Persistent Bacterial Infections

Researchers traditionally divide the biological relationships of equilibrium between coexisting species into three exclusive categories. The category best known to medical bacteriologists is parasitism, the biological state in which one organism benefits while the other is harmed. A second state, symbiosis, describes the situation in which both species derive benefit. The third state is commensalism, in which the host neither benefits nor is harmed. Recently, the spectrum of virulence has been categorized according to the ways in which the host facilitates the pathogenicity of the microbial biota. Ancient human populations typically consisted of small bands of perhaps several hundred individuals who interacted with other bands and tribes for purposes of warfare, commerce, and intermarriage. Researchers have classified the strategies used by the pathogen to circumvent complex host defenses as (i) sequestration, (ii) humoral evasion, and (iii) cellular evasion strategies. Certain persistent bacterial infections are characterized by the presence of a physical barrier between the microbe and the host. Careful analysis of the biology of persistence will permit new insights that can be broadly useful to science and ultimately to clinicians.

This chapter examines the persistent bacterial pathogens within the context of evolutionary ecology by addressing the functional and evolutionary aspects of the mechanisms that bacteria have developed to persist in mammalian hosts. The focus will be on antigenic variation, as this is one of the most widely studied aspects of persistence and also one of the most variable in terms of the extent to which extracellular pathogens have developed genetic mechanisms to enable variation to occur. A recent paper has provided an overview of the significance of antigenic variation in terms of the evolutionary forces driving host-pathogen coevolution. A distinction among conjugation, transduction, and transformation is that the first two processes are largely driven by factors external to the cell receiving the DNA while transformation is largely under the control of the recipient cell. Pathogenicity islands, consisting of large blocks of DNA encoding multiple, and frequently related, gene products, have been identified in some bacterial populations. The emergence of intracellular antigenic variation may be a multifactorial process, governed by the population biology of the organism, the response of the vertebrate host, and the specific bacterial interactions encountered within the host environment. Analysis of the mechanisms and population biologies of bacterial persistence within and between hosts is only one area in which a greater understanding of microbial evolution will facilitate proactive approaches for the control of infectious disease.

The strategies utilized by intracellular pathogens to propagate their genomes are discussed in this chapter in the context of recent developments in one's understanding of cell biology. The strategies employed by intracellular bacteria, defined by the establishment of replication-permissive niches, include (i) survival and replication within a phagolysosome , (ii) escape from the phagosome and replication within the cytoplasm, (iii) modulation of progression along the endocytic cascade, and (iv) exit from the endocytic cascade by entry into alternative pathways of membrane traffic within the host cell. The study of the cell biology of Ehrlichia-containing vacuoles is in its infancy, but it does suggest differences among species. Interestingly, while avoiding interactions with the endocytic cascade, Legionella pneumophila, Brucella abortus, and some strains of Chlamydia psittaci display intimate interactions with other organelle systems, most notably the endoplasmic reticulum (ER) and mitochondria. The acquisition of the lysosomal marker LAMP, while bypassing the late-endosomal compartment, is explained by the localization of brucellae within vacuoles surrounded by the ER, identified by staining with Sec61β. In summary, the data suggest that B. abortus-containing phagosomes exit the endocytic pathway and transit through the autophagous pathway en route to their replication-permissive niche, the ER. The study of the mechanisms by which the pathogens infect mammalian cells will undoubtedly yield fascinating insights into both microbiology and cell biology at the most intimate interface between the pathogen and host.

This chapter examines the increasingly common scenarios in which bacteria actively modulate the immune response to ensure their persistence. The mechanisms of protection against infectious agents may be divided into two complementary components: innate, or natural, immunity and specific, or acquired, immunity. It is important to mention that nonprotein antigens, such as microbial (particularly mycobacterial) lipid and glycolipid antigens, can be presented to T cells in a restricted fashion by nonclassical major histocompatibility complex (MHC) molecules, such as CD1, that are differentially expressed on antigen-presenting cells (APC) and cells of the gastrointestinal epithelium. Inhibition of mitogen-induced lymphocyte proliferation by whole live bacteria, soluble or particulate preparations of the bacteria, and sometimes purified molecules is arguably the most common observation in the literature concerning inhibition of the host's immune response. The induction of apoptosis in monocytes/macrophages has been shown to be triggered by a number of bacteria or purified bacterial products in vivo, as well as in vitro in primary and short-term cultures. Through an in-depth examination of the mechanisms used by the microbial adversaries to evade or otherwise subvert the host's immune response, we may be able to devise more effective live vaccines and develop novel therapeutic strategies for the treatment of persistent infections, as well as to discover valuable immunomodulatory agents.

