Chapter 1 – Molecular Medical Microbiology – The Expanding Concept
Though the molecular aspect of microbiology has long been recognized, it has greatly expanded in recent years. The molecular study of medical microbiology has revealed conceptual insights and technical approaches that have advanced the subject almost beyond recognition. The biological experiments by Fred Griffiths that identified the pneumococcal transforming principle were the prelude to its identification as DNA, in turn eventually leading to the recognition of genetics as the foundation of molecular microbiology. Similarly, the understanding of DNA at the structural level led to the discovery of the polymerase chain reaction (PCR). The discovery of host-controlled restriction-modification and restriction enzymes was the foundation of genetic manipulation. This molecular approach has provided information about the pathogenesis and prevention of bacterial diseases. In the case of Haemophilus influenzae type b these advances have brought into focus the possible elimination of this virulent childhood pathogen. Since 1995 the whole genomes of an increasing number of bacterial species have been described and this has opened up the omics technologies. These fundamental approaches have opened up new vistas in the diagnosis, treatment and prevention of diseases due to bacteria.
Keywords
- bacterial ‘variation’;
- diagnosis;
- DNA;
- DNA ligase;
- fluxomics;
- host-controlled restriction-modification;
- metabolomics;
- metagenomics;
- omics technologies;
- polymerase chain reaction (PCR);
- proteomics;
- restriction endonucleases;
- transformation
Chapter 2 – Bacterial Ultrastructure
Our understanding of bacterial ultrastructure has changed with improvements to light and electron microscopy technologies coupled with the introduction of novel preparative procedures. This chapter, whilst reviewing the variety of structures found in bacteria, also introduces some of the new structural findings that these improvements have revealed and the concepts that they have led to.Keywords
- bacterial morphology;
- bacterial structure;
- electron microscopy;
- immunomicroscopy
Chapter 3 – Bacterial Capsules
Capsules are the outmost structures of bacterial and fungal cells. The capsules protect microbial cells from immune recognition and killing during infection of mammalian hosts. Except for the poly-γ-glutamate (PGA) capsule of Bacillus anthracis, other known capsules are all composed of polysaccharides. Certain bacteria (e.g. B. anthracis and Streptococcus pyogenes) produce only one capsule structure, whereas many other bacteria express capsules with great biochemical, structural and immunological diversity within the same species. This diversity is driven by immune selection from the mammalian hosts. The genes for capsule synthesis are typically clustered in a single locus of bacterial chromosome. The number of genes associated with capsule synthesis ranges from one in serotype 37 Streptococcus pneumoniae to >20 in serotype 38 S. pneumoniae. Different bacterial species can share similar genes or mechanisms for capsule synthesis. The expression of the capsule genes is often regulated by environmental conditions. The capsular polysaccharides are the antigens of the current polysaccharide-based vaccines for S. pneumoniae, Neisseria meningitidis, Haemophilus influenzae and Salmonella enterica serovar Typhi. Certain capsule polymers also have important industrial applications.Keywords
- bacteria;
- capsule;
- gene regulation;
- pathogen;
- microbiology;
- polysaccharide;
- vaccine;
- virulence
Chapter 4 – Genetics and Biosynthesis of Lipopolysaccharide
Lipopolysaccharide (LPS), an integral component of the outer membrane in Gram-negative bacteria, consists of lipid A, core oligosaccharide (core), and O-specific polysaccharide or O antigen (OAg). LPS protects Gram-negative bacteria from environmental chemical and physical stress and is also recognized by the innate immune system upon infection. LPS biosynthesis, export and assembly require a large number of enzymes and structural proteins encoded by numerous genes. Both the lipid A and core are assembled on the cytoplasmic side of the inner membrane and translocated across the inner membrane. The OAg is independently assembled in a separate pathway, also translocated to the periplasmic side of the cell membrane, and ligated to the lipid A-core. Newly formed LPS is then shuttled across the periplasm by a complex multiprotein pathway, which also mediates the insertion of LPS into the outer leaflet of the outer membrane. This chapter discusses current mechanistic understanding of the synthesis and assembly of the LPS molecule.Keywords
- ABC transporter;
- acylation;
- flippase;
- glycosyltransferases;
- lipid A;
- lipopolysaccharide;
- lipoproteins;
- membrane proteins;
- O antigen;
- outer membrane
Chapter 5 – Teichoic Acids, Lipoteichoic Acids and Other Secondary Cell Wall and Membrane Polysaccharides of Gram-Positive Bacteria
Teichoic acids and similar molecules can make up 50% of the cell wall of Gram-positive bacteria, and their lipid-linked analogues are bound to the cytoplasmic membrane, expressed on the surface and are essential for viability. Their main function is to bind cations for use by the bacterial cell. They also function as pathogen-associated molecular patterns stimulating the host innate immune system. As antigens they are often important in serodiagnostics. The capsular polysaccharides of some Gram-negative bacteria have teichoic acid like structures.Keywords
- anionic polymers;
- bacterial cell envelope;
- capsular teichoic acids of Gram-negative bacteria;
- innate immunity;
- poly(glycerol phosphate);
- polyribitol phosphate
-
Peptidoglycan is an essential component of the bacterial cell envelope and protects the cell from bursting due to turgor and maintains cell shape. Composed of glycan chains connected by short peptides, peptidoglycan forms a net-like macromolecule around the cytoplasmic membrane. There is significant structural variation in the peptidoglycans of different bacteria. Pathogens modify the peptidoglycan to become resistant to lysozyme. Peptidoglycan carries covalently attached cell surface components like teichoic acid, capsular polysaccharide and cell wall proteins. Peptidoglycan precursors are synthesized in the cytoplasm and linked to a polyprenyl phosphate lipid for transport across the cytoplasmic membrane. Presumably, peptidoglycan synthases and hydrolases form dynamic multi-enzyme complexes which polymerize new peptidoglycan and insert it into the existing cell wall, concomitant with the release of old material. The peptidoglycan synthesis complexes are controlled by components of the bacterial cytoskeleton. Gram-negative bacteria also regulate peptidoglycan synthesis by outer-membrane proteins.Keywords
