Evolutionary Biology of Bacterial and Fungal Pathogens

This chapter focuses on aspects of the evolution of the bacterium-host interactions that cannot be readily accounted for by simple, advantage-to-the individual evolutionary scenarios. It discusses how the perversity of the immune system fits with current hypotheses for the evolution of virulence and the evolution of the so-called virulence factors, and speculates on the reasons natural selection has failed to or is unable to blunt the immune overresponse to bacterial infections. The chapter provides a brief discussion of the implications of this perspective on virulence for the treatment of bacterial infections. A section discusses how the observation that morbidity and mortality of bacterial infections can be attributed to the hosts’ immune overresponse fits each of these hypotheses for the evolution of the virulence of bacteria. The conventional wisdom is an observation rather than a mechanism, an observation that focuses on the interactions between bacteria and the individual hosts they colonize. Reasonable candidates for coincidental virulence due to an immune overresponse are diseases associated with Helicobacter pylori. As a consequence of their vastly shorter generation times, haploid genomes, and propensity to receive genes and pathogenicity islands by horizontal transfer, it seems reasonable to assume that bacteria would have an edge in an evolutionary arms race with their mammal hosts.

This chapter focuses on how selection at the microbial level may lead to changes at the infection level and examines some predictions one can draw from evolutionary theory. The major motivation for this review stems from the fact that natural selection among microbes can potentially have two opposite outcomes on pathogenesis. Pathogenic bacteria use a chemical communication system, called quorum sensing, to regulate within- host density. The chapter reviews the effects of within-host selection; as predicted by theoretical models and as empirically observed. There are three reasons why an allele favored within a host may nevertheless not go to complete fixation in the population at large. The first reason is that a locally favored variant may have partially, or completely, lost the ability to disperse from its host. This may be a direct result of selection on dispersal: evolutionary ecology shows that dispersal tends to be locally counter selected. Second, a locally favored variant, V, may have lost (completely or partially) the ability to survive and/or replicate in environments other than in the focal host itself. The third mechanism is that a pathogen variant, V, may increase in frequency within a focal host but not fix globally because the traits favored by within host selective pressures are not those that maximize the total infective output.

Studies relating to the epidemiology and evolutionary dynamics of pathogens are just beginning to emerge with some exciting insights. This chapter reviews some of the basic evolutionary approaches that might give insights into the population dynamics of infectious disease and that would link well with epidemiological data to give a more complete picture of population dynamics over time and insights into more appropriate intervention strategies. The author reviews these applications of evolutionary techniques through a series of examples throughout the chapter. The key factors in the evolutionary response of pathogens to their environments can be measured by assessing the genetic diversity, the impact of natural selection in shaping that existing diversity, and the impact of recombination in redistributing that diversity, sometimes into novel combinations. Natural selection at the phenotypic level is generally agreed upon, and there are abundant examples, perhaps the most famous of which are Darwin’s finches. One of the main goals of genomic science is to elucidate the relationships between genotypes and phenotypes. Population genetic approaches can provide a statistical framework within which one can test such associations. Phenotypes associated with each sequence can then be evaluated using a nested analysis of variance. In summary, a diversity of quantitative approaches are now available for analyzing microbial data and linking genetic diversity with epidemiological factors.

The contemporary social and environmental influences on infectious diseases can be better understood by exploring the long historical procession of disease risks, as human culture has evolved from early hunter-gatherer days. The main features of today's world that contribute to the increased probability of emergence and spread of infectious diseases in humans are shown in a table. Social-cultural factors are of great importance in influencing the contemporary changes in patterns of new and spreading infectious disease. Further, social circumstances are also a basic determinant of susceptibility to infection. The emergence of a zoonotic disease in humans requires a novel type or amount of contact between humans and the existing animal reservoir for the infectious agent. It is necessary to understand the particular circumstances that favor the “emergence” of bacteria, viruses, protozoa, and prions in response to these social and environmental changes. It is also forcibly reminded of the unpredictability and irrepressibility of infectious disease mobility and mutability by the devastating ongoing pandemic of HIV/AIDS since it emerged in the 1980s. Many infectious diseases are sensitive to climatic conditions, particularly insect-borne infections and infections that are spread person-to-person via contaminated food and water. Natural climatic variations and events can influence infectious disease emergence. Each of these infectious diseases is maintained, at least in part, in wild birds. Several recent scientific reports suggest that recent climate change has already begun to influence some infectious diseases.

Studies of multifactorial diseases, such as infectious diseases, must consider the complex interplay between environmental (microbial and nonmicrobial) and human (genetic and nongenetic) factors determining immunity to infection. There is increasing evidence that host genetic factors determine differences in host susceptibility to infection and contribute to the pattern of clinical disease. The diversity of the human genome varies according to the genomic region and human population considered. Two major population processes have a strong impact on the shaping of genetic diversity: founder effects and bottlenecks. The evolutionary dynamics of host-pathogen interactions lead to constant natural selection for adaptation and counter-adaptation in the two competing species. In the context of host-pathogen interactions, the first evidence of selection acting on a human gene was obtained for the sickle cell anemia Hb^S allele and malaria resistance. Selection pressure on the human genes involved in immune-related processes or, more generally, in host-pathogen interactions, is not limited to these instances. In this context variation in human genes conferring resistance to infectious diseases in the past may today provide us with information essential for improvements in our understanding of host-pathogen interactions. The identification of human genes that have been acted upon by natural selection, indicating an essential role in human survival over time, is therefore a complementary strategy for identifying genes that may be involved in different susceptibilities of the human host to infectious diseases today.

Bacterial infections pose a serious threat to human health; antibiotics are a powerful remedy. This chapter discusses the biological background for the evolution of antibiotic resistance and reviews some of the possible ways that humans can intervene. It focuses on a priori justification for the interventions, the difficulties in implementing them, and some evidence for their success. The problem of resistance to antibiotics in bacteria spans many academic disciplines, including ecology, microbiology, medicine, nursing, bacterial genetics, evolutionary biology, economics, statistics, and mathematics. Colonization and infection define the ecological and epidemiological background for the evolution of antibiotic resistance. The evolution of antibiotic resistance involves two processes: emergence and spread. Thresholds may be most important as motivating concepts for understanding the dynamics and control of antibiotic resistance. The chapter examines the way antibiotics are typically used and strategies for reducing use or manipulating antibiotic use to reduce the spread of resistance. The use of antibiotics is a major perturbation to microbial communities. The evolution of resistance has economic causes as well as biological ones. Sufficient investment in the types of solutions discussed here, and research to support their use could reduce resistance, but without an understanding of the current incentives and without structural changes, these solutions are not likely to reverse current trends.

Bacterial pathogens, the subject of this chapter, exhibit antigenic diversity both at the level of the pathogen population through allelic polymorphism and within individual bacteria, where the expression of antigenic loci may be switched on and off. The balance between the different effects of immune selection leads to the various patterns of antigenic diversity observed among bacterial pathogen populations. The chapter discusses some of these patterns and the theoretical frameworks that have been developed to try to understand them. The different population structures of meningococcal serogroups can be explained using simple mathematical models of immune selection. It introduces the basic frameworks used to understand the population dynamics of infectious diseases before describing models that incorporate pathogen population structure and the role of immune selection. For many bacterial pathogens, however, the immune response is generated to more than one antigen. Despite the complexity of the biological and epidemiological factors that affect the evolution of bacterial pathogens, a wide range of population structures can be accounted for using the simple theoretical models of immune selection. Understanding the role of immune selection for other bacterial pathogens will require similar large-scale projects that examine the structuring of antigenic determinants at a population level. As sequencing methods and other technological tools advance, examining pathogen population structures should become increasingly straightforward, enabling a deeper examination of the effects of immune selection on pathogen populations.