This chapter focuses on mathematical models of colonization and persistent bacterial infections. It reviews the modeling method and the state of the field and then focuses on three key areas where modeling has, and will continue to have, an impact: the ecology of the indigenous microflora and its plasmids, Helicobacter pylori colonization, and host-pathogen interactions with Mycobacterium tuberculosis. Mathematical models of host-pathogen dynamics are formulated on the basis of specific assumptions regarding the system's components and their interactions. Models of persistent viral infections, namely, human immunodeficiency virus (HlV)-host models, also have a successful recent history. H. pylori induces chronic gastric inflammation that results in peptic ulcer disease or gastric cancer in a small set of infected persons. This high ratio of mucus-living to adherent bacteria, although characteristic, is not necessary for colonization, as low concentrations of H. pylori may be present in the mucus during persistence. Incorporation of the dynamic host response into a model of H. pylori colonization is critical if one understands the initial features of the interactions between microbe and host, as well as the phenomena that permit persistence to develop. If we consider long-term associations between bacteria and humans a question of bacterial ecology, such as persistent infections or the homeostasis of an indigenous microflora, it becomes more logical to consider mathematical approaches to understanding these associations, as modeling has long been used by ecologists.

It has been estimated that the number of microorganisms living and thriving in or on an average human being exceeds the number of cells making up that human being by an order of magnitude. In order to gain an understanding of the complexity of the human-associated microflora it has to be seen as the complex ecosystem it is. The importance of being able to differentiate various clones in an investigation of the microbial ecology of a human site was amply demonstrated with the use of serotyping to study intestinal Escherichia coli. Corynebacterium xerosis is an accepted member of the normal flora of the human skin, as well as mucocutaneous membranes such as the nasopharynx. Lactobacillus acidophilus and L. fermentum are the most frequently described species inhabiting the human vagina, but there have also been reported isolations of other species, such as L. casei, L. plantarum, L. brevis, L. delbrueckii, L. lactis, L. bulgaricus, L. leichmannii, L. salivarius. This chapter provides an understanding of the microbiological and ecological principles that underlie the distribution of bacteria on the human body. Enterohemorrhagic E. coli, of which only a few bacteria are required to colonize and cause disease, utilizes the colonizing abilities of the commensal E. coli, but with disastrous effects for the host.

This chapter discusses the classical distinguishing characteristics of persistent pathogens, that they elicit host responses, including inflammation, and that they have the capacity to damage the host, are increasingly being associated with the so-called commensal flora as well. The focus is mainly on the normal flora of the gastrointestinal tract. All external and certain, but not all, internal body surfaces are covered by a film of microorganisms, a normal microflora. The upper respiratory tract, gastrointestinal tract, perineum, vagina, and distal urethra contain large resident bacterial populations, while the bronchi, alveolar spaces, urinary tract, and uterus are normally sterile. Macrophages from animals colonized by a microflora also display increased cytotoxic activity, secrete more oxygen radicals, have increased levels of cyclic AMP, and produce more interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha in response to lipopolysaccharide (LPS) than macrophages from germfree animals. Intestinal immune responses are mainly induced in the Peyer's patches, which are mucosal lymphoid nodules covered by a specialized epithelium, the follicle-associated epithelium. Oral tolerance also protects us from exaggerated responsiveness, including inflammatory responses to antigens which are not dangerous, and thus minimizes the risk of inflammatory states in the mucosa. Colon cancer is one of the most common neoplastic diseases in wealthy populations but is relatively uncommon in developing countries. Staphylococci have been implicated in the pathogenesis of atopic dermatitis. Evidently, the epidemiological connection between the intestinal colonization pattern and allergy development needs to be further studied.

This chapter focuses on bacterial infections in specific settings where fundamental relationships relevant to the persistence of bacterial infections in the human host may be observed and studied. Neonates usually acquire bacterial pathogens transplacentally through the umbilical vein or by aspiration from amniotic fluid or cervical secretions. The complement system is able directly to lyse some gram-negative bacteria even in the absence of specific antibodies, but circulating levels of complement in the sera of neonates are lower than those in adults. Both the infected fetus and the neonate are able to produce protective IgM antibodies in response to bacterial antigens, but the levels in serum are lower than those in adults. The primary T-cell deficiencies produce defects in the cytokine production that is required to activate macrophages to kill intracellular bacteria. A primary defect of superoxide synthesis or adhesion of neutrophils is related to severe infection with pyogenic bacteria. The extreme example of acquired immune deficiency is human immunodeficiency virus (HIV) infection, which produces AIDS. Persistent infections modulate all aspects of immune response, including both adaptive and natural immune functions, and have long-term consequences for immune defense. Although encounters with bacteria are a normal aspect of neonatal life, infants are also more susceptible to bacterial infections than are adults.

