much of the current information regarding S. pneumoniae genetics. Both transformation and conjugation have been described in S. pneumoniae. Transformation serves as the primary, and perhaps sole, means of transferring chromosomal genes. Conjugation occurs with plasmids that are capable of self-transfer or mobilization and with conjugative transposons that are integrated into the chromosome. S. pneumoniae can serve as both a donor and recipient in the conjugation of plasmid DNA and conjugative transposons. Generalized transduction has not been observed to occur, although a process termed pseudotransduction, which involves properties of both transduction and transformation, has been described for one pneumococcal bacteriophage. Some important similarities between many of the phages have been noted, and the complete nucleotide sequences have been determined for the genomes of phages Cp-1, Dp-1, EJ-1, and MM1. Replicating plasmids can be introduced into S. pneumoniae by electroporation. Unlike plasmids taken up via the natural transformation pathway, they are subject to restriction by the Dpn system, indicating that double-stranded DNA is transformed. The chapter discusses promoter activity of S. pneumoniae and the generation and analysis of mutants.

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• Chapter 24 : Pneumococcal Vaccines

  Authors: D. E. Briles, J. C. Paton, E. Swiatlo, M. J. Crain
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    This chapter provides a summary of issues critical to the development and application of pneumococcal vaccines. In the preantibiotic era, vaccination attempts utilized whole killed pneumococci injected parenterally. Although such vaccines were sometimes protective in humans, they were also highly reactogenic. These killed vaccines were mainly used to elicit antibody in animals for passive treatment of infected humans. In 1933 it was clearly demonstrated that antibody to type-specific capsular polysaccharides (PS) could be highly protective. However, it soon became apparent that different strains of Streptococcus pneumoniae each expressed one of many different PS. Most subsequent vaccine attempts focused on the use of mixtures of the isolated PSs to elicit protection. However, the inability of young children to make adequate responses to most soluble PS led to the development and licensing of an immunogenic PS-protein conjugate vaccine for children. The problem of poor vaccine immunogenicity in children is being addressed by conjugation of the PS to protein carriers, thereby converting the PS from T-cell-independent to T-cell-dependent antigens. These antigens include the pneumococcal surface protein PspA; autolysin (lytA), an enzyme on the pneumococcal cell wall; and pneumolysin, a cytoplasmic protein that is released when pneumococci are autolyzed.

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ENTEROCOCCI

• Chapter 25 : Pathogenicity of Enterococci

  Authors: Lynn E. Hancock, Michael S. Gilmore
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    This chapter focuses on the pathogenic mechanisms by which enterococci cause human disease. It discusses biology, epidemiology, and environmental persistence of the enterococci. For enterococci to cause disease several barriers must first be overcome. An initial barrier that these organisms confront is the ability to colonize the intestinal tract, where they must compete for nutrient resources in an intestinal milieu including several hundred unique bacterial species. From the site of colonization, the organism must translocate to infectious sites, evade host clearance, and ultimately produce pathologic changes in the host through direct toxic activity, or indirectly by inducing an inflammatory response. Our understanding of how these organisms continually evolve will need to keep pace with their evolution or we may potentially be on the wrong end of a public health nightmare, in which isolates evolve to higher levels of antibiotic resistance and ever increasing virulence.

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• Chapter 26 : Enterococcal Genetics

  Author: Keith E. Weaver
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    The majority of interest in enterococcal genetics has been generated in response to three landmark discoveries: (i) identification of the first conjugative plasmids whose transfer systems are induced by an identifiable signal, (ii) identification of the first "transposons" capable of intercellular (conjugative) transposition, and (iii) the acquisition of vancomycin resistance. Because the most prevalent vancomycin resistance genes are located on plasmids and transposons, most work on enterococcal genetics has focused on mobile genetic elements. Examination of the complete sequence of a vancomycin-resistant clinical isolate of Enterococcus faecalis reaffirmed the importance of such elements in the evolution of this species, revealing that over a quarter of the genome consists of mobile and/or exogenously acquired DNA. However, understanding of the basic mechanisms of DNA replication and repair, chromosomal segregation, cell division, and transcription in this genus remains limited. This chapter provides a review of what is known or can be discerned from the genome sequence about these basic genetic mechanisms. Next, it focuses on the known mobile genetic elements, which seem to play such a significant role in the evolution of the enterococci.

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ORAL STREPTOCOCCI

• Chapter 27 : Pathogenesis of Oral Streptococci

  Author: R. R. B. Russell
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    Species of streptococci are well represented among the bacteria found in the oral cavity, which has been estimated to harbor around 500 different species of bacteria, though there remain many taxa of uncertain status and many microscopically observable microbes that have not yet been isolated in laboratory culture. These oral streptococci seem to be ubiquitous among all the human populations studied. When they have been sought, identical or closely related streptococci have also been found in a wide variety of animal species, so streptococci are clearly part of the normal commensal flora of mammals; this chapter considers the problems that arise when this commensal relationship breaks down and the oral streptococci become opportunistic pathogens. The chapter talks about acquisition of oral streptococci, mechanism of colonization, immunological processes in the mouth, and metabolism of dental plaque. The oral streptococci are normal commensals of the human mouth and as such play a beneficial role in colonization resistance, excluding potentially pathogenic species oral streptococci as pathogens, systemic infections, virulence factors of oral streptococci.

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• Chapter 28 : The Virulence Properties of Streptococcus mutans

  Author: Howard K. Kuramitsu
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    This chapter focuses on virulence properties of Streptococcus mutans. Using primarily biochemical approaches, it was established almost thirty years ago that three principal properties distinguished S. mutans strains from the other oral streptococci isolated from the human oral cavity: (i) their ability to synthesize insoluble adhesive glucans from sucrose; (ii) their relative acid tolerance (aciduricity); and (iii) their rapid production of lactic acid from dietary sugars. Furthermore, the importance of these properties relative to cariogenicity was subsequently confirmed utilizing genetic approaches with defined mutants and rat model systems. The development of recombinant DNA techniques as well as gene inactivation strategies was crucial in this regard. These approaches identified a number of genes of the mutans streptococci that influenced the virulence of these organisms, including the gtf genes coding for glucosyltransferases (Gtfs), the gbpA and gbpC genes encoding glucan-binding proteins, spaP expressing a cell surface adhesion, and the glgR gene involved in intracellular polysaccharide storage. In addition, a number of other genes that have been shown to affect potential virulence properties in vitro were also characterized, including some involved in the stress responses of S. mutans (ffh, dgk, gbpB, and an apurinic-apyrimidinic endonuclease gene).

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• Chapter 29 : Genetics of sanguinis Group Streptococci

  Authors: Howard F. Jenkinson, M. Margaret Vickerman
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    Historically, the characteristics of sanguinis group organisms that have been most studied are competence for DNA-mediated transformation and adhesion to saliva-coated surfaces. With the recent completion of the genome sequence of Streptococcus gordonii Challis (CH1), and the recognition of streptococci as critical components in the development of oral biofilms, the main focus of this chapter is to consider the genomics, genetic control, and molecular mechanisms of processes associated with intercellular communication (mediated by peptide pheromones and quorum-sensing molecules), adhesion to surfaces, and host colonization. Upon gaining access to the blood stream, sanguinis group streptococci can infect the heart valves and endocardium. Strains of sanguinis group streptococci vary in their abilities to adhere to platelets and to induce platelet aggregation. Genetic studies of sanguinis group streptococci have significantly advanced one's understanding of the processes involved in transformation, adhesion, and virulence. This knowledge will ultimately lead to new ways of controlling infections caused by these bacteria and by related streptococci. Paradoxically, it is conceivable that organisms such as S. gordonii may, in the future, be considered therapeutic products in novel strategies to combat other infectious diseases.