- bacterial cytoskeleton;
- cell division;
- lysozyme resistance;
- Mur enzymes;
- penicillin-binding protein;
- peptidoglycan;
- peptidoglycan hydrolyase
-
The bacterial flagellum is an apparatus of motility commonly found among motile species. The flagellum is a supramolecular structure composed of about 20 protein components and divided into three substructures: the filament, the hook and the basal body. The filament is a helix, which takes on several distinct forms under various conditions. Helical forms of peritrichous, polar and lateral flagella are independent from each other and belong to different flagellar families. The basal body contains the rotary motor, which is powered typically by a proton motive force. The C ring is a cup-shaped structure attached to the cytoplasmic side of the basal body and works as the rotor of the motor and as a part of the secretion apparatus. About 40 genes required for the flagellar assembly are ordered in a hierarchical manner at the transcriptional level. The flagellar assembly is also regulated at the secretion gate, which does not allow the secretion of filament proteins until the hook-basal body is completed. The flagellar basal body shares common features with the secretion apparatus for virulence factors, indicating that the two systems were derived from a common ancestor.Keywords
- bacterial flagellum;
- gene regulation;
- morphology;
- pathogenicity;
- protein export;
- rotation;
- self-assembly;
- supramolecular structure
Chapter 8 – Pili and Fimbriae of Gram-Negative Bacteria
Research on the function and assembly of extracellular fibres in Gram-negative pathogenic bacteria has provided insights into some of the most basic principles of molecular biology: how a protein folds into domains that serve as assembly modules for building up large supramolecular structures. Studies of the chaperone–usher pathway (CUP) pili have elucidated a reaction called donor strand complementation, in which the chaperone mediates pilus subunit folding, and a reaction called donor strand exchange, in which subunits of a pilus polymerize into a fibre with the aid of the usher, an outer-membrane-gated channel. CUP pili are ubiquitous in Gram-negative bacteria, with many genomes encoding ten or more types, all containing dedicated adhesins that function in adherence, invasion of host tissues, and biofilm formation on medical devices and in various niches and body habitats. Many Gram-negative bacteria also use specific molecular machinery to direct production of amyloid fibres called curli, which can provide structural, adhesive and protective properties to a biofilm. Frequent and long-term prophylactic use of antimicrobial agents has contributed to a looming worldwide crisis of multi-drug resistance that has spawned the need for new ways of thinking about drug development, including the targeting of bacterial molecular machines that catalyse the biogenesis of virulence-associated extracellular fibres.Keywords
- bacterial infection;
- biofilms;
- chaperone–usher pathway;
- curli;
- pili biogenesis
Chapter 9 – Endospores, Sporulation and Germination
Endospore-forming bacteria cause a range of important clinical infections. In this chapter, recent advances in endospore research are summarized. The function of endospore structures and their relation to resistance are discussed and the latest advances in understanding the biological pathways that lead to sporulation and endospore germination are reviewed. The targeting of endospore germination to prevent infections is emphasized. From a more clinical perspective, the most important endospore-borne diseases are highlighted. The need to develop new methods for endospore detection is discussed. Finally, practical applications for exploiting the inert properties of endospores as platform for probiotics, insecticides, food technology and protein display are discussed.Keywords
- anthrax;
- Bacillus;
- botulism;
- CDI;
- Clostridium;
- endospore;
- germination;
- sporulation;
- tetanus
Chapter 10 – Bacterial Growth, Culturability and Viability
For most of the 20th century ideas of the growth and life cycles of bacteria were dominated by the concepts of lag, exponential, stationary and death phases and analogies with the eukaryotic cell cycle were largely rejected. While the classical growth phases remain key points of reference, the last 20 years have seen an explosion of molecular and cytological results showing the diversity of bacterial physiological adaptive states and indicating cyclical events beyond a headlong accumulation of biomass; there is clearly more to bacteria than growing, not growing or dying. Since growth is integral to infection we would like to know the growth state of bacteria in a medically relevant sample. In this chapter, bacterial growth is reviewed from a molecular perspective considering the signals that might indicate the status of cells in a sample. Major advances have been made in describing cell replication and division and, in particular, the development of microfluidic systems linked to imaging has made it possible to follow the fate of cells through many generations. We are beginning to appreciate the consequences of asymmetric cell division and how this further underpins the diversity of cells in a bacterial population where once all those comprising a balanced exponential phase culture were considered identical with respect to their time since division. The concepts of viability and culturability remain a challenge and it is necessary now to link them up with the avalanche of data emerging from microbiomic studies applied to human samples.Keywords
- asymmetric division;
- bacterial growth phases;
- cell cycle stages (B, C, D);
- culturability;
- molecular correlates of growth;
- microfluidics and bacterial imaging;
- signals for growth;
- viability
Chapter 11 – Bacterial Energy Metabolism