Human beings live in close association with vast numbers of microorganisms that are present on the skin, mouth, and gastrointestinal tract. Although the small bowel and stomach can become heavily colonized by pathogenic bacteria and yeasts under some circumstances, the colon is the principal region of bacterial colonization in the healthy gastrointestinal tract, owing to the antimicrobial effects of gastric acid and bile salts in the upper gut and the rapid passage of digestive materials that prevents microbial overgrowth. While the effects are often subtle, intestinal microorganisms exert their influence on the host in many ways. Culturing studies have shown that the microbiota comprises several hundred bacterial species, subspecies, and biotypes, and that some organisms occur in higher numbers than others, although about 40 species constitute approximately 99% of all isolates. Pathogenic bacteria invading the body are affected by, and deal with, mucus barriers in different ways. Ulcerative colitis (UC) is one of the two major forms of idiopathic inflammatory bowel disease, and is an acute and chronic disabling condition that is essentially incurable and treated primarily with anti-inflammatory drugs and steroids. Many reactions are known in which enzymes produced by intestinal microorganisms form carcinogens from dietary precursors. Fecal bile acids are also related to the risk of colon cancer, because they are converted to steroids by intestinal microorganisms, which have procarcinogenic properties.

Opportunistic pathogens are particularly important at hospitals, as they are significantly responsible for nosocomial infections. In trying to understand the underlying reasons for the acquisition and evolution of virulence of opportunistic pathogens, this chapter addresses two topics. The first one is to know whether the environment where they grow during infection is changing through time and thus forcing their evolution. In other words, during infection, the infected patient is both the habitat and the food source of the infecting bacteria. The second topic addressed is the mechanisms of evolution, which allow bacteria that usually do not infect humans to produce a disease in sick people. The study of Pseudomonas aeruginosa isolates from chronic and acute infections has demonstrated that transition to chronicity involves an increase in the ability to form biofilms and a concomitant reduction in the expression of the type III secretion system. This transition does not consist of phenotypic changes, but consists of the accumulation of adaptive mutations during the in-host evolution of this bacterial opportunistic pathogen. Concerning bacterial-protozoa interactions as forces shaping the evolution of bacterial opportunistic pathogens, it is important to recall that protozoa are major grazers of bacteria in natural environments. If natural nonclinical environments are relevant for the evolution of bacterial opportunistic pathogens, modifications of these environments can also produce changes in bacterial populations.

In the multilocus Fisher-Wright model allele frequencies and the combinations of alleles (STs) fluctuate solely through genetic drift. Using multiple loci prevents recombination from obscuring the relationships between strains, because a change at one locus will not alter the information about relationships between strains present at the others. Computer simulation of bacterial populations is complementary to an analytic approach. Furthermore, simulation of the evolution of bacterial populations provides an alternative means of testing hypotheses: the effects of various evolutionary scenarios may be explored by introducing them into a simulation based on the neutral model. An example of this type of approach is discussed in this chapter, where the conditions under which an evolving bacterial population splits into separate clusters, mimicking speciation, are explored. In sum, while coalescent analyses may be the most appropriate for individual loci, and certainly have a much wider body of theoretical work to draw on than the methods discussed in the chapter, the authors are excited by the potential of methods such as multilocus sequence typing (MLST) that consider the genetics of populations of strains rather than of individual loci, to demonstrate forces other than neutrality influencing the structure of populations. The chapter also talks about the modeling of alleles and their association using recently developed techniques designed to simulate and analyze data sets obtained from MLST studies.

The fungal cell wall surface, which represents the interface between the host and the infective microbe, emerges as the complex structure that harbors many of the relevant components that interact with the host tissues and defenses. Knowledge of cell wall biogenesis and the functions that control dynamics of this interface may provide significant clues for the development of novel therapeutic strategies. This chapter summarizes some structural and functional aspects of the cell wall of the fungal pathogen Candida albicans. It also highlights recent findings that indicate its relevance in the interaction with the host immune cells, as this process is essential to prime and develop a protective immune response and contributes significantly to the control and pathology of the infection. The cell wall is an external structure that confers the typical morphology to almost all microbes. As the most external cellular structure, it mediates adhesion to the host tissues, being crucial to initiate colonization and, therefore, causes disease. Phagocytosis of C. albicans is carried out by different types of cells, mainly neutrophils and macrophages. This process occurs presumably by the action of complement and antibody receptors, following the routes of microbial ingestion after opsonization by either the complement, antibodies, or both. The identification of permanent dialog among the microbe and the host cells at the molecular level (signals, mechanisms, and responses) will have important consequences from which both basic and applied research may benefit in the near future.

Bacterial genomes are usually packed with genes occupying around 90% of their DNA, and no structures resembling isochores or telomeres have been identified. A second source of heterogeneity could come from structural domains in the genome, which would make some areas of the chromosome more accessible than others to foreign DNA sequences and influence intrachromosomal recombination. With the advent of genomic data and the improvement of methods for replication origin and terminus detection, it is now possible to review the genome balance hypothesis. The mutation probability has been proposed to vary within a bacterial chromosome for genes located at different positions relative to the replication origin. Electron micrographs of the Escherichia coli chromosome displayed a rossette-like organization with loops of supercoiled DNA distributed around a central node. Species such as Staphylococcus aureus and E. coli display an increase in horizontally acquired genes closer to the terminus, whereas transposable elements seem unaffected. Sequence repeats, in all their variants, are one of the main evolutionary tools to generate variability, this being structural (rearrangements) or functional (generation of new genes). The plethora of genomic data has unexpectedly suggested that the traditional view of IS Elements (ISs) as detrimental selfish elements is probably an oversimplification. In relation to bacterial pathogens, the data suggest that their genomes are more flexible than related nonpathogenic species, as if their need for fast adaptation and plasticity had relaxed organizational constraints.

This chapter shows that genetic variability drives the evolution of microbes. During evolution, bacterium-host interactions have developed, and different types of associations have evolved. The chapter describes major facets of the genetic basis of host-microbe interaction, with particular attention to newly discovered genetic elements, so-called genomic islands (GEIs), which are parts of the genomes of many bacteria, pathogenic as well as nonpathogenic. The majority of GEIs differ with respect to the G+C content and codon usage from the rest of the core genome. The first pathogenicity islands (PAIs) were described in pathogenic Escherichia coli strains. Enterobacteria represent a diverse group of microbes; the majority of them have their habitat in the intestine of humans and many animals. The majority of genes of Buchnera and Blochmannia genomes are identical to genes that are present in the genome of E. coli. PAI I and PAI II encode α-hemolysin, and PAI I to PAI III carry genes for different fimbrial adhesins. PAI IV is identical to the so-called high-pathogenicity island that was first described for Yersinia pestis and encodes an iron uptake system, termed yersiniabactin. The capsular antigen K15 is encoded by island V. Based on the complete genome sequence of E. coli strain 536, the authors were recently able to detect four additional genomic islands, GEI VI to GEI IX. There is no doubt that the gene products of PAI I to PAI V contribute to urinary tract infections.