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LACTOCOCCI

• Chapter 30 : Genetics of Lactococci

  Authors: Philippe Gaudu, Yuji Yamamoto, Peter Ruhdal Jensen, Karin Hammer, Alexandra Gruss
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    This chapter presents current information in different areas of lactococcal genetics, keeping in mind (where possible) pertinence of findings to related pathogens. It highlights major recent work in lactococci, including surprising metabolic capacities, physiology, stress response, and studies leading to novel successful uses of lactococci for protein delivery. Lactococcal metabolism has been intensively studied for its industrial importance in fermentation processes, with a focus on metabolic pathways and their engineering. However, basic metabolic functions may have far-reaching effects, metabolic shifts can result in dramatic changes in Lactococcus lactis growth characteristics and survival. Researchers confirmed and developed a 1970 study showing that lactococci not only ferment sugars, but are also capable of forming an active electron transport chain to generate respiration metabolism. Laboratory results demonstrate that respiration metabolism in lactococci is an efficient means of eliminating oxygen, compared to fermentation, leading to good survival in stationary phase. Some of the most spectacular applications of lactococci concern their use in "bioprotein" delivery. Some tools developed in L. lactis are adaptable to other gram-positive bacteria. The development of surface display systems in lactic acid bacteria (LAB) will be potentially useful in the development of oral vaccines based on the nontoxic LAB. As an organism present on plants, in milk, in dairy products, and in the gut, L. lactis may be the organism of choice for studies on the influence of environmental stress on evolution.

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• Chapter 31 : Diagnostics, Typing, and Taxonomy

  Authors: Richard P. Novick, Wolfgang Witte, Birgit Strommenger, Guido Werner
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    Speciation in bacterial systematics is based on a comparison of organismal characteristics in order to arrange microorganisms in groups sharing common properties. The purpose of speciation is identification of a microorganism as belonging to a basic taxon, which, for example, has a particular ecological or clinical significance. In general, the purpose of typing is the study of population dynamics and the spread of microorganisms that undergo clonal (nonsexual) reproduction. Thus typing is an important tool of epidemiology for tracing the spread of particular strains and discovering routes of transmission and reservoirs. Clumping factor causes agglutination in the presence of human plasma and is therefore very easy to detect. The accuracy of commercial slide agglutination tests has recently been improved by the addition of antibodies against capsular antigens, which provides sufficient sensitivity for speciation of methicillin-resistant Staphylococcus aureus (MRSA). The chapter talks about requirements for typing systems, and discusses phenotypic typing methods and genotyping methods applied to S. aureus. Polymorphisms detected by PCR can be based on genetic events taking place between the location of primer binding sequences, thus leading to different length of amplimers. Multilocus sequence typing (MLST) requires the highest workload and is not discriminatory enough for epidemiological typing, but is highly predicative in analysis of evolutionary relationships. Finally, the chapter discusses combined use of different molecular typing methods, and compares different genotypic typing systems.

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• Chapter 32 : The Staphylococcus aureus NCTC 8325 Genome

  Authors: Allison F. Gillaspy, Veronica Worrell, Joshua Orvis, Bruce A. Roe, David W. Dyer, John J. Iandolo
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    By focusing on the Staphylococcus aureus phage group III strain NCTC 8325, Pattee’s laboratory utilized transduction and transformation to identify a series of linkage groups that were organized into a rough genetic map. Once physical techniques for genome mapping (restriction endonuclease digestion and pulsed field gel electrophoresis) became feasible, the researcher and his coworkers attempted to fit the genetic linkage data to the physical maps of the genome. The genome sequence and annotation are now complete and are the main focus of this chapter. In addition to strain NCTC 8325, the genome sequence and annotation for at least six other S. aureus strains (COL, N315, Mu50, MW2, MRSA252, MSSA476) have been completed in recent years. The complete circular genome map of NCTC 8325 shows the position of each predicted open reading frame (ORF) within the genome and the predicted functional role for each coding sequence. The current map for the NCTC 8325 genome shows the position of each of the 2,892 ORFs designated by color based on their predicted functional roles. Strain 8325 appears to contain components of at least nine potential insertion elements (IS). S. aureus NCTC 8325 appears to contain fewer of the previously identified genomic islands, pathogenicity islands, or transposons than other sequenced S. aureus strains.

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• Chapter 33 : Genetics: Accessory Elements and Genetic Exchange

  Authors: Neville Firth, Ronald A. Skurray
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    It is clear that the acquisition, maintenance, and dissemination of accessory elements have been central to the ongoing success of staphylococci as pathogens. Staphylococci represent a salient illustration of the adaptability afforded to microorganisms by access to additional functions through gene transfer mechanisms. Although DNA can be introduced into staphylococci in the laboratory via each of the three traditional bacterial gene transfer mechanisms—transformation, transduction, and conjugation—the latter two are believed to be the most significant mediators of natural genetic exchange. Most staphylococcal plasmids can be categorized as one of three main classes based on physical/genetic organization and functional characteristics, although another group, the pSK639 family plasmids, should be considered a fourth class. Staphylococcal insertion sequences and transposons are discussed in this chapter. Gene transfer mechanisms, together with accessory elements such as plasmids, transposable elements, prophages, and pathogenicity and resistance islands, serve as catalysts for microbial evolution by providing access to a shared reservoir of niche-adaptive functions. However, just as importantly, the combination of comparative genomics and sequence-based strain typing is clarifying the relationships between clinical Staphylococcus aureus strains. These studies are providing a new perspective on the scope and importance of the accessory genome, demonstrating that variation in pathogenic potential largely does not reside in the core genome, but rather that it is primarily attributable to the complement of mobile accessory elements that are present.

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• Chapter 34 : Carbohydrate Catabolism: Pathways and Regulation

  Authors: Reinhold Brückner, Ralf Rosenstein
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    The central pathways of carbon metabolism are conserved in virtually all organisms, but details of specific biosynthetic and degradative pathways vary considerably between bacteria, plants, and animals. In Staphylococcus aureus and other staphylococcal species, relatively few molecular details are known about carbohydrate utilization, biosynthetic pathways, and nutritional requirements. The limited knowledge on sugar utilization systems is especially surprising, because S. aureus was the first gram-positive bacterium in which the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS) was described. The phosphoryl-transfer chain begins with enzyme I (EI) and PEP and proceeds via the phosphocarrier protein HPr to the EIIA and EIIB domains of the PTS permeases. The uptake of glucose, mannose, mannitol, glucosamine, N-acetylglucosamine, sucrose, lactose, galactose, and β-glucosides is reported to be PTS-dependent. Glucose-6-phosphate, produced by a glucose kinase, enters the EMP pathway, the main glycolytic pathway in staphylococci. Utilization of lactose and galactose in S. aureus relies on the PTS-dependent uptake and phosphorylation of the sugars, resulting in lactose-6-phosphate and galactose-6-phosphate, respectively. The system consists of an EIICB enzyme, encoded by mtlA, and EIIA, encoded by mtlF, which together form the mannitol-specific PTS permease. The sucrose PTS permease, analyzed in S. xylosus and encoded by scrA, is composed of fused EIIBC domains. Maltose utilization in S. xylosus is dependent on an α-glucosidase or maltase, whose gene, malA, is the second gene of the malRA operon. The availability of carbohydrates, especially of glucose, leads to regulatory processes often referred to as glucose effect or carbon catabolite repression.