The bacteria described in this book on molecular medical microbiology are chemoorganotrophs which gain energy by utilization of organic substrates using either aerobic respiration, anaerobic respiration or fermentation. The theory of chemiosmosis is the essential link between energy generation, its coupling to ATP synthesis and the utilization of energy for metabolism. The chapter discusses the principles of chemiosmosis, the respiratory components of electron transport chains and the H+- and Na+-translocating ATPases. Fermentation is covered, with emphasis on fermentation in the human gut. As oxygen availability in host tissue is both variable and often limited, discussion of aerobic recitation is focused on cytochrome oxidases whilst for anaerobic respiration denitrification is the main topic. Regulation of the switch between aerobic and anaerobic metabolism regulation covers the sensory and regulatory functions of regulators, illustrated with examples from Escherichia coli and Paracoccus denitrificans.This is extended into our current understanding of regulatory networks. The energy metabolism of five pathogens is discussed in relation to their cell physiology and their growth and survival in their host.Keywords
- ATPases;
- chemiosmosis;
- component systems;
- cytochrome oxidases;
- denitrification;
- fermentation;
- regulatory networks;
- two aerobic/anaerobic switch
Chapter 12 – Biofilms, Quorum Sensing and Crosstalk in Medically Important Microbes
Biofilms are heterogeneous communities of microorganisms firmly attached to a biological or abiotic surface. They are the causative agents of chronic infection in over 25 different diseases and the microbes that comprise biofilms are typically highly tolerant to antimicrobials. Biofilm formation is a highly coordinated process that relies heavily on a cell-density-dependent form of microbial communication called ‘quorum sensing’ (QS). QS is facilitated by the production of small-molecule signals that induce shifts in gene expression and metabolic activity in the organisms residing in and around biofilms. Quorum signals can elicit specific responses in a wide range of both prokaryotic and eukaryotic cells, and this ‘crosstalk’ or ‘interkingdom signalling, (IKS) travels both directions, as some mammalian hormones can directly enhance bacterial virulence. Since QS controls the pathogenic processes of so many medically important microbes, it holds significant promise as a future target for therapeutics.Keywords
- acylated homoserine lactone;
- antibiotic tolerance;
- biofilm;
- crosstalk;
- exopolymeric substance (EPS);
- interkingdom signalling;
- PQS;
- quorum sensing
Chapter 13 – Oxidative Stress Responses and Redox Signalling Mechanisms in Bacillus subtilis and Staphylococcus aureus
Life evolved from transition of anaerobic to aerobic conditions and microorganisms have to adapt to the consequences of oxygen toxicity as well as to changes in oxygen tension in the environment. ROS are generally produced in microorganisms during respiration but pathogens also are exposed to the oxidative burst produced by activated neutrophils. Bacteria further encounter reactive electrophilic species (RES), such as quinones and aldehydes, antimicrobials, antibiotics and environmental contaminants (xenobiotics) in their natural habitat which can modify the cellular redox status. Thus, bacteria have to adapt to different redox active compounds, such as ROS, RES, antibiotics and changes in oxygen tension using specific redox-sensing mechanisms that control the expression of specific detoxification pathways. In addition, the reduced state of the cytosol is maintained by low-molecular-weight (LMW) thiol redox buffers and thiol-disulphide reducing systems, including the Trx and Grx systems. Here, we review the O2, ROS and RES-specific redox-sensing mechanisms that have been characterized in two related model Gram-positive Firmicutes bacteria, the important human pathogen Staphylococcus aureus and the industrially important Bacillus subtilis. We further pay particular attention to the function of the redox buffer bacillithiol for the redox balance of the cell, redox regulation and virulence. The redox-sensing regulators of B. subtilis and S. aureus employ various mechanisms to sense and respond to ROS, RES or O2 by using classical thiol redox switches or S-thiolations (OhrR, HypR, YodB, Spx), the recently discovered Cys-phosphorylation (SarZ, MgrA, SarA), thiol-S-alkylation (QsrR), His-oxidation (PerR), FeS-cluster disassembly (FNR, NreB) or they can sense changes in the NAD+/NADH ratio upon switch from aerobic to anaerobic conditons (Rex). In S. aureus, the ROS and RES resistance mechanisms controlled by PerR, SarZ, MgrA and QsrR are important for the efficient adaptation to the host environment that contributes to the virulence of this major pathogen of community-acquired infections.Keywords
- bacillithiol;
- Bacillus subtilis;
- oxidative stress;
- redox sensing;
- Staphylococcus;
- virulence
Chapter 14 – Bacterial Proteomics in the Study of Virulence: An Overview
Advances being made in proteomics methodologies, including sample fractionation and mass spectrometry, are beginning to allow the bacterial proteome to be characterized in the context of infected tissues although much progress relies on carefully constructed in vitro experimentation. A spectrum of approaches is available for characterizing and comparing proteomes of bacterial pathogens from ‘shotgun’ sampling of protein populations to sensitive and sophisticated quantitative proteomics. Each approach offers means to define virulence components through targeting of pertinent bacterial compartments, relevant environmental cues and appropriate infection models. Recent developments in advancing understanding of bacterial pathogens and their virulence determinants have come via focus on post-translational modifications of proteins, by combining several omics approaches (‘polyomics’ or ‘multi-omics’) or through sampling of bacteria directly from infections. Data generated are vast and complex, hence sophisticated analytical tools are required to assist interpretation in a biological context. Proteomics studies of bacterial pathogens have identified numerous virulence-associated determinants and functional analyses – including relevant virulence models – are important components in advancing proteomics into further understanding of disease-causing bacteria and their virulence characteristics and processes.Keywords
- bacterial pathogens;
- in vivo models;