Integrons were only formally identified as agents of antibiotic resistance gene recruitment in the late 1980s following the observation that transposons and R-plasmids expressing different antibiotic resistance phenotypes shared the same genetic backbone and differed only in the resistance genes they harbored. Integrons can be divided into two distinct subsets, the mobile integrons (MIs), linked to mobile DNA elements and primarily involved in the spread of antibiotic-resistance genes, and the superintegrons (SIs). Integrons are undoubtedly ancient entities, as indicated by the species-specific clustering of the respective SI integrase genes in a pattern that adheres, in several cases, to the line of descent among the bacterial species in which they are located. Thus, the establishment of SIs likely predates speciation within the respective genera, indicating that integrons are ancient structures that have been impacting on the evolution of bacterial genomes for hundreds of millions of years. The determination of the diverse number of metabolic activities associated with SI cassettes (other than antibiotic resistance and virulence) indicates that integrons operate as a general gene capture system in bacterial adaptation. Integrases encoded by integrons mediate recombination involving two types of sites-their specific attI site and the cassette-associated attC site-and are able to recombine distantly related DNA sequences. With the discovery of SIs, and of the thousands of gene cassettes associated with integrons that are located in the genomes of environmental bacterial species, the importance of these elements clearly extends beyond the phenomenon of antibiotic resistance.

This chapter discusses the characteristics and features of plasmids, providing limited but significant and often cited examples of the evolution of natural plasmids. The evolution of plasmid-mediated antibiotic resistance is illustrated through the description of the IncFIme plasmid, a well-studied virulence and resistance plasmid, and of other broad-host-range resistance plasmids. Many natural plasmids are stably maintained at their characteristic copy number within the growing bacterial population. The study of how the evolution of virulence plasmids happens may allow a more complete understanding of how pathogens evolve, and the analysis of those sequences offers the opportunity to compare virulence plasmids from closely related or distant species to better understand the origin of the pathogenic traits. Furthermore, the virulence plasmid might occasionally be replaced or driven away by incoming plasmids of the same Inc group. A model developed to describe the evolution of the iteron-based replication system is that of the IncQ plasmids. Bacterial conjugation is an essential property for plasmid dissemination. Conjugative systems (Tra systems) in gram-negative bacteria support transfer between different genera and kingdoms, regardless of their replication mechanisms. They consist of three components: the transferosome, the relaxosome, and the coupling protein. The presence of multiple physically linked resistance genes on the same plasmid, conferring resistance to different classes of antibiotics, may confer a selective advantage to the bacterial host when several antimicrobials are simultaneously administered. Such synergy between different coexpressed resistance genes would allow the recipient host to be positively selected by each individual class of antibiotics.

Bacteriophages (so-called phages), viruses that infect bacteria, are the most abundant and varied biological group on the planet, with ~10⁸ different phage species representing an estimated global population on the order of ~10³¹ viral particles. The field of phages as biotherapeutic agents has been rediscovered, as several experiments have demonstrated their potential for treatment of antibiotic-resistant bacteria, such as vancomycin-resistant Enterococcus faecium. In addition to the classical virulence factors described in prophages, the “genomic era” has revealed new pathogenicity factors linked to prophages. Several examples of antibiotic resistance genes spread by generalized or specialized transduction have been described. For instance, resistance to imipenem, aztreonam, and ceftazidime in Pseudomonas aeruginosa can be transduced by two phages, AP-2 and AP-12. Phages can also transduce resistance to chloramphenicol in E. coli, methicillin in Staphylococcus epidermidis, novobiocin or tetracycline in Staphylococcus aureus, or tetracycline and chloramphenicol in Actinobacillus actinomycetemcomitans. There are few examples of bacteriophages harboring antibiotic resistance genes. An interesting example is the presence of the ars operon in the skin element of Bacillus subtilis, which confers resistance to arsenate. One of the mechanisms of macrolide resistance found in streptococci is mediated by the proton-dependent efflux pump encoded by mef (A). Two main lines of research have been opened to find novel anti-infectives. The first is using lytic enzymes to weaken the bacterial cell wall of specific bacteria. The second line of research consists of using bacteriophages to design protein-like chemical compounds that arrest critical cellular processes.

The tremendous genetic diversity observed among pathogenic bacteria mirrors their different lifestyles and physiological versatility; their evolution has enabled them to adapt to specific niches and growth conditions. A remarkable aspect of the major gram-positive community-acquired pathogens infecting humans is the extent of their clonality. A number of factors may influence the emergence, spread, and extinction of clones. These are discussed sequentially for Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, and the common themes and differences between these organisms are explored. A series of methicillin-susceptible S. aureus isolates of phage type 80/81 isolated between 1962 and 1998, most of which were multilocus sequence type (ST) 30, have been shown to harbor the genes encoding Panton-Valentine Leucocidin (PVL), a common virulence factor among many isolates of contemporary community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Infectious disease epidemiology is characterized by patterns of emergence, spread, and evolution of strains, with phases of reduction and apparent extinction or stabilization of genetic types, and the emergence of new pathogenic or antimicrobial-resistant clones. A major determinant in the stability of bacterial clones is the relative impact of point mutations, DNA rearrangements and horizontal gene transfer, and bacteriophage acquisition, although other factors are undoubtedly important. An expansion in the implementation of sequencing methodologies and clustering algorithms to examine clonal emergence and spread will be important for our continued understanding of how bacteria evolve to become virulent and the response of bacterial populations to antibiotics and vaccines.

This chapter reviews the examples of Candida albicans survival resulting from specific chromosome alterations in vitro, as well as recent advances in understanding this regulation. Previously classified as asexual, C. albicans belongs to the genus Candida, which is composed of approximately 159 species of lower fungi reproducing by multilateral budding. Development of fluconazole resistance has been extensively studied in clinical settings and in laboratories. Series of matched clinical isolates became an important tool in these studies. Matched series are usually represented by the isolates, which were sampled from the same patient either at a certain time during infection or during recurrent episodes and which were derived from the same strain. Study of the deletion in one of the mutants confirmed the loss of at least a 305-kbp portion adjacent to the right telomere. The reason this deletion confers the Sou⁺ phenotype is discussed in the section Ch5 carries functionally redundant regulatory genes. In fact, an uncharacterized rearrangement of one mutant of strain CAF4-2 is probably the only true exception from the Ch5 monosomy. The major control of sorbose utilization (CSUs) on Ch5 that are controlled by this chromosome copy number, apparently can 'override' secondary CSUs, thus allowing ready adaptation to sorbose. Universality of the control caused by aneuploidy implies that C. albicans regulatory and metabolic genes are distributed nonrandomly over the chromosomes.

In 1999, researchers identified in the emerging Candida albicans genome the mating-type locus, and one year later two reports documented cell type-dependent mating. With these discoveries, C. albicans had become a sexual organism, but the sequence of discoveries did not stop there. If switching and the mating process proved to play important roles in biofilm formation and pathogenesis, then there could exist strong selection pressure to maintain them. To test biofilm hypothesis, researchers collected spontaneous mating type-like (MTL)-homozygous offspring from natural a/α strains that exhibited high frequencies of MTL-homozygosis and compared virulence of offspring and parent strains in the murine model for systemic infection in two ways. First, they simply followed the survival curves of outbred mice injected individually with each strain. Second, they coinjected parent and offspring with equal numbers of parent and offspring, and at the time of death or extreme morbidity quantitated the concentration of the two strains in the kidney. In the great majority of cases, the parent was more virulent than the offspring in both tests, supporting their hypothesis for a/α maintenance. The intricate relationships between switching, mating, and pathogenesis in C. albicans have therefore provided not only explanations for some of the unique characteristics of each of the component processes, but insights as well into how the basic biology of an organism is manipulated in the evolution of hostpathogen interactions.