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• Chapter 35 : Respiration and Small-Colony Variants of Staphylococcus aureus

  Author: Richard A. Proctor
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    Staphylococcus aureus respiration and carbohydrate metabolism follow patterns very similar to those in Escherichia coli, with glycolysis in staphylococci following the nearly universal patterns found in all biological systems. Reactions that are favored in respiratory-deficient small-colony variants (SCVs) are shown in a table, and it is apparent that these variants are optimizing reactions that produce ATP and oxidize NADH by non-oxygen-dependent mechanisms. Although mutations in any essential gene can cause slow growth and small colonies, this chapter considers a subset of these mutants that was first observed in patients who had unusually persistent infections and were later found to have defects in oxidative metabolism and electron transport. This chapter focuses on multiple phenotypic changes associated with interrupted electron transport. It was also found that bacterial electron transport inhibitors Z69 and Z90 are able to reproduce the SCV phenotype. S. aureus JB-1 is a menadione auxotrophic SCV that was selected with gentamicin from its parent, S. aureus 6850. Some hemin auxotrophs selected by resistance to gentamicin have also been characterized in detail. Although many different mutations might produce slow growth, menadione and hemin auxotrophs predominate among clinical S. aureus SCV isolates. Finally, the chapter talks about instability of the SCV phenotype, SCVs and antibiotic resistance, and electron transport, respiration, and toxin production in S. aureus.

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• Chapter 36 : The Staphylococcal Cell Wall

  Author: Alexander Tomasz
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    The history of interest in the staphylococcal cell wall reflects the history of success and failure of the antibiotic era. Elucidation of the mode of action of several important antibiotics in the 1960s and 1970s has been intimately linked to studies on the biosynthesis of staphylococcal cell walls. In addition to the reemergence of interest in cell walls in the context of modern microbial cell biology, two approaches have been making great impact on discoveries in this field: the introduction of high-resolution analytical techniques (high-pressure liquid chromatography [HPLC] and mass spectrometry) and the increasing application of molecular genetic approaches. This chapter includes a brief reminder of the anatomy of staphylococcal cell walls, and reviews new information under four headings: high-resolution analysis of the Staphylococcus aureus peptidoglycan; variations in peptidoglycan composition; genetic determinants and enzymes in cell wall synthesis; and complex functions of cell walls. Unlike in streptococci, in S. aureus consecutive cell divisions occur in three division planes, each at right angles to one another, and proper orientation of cell wall septa must involve a complex and superbly controlled mechanism. Progress in the high-resolution chemistry of the S. aureus cell wall came from the introduction of HPLC and mass spectrometric methods for the analysis of the peptidoglycan.

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• Chapter 37 : Staphylococcal Capsule

  Authors: Chia Y. Lee, Jean C. Lee
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    This chapter focuses on major advances in staphylococcal capsule research. The cap5 and cap8 genes required for the synthesis of CP5 and CP8, respectively, have been cloned and characterized. The cap5(8) locus was replaced by IS257 in a small subset of bovine isolates of S. aureus from Argentina. Several findings reveal that lack of capsule expression in nontypeable (NT) Staphylococcus aureus can be explained by multiple mechanisms, and these data argue against the existence of capsule serotypes other than 1, 2, 5, and 8. The arl system has been shown to be a key regulator of autolysis and the synthesis of several S. aureus extracellular virulence factors. SigB is an S. aureus alternative stress sigma factor that regulates many staphylococcal genes, including some involved in bacterial virulence. A cap5O mutation created in S. aureus Reynolds rendered the bacterium negative for CP5; capsule expression was restored when cap5O was provided to the mutant in trans. Staphylococcal adherence to the damaged heart valve is critical to initiate infection in the endocarditis model of infection. The conjugate vaccines were highly immunogenic in mice and humans and induced antibodies that opsonized encapsulated S. aureus for phagocytosis.

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• Chapter 38 : Staphylococcus aureus Exotoxins

  Author: Gregory A. Bohach
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    This chapter talks about Staphylococcus aureus exotoxins fall into three general groups: (i) membrane-active agents, (ii) pyrogenic toxin superantigens (PTSAgs), and (iii) exfoliative toxins (ETs). Researchers proposed the existence of delta-toxin as the fourth cytolytic S. aureus toxin in 1947. Panton-Valentine leukocidin (PVL) and gamma-toxin are two prototypic bicomponent toxins. Unfortunately, the rapid rate of new toxin discovery has resulted in more than one SE being given the same designation in the literature. Therefore, it is now recommended that nomenclature for new PTSAgs be assigned by the International Nomenclature Committee for Staphylococcal Superantigens prior to publication. The major cytokines induced initially include IL-1, tumor necrosis factors alpha and beta, interferon-γ, and IL-2. The ETs have been conclusively implicated in staphylococcal scalded-skin syndrome (SSSS). Two antigenically distinct forms, designated ETA and ETB, are the best characterized ETs and are produced most frequently by phage group II by S. aureus isolates; strains expressing ETs constitute agr group IV staphylococcal isolates. Lesions in SSSS and mice are characterized by separation of stratum granulosa cells causing intraepidermal skin peeling.

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• Chapter 39 : Extracellular Enzymes

  Author: Staffan Arvidson
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    Staphylococcus aureus produces a large number of extracellular enzymes, many of which are regarded as important virulence factors. Some extracellular enzymes contribute to the virulence of staphylococci by attacking molecules involved in host defenses against infection. Searches in the genome sequences of several S. aureus strains identified 22 genes coding for extracellular enzymes. Coagulase production is the principal criterion used in the clinical microbiology laboratory for the identification of S. aureus. Several reports have indicated that site-specific inactivation of the coagulase gene does not impair virulence in experimental endocarditis, subcutaneous, or mammary infections of mice. The streptokinase-plasmin complex, on the other hand, is insensitive to inhibition by α2-antiplasmin. Staphylokinase (Sak) also has a higher affinity for plasmin(ogen) bound to fibrin than for free plasmin(ogen). Most lipases are also active against acyl p-nitrophenylesters, Tweens (polyoxyethylenesorbitan), and sometimes phospholipids. A role in virulence has also been suggested, based on the observation that staphylococcal lipase impairs granulocyte function. Staphylococcal abscesses contain long-chain free fatty acids and other neutral lipids that are bacteriocidal to S. aureus. Fatty acid-modifying enzyme (FAME), which is found in culture supernatants of about 80% of S. aureus strains, can inactivate these bacteriocidal lipids by catalyzing the esterification of these lipids to alcohols, preferably cholesterol. Hyaluronic acid is a ubiquitous component of the extracellular matrix of vertebrates. Extracellular enzymes that could hydrolyze hyaluronic acid were therefore among the first enzymes to be implicated in bacterial pathogenesis.

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• Chapter 40 : Staphylococcal Sortases and Surface Proteins

  Authors: Andrea C. Dedent, Luciano A. Marraffini, Olaf Schneewind
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    This chapter reviews what is known about surface proteins of Staphylococcus aureus, their mechanisms of anchoring to the cell wall envelope, and their contributions to the pathogenesis of staphylococcal infections. Protein A amino acid sequence, gene sequence, and three dimensional nuclear magnetic resonance and X-ray diffraction structures revealed a molecule comprised of five nearly identical Ig-binding domains as well as the molecular elements involved in binding Ig. S. aureus strains clump in the presence of plasma; this phenomenon, which has been exploited for diagnostic purposes, is the product of a molecular interaction between two microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), clumping factor A and B, with fibrinogen. Both S. aureus and S. epidermidis strains encode for multiple cell wall-anchored surface proteins with large serine-aspartate repeat (Sdr) domains. In addition to the subset of S. aureus sortase-anchored cell wall surface proteins that are covalently attached to the cell wall, there are also a number of surface proteins that lack a C-terminal cell wall sorting signal, yet remain in one way or another cell wall associated. The current model for the uptake of heme-iron by the Isd proteins states that IsdA, IsdB, and IsdH would interact with host hemoproteins such as hemoglobin (Hb), haptoglobin (Hpt), and/or hemopexin, which are released after erythrocyte lysis. The study and characterization of these and other cell wall-associated proteins has provided much insight into the understanding of the interactions of S. aureus with its environment.