- mass spectrometry;
- multi-omics/polyomics;
- post-translational modification;
- proteome/proteomics
Chapter 15 – Mechanisms of Horizontal Gene Transfer and DNA Recombination
Comparative genomics is revealing extensive diversity within many bacterial species. The pan-genome of a species is composed of core genes present in all strains and dispensable genes that provide a selective advantage under specific conditions. Movement of these dispensable genes between species, genera and kingdoms is known as horizontal gene transfer (HGT). There are three primary mechanisms of HGT in bacteria. Transformation: uptake of naked DNA from the environment by naturally competent cells. Transduction: transfer of bacterial DNA between cells using bacteriophages as vectors. Conjugation: intimate cell-to-cell contact with transfer of single-stranded DNA by a type-IV-like secretion system.Horizontally acquired DNA that cannot replicate autonomously must be integrated into the genome of the recipient if it is to be maintained. Incoming DNA with significant similarity to the recipient genome can integrate by homologous recombination. Mobile genetic elements, such as integrative and conjugative elements, that have limited homology to the host genome use site-specific recombination to integrate at target sequences. Understanding these processes provides insight into the evolution of bacteria and emerging pathogens.Keywords
- conjugation;
- homologous recombination;
- horizontal gene transfer;
- site-specific recombination;
- transduction;
- transformation
Chapter 16 – Pathogenicity Islands: Origins, Structure, and Roles in Bacterial Pathogenesis
Bacterial pathogens possess virulence factors that distinguish them from their non-pathogenic counterparts, and enable them to induce pathogenesis. Typically, these unique genes are encoded on specialized regions of the bacterial chromosome termed pathogenicity islands. Acquired through horizontal transmission, pathogenicity islands are large sections of the chromosome that differ in nucleotide content and in the presence of genetic elements compared to the core genome, and contain genes that promote pathogenesis. Pathogenicity islands were first discovered in bacteria belonging to the Enterobacteriaceae family, but are now known to exist in various animal and plant pathogens. As pathogenicity islands are unique to pathogenic bacteria, it is likely their presence permitted the emergence of bacterial pathogens and continues to shape their evolution. This chapter highlights the genetic organization and content of pathogenicity islands, their regulation, and their impact on the evolution of pathogenic bacteria.Keywords
- horizontal transmission;
- pathogenesis;
- pathogenicity island;
- secretion system;
- toxin;
- virulence
Chapter 17 – Coordination of Bacterial Virulence Gene Expression
Coordination of virulence is achieved by bacterial pathogens, in part, through the control of gene expression. The nucleoid is the site where this is controlled and its architecture has an important influence on gene expression. These structural and regulatory features are also discussed in the context of bacterial evolution. Three ‘case studies’ involving Salmonella enterica, Shigella flexneri and Vibrio cholerae are used as examples to illustrate coordination of virulence.Keywords
- horizontal gene transfer;
- nucleoid;
- nucleoid-associated proteins;
- Salmonella enterica;
- Shigella flexneri;
- Vibrio cholerae
Chapter 18 – Towards a Synthesis of Population Genomics and Epidemiology: Next-Generation Sequencing of Bacterial Pathogens
The advent of next-generation sequencing technology has provided unprecedented detail as to the micro-evolutionary processes impacting on bacterial genomes. These data are also leading to a powerful synthesis between evolutionary genetics, population biology, ecology and epidemiology: such a holistic approach is necessary to understand the emergence and spread of bacterial pathogens. Here the recent advances made in understanding genome dynamics within this broader epidemiological context are reviewed, and the challenges and opportunities bestowed by next-generation sequencing in the future are discussed.Keywords
- ecology of pathogens;
- evolutionary genetics;
- genome dynamics;
- population genetics
Chapter 19 – The Human Microbiota and Pathogen Interactions
Human beings are colonized by abundant and diverse microbial communities, collectively termed the human microbiota. These microbes inhabit niches at sites throughout the body and are an important aide in the fight against infectious disease. They protect the host by acting as a significant barrier against invasion by extrinsic pathogens, many of which have evolved sophisticated mechanisms to outcompete the indigenous microbiota. Conversely, a shift in microbiota composition towards less beneficial species (‘dysbiosis’) may act to initiate or worsen disease by compromising the barrier effect. A dysbiotic microbiota may also influence the potency of pathogenic invaders via the transfer of virulence and antibiotic resistance genes. Understanding the complex communities that share our bodies, and the ways in which they interact with pathogens, are therefore important and emerging research goals, which have the potential to inform clinical practice and generate novel therapeutics.Keywords
- bacteriotherapy;
- colonization resistance;
- metagenomics;
- microbe–microbe interactions;
- microbiome;
- microbiota
Chapter 20 – Bacterial Whole-Genome Determination and Applications
More and more bacterial whole genomes have been sequenced and used for different applications. Compared to the traditional Sanger method, the next-generation sequencing technologies significantly decrease the cost and time of genome sequencing. After the whole genome sequence is obtained, the genes and their functions are predicted by computational methods. The genome sequence with gene annotations are then submitted to a repository such as GenBank. Many genomes within a specific bacterium can be used to run pan-genome and phylogenetic tree analyses. Furthermore, the genome sequences can be used to predict different features including subcellular localization and adhesin probability. Reverse vaccinology starts with bioinformatics analysis to predict virulence factors and vaccine candidates. Genome sequencing-based methods have also frequently been used for clinical diagnosis, genomic epidemiology, metagenomics and microbiome profiling. Many challenges and opportunities co-exist in genome sequencing technology and applications.Keywords