Variation that occurs in pathogens is based firmly upon one of three prime mechanisms of genetic variability: point mutations, genetic rearrangements, or lateral gene transfer (LGT). As LGT recently has been the focus of significant new insights into pathogen evolution it will be highlighted in this chapter. The intensive sequencing of microbial genomes and the comparisons made among them expanded our understanding of the scope of LGT in bacterial gene evolution. The main distinction is that the environment and ecology of pathogens is played out within another living organism-the host. The pan-genome represents the most accessible set of building blocks available for use in LGT. In the current environment, however, the widespread selective adaptation has spread because antibiotics are added to everything from cutting boards to cattle feed. The discussion of pathogenic gene content, style, and core distribution is intimately linked to the problem of defining both the species in bacteria and the events most likely to lead to new speciation. Multilocus sequence typing (MLST) involves the sampling of DNA sequences for a subset of housekeeping genes and the assessing of alleles for the purpose of studying the extent of recombination. LGT can cause shifts in the immediate pathogenesis of a clonally evolving group such as the clonal complex. The chapter reviews the way in which LGT has been an architect in the virulence, the genes, the genome, and the pan-genome of pathogens.

In the real natural world, the term evolvability is used to mean the actual propensity for any biological structure to evolve-evolutionary rates. Antibiotic resistance is not only a clinical problem, but also a unique opportunity of observing “evolution in real time,” and therefore constitutes a privileged meeting point for clinical and evolutionary microbiologists. This chapter stresses the notion of modules as anatomical, structural genetic modules. Modules are also evolutionary entities, and a general view on modular evolution is presented. Modularization might be first understood as an increasing-variability process that adds modular units within a given local genetic structure. This type of first-order (essentially quantitative) modularization has a limit, because modules might tend to be either deleted or fused to other modules in a reducing-variability process. Second-order modularization might be the result of selective events acting on groups of modules produced by the first-order process. Most observed second-order modularization is possibly the result of selective events. Plasmids made by second-order modularization of other plasmid modules are probably frequent. Techniques of comparative genomics have been used to infer functional associations between proteins, based on common phylogenetic distributions, conserved gene neighborhood, or gene fusions. Similar types of methods could be developed to predict functional associations between modules involved in the emergence, expression, mobilization, or evolution of antibiotic resistance. Probably the evolutionary consequences of modularization are far more significant than those related to mutation in terms of genetic innovation, including antibiotic resistance.

Genes participating in the synthesis and metabolism of the cell wall might have a common ancestor with well-disseminated beta-lactamase genes (bla genes) in pathogenic bacteria. Moreover, some of the most successful beta-lactamases have evolved by mobilization from chromosomal bla genes in environmental bacteria, and their evolution in pathogenic bacteria has been accelerated by the extensive use of beta-lactams in the clinical field over the past 70 years. The most widespread plasmid-mediated beta-lactamases are also represented in some of these subgroups such as penicillinase from Staphylococcus aureus (group 2a) or TEM-1 and SHV-1 enzymes (group 2b) and those that have suffered important evolutionary processes in the past years such as extended spectrum beta-lactamases (ESBLs) (group 2be). The ancient evolution of beta-lactamases can now be hypothesized with the aid of structural phylogenetic analysis. The majority of bla genes encoding B1 and B2 beta-lactamases are located on the chromosome but have been recently mobilized at least twice to plasmids, once for the VIM-group and once for the IMP group. The number of studies of phylogenetic groups in Klebsiella pneumoniae isolates is scarce and has mainly focused on their relationship with chromosomal beta-lactamases. The production of virulence markers has been associated with specific clones. This is the case of extraintestinal pathogenic CTX-M-producing Escherichia coli isolates that were compared in a recent study with non-ESBL producers.

In developing countries, the levels of quinolone resistance among gram-negative bacteria are very low, probably because of the low use of these antimicrobial agents, in association with their high cost. One of the most fundamental measures that could be taken to minimize quinolone resistance and antibiotic resistance in general is to eliminate supplementation of animal feeds with antibiotics, including tetracycline, macrolide, and quinolone derivatives. Escherichia coli is likely the Enterobacteriaceae in which the increase in quinolone resistance has been most evident. This trend in fluoroquinolone resistance has also been shown in Enterobacteriaceae from bacteremias from hospitals in England and Wales. Pseudomonas aeruginosa and Acinetobacter baumannii are the two most relevant nonfermentative gram-negative bacteria. They share similar characteristics, one being the ability to develop resistance to multiple antimicrobial agents. Quinolone resistance in enterococci has developed rapidly, and there are high rates of resistance, especially among vancomycin-resistant enterococci. In 1998, plasmid-mediated resistance in Klebsiella pneumoniae, caused by the qnrA gene, was reported. The future of the increase of quinolone resistance is difficult to predict, but it will clearly continue to increase unless steps are taken to reduce it, such as the development of SOS inhibitors.

This chapter briefly reviews the mode of action and the mechanism of bacterial resistance to glycopeptides, as exemplified by the VanB type, and discusses its diversity, regulation, evolution, origin, and recent dissemination to methicillin-resistant Staphylococcus aureus. Classification of glycopeptide resistance is based on the primary sequence of the structural genes for the resistance ligases. Although the six types of resistance involve related enzymic functions, they can be distinguished by the location of the corresponding genes and by the mode of regulation of gene expression. An interesting phenomenon that has developed in some VanB- and VanA-type enterococci is vancomycin dependence. These glycopeptide-dependent strains are also able to grow in the absence of glycopeptides if supplied with the dipeptide D-Ala-D-Ala, confirming that they are unable to produce the ligase encoded by the chromosomal ddl gene. The vanF operon is composed of five genes (vanY_F, vanZ_F, vanH_F, van_F, and vanX_F) encoding homologues of VanY, VanZ, VanH, VanA, and VanX, and the genes essential for resistance (vanH_F, vanF, and vanX_F) are organized and oriented as in VanA-type strains. The evolutionary lineage of these groups of homologous genes is not clear, but they may have a common ancestor, or Paenibacillus could be a progenitor of the resistance operons acquired by enterococci. Conjugal transfer of plasmids that have acquired Tn1546-like elements by transposition appears to be responsible for the spread of glycopeptide resistance in enterococci.

The intent of this chapter is to take a broader perspective, extending our understanding of acquired resistance in a few fungi to the problem of intrinsic (primary) resistance throughout the fungal kingdom. In diploid fungi heterozygous mutations associated with antifungal resistance may be dominant or recessive; under selective pressure the latter may undergo mitotic gene conversion to homozygosity. A role for genome instability (chromosome rearrangements or aneuploidy) in antifungal resistance was illustrated by recent studies of Candida albicans. The first generation of azole antifungals were imidazoles such as clotrimazole and miconazole. As with PDR1, TAC1 is evolutionarily divergent, and there are no unambiguous orthologs outside of the C. albicans-containing CTG clade. The first generation of research on antifungal resistance has focused on understanding the basis for acquired resistance in the experimentally tractable fungi Saccharomyces cerevisiae, C. albicans, and C. glabrata. The second generation of antifungal resistance research will explore the basis for intrinsic resistance in the diverse fungal pathogens that increasingly threaten immunocompromised patients, particularly Aspergillus, Fusarium, Scedosporium, and zygomycete species. Ultimately, understanding the molecular basis for intrinsic antifungal resistance will facilitate the design of second-generation inhibitors with an extended spectrum of activity.