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• Chapter 41 : Staphylococcal Pathogenesis and Pathogenicity Factors: Genetics and Regulation

  Author: Richard P. Novick
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    Several gene products have been implicated in internalization, and the function and regulation of these and other pathogenicity factors in the intracellular environment are discussed in this chapter. Traditionally, bacterial pathogenicity or virulence factors are products whose role in the disease process is either clearly demonstrable, e.g., toxins, or more or less obvious on the basis of biological properties, e.g., enzymes that degrade tissue components. The chapter outlines current understanding of genetics and regulation of staphylococcal virulon. Staphylococcal pathogenesis is multifactorial, involving three classes of factors: secreted proteins, including superantigens (SAgs), cytotoxins, and tissue-degrading enzymes; cell surface-bound proteins, including fibrinogen-binding protein, fibronectin-binding protein, collagen-binding protein, other adhesins, and antiopsonins; and cell surface components, including the polysaccharide capsule and components of the cell wall peptidoglycan. Considering first the genetics of staphylococcal virulence factors, there would appear to be two classes—those encoded by constant chromosomal genes, present in most or all strains, and those encoded by variable genes, present in a minority of strains, and usually belonging to accessory genetic elements, including plasmids, transposons, prophages, and pathogenicity islands (SaPIs), some of which are mobile. The first evidence for virulence gene regulation was the isolation of pleiotropic staphylococcal mutants defective in the production of hemolysins and other virulence factors. A table in the chapter gives a summary of the known genes and environmental conditions that affect the expression of pathogenicity factors by Staphylococcus aureus.

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• Chapter 42 : Staphylococcus aureus—Eukaryotic Cell Interactions

  Authors: Carlos Arrecubieta, Franklin D. Lowy
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    The pathogen Staphylococcus aureus causes a diversity of diseases that range from minor skin and soft tissue infections to life-threatening systemic infections. S. aureus-endothelial cell interactions have been the most extensively studied and are among the most important events in the pathogenesis of invasive systemic disease. While this chapter primarily focuses on S. aureus-endothelial cell interactions as a model of staphylococcal interaction with eukaryotic cells, reference is also made to more recent publications describing staphylococcal interactions with other cell types. S. aureus adherence to endothelial cells is the critical first step in the invasion process. It was demonstrated that staphylococci adhere to endovascular tissue and endothelial cells grown in tissue culture more avidly than do other bacterial species. In general, the bacterial species most commonly associated with acute bacterial endocarditis were also the most adherent. Variation in endothelial cell growth conditions altered adherence of staphylococci to endothelial cells. A variety of cellular changes occurs as a result of S. aureus invasion. Surface expression of proteins, such as Fc receptors and adhesion molecules, as well as secretion of cytokines, all occur in response to staphylococcal invasion. A prevailing concern regarding the in vitro observations of S. aureus invasion of eukaryotic cells has been the limited amount of supporting clinical and experimental in vivo data. S. aureus appears able to partially modulate the host cell-mediated immune response by eliciting or inhibiting its inflammatory response, which could account for differences in the outcomes of the infective process.

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• Chapter 43 : The Epidemiology of Staphylococcus Infections

  Authors: Fred C. Tenover, Rachel J. Gorwitz
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    This chapter describes some of the common illnesses caused by staphylococci, particularly Staphylococcus aureus. Colonization affords organisms, such as S. aureus, the opportunity to gain access to skin sites, which, when infected, can serve as a source for more serious diseases, such as bacteremia, endocarditis, or toxemias. Approximately one-half of all skin infections are caused by S. aureus. Infections include carbuncles, cellulitis, folliculitis, furuncles, hydradenitis suppurtiva, impetigo, mastitis, pyodermas, and pyomyositis. Staphylococcal osteomyelitis is classified as either acute or chronic. Acute hematogenous osteomyelitis is usually a disease of children, primarily neonates, in whom it affects the long bones of the lower extremity. Several staphylococcal diseases are mediated by toxins, including impetigo, food poisoning, necrotizing pneumonia, and toxic shock syndrome. Community-acquired pneumonia caused by S. aureus is not common but does occur, often as a consequence of influenza. Staphylococci are among the most common causes of health care-associated infections, including bacteremia, surgical site infections (SSIs), and pneumonia. Several reports suggest that the prevalence of S. aureus strains resistant to methicillin, oxacillin, or nafcillin is increasing in the United States and abroad and that such strains can cause outbreaks. Transmission of infection in health care settings requires three elements: a source of infecting microorganisms, a susceptible host, and a means of transmission for the microorganism. Contact transmission is the most important and frequent mode of transmission for S. aureus. Several antimicrobial regimens have been used to eradicate carriage of S. aureus.

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• Chapter 44 : Animal Models of Experimental Staphylococcus aureus Infection

  Authors: L. Vincent Collins, Andrzej Tarkowski
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    This chapter discusses experimental models of Staphylococcus aureus infections, including toxic shock, sepsis, endocarditis, colonization of joints and bones, mastitis, eye and skin infections, and septic arthritis. It concentrates on models that provide an insight into the pathogenesis of S. aureus. As an example of a model for studying staphylococcal disease, the chapter presents the murine model of septic arthritis and sepsis and discusses how models such as this might be used to formulate treatment and prophylaxis regimens. The various disease entities associated with S. aureus infections and some proposed animal models are listed in a table. Cytokines play a critically important role in the pathogenesis of S. aureus infection, and the modulation of specific cytokines is attracting substantial interest as a means of treating disease. The emergence of antibiotic-resistant staphylococci and in particular the methicillin-resistant S. aureus strains has stimulated a resurgence in the development of new antibiotics with antistaphylococcal activities and alternatives to classical antibacterial therapies. During the last decade, the use of experimental models of staphylococcal infections has clarified the involvement of several bacterial virulence factors as well as many hematopoietic cell types and their products in the pathogenesis of infection. Animal models that mimic the etiology, progression, and pathology of the disease in the natural host are a crucial component in the development of therapeutic strategies.

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• Chapter 45 : Cellular and Extracellular Defenses against Staphylococcal Infections

  Authors: Jerrold Weiss, Arnold S. Bayer, Michael Yeaman
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    Breaches of the skin and mucosal barriers greatly increase the likelihood of invasive staphylococcal infections, affirming the importance of these peripheral barriers in maintaining a normally asymptomatic host-bacterial relationship. Deficiencies in the mobilization or function of polymorphonuclear leukocytes (PMN) are associated with increased susceptibility to infection by many extracellular bacterial pathogens, including staphylococci. However, more recent studies have revived interest in secretion-based extracellular defenses against staphylococci and other gram-positive bacteria. Secreted antistaphylococcal agents may act alone, providing host defense against bacteria that resist or exceed phagocyte-based defenses, and may also act in concert with resident and mobilized phagocytes to increase antibacterial cytotoxicity of host defenses. In general, the action of PMN at extravascular sites of infection requires a highly regulated series of PMN responses resulting in the directed migration of PMN from blood to sites of infection, sequestration of bacterial prey, and intracellular cytotoxic action. Staphylococcal infections in chronic granulomatous disease (CGD) are overwhelmingly of extravascular nature. This is consistent with the retention of normal clearance function of phagocytes in this disease but also raises the possibility that other mechanisms of host defense against intravascular infections are operative. The acute inflammatory response mobilizes both PMN and extracellular antistaphylococcal activity at the site of bacterial invasion. The chapter focuses on the agents whose mechanism of action against S. aureus has been most extensively studied. These agents are group IIA phospholipase A2 (PLA2), platelet microbicidal proteins (PMPs) and platelet kinocidins, defensins, and cathelicidins.