- adhesin;
- bacterium;
- gene annotation;
- gene prediction;
- genome;
- genomic epidemiology;
- metagenomics;
- microbiome;
- next-generation sequencing;
- pan-genome;
- reverse vaccinology;
- sequencing;
- subcellular localization;
- whole-genome sequencing
Chapter 21 – Molecular Taxonomy
Taxonomy is the branch of science concerned with the classification of organisms. A taxonomic designation is more than just a name. Ideally, it reflects evolutionary history and the relationship between organisms. Traditionally, taxonomic classification has relied upon morphological features and physiological characteristics. However, for bacterial taxonomy, phenotypic approaches have proven insufficient. Unrelated bacteria can exhibit identical traits, closely related bacteria can have divergent features, and methods for accurate identification may be too cumbersome for routine use. In contrast, molecular taxonomy approaches use data derived from hereditary material and provide a robust view of genetic relatedness. Advances in technology have been accompanied by improvements in the cost, speed and availability of molecular methods. Here, we provide a brief history of approaches to prokaryotic classification and describe how molecular taxonomy is redefining our understanding of bacterial evolution and the tree of life.Keywords
- classification;
- molecular epidemiology;
- ribosomes;
- sequence analysis;
- taxonomy
Chapter 22 – Principles and Applications of Genomic Diagnostic Techniques
Genomic methods such as polymerase chain reaction (PCR) have evolved into faster, more accurate technologies which can not only accurately detect microbial nucleic acids in a wide variety of body samples, but also quantify target genetic material, and in some cases detect and measure microbial gene expression. This has allowed clinicians to better characterize microbial behaviour and interactions during states of health and disease, and to identify unique phenotypic and genotypic markers for fingerprinting in outbreaks or epidemics. As these new technologies become available, a judicious and careful assessment of these approaches will be necessary to better understand their clinical diagnostic value. Although these tests have greatly empowered clinicians and microbiologists to diagnose infections in a manner never before possible, correct interpretation of the results has never been more important. In this chapter we review current genomic technologies available, with attention to clinical applications, limitations, and interpretation in the medical setting.Keywords
- hybridization;
- mass spectrometry;
- microarray;
- molecular microbiology;
- molecular typing;
- next-generation sequencing;
- polymerase chain reaction
Chapter 23 – Non-genomic Omic Techniques
‘Omic’ technologies, which adopt a holistic view of the molecules that make up an organism, are aimed primarily at the global detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics) and metabolites (metabonomics) in a given biological sample. Application of genomic techniques in the field of diagnostic microbiology has limitations as genomics targets microbial organism-specific nucleic acids; therefore, a positive result can occur with both active and inactive microorganisms. Transcriptomics has progressed along with advances in microarray and real-time reserve transcriptase PCR technology, while proteomics and metabonomics have benefited greatly from the increasing sophistication of mass spectrometrical techniques that detect protein and metabolic analytes. Together, transcriptomics, proteomics and metabonomics are helping to address questions about gene expression, thereby providing ‘functional’ diagnosis and assessment of microbial infections.Keywords
- genomics;
- metabonomics;
- proteomics;
- transcriptomics
Chapter 24 – Adhesion and Colonization
The binding of bacterial adhesins to host receptors is a dynamic process occurring in several steps, which involve complex bacteria–host cell interaction. Initial weak physical interactions lead to more specific adhesion mechanisms that may be shared by several organisms, but eventually to species-specific adhesins that may elicit both bacterial and host factors leading to host cell damage, induction of inflammation and disease. Species-specific fimbrial adhesins may be viewed as direct mediators of bidirectional signalling between bacteria and host cells. Understanding of this process has been highly informative for the design of novel strategies to modulate these signalling pathways and to curb bacterial infections and their harmful sequelae. Development of mixtures of inhibitors or a polyvalent inhibitor is under investigation, since many infectious agents express multiple specificities. Multiple molecular mechanisms of adhesion are required to initiate infection, and effective anti-adhesion strategies will need to address both bacterial and host site particularities.Keywords
- adhesins;
- adhesion;
- colonization;
- fimbriae;
- pili;
- glycoconjugate;
- lectin;
- lipopolysaccharide;
- Escherichia coli;
- Klebsiella pneumoniae;
- Neisseria meningitidis
Chapter 25 – Invasion
- Kingmed Center for Clinical Laboratory, Guangzhou, China and Tianjin Medical University, Tianjin, China
- Available online 29 September 2014
Among pathogenic bacteria, a group of species causes infections relying on their ability to enter host cells at an early stage of inflammation, including Salmonella typhimurium, Shigella flexneri, Campylobacter jejuni, Yersinia pseudotuberculosis, Listeria monocytogenes, Neisseria gonorrhoeae, Streptococcus pyogenes and others. Entry of the microorganisms into non-phagocytic cells, such as epithelial, endothelial or stromal cells, is an invasive process that benefits the pathogenic bacteria. The entry process is often called ‘invasion’ and such pathogens are termed invasive bacteria. Invasion results in internalization and allows bacterial colonization within or translocation across the mucosal barrier. In some cases, the pathogen becomes sequestered within an infected organ or gains further access to deep tissues by way of blood or lymphatic vessels. Thus, the ability to invade non-phagocytic cells is a prominent feature of bacterial pathogenesis. In addition, entry into host cell cytoplasm offers bacteria a safe environment or a niche for multiplication, where the pathogen has no competition for growth and may evade the host immune system or avoid killing by antibiotics. The nutrient supply in intracellular environments is limited, therefore invasive bacteria employ various sensing systems that respond to environmental changes and obtain necessary nutrients from the host.Keywords