This chapter deals with study of antibiotic resistance and its fitness costs has had several interesting evolutionary implications. The effect of antibiotic resistance on the virulence and disease pathology of pathogenic bacteria can be assessed by measuring 50% lethal dose LD₅₀ values in experimental animals or by examining the specific pathology of the disease. An interesting question is whether antibiotic resistance is associated with an altered rate of bacterial transmission between hosts. The most commonly occurring katG mutation in Mycobacterium tuberculosis, Ser315Thr, is both highly resistant to isoniazid and virulent in the mouse model of the disease. A meta-analysis of the literature on isoniazid resistance resulting from the S315T mutation in clinical isolates in relation to ecological factors supports this, suggesting that this mutation provides high-level resistance without diminishing virulence or transmissibility. For the models to be most useful in the context of antibiotic resistance, one needs to know if the transmission rates for susceptible and resistant bacteria differ significantly. The limited amount of data currently available on the biological costs suggests that antibiotic resistance might be less easily reversed than previously anticipated, and the expected rate and extent of reduction are predicted to be at best moderate in community settings. In particular, one needs measurements under conditions that are as similar to the clinical situation as possible. Thus, competition, colonization, and transmission studies in human volunteers are needed, as they are likely to give us the most relevant parameter values.

Antibiotic resistance may be regarded as the paradoxical consequence of the success of antibiotic therapy. Bacteria develop antibiotic resistance in two main ways: horizontal gene transfer and mutation in different chromosomal loci. The Mycobaterium tuberculosis species has to acquire antibiotic resistance by mutational events exclusively. Mutation is the raw material of evolution and is the ultimate source of heritable variation on which natural selection acts. The importance of recombination in the evolution of bacterial pathogens has become increasingly apparent. Recombination probably mediates genetic change in all bacterial species and is likely to have been crucial in allowing bacteria to avoid the immune response, in distributing among the population genes that increase virulence or transmission between hosts, and in providing increased resistance to antibiotics. A pathogen microorganism may be the paradigmatic example of a relationship between the stable hypermutation/hyperrecombination status and antibiotic resistance acquisition. This is the case of Streptococcus pneumoniae, where transformation and recombination seem to be the major sources of genetic variability. Microorganisms harboring an antibiotic-resistance mechanism, acquired either by horizontal transfer or mutation, will be positively selected in the presence of the antibiotic. It has been found that environmental and physiological stress conditions can transiently increase the mutation rate in bacteria. A number of studies strongly suggest a possible association between bacteria with high mutation rates and antibiotic-resistance acquisition.

The appearance of methicillin-resistant Staphylococcus aureus (MRSA) represents a fascinating detective story of evolution in multiple stages beginning with the original source of the resistant gene mecA followed by its mobilization and association with the unique staphylococcal chromosomal cassettes (SCC), which appear to have their own independent evolutionary history. In this chapter the authors have concentrated on the stages of this evolutionary process that are relatively rarely discussed in reviews. A DNA probe generated from the S. aureus mecA sequence was used to search for a staphylococcal species in which all epidemiologically unrelated isolates produced a hybridizing signal under stringent conditions even if the isolates were susceptible to methicillin. The high-level methicillin resistance of the S. aureus transductants had an absolute dependence on the S. sciuri gene. The evolution of the mecA gene may have occurred on a much longer timescale and under the selective pressure of penicillin in a staphylococcal species such as S. sciuri, which appears to be free of the penicillinase plasmid. Ongoing studies in several laboratories of structural variants of SCCmec in various staphylococcal strains should eventually shed some light on the stages of molecular evolution between the original source of mecA and the construction of an SCC vector capable of capturing and delivering the chromosomal mecA determinant to an S. aureus recipient. Characterization of MRSA clones by molecular and microbiological techniques indicates that the evolution of MRSA does not stop after the acquisition of the SCCmec determinant.

Human disease is mainly caused by Salmonella serotypes belonging to subspecies I. Salmonella is capable of causing a variety of disease syndromes: typhoid (enteric) fever, gastroenteritis, septicemia, and focal infections, depending on the serotype and the host susceptibility. Speciation of the genus Salmonella are correlated with the acquisition of virulence genes, mainly by horizontal transfer. Acquisition of SPI-1 is believed to be an essential event for the separation of the Escherichia and Salmonella genera and permitted the latter to invade intestinal epithelial cells. The invasion step in which the enzymatic activity of SigD is essential is the biogenesis of the Salmonella-containing vacuole (SCV) after membrane ruffling. A new step in the evolution of Salmonella could be gained by acquisition of the pathogenicity island SPI-2, a region of 40 kb composed of at least two distinct elements. Several of them are involved in the control of trafficking and evolution of these vesicles, but details about their function are still scarce. The evolution of all these, relatively recent, serotypes has involved acquisition and loss of a substantial number of genes. The nature of many pseudogenes and some phenotypic traits is different in the two serotypes, indicating an independent evolution pathway. Currently, ceftriaxone is considered the most effective antimicrobial agent for Salmonella infections, and the fluoroquinolones are an alternative option.

Of more than 200 known serogroups of Vibrio cholerae, only strains of O1 or O139 serogroups cause cholera epidemics. This chapter summarizes information on the evolution of pathogenic V. cholerae with an insight into the significance of virulence factors being encoded by accessory genetic elements, and phages, and into factors associated with the generation of diversity among virulent strains. Although this amazing transformation of V. cholera strains associated with epidemics was observed only once in nearly two centuries of recorded history of cholera, this period is not too long in an evolutionary time frame. Paradoxically, besides virulence genes and genetic elements mediating their transfer, the most important contributor to the evolution of pathogenic V. cholera is the human host itself, which supports the selective enrichment of diverse pathogenic strains. Overall, these molecular epidemiological studies indicated that there are temporal variations in the clones involved with cholera epidemics in particular geographical regions. Phage predation in the environment influences the temporal dynamics of cholera epidemics. The continual emergence of new strains of toxigenic V. cholera and their selective enrichment during cholera outbreaks constitute an essential component of the ecosystem for the survival and evolution of V. cholera and the genetic elements that mediate the transfer of virulence genes. Such studies offer the opportunity to expand our understanding of the phylogenic relationships between pathogenic and nonpathogenic strains of V. cholera and the discovery of new genes that may be involved directly or indirectly in the evolution of pathogenic V. cholera.

The structure of the genus Haemophilus has been studied by examining the phylogeny of housekeeping genes such as the adenylate kinase gene (adk), the glucose-6-phosphate isomerase gene (pgi), and the recombination protein gene (recA). Housekeeping gene similarity supported previous DNA-DNA hybridization data for the genus rather than the phylogeny inferred from 16S rRNA gene sequence comparison. Escherichia coli and Haemophilus influenzae appear to be closely related. However, their different ways of life and their different genome sizes (from 4.7 Mb in E. coli to 1.8 Mb in H. influenzae) suggest that these two bacteria followed different paths in their recent evolutionary history after having diverged from their last common ancestor. Virulence determinants are often highly mutable compared to genes for housekeeping functions; their variability can provide clues to the nature of the selective forces driving pathogen evolution. As in other bacteria, horizontal evolution in H. influenzae occurs by the acquisition of new genetic material from transformation of native DNA, transduction by phages, or conjugation by plasmids; this new genetic material is then passed on to subsequent generations through vertical evolution. Polymorphisms in variable number of tandem repeats (VNTRs) and microsatellites may be an efficient procedure to enhance phenotypic variation in pathogenesis and the evolution of bacterial H. influenzae strains. Bacteriophages may influence the chromosomal evolution of their bacterial hosts, mediating rearrangements and the acquisition of novel genes.