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• Chapter 46 : Biology and Pathogenicity of Staphylococcus epidermidis

  Authors: Christine Heilmann, Georg Peters
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    This chapter deals with the current knowledge about Staphylococcus epidermidis. It especially focuses on the pathogenicity of S. epidermidis, the underlying biological properties, and how these properties contrast with those of S. aureus. The brief overview of the disease spectrum is given to place the studies on pathogenesis in perspective. The last part of the chapter deals with one ecological aspect, lantibiotics, which are potentially important for bacterial interference on skin and mucous membranes. Non-aureus staphylococci (NAS), particularly S. epidermidis, are among the most frequently isolated microorganisms in the clinical microbiology laboratory. The most important step in the pathogenesis of S. epidermidis foreign body-associated infectious diseases is the colonization of the polymer surface by the formation of multi-layered cell clusters, which are embedded in an amorphous extracellular material. The relationship of polysaccharide/adhesin (PS/A) to other polysaccharides of S. epidermidis is discussed in the chapter. The establishment of an infection and the survival of the bacteria in the host depend on the ability to invade host tissues and to evade host defense systems, respectively. Although studied in more detail with S. aureus, knowledge about the regulation of S. epidermidis virulence factors has been increased significantly in recent years. Lantibiotics are antibiotic peptides that contain the rare thioether amino acid lanthionine and/or methyllanthionine. Improvement in the armamentarium of molecular methods will enable us to analyze not only the genome but also the proteome of S. epidermidis in the future.

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• Chapter 47 : Biology and Pathogenicity of Staphylococci Other than Staphylococcus aureus and Staphylococcus epidermidis

  Authors: Anne Tristan, Gerard Lina, Jerome Etienne, François Vandenesch
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    This chapter focuses on species of non-aureus species of staphylococci (NAS) selected for their pathogenic potential and those for which recent discoveries have had a significant outcome on the general knowledge of staphylococcal biology. S. lugdunensis is a human commensal more pathogenic than most other NAS, causing primary infections of the human. S. saprophyticus is known as a frequent cause of acute urinary tract infection among female outpatients. S. haemolyticus is regarded as an emerging nosocomial pathogen with a tendency to develop antibiotic resistance. S. schleiferi is a rather genetically homogeneous species, since a very limited polymorphism is detected by pulsed-field gel electrophoresis (PFGE) or plasmid analysis. S. caprae was originally isolated from goat milk; it is the most prevalent NAS in mastitis-free goat milk and has not been isolated from cow or sheep milk. S. warneri has the usual characteristics of the NAS and can cause significant infection both in the community and in the hospital. S. pasteuri strains are phenotypically similar to S. warneri strains, but a clear-cut distinction between these two species may be obtained by comparing their rRNA gene restriction patterns. S. simulans has been isolated as a rare cause of human infection including urinary tract, wound, bone, and joint infections; vertebral osteomyelitis; septicemia; and native valve endocarditis. S. intermedius is the most important NAS that produces coagulase; it is, however, taxonomically distinct from S. aureus. It is the predominant coagulase-positive staphylococcus in the mouth and in skin infections of dogs.

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• Chapter 48 : Antibiotic Resistance in the Staphylococci

  Authors: Steven J. Projan, Alexey Ruzin
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    This chapter summarizes specific resistance mechanisms found in the staphylococci. When discussing resistance among the staphylococci, it is important to draw a distinction between community-acquired versus hospital-acquired (nosocomial) infections. Strains resistant to arsenicals and mercury were identified well before what is now known as the antibiotic era. From the genetic point of view, resistance falls into one of two classes: mutation of a bacterial gene or acquisition of a dedicated resistance gene from some other organism by some form of genetic exchange (transduction, conjugation, or transformation). In the history of antimicrobial chemotherapy, the most useful of antistaphylococcal agents have been the beta-lactam antibiotics, the prototype of which is penicillin. These agents, which include several structural classes, all contain one common structural feature: the beta-lactam ring. Rifampin, a member of the rifamycin class of antibiotics, inhibits transcription by attacking the beta-subunit of RNA polymerase. The fluoroquinolone antimicrobials are one of the few classes of antibacterial agents that are not based on a natural product. Sulfonamide resistance in the staphylococci, which arose soon after the introduction of the sulfa drugs, is chromosomally encoded (by the sulA gene) and is attributed to the overproduction of p-aminobenzoate. Mupirocin (formulated with the trade name Bactroban) has come into wide use as a topical agent for the treatment of gram-positive infections and more recently has been employed successfully to treat nasal carriers of methicillin-resistant S. aureus (MRSA), especially those in chronic care settings (e.g., nursing homes) and hospital staff.

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• Chapter 49 : Epidemiology and Clinical Manifestations of Listeria monocytogenes Infection

  Author: Walter F. Schlech, III
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    Enhancement of organism specific virulence factors may play a role in epidemic disease, although all isolates of Listeria monocytogenes have the constitutive ability to produce all the virulence factors characteristic of the species. The current understanding of the epidemiology of human listeriosis suggests that the organism is a common contaminant of food products and that ingestion of small numbers of L. monocytogenes occurs frequently in human populations. A wide variety of clinical syndromes have been associated with L. monocytogenes infection in both animals and humans. The clinical syndromes associated with listeriosis in humans have been more recently elucidated. Autopsy findings in cases of early-onset listeriosis show significant chorioamnionitis in placental remnants and granulomas in multiple organs, particularly the liver and spleen, of infected infants. L. monocytogenes is an uncommon cause of bacterial meningitis in adults. There are two major clinical presentations. The first is a typical subacute bacterial meningitis characterized by fever, headache, and neck stiffness. The second form of central nervous system listeriosis in adults is a rhombencephalitis that has features characteristic of the same illness in animals described as circling disease. Listeria sepsis, or bacteremia without central nervous system involvement, represents one-third of adult cases of invasive listeriosis. The chapter also talks about other clinical syndromes.

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• Chapter 50 : Listeria monocytogenes Infection of Mice: an Elegant Probe To Dissect Innate and T-Cell Immune Responses

  Authors: Jodie S. Haring, John T. Harty
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    Although infection with the intracellular bacterium Listeria monocytogenes can result in severe illnesses such as sepsis and meningitis in immunocompromised people, a much more common outcome is control and clearance of the organism without serious malady. L. monocytogenes is able to infect common laboratory animals such as the mouse, immunologists have been able to dissect this host-pathogen interplay and elucidate the immune functions required to contain and eliminate L. monocytogenes infection. Importantly, the mouse model of L. monocytogenes infection has yielded discoveries that are not only specific to this host-pathogen interaction but also help define fundamental concepts of innate and adaptive immunity. The natural route of L. monocytogenes infection in humans is via the gut after consumption of contaminated food products. However, infection of mice in this manner requires an extremely large inoculum and often results in asynchronous systemic infections, which complicate experimental design. Once taken up into a cell, L. monocytogenes is contained within a phagosome. Several components of the innate immune system are critical in controlling L. monocytogenes infection; however, they are not enough to accomplish clearance of virulent bacteria. T cells are required to achieve sterilizing immunity to L. monocytogenes. Gamma interferon (IFN-γ) is essential in orchestrating innate defenses against L. monocytogenes; however, its role in regulating T-cell homeostasis was not predicted. Both the innate and T-cell components of the mammalian immune system work in concert to rid the body of bacteria before the development of serious pathology.