- colonization;
- filopodia;
- infection;
- internalization;
- invasion;
- pathogenic bacteria;
- T3SS;
- translocation;
- trigger-like;
- virulence;
- zipper-like
Chapter 26 – Pattern Recognition Receptors and the Innate Immune Network
The distinction originally made between innate and adaptive immunity has blurred. Innate immunity is an ensemble of cells and molecules that have evolved over time to perform a critical first responder’s function during the early stages of infection. Innate immunity holds the line, gathers information for ‘communication’ with more systemic elements of the adaptive immune system. Assessing the type of infection, first responders can call in effectors able to deal with the incursion. A major advance in understanding how changes in tissue status was recognized was the discovery of pattern recognition receptors (PRR) such as Toll-like receptors (TLR) that bind features of pathogens, as well as components of host cells that have died under duress. Barrier cells (keratinocytes, gut epithelia), along with WBC that are positioned beneath the barriers use a diverse set of PRR to recognize and differentiate different types of viruses, bacteria, fungi and parasites. PRR occupy different cellular compartments to ensure that they can monitor all possible locations that pathogens can replicate or exist in. The recognition is based on binding an integral part of the pathogen, a pattern: a consistently recognizable molecular signature of that type of pathogen. With the PRR strategy, if the pattern is there, then the pathogen is there and the battle is engaged.Keywords
- B lymphocyte;
- chemokine inflammosome;
- collectin;
- dendritic cell;
- inflammation;
- innate immunity;
- interferon;
- interleukin;
- macrophage;
- pattern recognition receptor;
- pentraxin;
- pro-inflammatory cytokines;
- surveillance;
- Toll-like receptor;
- T lymphocyte
Chapter 27 – Survival Strategies of Extracellular Bacterial Pathogens
Classic extracellular bacterial pathogens are endowed with an array of mechanisms that afford their survival in the inhospitable environment that is the human body. These mechanisms can be loosely grouped into those that defend the offending extracellular bacterial organisms from the devastating effects elicited by the host immune system, and those that are elaborated by extracellular bacterial pathogens to actively attack and override the human body. While extracellular bacterial pathogens employ different stratagems to subvert the human host, they are all unified by their exquisite ability to appropriate host cellular components and destroy target cells. In this chapter, we review, by means of prototypical examples, the different tactics utilized by extracellular bacterial pathogens to proliferate in the human body.Keywords
- antibacterial peptide;
- bacterial;
- capsule;
- complement;
- endotoxin;
- exotoxin;
- extracellular;
- human;
- pathogen;
- survival;
- superantigen
Chapter 28 – Survival Strategies of Intracellular Bacterial Pathogens
Many pathogens use the intracellular compartment of the host as a niche for immune evasion and replication. Avoidance of the microbicidal lysosome is central to all intracellular survival strategies. This is achieved by either subverting endosomal trafficking and remodelling of the phagosome into a hospitable vacuole, or by promoting phagosomal membrane disruption and escaping to the cytosol. This chapter reviews the general mechanisms used by bacterial pathogens to gain access to the intracellular habitat and to overcome the specific challenges imposed by the vacuolar and cytosolic lifestyles. Other general intracellular survival strategies are discussed.Keywords
- actin-based motility;
- cell egress;
- cell invasion;
- cell-to-cell spread;
- Chlamydia;
- Coxiella;
- cytosolic pathogens;
- Francisella;
- host cell subversion;
- intracellular pathogens;
- intracellular survival;
- vacuolar pathogens;
- intracellular replication;
- Legionella;
- Listeria;
- Mycobacterium;
- Rickettsia;
- Salmonella;
- Shigella;
- vacuole escape
Chapter 29 – A Phylogenetic Perspective on Molecular Epidemiology
Molecular epidemiology is a discipline that uses molecular or genetic markers to trace the development of a disease in a population and to understand transmission, as well as the population structure and evolution of bacterial pathogens. Phylogenetic analysis of molecular markers allows the determination of the genetic relatedness of strains from different sources, geographic locations and/or even different time periods and inferring evolutionary relationships. Molecular epidemiology has grown rapidly in the past couple of decades with the advances in DNA-based molecular typing techniques. Next-generation sequencing technologies have allowed remarkable advances in molecular epidemiological studies. This chapter reviews classical molecular typing schemes and introduces more advanced typing tools. Exemplar applications on a few extensive phylogenetic studies are also provided to demonstrate the usefulness of molecular typing and phylogenetic analysis in epidemiological investigations of bacterial infectious disease.Keywords
- 16S rRNA gene;
- DNA sequencing;
- epidemiology;
- genetic variations;
- molecular typing;
- phylogenetic analysis;
- PCR
Chapter 30 – Mammalian Antimicrobial Peptides; Defensins and Cathelicidins
Antimicrobial peptides (also known as host defence peptides) are an increasingly well-characterized, central component of host defence against infection. These peptides are an ancient form of innate immunity, conserved across evolution; found in animals, plants and even produced by microorganisms themselves. As antibiotic resistance becomes an ever-greater concern for our ability to treat infectious diseases, the study of antimicrobial peptides is providing new insights into the functioning of innate immunity and providing templates for the development of novel therapeutics. As knowledge of the properties of these peptides has developed, it has also become clear that, in addition to broad-spectrum direct microbicidal potential, they have modulatory effects on innate and adaptive immune processes in mammals, as well as some apparently non-immune functions. This chapter focuses on two families of mammalian peptides; the defensins and the cathelicidins, concentrating primarily on human peptides, but with reference to homologous peptides in mouse models.Keywords