This chapter reviews emerging themes from genome sequence data and microarray whole genome comparisons of the pathogenic yersiniae and discusses how this information is guiding hypotheses on the evolution of Yersinia. The factors influencing the rise and fall of plague epidemics remain obscure, but it is possible that severe epidemics may be preceded by subtle genetic changes in Y. pestis resulting in a highly virulent strain. A large-scale comparison of the new Y. pestis and Y. pseudotuberculosis sequences should allow the comparative evolution of Y. pestis to be investigated in great detail and key acquisitions and mutations to be identified. A striking feature of the Y. pestis CO92 genome sequence is the large number of insertion sequence (IS) elements. A total of 140 IS elements comprises 3.7% of the genome. Recombination between two IS100 elements is likely to be responsible for the extreme in vitro instability of the hms locus in Y. pestis. Several genetic mechanisms account for the accumulation of pseudogenes in Y. pestis, including IS element expansion, deletion, point mutation, and slippage in tracts of single-nucleotide repeats. When pathogenic Shigella strains arose from a nonpathogenic Escherichia coli ancestor, the loss of ompT and cadA genes (so-called black holes) may have contributed to their virulence and evolution.

The genus Bordetella contains three widely studied species: Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These gram-negative coccobacilli cause respiratory disease in humans and other mammals. The significance of insertion sequence (IS) elements to the evolution of B. pertussis and B. parapertussis is discussed. The bordetellae provide an opportunity to propose hypotheses to answer questions about speciation, host restriction, and differences in genotype that could account for virulence phenotypes. This was the rationale behind the Bordetella genome sequence project in which the genome sequences of representative strains of B. bronchiseptica, B. pertussis, and B. parapertussis were generated and analyzed. The evolution of any bacterium is shaped by the selection pressures faced by the organism. The evolution of species toward causing highly contagious infections, but with a low infectivity period, from a progenitor that is much less contagious but with a very prolonged infectious period is a recurrent theme in the evolution of human pathogens. A number of putative mutations are predicted to increase the level of expression of the pertussis toxin (PT) genes in B. pertussis compared to B. bronchiseptica or B. parapertussis, and this might have contributed to the change from chronic to acute infections during B. pertussis evolution. Analysis of B. parapertussis reveals that it appears to have evolved in the face of selection pressure against the expression of factors that might be recognized by existing host immunity, but in this case directed against B. pertussis.

In this chapter, among the various Escherichia coli strains, the authors focus on strains belonging to Enterohemorrhagic E. coli (EHEC), particularly the O157:H7 serotype, and review the recent advancement in understanding of their evolution and diversification. Although the importance of horizontal gene transfer in the evolution of pathogenic E. coli strains was noticed before, its genome-wide view was first obtained by the genome sequence determination of two O157:H7 strains and following genomic comparison with K-12. To fully understand the genomic differences between O157 and non-O157 EHEC and the evolution pathways of each non-O157 EHEC, the genome sequencing of non-O157 EHEC strains is also required. The genome sequencing of O157 EHEC revealed that the O157 genome is a huge genetic mosaic generated by insertion of an extremely large amount of foreign DNA into the chromosome backbone shared by a benign strain K-12. A large number of virulence-related genes are encoded on this strain-specific DNA. These findings highlighted a surprisingly high level of genome plasticity in bacteria and reinforced the importance of horizontal gene transfer, especially that by bacteriophages, in the evolution of pathogenic bacteria. It was also revealed that gain and loss of foreign genes mediated by bacteriophages are still actively ongoing and continuously producing a wide variety of O157 strains and recombinant phages.

Shigellosis in humans is characterized by the destruction of the colonic epithelium provoked by the inflammatory response that is induced upon invasion of the mucosa by bacteria of Shigella spp. and enteroinvasive Escherichia coli (EIEC). Numerous phylogenetic analyses based on multilocus enzyme electrophoresis, ribotyping, and sequence comparison established that all members of the genus Shigella and EIEC strains belong to the species E. coli. Sequence analysis of chromosomal genes indicates that Shigella and EIEC strains belong to at least six phylogenetic groups, designated S1, S2, S3, SD1, SS, and EIEC. Informative sites used for the phylogenetic analysis of the virulence plasmid were clustered mostly in two genes (ipaD and ipgD), and a more complete view might come from the analysis of sequences of whole virulence plasmids. For the time being, there are two possible scenarios for the origin of Shigella and EIEC groups: (i) the arrival (or construction) of the virulence plasmid in an ancestral E. coli strain from which all Shigella and EIEC groups descend or (ii) multiple arrivals of the virulence plasmid(s) in different E. coli strains. The genomic sequence of five Shigella strains, including strains of S. flexneri 2a (Sf301 and 2457T), S. dysenteriae 1 (Sd197), S. boydii 4 (Sb227), and S. sonnei (Ss046), has been determined. The large number of genes deleted in Shigella and EIEC genomes, compared to the E. coli K-12 genome, is confirmed by comparative genomic hybridization analyses.

This chapter focuses on the analysis of the factors driving the evolutive transition of Pseudomonas aeruginosa populations from the acute to the chronic infection scenario and the underlying consequences in terms of virulence, persistence (adaptation), and antimicrobial resistance. The transcendence of P. aeruginosa biofilms in the persistence of chronic infections demonstrates both its increased resistance to the host defense mechanisms, including the mechanical clearance and that mediated by complement, antibodies, or phagocytes, and its increased resistance to the antimicrobials action, reported to be over 100-fold higher than in planktonic cells. A nice example of how mutator cells can be amplified in a bacterial population by hitchhiking with adapting mutations was provided by Mao et al. In this work it was found that when E. coli populations are subjected to a one-step mutation selection process, hypermutable variants were amplified in the population from approximately 0.001 to 0.5%, and when they were subjected to two consecutive steps of mutants selection, the amplification reached 25 to 100%. A link between P. aeruginosa hypermutation and antibiotic resistance has also been documented for other chronic infections such as those occurring in patients with bronchiectasis or chronic obstructive pulmonary disease (COPD). The establishment of P. aeruginosa chronic infections is the result of a complex adaptation process that includes both physiological and genetic changes, ultimately leading to the selection of highly persistent bacterial populations.

Skin, ears, eyes, vagina, mouth, and gastrointestinal mucosa are colonized with diverse microbial communities of bacteria, archaea, fungi, protozoa, and helminths. We know little about these colonizers and even less about the indigenous viruses and their ecologic role in microbial communities. Mechanisms that may permit lactic acid bacteria to reduce intestinal infections include lowering the gut pH to inhibit competing pathogens, secretion of natural antibiotics (e.g., lactobacilli and bifidobacteria species), improved immune stimulation, and blocking of adhesion sites in the gut needed by the pathogens. Competition for resources in the face of host responses, population density, and strain dominance surely affect the evolution of Helicobacter pylori as well as the downstream risks of disease. Host physiology allows higher density and more diverse colonization in some parts of the body than in others. A list of Helicobacter species isolated from animals and humans is provided in this chapter. H. pylori has numerous strain-specific restriction-modification (RM) systems that vary in activity and are regulated by mutation and recombination. A list of restriction endonucleases isolated from three H. pylori strains is also provided in the chapter. Transformation and mutation are important sources of variation of H. pylori to provide genetic substrate to respond to selection forces. In total, through endogenous mutation and horizontal gene transfer H. pylori is capable of maintaining great variability that confers the ability to colonize numerous different gastric niches and to survive gastric changes.