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• Chapter 51 : Genetic Tools for Use with Listeria monocytogenes

  Authors: Darren E. Higgins, Carmen Buchrieser, Nancy E. Freitag
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    The number and sophistication of genetic tools that have become available in recent years for the molecular characterization of Listeria monocytogenes have continued to increase. Plasmid vectors, reporter genes, systems designed for transposon mutagenesis, heterologous expression systems, integration vectors, and transducing phage have all greatly advanced the experimental capacity to generate, characterize, and complement mutations within L. monocytogenes and to define functional roles of gene products. This chapter provides a brief description of the genetic tools currently available for use with L. monocytogenes. Key references are given throughout this description to provide sources for expanded details on plasmid constructions, assay conditions, and other technical aspects. The variety of genetic tools described is meant to be representative of the resources available to those interested in L. monocytogenes genetics. More widely used tools for studying expression profiles of bacteria are whole-genome DNA macro- and microarrays, which provide a comprehensive transcriptional analysis enabling researchers to view the organism as a system. Several reporter genes developed for use in other systems have proven useful for monitoring L. monocytogenes transcriptional gene regulation. Transcriptional fusions to reporter genes such as lacZ, gus, gfp, lux, and cat have all been constructed in L. monocytogenes and have been used successfully to monitor patterns of bacterial gene expression in culture and within infected cells and animals. The advantages and/or disadvantages of some of these reporter systems are discussed briefly.

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• Chapter 52 : Regulation of Virulence Genes in Pathogenic Listeria spp.

  Authors: Werner Goebel, Stefanie Müller-Altrock, Jürgen Kreft
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    Regulation of virulence genes in pathogenic bacteria must occur by mechanisms allowing the coordinate and differential expression of the virulence factors during infection. Although positive regulatory factor A (PrfA) and (to some extent) sigma factor B (SigB), are not specific for the pathogenic Listeria species, they have been so far shown by genetic and biochemical studies to be involved in the regulation of virulence genes. This chapter focuses on gene regulation by PrfA. Extensive molecular studies have been carried out with the cyclic AMP (cAMP)-binding factor Crp; since PrfA shares extended sequential and structural similarity with Crp, a short overview of the most essential features of Crp has been provided in this chapter for the understanding of PrfA. The three-dimensional structure of PrfA shows high structural similarity with Crp. The chapter discusses some evidence for the involvement of a low-molecular-weight effector(s) for PrfA that is distinct from cAMP. The results described in the chapter suggest that regulation of virulence genes mediated by PrfA involves environmental parameters, as well as additional bacterial factors. The transcriptional activator of the pathogenic Listeria species L. monocytogenes and L. ivanovii shares common properties with other members of the Crp/Fnr family to which it belongs, but it also possesses unique features. The precise knowledge of the mechanisms involved in the regulation of the listerial virulence genes will be crucial for the understanding of the pathogenesis of infections by pathogenic Listeria spp.

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• Chapter 53 : Cell Biology of Invasion and Intracellular Growth by Listeria monocytogenes

  Authors: Javier Pizarro-Cerdá, Pascale Cossart
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    It is recognized that Listeria monocytogenes grows in a broad variety of cell types in animal models and in cell culture systems. This chapter reviews the molecular mechanisms involved in host-cell invasion, intracellular growth, and cell-to-cell spread. The process of entry of L. monocytogenes into nonphagocytic cells has been examined by scanning and transmission electron microscopy. In the L. monocytogenes genome, 41 genes encoding LPXTG proteins are detected. Several lines of evidence indicate that internalin is sufficient for entry in cells expressing its receptor. Indeed, expression of inlA, the gene encoding internalin, in L. innocua and also in the more distantly related grampositive bacterium Enterococcus faecalis, confers invasiveness to these noninvasive species. Externally added InlB is also able to associate with L. monocytogenes and several other gram-positive bacteria. Several autolysins have been shown to contribute to infection, and it has been hypothesized that they could have functioned as primitive colonizing factors, allowing bacteria to interact with surfaces that express molecules analogous to their natural receptors. FbpA was identified through a signature-tagged mutagenesis screening designed to identify new L. monocytogenes virulence factors. A fusion molecule of the E-cadherin ecto-domain and of the a-catenin actin-binding site restores invasion, suggesting that L. monocytogenes exploits the same molecular scaffold as the one involved in adherens junctions function to induce its entry into target cells. Intracellular pathogens can be divided into those that reside within a host vacuole and those, like L. monocytogenes, that escape and grow directly in the host cytosol.

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• Chapter 54 : Bacillus anthracis

  Author: Theresa M. Koehler
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    Prior to the extraordinary interest in Bacillus anthracis generated by the recent bioterrorism events in the United States, much of microbiologists' awareness of the bacterium resulted from its historical significance. B. anthracis can infect all mammals, some birds, and possibly even reptiles. Systemic anthrax, generally resulting from inhalation or ingestion of B. anthracis spores, has a high fatality rate. B. anthracis is a facultative anaerobe and grows in most rich undefined media with a doubling time of approximately 30 min. Experimental studies of anthrax toxin are summarized in this chapter. The majority of animal models for anthrax have been used to assess pathophysiological effects of purified toxin and to test efficacy of vaccines against anthrax. When B. anthracis is grown under appropriate conditions, the outermost surface of vegetative cells is covered by a capsule. As is true for numerous pathogens, the capsule is an important virulence factor. Toxin and capsule synthesis by B. anthracis represents an intriguing example of coordinate expression of virulence genes in response to host-related cues. Major advances in understanding of structure and function of the "classic" virulence factors of B. anthracis, the anthrax toxin proteins and the poly-D-glutamic capsule, combined with new information regarding the anthrax toxin receptors are fueling new strategies for anthrax therapeutics and improved human vaccines. Molecular genetic analyses involving multiple B. anthracis strains will continue to facilitate epidemiological studies and development of advanced methods for detection and identification.

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• Chapter 55 : Clostridial Genetics

  Authors: Dena Lyras, Julian I. Rood
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    This chapter focuses on the genetics and genomics of the pathogenic clostridia, dealing exclusively with the major clostridial pathogens Clostridium perfringens, C. difficile, C. botulinum, and C. tetani. There are seven distinct toxin types of C. botulinum, the causative agent of both human and animal botulism. These types are distinguished by their ability to produce antigenically distinct botulinum neurotoxins (BoNTs). Phylogenetically, these isolates represent at least three quite distinct strains, which in any other genus would be classified as separate species. The complete genome sequence of C. tetani strain E88, which is a variant of the vaccine strain Massachusetts, has been determined. The study of erythromycin or macrolide-lincosamide-strep-togramin B (MLS) resistance in C. perfringens has predominantly focused on the Erm(B) determinant. Many different types of plasmids have been found in C. perfringens, including plasmids that encode antibiotic resistance, bacteriocin production and immunity, and virulence factors or toxins. Importantly, the sequenced toxigenic C. difficile strain 630 has been shown to lack restriction endonucleases, despite the presence of five methylase genes, thereby making feasible the genetic analysis of virulence factors in this strain. Unfortunately, the analysis of this C. difficile strain is complicated by the fact that it is resistant to both erythromycin and tetracycline, which are commonly used as selectable makers in clostridial genetics.