- antimicrobial peptide;
- bacteria;
- cathelicidin;
- defensin;
- host defence;
- host defence peptide;
- immunomodulation;
- infection;
- innate immunity;
- virus
Chapter 31 – Antimicrobial Phages
Since their discovery near the beginning of the 20th century lytic viruses of bacteria known as bacteriophages (phages), have been widely used as antimicrobial agents even prior to their characterization as viruses. Early studies demonstrated their potential to treat infections in large but poorly described trials in cholera and dysentery patients. Phage therapy was commercialized by several large companies in the 1920s and 1930s but preparations were heterogeneous in quality and were recommended for the treatment of diseases that did not have a bacterial aetiology. The introduction of antibiotics into human and veterinary medicine side-lined the use of phage to pockets of use in countries in Eastern Europe and the countries of the former USSR where they are still used today. Phages were central to early discoveries in molecular biology and this and studies in animal models of infection in the 1980s led to a revival in interest as therapeutic agents. More recently, several early-stage clinical trials have been performed to demonstrate their safety in topical applications and a small phase 2/2a study showed efficacy in the treatment of antibiotic-resistant otitis. Phage genomics is still at an early stage and most phage genes are of unknown function, however expanding antibiotic resistance and a paucity of novel antibiotics has generated recent, further interest in their therapeutic development built upon genetically characterized bacteriophages.Keywords
- animal models;
- bacteriophage;
- biofilms;
- clinical trials;
- genomics;
- lytic
Chapter 32 – Modes of Action of Antibacterial Agents
This chapter describes the modes of action of the major antibiotics and synthetic agents used to treat bacterial infections. Particular attention is given to the biochemical mechanisms by which the agents interfere with biosynthetic processes and the basis for their selective antibacterial action. Interference with the biosynthesis and assembly of structural components of the bacterial cell wall provides the basis for many important groups of antibiotics, including the agents targeting steps in peptidoglycan synthesis. Other agents exploit more subtle differences between bacteria and mammalian cells in fundamental processes such as DNA, RNA and protein synthesis.Keywords
- action;
- antibacterial agents;
- antibiotics;
- mechanism;
- selectivity
Chapter 33 – Molecular Epidemiology of Antibiotic Resistance in Humans and Animals
The spread of antibiotic resistance is often the dissemination of individual resistant clones passing from one patient to another. The progenitors of most resistance genes are far older than the antibiotic era and many genes have migrated from their original location, through multiple episodes of transposition and plasmid transfer. During this journey they have been evolving to combat modern antibiotics, the extended-spectrum β-lactamases epitomise this. Multiresistant bacterial clones responsible for resistance are a combination of a ‘fit’ bacterial clone with a favourable combination of evolved resistance genes; examples include Staphylococcus aureus, Streptococcus pneumoniae and Acinetobacter baumannii. The use of molecular genotyping techniques such as pulsed-field gel electrophoresis and multilocus sequence typing has shown that as bacteria evolve into multiresistance, they lose their diversity. Whether the use of antibiotics in animals causes resistance in human bacteria is controversial but molecular techniques suggest it is not the major source of resistance genes.Keywords
- Acinetobacter baumannii;
- carbapenem resistance;
- clonal spread;
- extended-spectrum;
- β-lactamase;
- genotyping;
- MRSA;
- plasmid;
- transposon;
- Streptococcus pneumoniae;
- resistance gene origins
Chapter 34 – Design of Antibacterial Agents
The ‘design’ of a new antibacterial agent is utterly different from the design of a product in ‘macro’ engineering, such as aeronautical engineering. There is only a partial understanding of how the properties of the materials used in the design of an antibacterial agent (core structures, functional groups) determine its biological activities: from inhibitory potency at the bacterial target to bacterial cell envelope permeation to human pharmacokinetics, all of which are complex and multifactorial. Nevertheless, design elements can be used, including biophysically directed structure-based design. A case history is presented that describes the progression from a small compound or ‘fragment’ identified by nuclear magnetic resonance (NMR) as a relatively weak ligand of the GyrB protein of bacterial DNA gyrase to a derivative compound that displayed efficacy in an animal infection model and underwent phase 1 investigation in humans.Keywords
- antibiotic discovery;
- chemistry;
- design;
- drug development;
- DNA gyrase;
- drug discovery;
- topoisomerase
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When the body is exposed to a pathogen, the immune system responds to fight the infection. The immune system retains a memory immune response for many years which will provide protection in the event of future encounters with the same pathogen. This phenomenon has been exploited to protect against infection by exposing the body to safe derivatives of the pathogen to induce a protective immune response: these formulations are termed vaccines. An improved understanding of host–pathogen interactions, protective immune responses and novel adjuvants and delivery systems is helping produce safe, effective vaccines against infectious disease. These developments are reviewed in this chapter, along with some of the challenges presented in developing effective, protective vaccines.Keywords
- adjuvants;
- attenuated strains;
- delivery;
- DNA vaccines;
- immune responses;
- subunits;
- vaccines