Legionella pneumophila, the causative agent of Legionnaires’ disease and related respiratory ailments, is a facultative intracellular pathogen. Many bacterial pathogens use secretion systems as part of their pathogenesis machinery. Bacteria such as Agrobacterium tumefaciens, Helicobacter pylori, Bordetella pertussis, Brucella sp., L. pneumophila, Coxiella burnetii, and others utilize a type IV secretion system for pathogenesis. The icm/dot type IVB secretion system was initially identified in L. pneumophila by the use of several genetic screens aimed at identifying genes required for intracellular growth and host cell killing, as well as other screens including complementation of salt-resistant mutants. Most of the icm/dot genes were found to be completely required for intracellular growth in amoebae hosts, and most of these genes are also completely required for intracellular growth in macrophage cell lines. The DotA protein was shown to be secreted in an Icm/Dot dependent manner into culture supernatants. IcmS and IcmW are unique proteins in the icm/dot system mainly because of several classical icm/dot phenotypes to which they were found to be dispensable. The current knowledge of the Legionella type IV secretion system probably indicates that components from at least three different evolutionary origins were put together to result in a functional pathogenesis system. The majority of the components of the translocation apparatus probably originate from a conjugative plasmid such as R64, and the IcmR and IcmQ proteins are probably involved in the initial step of host recognition, which might explain the high diversity of the FIR protein family.

The N. meningitidis portal of entry is the nasopharynx. Asymptomatic carriage is the most frequent outcome of bacterial colonization, with transmission from host to host occurring through airborne salivary droplets. Secondary localization may occur if the bacteria crosses body barriers such as the blood-brain barrier to infect the subarachnoidal space, leading to meningitis, the most frequent meningococcal disease. As with other bacterial species, Neisseria undergo asexual haploid reproduction, although the frequent DNA transformation and recombination that occur between Neisseria species are responsible for localized gene exchanges. The development of this "sexual behavior" may be the result of the coevolution of pathogenic Neisseria as an obligate parasite of humans. The critical step in the development of pathogenic Neisseria infection takes place at the surface of epithelial cells. Pathogenic Neisseria strains usually release outer membrane vesicles (blebs) that contain large amounts of surface bacterial components. Contact between pathogenic Neisseria strains and viable target cells promotes the effective adhesion process through type IV pili, filamentous structures at the bacterial surface, crossing the N. meningitidis capsule. Iron acquisition on mucosal surfaces and in blood is crucial for neisserial virulence. The continuous shuffling and recombination of genes instead of the appearance of mutations are major traits in neisserial evolution. Evolution by tinkering should lead to genomic plasticity and the differential use of the same genes.

Organisms of the order Chlamydiales comprise a group of obligate intracellular pathogens of ever-growing importance and number. Over the past six years, comparative analyses of complete chlamydial genome sequences have provided an explosive amount of data that have led to important new hypotheses regarding the molecular origins of Chlamydiales. It was surprising that Chlamydiales had many proteins that were similar to plant proteins targeted to the chloroplast. Protease-like activating factor (CPAF) is unique to Chlamydiales and likely critical to host adaptive immunity. Many of the gene differences identified are clustered into discrete regions including the replication termination region terminus or the plasticity zone (PZ), the two clusters plus PmpD of polymorphic membrane proteins (Pmp), the transmembrane head (TMH)/Inc protein cluster, and the biotin biosynthetic operon. Some of the most interesting data on Inc proteins has come from Chlamydiales genomic comparisons, where 20 open reading frames were identified for the genus Chlamydia. Studying the genetic variability of multiple genes encoded by chlamydial genomes has resulted in insights into the molecular evolution of this organism. Researchers have focused on the comparative genetics of genes with sufficient genetic variation or those with specialized structural or housekeeping functions. From these studies, a number of findings are revealing the mechanisms by which Chlamydiales diverge.

In the mid-1980s, molecular biology coupled with bacterial genetics and cell biology approaches allowed detailed investigations of the genetic basis of Listeria monocytogenes virulence. L. monocytogenes infects humans and animals, although its presence has been reported in an impressive number of animal species, which are probably healthy carriers. Southern blot hybridization highlighted that the virulence gene cluster and the internalin locus were absent in L. innocua. The most abundant class of surface proteins are lipoproteins, a class of bacterial surface proteins that may be implicated in adherence to different substrates, host tissues, or other bacteria, as well as in conjugation, signalling, or metabolic functions. The origin of the known virulence genes is still unclear, but comparative sequence analysis gives insight into the possible evolution of pathogenesis in Listeria. Further analysis of the gene content of these strains with respect to virulence genes revealed that all known virulence factors (inlAB, prfA, plcA, hly, mpl, actA, plcB, uhpT, and bsh) are present in all L. monocytogenes strains tested. Interestingly, the distribution of surface proteins among the L. monocytogenes strains mirrored the three lineages, as each lineage and each subgroup within a lineage is characterized by a specific surface protein combination. The many fascinating strategies used by Listeria to invade cells, escape from the internalization vacuole, spread from cell to cell, and escape the host as early defense mechanisms are providing unexpected clues to how microbes can establish an infection.

This chapter focuses on the properties and ecological features that might contribute to enterococci behaving as a human pathogen. The study of the molecular bases of the host-pathogen interactions constitutes the cornerstone of the new discipline of ecogenomics that is expected to provide insights for disease suppression. The wide dissemination of cytolysin in enterococci from different origins might reflect the selection of additional antieukaryotic activities for enterococci in water or soil niches. Enterococci are ubiquitous microorganisms associated with most mammals and birds and are recovered from reptiles, insects, and natural environments apparently lacking exposure to heavy fecal contamination. Lateral gene transfer (LGT) seems to play a critical role in the evolution of Enterococcus faecalis and E. faecium and their adaptation to specific environments. In addition, the multiplicity of mobile and foreign elements found in available sequenced genomes might explain the lack of synteny between genomes of enterococci and any sequenced low-GC organisms. The growing knowledge about transfer and sequences of elements and genomes has revealed a multitude of strategies for bacterial adaptation. A section summarizes the available knowledge on mobile genetic elements (MGE) found among enterococci and their distribution among other microorganisms belonging to the same exchange communities. The 154-kb E. faecalis PAI encodes well-known putative enterococcal virulence traits. One-third of the pathogenicity island (PAI) consists of structural genes from the enterococcal pheromone-responsive plasmids pAD1 and pAM373, indicating that the evolutionary building-up of this PAI was based on plasmid-integration events.

Bacillus anthracis is the causative agent of anthrax, primarily a disease of herbivores but also a serious threat to humans when introduced into their environment by natural or nefarious means. The anthrax toxin complex is separated into three protein components called edema factor (EF), lethal factor (LF), and protective antigen (PA). It is clear that the classical anthrax-associated strains are monophyletic and that their global distribution is due to a clonal expansion. Important anthrax virulence factors are likely to have evolved before this phylogenetic bifurcation and are thus found in non-B. anthracis strains, some of which are even associated with severe pathological conditions. B. cereus sensu lato contains numerous pathogenic types whose attributes are controlled or greatly modified by virulence genes on large plasmids. The monophyletic population structure of B. anthracis sensu stricto contains at least three major and multiple minor clonal genetic divisions, estimated by several different molecular genetic methods that largely agree. Full virulence of B. anthracis and the manifestation of anthrax requires both capsule production, pXO2, and the tripartite toxin on pXO1. The pleiotropic effects of the plcR inactivation result in numerous phenotypic deficiencies that would impair strains from existing opportunistic pathogens in the environment.