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• Chapter 56 : Neurotoxigenic Clostridia

  Author: Eric A. Johnson
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    This chapter describes the microbiological properties of Clostridium botulinum and C. tetani, with an emphasis on pathogenesis and new findings on the organisms and their clostridial neurotoxins (CNTs). Botulism is a rare but often severe paralytic disease caused by the extremely potent botulinum neurotoxins (BoNTs) produced by C. botulinum and certain other clostridia. The major treatment of botulism is supportive care, with careful attention being given to respiratory status. Tetanus is a vivid neurological disease characterized by violent and persistent spasms of the head, trunk, and limb muscles. Neurotoxigenic clostridia tend to grow as consortia, and pure cultures are often difficult to achieve and maintain. Owing to the complex nutrient requirements of neurotoxigenic clostridia, rich media are commonly used for cultivation. Bioassays are currently the most important laboratory tests used to identify neurotoxigenic clostridial species. Although BoNTs and tetanus neurotoxin (TeNT) are the primary determinants of virulence in neurotoxigenic clostridia, wound or intestinal infections also require additional virulence processes. The availability of genomic sequences and comparative genomic analyses, together with the development of genetic tools such as gene replacement and vectors for controlled gene expression, will be invaluable in elucidating pathogenic mechanisms of neurotoxigenic clostridia. To prevent further human illness and deaths by neurotoxic clostridia, antidotes are urgently needed that can reverse the detrimental effects of BoNTs and TeNT once they are bound and internalized in nerves.

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• Chapter 57 : Enterotoxic Clostridia: Clostridium perfringens Type A and Clostridium difficile

  Authors: Bruce A. McClane, David M. Lyerly, Tracy D. Wilkins
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    This chapter discusses two enterotoxin-producing clostridia that rank among the most important enteric pathogens of humans, Clostridium difficile and enterotoxin-positive type A strains of C. perfringens. The major lethal toxins (LTs) are not the only biomedically important C. perfringens toxins; some C. perfringens isolates, mostly belonging to type A, express C. perfringens enterotoxin (CPE). Recognized outbreaks of C. perfringens type A food poisoning are usually very large, averaging about 100 cases. C. perfringens type A food poisoning is acquired by ingestion of a food item containing vegetative cells of a CPE-positive C. perfringens type A strain. C. perfringens isolates associated with non-food-borne human gastrointestinal (GI) disease consistently carry a plasmid-borne cpe gene, which distinguishes them from food poisoning isolates carrying a chromosomal cpe gene. C. difficile is an opportunistic pathogen that causes nosocomial diarrhea and colitis after the normal GI flora has been altered, most typically by antibiotics. C. difficile-mediated disease develops from the production of two toxins, toxin A and toxin B, which in some papers are referred to as the enterotoxin and cytotoxin, respectively. Toxin production occurs during the stationary phase, under conditions that limit the growth of the organism. Certain basic precautions should be taken to help control outbreaks of C. difficile disease. In most instances, the incidence of disease can be reduced simply by educating health care workers about the disease and how it is spread.

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• Chapter 58 : Histotoxic Clostridia

  Authors: Dennis L. Stevens, Julian I. Rood
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    Histotoxic clostridial infection is a general term coined over a century ago that referred to gas gangrene and malignant edema in humans and blackleg in cattle. Histotoxic infections are rapidly progressive, are associated with gas in tissue, and manifest impressive tissue destruction, shock, and frequently death. Although the histotoxic clostridia are classified as grampositive, spore-forming, anaerobic bacilli, not all of them are definitely so. The main habitats of all of the histotoxic clostridia are soils and the intestinal contents of humans and animals. Clostridium perfringens is the most widespread of the histotoxic clostridia, with the quantity of organisms in soil being proportional to the degree and duration of animal husbandry in the region. The major C. perfringens extracellular toxins implicated in gas gangrene are alpha-toxin and theta-toxin. Two independent studies have shown that the alpha-toxin is an essential toxin in the disease process. First, vaccination with a purified recombinant protein consisting of the C-terminal alpha-toxin domain (amino acids 247 to 370) has been shown to protect mice from experimental C. perfringens infection. Second, an alpha-toxin (plc) mutant constructed by allelic exchange has been shown to be avirulent in a mouse myonecrosis model. Toxin production has been documented in vivo by demonstrating the progressive appearance of both theta- and alpha-toxins at the site of the experimental infection by 4 h. Immunization with the C-domain of alpha-toxin is a viable strategy for the prevention of gas gangrene caused by C. perfringens.

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ACTINOMYCETALES

• Chapter 59 : Corynebacterium diphtheriae: Iron-Mediated Activation of DtxR and Regulation of Diphtheria Toxin Expression

  Authors: John F. Love, John R. Murphy
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    A detailed understanding of the molecular biology and genetics of both the regulation of diphtheria toxin expression and the structure-function relationships and mode of action of the toxin is known. The existence of virulence determinants in C. diphtheriae beyond those associated with diphtheria toxin is demonstrated by the recently reported outbreak of invasive disease caused by a clonal focus of nontoxigenic C. diphtheriae among intravenous drug users in Switzerland. The regulation of expression of the tox gene, as well as the genes involved in iron acquisition and utilization, is under the control of the C. diphtheriae-encoded iron-activated repressor diphtheria toxin repressor (DtxR). The protein-protein interactions stabilizing DtxR dimers arise mostly from hydrophobic associations. The X-ray structure of the C-terminal domain of DtxR shows that this region of the repressor is composed of five antiparallel β-sheets and two short α-helices. Diphtheria toxin is the primary virulence factor expressed by toxigenic strains of C. diphtheriae. The structural gene encoding diphtheria toxin is carried by a family of closely related corynebacteriophages, the best studied of which is corynephage. The C. diphtheriae genome sequence has also permitted examination of microbial speciation and evolution. Outbreaks of clinical diphtheria almost always occur in individuals who have not become immunized and who have been exposed to a carrier. Molecular epidemiologic analysis of toxigenic strains of C. diphtheriae isolated from this epidemic has provided further insight into the virulence of this pathogen.

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• Chapter 60 : Actinomyces and Arcanobacterium spp.: Host-Microbe Interactions

  Authors: B. Helen Jost, Stephen J. Billington
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    This chapter describes the current knowledge of the mechanisms of Actinomyces and Arcanobacterium spp. host-microbe interactions, with an emphasis on recent advances in the molecular analyses of toxins and factors involved in bacterial adherence. Members of the genus Actinomyces are nonmotile and non-spore-forming and although facultative anaerobes, most species grow best under anaerobic conditions. Numerous in vivo and in vitro studies have demonstrated the significance of Actinomyces spp. in the initiation and progression of plaque development. Adhesion of Actinomyces spp. to host epithelial, phagocytic, and red blood (hemagglutination) cells requires the action of neuraminidase to expose cryptic host cell receptors. The major receptor on host polymorphonuclear leukocytes (PMNs) was identified as CD43 (leukosialin). The levansucrases (fructosyltransferases) from Actinomyces spp. catabolize sucrose to form ß2,6-linked (levans or fructans) or β2,1-linked (inulin) homopolymers of fructose. Three recently described Actinomyces spp., Actinomyces turicenesis, Actinomyces radingae, and Actinomyces europaeus, are also associated with skin, genital, urinary, and reproductive tract infections. The genus Arcanobacterium is closely related to that of Actinomyces, so much so in fact that until recently, members of these genera were intermingled with each other phylogenetically. The two predominant species within the arcanobacteria are the ubiquitous animal commensal and opportunistic pathogen A. pyogenes and the human pathogen Arcanobacterium haemolyticum. Further research investigating the mechanisms and regulation of the adhesins of Actinomyces and Arcanobacterium spp., as well as their interactions with other bacteria in polymicrobial infections, will be necessary to complete the understanding of disease pathogenesis.