Chapter 36 – Disseminated Infections: A Clinical Overview
The ability of microorganisms to cause infection is governed by multiple different factors – some linked to the microorganisms itself and others to the host or environment. In this chapter we explore some of these factors and provide an overview of disseminated infections examining some important manifestations such as toxic shock syndrome and sepsis as well as examining some of molecular aspects including host receptors and bacterial antigens. A brief discussion will also follow about disseminated infections in specific populations such as neonates and the immunosuppressed.Keywords
- colonization;
- disseminated infections;
- E. coli;
- endocarditis;
- humoral immune deficiency;
- immunocompromised host;
- lipopolysaccharide;
- neutropenia cell-mediated immunodeficiency;
- opsonization;
- pathogenesis;
- sepsis;
- staphylococci;
- toxic shock syndrome
Chapter 37 – Staphylococcus aureus
Staphylococcus aureus persistently colonizes the nares of approximately 20% of humans. The organisms express a plethora of secreted and surface proteins that promote colonization and evasion of immune responses. Surface proteins promote adhesion to tissue components and invasion into host cells. Cytolytic toxins damage host epithelial cells and neutrophils. Extracellular enzymes and zymogen activators contribute to immune evasion and tissue damage. Several small secreted proteins interfere with complement and inhibit neutrophil activation and migration. The core genome harbours genomic islands that encode virulence factors and restriction systems. Mobile genetic elements (MGE) are commonplace. Most strains carry prophages, insertion sequences, transposons and pathogenicity islands. Strains that are resistant to β-lactam antibiotics (methicillin-resistant S. aureus) cause hospital-acquired infections while hypervirulent community-associated MRSA are increasing prevalent. β-Lactam resistance is encoded by an MGE called SCCmec of which there are at least 11 distinct types.Keywords
- adhesion;
- biofilm;
- immune evasion;
- mobile genetic elements;
- MRSA;
- Staphylococcus aureus;
- toxins;
- virulence
Chapter 38 – Streptococcus pyogenes
Streptococcus pyogenes, also known as group A streptococcus (GAS), is most commonly associated with mild, self-resolving infections of the skin and oropharynx. However, dissemination of the bacteria to normally sterile sites within the body can lead to a variety of invasive conditions that are associated with high morbidity and mortality. In addition, the generation of human cross-reactive antibodies in response to lingering GAS infection can result in the development of post-streptococcal autoimmune sequelae that afflict the organs, joints and CNS.GAS pathogenesis is mediated by an extensive repertoire of extracellular virulence factors. Initial colonization of the skin and oropharynx is facilitated by cell-associated adhesins that bind to multiple components of the host extracellular matrix. While a battery of antiphagocytic molecules allow the organism to persist at the initial site of infection, the production of multiple toxigenic and tissue-destructive virulence factors facilitates the transition from a superficial to an invasive disease phenotype.Despite the continuing susceptibility of GAS to β-lactam antibiotics a resurgence of serious streptococcal disease has been observed over the past 30 years. While the cause of this resurgence is incompletely understood it has been tentatively attributed to the reappearance and/or increased circulation of a highly invasive clone of serotype M1T1 GAS. This shift in the epidemiology of GAS infection highlights the need for increased surveillance of GAS in the community, faster, more reliable diagnostic tests for GAS infection in a clinical setting, and more targeted treatments of invasive GAS disease. Above all, the development of a safe, effective GAS vaccine would prove invaluable.Keywords
- epidemiology;
- group A streptococcus;
- pathogenesis;
- virulence factors
Chapter 39 – The Enterococci
The enterococci, microbes well-adapted to existence as commensals of the gastrointestinal tracts of organisms from man to insects, have emerged over the last several decades as leading hospital pathogens. This evolution stems in part from their intrinsic resistance to harsh conditions in the hospital environment, including host mucosal defences, disinfectants and desiccation, often resulting in their occurrence with other antibiotic-resistant microbes. In hospital and agricultural environments, enterococci have served as a collection point for antibiotic resistance factors, and with the emergence in the 1980s of vancomycin-resistant strains, few bactericidal therapies remain. In an ominous development, they have begun to transmit this resistance to meticillin-resistant Staphylococcus aureus. As a result, multidrug-resistant enterococci have become a leading public health concern.Keywords
- antibiotic;
- commensal;
- Enterococcus;
- faecalis;
- E. faecium;
- microbiome;
- plasmid;
- resistance;
- transposon;
- vancomycin
Chapter 40 – Nocardia and Actinomyces
Nocardiosis is an opportunistic infectious disease caused by a ubiquitous aerobic bacterium of the genus Nocardia that can be found in the environment. These intracellular bacteria are held responsible for many infections affecting the lungs, the brain or the skin, especially in immunocompromised patients. The taxonomy of this bacterium is complex and a multilocus sequence analysis may sometimes be necessary for a correct identification. Only three complete genomes of the genus Nocardia have so far been fully sequenced and referenced which constitutes an important stage in the study of this bacterium.Actinomyces belong to the normal indigenous microflora, so that they are considered as facultative pathogens. Actinomycosis is usually associated with the breakdown of normal physical barriers, such as disruption of mucosal membranes.Actinomycosis and nocardiosis are distinct diseases that respond to very different forms of therapy. Actinomyces can be readily distinguished from Nocardia by their distinct anaerobic versus aerobic patterns of growth after isolation from a clinical sample.Keywords
- Actinomyces;
- actinomycosis;
- genome;
- Nocardia;
- nocardiosis;
- nucleic acid methodology;
- pathogenic proteins
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