Tuberculosis (TB) was declared a global emergency by the World Health Organization in 1993, and the international scientific community is now making tremendous efforts to develop new antimycobacterial drugs and a new anti-TB vaccine, more effective than the Mycobacterium bovis BCG vaccine currently used in most parts of the world. Some actinobacteria form branching filaments resembling the mycelia of fungi and were originally classified as ''Actinomycetes.'' The future development of a new anti-TB vaccine, and of new antimycobacterial drugs, would probably benefit from a more detailed understanding of Mycobacterium tuberculosis virulence and evolution, including an explanation of how a bacterium that was probably initially saprophytic evolved to become a major, potentially lethal, microbial pathogen. Two single nucleotide polymorphisms (SNPs), in gyrA codon 95 and in katG codon 463, have been used to reconstruct the evolutionary history of the M. tuberculosis complex. The evolution of the M. tuberculosis complex thus appears to have been mostly clonal, with DNA duplications, inversions, and deletions. Restriction fragment length polymorphism studies take advantage of the presence in M. tuberculosis of insertion elements located at various positions in the genome in different strains. DNA fingerprinting techniques have been extensively used for clustering mycobacterial genotypes, studying M. tuberculosis transmission, characterizing outbreaks, and improving clinical management. Global searches for mycobacterial virulence genes have been carried out by screening libraries of M. tuberculosis mutants in mice and in macrophages, at both the cellular and the subcellular levels.

In the mid-1970s, two models of mycoplasma evolution were proposed. The first model considered that mycoplasmas were polyphyletic and had arisen by degenerate evolution and diversification of different bacterial lineages, with different mycoplasmas originating from different branches of the bacterial phylogenetic tree. The second model was that mycoplasmas arose very early in the evolution of living forms on Earth and ancestral mycoplasmas were precursors of bacteria. Earlier phylogenetic work indicated that genome size reductions must have occurred on at least two and possibly four separate occasions in the course of Mollicutes evolution. Mycoplasma with smaller genome sizes (<1,00 kb) are on three of the most rapidly evolving mycoplasma phylogenetic branches: the Mycoplasma pneumoniae, Ureaplasma, and M. hominis group branches. Complete genome analysis of mycoplasmas fails to detect orthologues of most genes in the two systems for homologous recombination. Phylogenomic studies suggest that many genes involved in a variety of DNA repair pathways have been lost in Mycoplasma. Defects in mismatch repair might increase pathogenicity because of an increase in the mutation rate, which allows a faster evolutionary response to immune systems and other host defenses. Sequence repeats leading to recombination events constitute evolutionary reservoirs that can be recruited by adaptive strategies involving sequence varation. Antigenic epitopes that are shared by different mycoplasmas and host cells have been proposed as possible factors involved in the evasion of host defense mechanisms and in the induction of auto-antibodies observed during infection.

Streptococcus pneumoniae disease has been a major cause of mortality throughout human history, causing serious invasive diseases such as pneumonia, bacteremia, septicemia, and meningitis. In common with other bacteria colonizing the nasopharynx, S. pneumoniae rarely causes invasive disease despite its prevalence in the population. The precise regulation of virulence factors in pneumococci is essential as the organism changes from colonizing the nasopharynx and surviving in and invading the lung before entering the bloodstream and cerebrospinal fluid. Studies have found 13 putative two-component signal-transduction systems in S. pneumoniae, and early studies suggest that these systems regulate expression of virulence loci in response to environmental stimuli as has been found in other bacterial pathogens. The S. pneumoniae capsule is an obvious feature of the organism when viewed on blood agar, and it serves a key role in both virulence and immune evasion. Increasing rates of antibiotic resistance have been found in studies in many countries, prompting the most alarmist of commentators to speculate about a return to the preantibiotic era. The pneumococcal capsular polysaccharide was the first virulence factor identified in the species. Pneumococcal capsular polysaccharide genes are not always reliable markers of the strain genetic background, as early studies using multilocus enzyme electrophoresis (MLEE) showed. MLEE examines allelic diversity at a number of housekeeping gene loci by comparing the mobility of their gene products on starch gels. The development of automated DNA sequencing allowed the development of multilocus sequence typing (MLST), which is based on the sound evolutionary theory underlying MLEE.

The evolutionary relationships between Candida albicans and C. dubliniensis are obviously very close, since the species are morphologically indistinguishable, and C. dubliniensis was discovered mainly as a consequence of molecular phylogenetic approaches. The pathogenic Candida species share a few common properties beyond their status as budding, fermentative yeasts. In this chapter, separate descriptions of each species is followed by a general overview of the evolutionary history of yeasts and of pathogenic Candida species. The chapter ends with a consideration of the evolutionary significance of interspecies differences to the clinically important considerations of pathogenicity and of antifungal susceptibility and resistance. In evolutionary terms C. glabrata is a close relative of Saccharomyces cerevisiae and thus differs from most other pathogenic Candida species, which diverged from the S. cerevisiae lineage before the whole-genome duplication event. The presumed evolutionary changes that led to the emergence of the pathogenic Candida species have mainly to do with adaptation to commensal existence in warm-blooded animals, rather than with causation of invasive infection.

Cryptococcus neoformans has several well-established virulence factors: a polysaccharide capsule that surrounds the cell body, melanin production, urease, phospholypase, and growth at 37°C. Among these factors, the best characterized are the capsule and melanin. The fact that the size of the capsule increases after infection suggests that it is required for survival in the host, and mutants unable to undergo this process are less virulent. The ability of C. neoformans to grow at 37°C and in slightly alkaline pH, which are conditions that the yeast encounters during infection in mammalian hosts, is a main feature that allows this yeast to be a pathogenic fungus. Furthermore, the interaction of C. neoformans with some of other hosts such as bacteria, protozoa and slime molds can enhance virulence by affecting what we consider to be the virulence factors of this yeast and the ability to kill mice. This chapter reviews the interactions and discusses how they may influence the virulence of the organism during evolution. Other virulence factors, such as phospholipase, are important for survival of the yeast cells. The chapter reviews how C. neoformans can infect and interact with many different hosts, including amoebas, nematodes, insects, and many mammals. At the same time, C. neoformans increased virulence by exploiting some of the features that this fungus uses for survival in the environment, such as the capsule and melanin production.

The opportunistic mould Aspergillus is one of the most ubiquitous filamentous fungi in the world. Aspergillus fumigatus is the most prevalent airborne fungal pathogen in humans and obviously the most common cause of aspergillosis, causing around 90% of infections. This chapter provides a general overview of the pathogenesis, clinical aspects, and epidemiology of aspergillosis. Invasive aspergillosis is uncommon in the healthy host, and hence there are doubts regarding the virulence of Aspergillus spp. The most significant pathogenic factor of A. fumigatus is its ubiquitous nature that increases the chance to interact with hosts presenting factors that predispose them to infection. Aspergillus is an unusual cause of onychomycosis, particularly Aspergillus sydowii and Aspergillus versicolor. The complete recognition of Aspergillus virulence could lead to advances in one's understanding of the disease and in the management of diverse clinical presentations. During the past decade the incidence of invasive infection by molds and resistance to antifungal agents have been increasing. The azole-derived antifungal agents inhibit the ergosterol biosynthesis pathway via the inhibition of 14-αsterol demethylase, the enzyme that removes the methyl group at position C-14 of precursor sterols. The study of molecular mechanisms of antifungal drug resistance is the most valuable strategy for controlling the progress of resistance and in helping develop safer and more active molecules able to avoid them.