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• Chapter 61 : The Pathogenesis of Nocardia

  Author: Blaine L. Beaman
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    Nocardiae are gram-positive, partially acid-fast, filamentous bacteria that grow by apical extension, forming elongated cells with lateral branching. Most species of Nocardia have been recovered from soil, plant material, and water in most regions of the world. Diseases in humans caused by nocardiae may be divided into at least six general categories based on the route of infection, site of disease, and subsequent pathological responses. They are pulmonary nocardiosis, extrapulmonary nocardiosis, systemic nocardiosis, central nervous system (CNS) nocardiosis, cutaneous, subcutaneous, and lymphocutaneous nocardiosis. A mycetoma is a chronic, progressive, pyogranulomatous disease that usually develops at the site of a localized injury such as a thorn prick. Nocardia asteroides, Nocardia farcinica, and Nocardia otitidiscaviarum have caused significant outbreaks worldwide in dairy cattle, usually in the form of mastitis. Nocardiae are facultatively intracellular pathogens that resist the microbicidal activities of polymorphonuclear neutrophils (PMNs). Concentrated culture filtrates from GUH-2 grown in a chemically defined medium also induced apoptosis, as well as dopamine depletion. Cells of N. asteroides GUH-2 in the log phase of growth adhered by way of the filament tip to the surface of both pulmonary epithelial cells (Clara cells) and brain capillary endothelial cells in mice. The differential and selective adherence displayed by nocardiae both in vitro and in vivo suggested distinct multiple ligands for host cells on the nocardial surface.

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• Chapter 62 : Mechanisms of Resistance to β-Lactam Antibiotics

  Author: Douglas S. Kernodle
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    The introduction of penicillin into clinical use in 1941 had a profound impact on the treatment of diseases caused by gram-positive pathogens. Antibiotic degradation by ß-lactamase and alterations in penicillin-binding membrane proteins remain the major mechanisms by which gram-positive pathogens express resistance to ß-lactam antibiotics. The penicillin-interactive enzymes involved in cell wall biosynthesis are specialized acyl serine transferases localized on the outer face of the cytoplasmic membrane. The strong antibacterial efficacy of β-lactams, combined with their low toxicity for eukaryotic cells, has helped to make them the most highly developed class of antibacterial agents in clinical use. The resistance phenotype in β-lactamase-producing gram-positive bacteria differs from that observed with gram-negative species and is associated with an inoculum effect in which the MIC depends upon the number of bacteria tested. Around 95% of Staphylococcus aureus isolates recovered from clinical specimens produce ß-lactamase. The production of large amounts of ß-lactamase in isolates possessing the normal penicillin-sensitive penicillin-binding proteins (PBPs) has been associated with borderline susceptibility to the antistaphylococcal penicillins. External factors such as temperature, osmolality, and light influence the proportion of the bacterial cell population that exhibits resistance. Most penicillin-resistant clinical isolates exhibit a PBP pattern more complex than just a combination of point mutations. In clinical isolates, tolerance appears to be more prevalent among gram-positive than gram-negative species.

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• Chapter 63 : Resistance to Glycopeptides in Gram-Positive Pathogens

  Authors: Henry S. Fraimow, Patrice Courvalin
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    Glycopeptide antimicrobials are natural products that are produced by various soil-dwelling species of the order Actinomycetales such as Amycolatopsis orientalis and Amycolatopsis coloradensis. With the recent emergence of glycopeptide-resistant enterococci and staphylococci, there has been renewed interest by the pharmaceutical industry in the development of modified semisynthetic glycopeptides with enhanced activity against resistant gram-positive pathogens including glycopeptide-resistant organisms. Due to glycopeptides' unique mechanism of action and ability to interfere with multiple critical reactions in peptidoglycan synthesis, acquired resistance to glycopeptides was deemed unlikely. The mechanisms of acquired resistance to glycopeptides in enterococci have been well characterized and reflect changes in target analogous to those in intrinsically resistant organisms. The newer, semisynthetic glycopeptides oritavancin and telavancin have enhanced activity against some strains of enterococci with acquired glycopeptide resistance. The basis of both intrinsic and acquired glycopeptide resistance in enterococci involves alteration of the composition of the terminal dipeptide in muramyl pentapeptide cell wall precursors, resulting in a structure with decreased binding affinity for glycopeptides. Two additional enterococcal resistance genotypes, vanE and vanG, have also recently been described. Both are characterized by low-level vancomycin resistance mediated through the synthesis of precursors terminating in D-Ala-DSer analogous to the mechanism of VanC-type resistance. The chromosomally located five-gene vanE resistance cluster from Enterococcus faecalis BM4405 is organized very similarly to the vanC operon and includes genes encoding the VanE ligase, the VanXYE D,D-dipeptidase-D,D-carboxypeptidase, and the VanTE serine racemase, as well as the VanRESE two-component regulator.

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• Chapter 64 : Tetracycline Resistance Determinants in Gram-Positive Bacteria

  Authors: Laura M. McMurry, Stuart B. Levy
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    The emergence of tetracycline resistance in the mid-1950s, initially in gram-negative bacteria and then in gram-positive bacteria, resulted in the declining usefulness of tetracyclines. There are three different tetracycline resistance mechanisms of clinical importance found among gram-positive organisms. They are active efflux, ribosomal protection, and mutated rRNA. Multidrug transporters are able to efflux substrates that are chemically diverse, and some from gram-negative bacteria include tetracycline in their repertoire, for example, AcrAB (Escherichia coli) and MexAB/OprM (Pseudomonas sp.). The only multidrug transporter from gram-positive bacteria known to transport tetracycline is the TetAB transporter from Corynebacterium. The emphasis in this chapter is on proteins found in gram-positive organisms. The Asp-66–Ala mutation may directly or indirectly prevent tetracycline binding, or it may prevent a conformational change in motif A caused by binding elsewhere. In the case of the gram-positive Tet(L) and Tet(K) proteins, substitutions at Asp-74, corresponding to the essential Asp-66 in TetA(B), only partly decrease tetracycline resistance, although efflux activity is more severely affected. Tetracycline resistance determinants are widely spread among different gram-positive genera. Tetracycline resistance spreads because the determinants are often located on conjugative elements, either plasmids or transposons.

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• Chapter 65 : Mechanisms of Quinolone Resistance

  Author: David C. Hooper
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    With the increasing use of quinolones for the treatment of gram-positive bacterial infections, an understanding of the mechanisms of quinolone resistance in gram-positive bacteria is of considerable importance. This chapter summarizes the current understanding of established mechanisms of resistance to this class of antimicrobial agents in gram-positive bacteria. There are important differences between gram-positive and gram negative bacteria both in target enzyme sensitivity and in the means by which efflux resistance mechanisms operate that are of clinical and fundamental importance. Quinolones interact with both of the two type 2 topoisomerases in eubacteria, DNA gyrase and topoisomerase IV, which are essential for bacterial DNA replication. Quinolone-resistant clinical and laboratory strains of Streptococcus pneumoniae have been shown to have reduced accumulation of quinolones that is reversible with reserpine, suggesting the involvement of an efflux system(s) in quinolone resistance. Quinolone-resistant clinical isolates of viridans streptococci have been shown to have an efflux phenotype defined as lower MICs of quinolones in the presence of reserpine. DNA from such strains of S. mitis and S. oralis was able to transform S. pneumoniae to efflux phenotype in the laboratory. Overexpression of norA and genes for topoisomerases from plasmids are known, however, to have toxic effects on the cell that may limit the fitness of resistant bacteria containing them. Thus, at present quinolone resistance in gram-positive bacteria is attributable exclusively to chromosomal mutations that affect quinolone targets or quinolone permeation to these targets.

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