The discovery of γ δ T cells originally occurred as an unexpected result of attempts to identify and clone the T-cell receptor (TCR). This chapter presents evidence that γ δ T cells are required in order to prevent the development of chronic disease, which in turn may be a contributing factor in determining whether a microbial pathogen is eradicated or becomes persistent. At first consideration, the numerous accounts of large numbers of γ δ T cells within the inflammatory lesions associated with autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis and its mouse model, experimental autoimmune encephalitis, would appear to indicate a pathogenic rather than immunoregulatory role for γ δ T cells. However, rather than being an indication of involvement in the pathogenesis of these disorders, it can be said that the presence of γ δ T cells is an attempt to downregulate the extent of inflammation associated with these diseases. It has been suggested, however, that γ δ T cells promote the influx of macrophages into the site of infection, resulting in the generation of granulomas, which are essential for bacterial clearance. This hypothesis is based on the finding that the production of macrophage-specific chemokines, such as macrophage inflammatory protein-1α (MlP-1α), MIP-1β, MIP-2, and methyl-accepting chemotaxis protein 1, is reduced in TCR-δ-/- mice following infection. In other cases, however, the bacteria can be reactivated and cause clinical disease.

Superantigens are usually regarded as potent inducers of an overwhelming (unmodulated) immune response that manifests as a rare event, such as toxic shock syndrome or food poisoning. This chapter presents evidence that immune response to superantigens may well trigger or exacerbate autoimmunity. Multiple sclerosis (MS) symptomology can often be observed to occur in a relapsing-remitting manner. Superantigen effects are suggested by studies of peripheral and synovial Vβ14+ T cells from patients with rheumatoid arthritis versus those from controls and of B-cell production of rheumatoid factor upon stimulation with SED. Skin lesion eruptions in guttate psoriasis have been linked with throat infections and increased antibody titers to streptococcal antigens. Initial studies of the infectivity of mouse mammary tumor virus (MMTV) indicated that an intact immune system was required for infection. Antibodies to IL-10 block the protective effects of IL-10 against experimental allergic encephalomyelitis (EAE). Focusing on cell cycle events, the authors have determined the effects of IL-10 on the entry of quiescent CD4+ T cells into the cell cycle upon stimulation with the staphylococcal superantigen staphylococcal enterotoxin B (SEB). The long-term effects of superantigen activation of T cells can include the induction of autoreactive cells, leading to an autoimmune state. Superantigens can also cause relapses into disease in patients with remission.

Uncomplicated gonorrhea is most commonly an acute urogenital infection involving the urethra in men and the endocervix in women of reproductive age. Upon entering the urogenital tract, Neisseria gonorrhoeae adheres to columnar epithelial cells, a step that presumably enables the gonococcus to withstand the flushing force of urine and the constant shedding of cervical mucus. Transcriptional regulation of genes in response to environmental stimuli in the mucus layer may coordinate adaptation of this pathogen to nutritionally different microenvironments and to physiological stress induced by nonspecific host defenses. The major pilin subunit, PilE, is encoded by one or two expression loci (pilE) on the chromosome. This chapter focuses on the survival mechanisms utilized by the gonococcus specifically in the context of the urogenital mucosa. The isolation of a gonococcal fur mutant by Thomas and Sparling facilitated the identification of numerous fur-dependent iron-induced (Fip) and iron-repressed (Frp) proteins by two-dimensional electrophoresis; from this work one might predict the existence of gonococcal virulence genes that are regulated by iron but not involved in iron uptake. Insights from these studies have fostered theories regarding the functions of these molecules and the role of variable gene expression in gonococcal adaptation and transmission. The development of animal models for studying specific aspects of gonococcal infection in a female background should also be pursued, incorporating transgenic mice when possible to better mimic human infection.

The genus Chlamydia spp. consists of highly specialized prokaryotic bacteria that exhibit a unique biphasic developmental cycle that ensures their survival. Along with Mycoplasma pneumoniae it is probably the most common cause of community-acquired pneumonia in school-aged children and young adults. This agent has been implicated in the development or acceleration of atherosclerosis, asthma, and chronic obstructive pulmonary disease, and recently, it has been linked to Alzheimer's disease (AD) as well. Recent studies using transmission electron microscopy reveal morphologically atypical chlamydial forms in sites of chronic tissue pathology in vivo, concurrent with reverse transcription (RT)-PCR detection of chlamydial rRNA and mRNA transcripts. Although productive chlamydial infection is the norm, multiple studies suggest the presence of nonculturable persistent chlamydial organisms in host tissues. More importantly, these persistent forms are often found in sites of chronic disease. Several studies report the delayed appearance and prolonged carriage of genital tract C. trachomatis strains acquired perinatally. The fact that all species and strains of chlamydiae produce productive infection of appropriate host cells is evidence of their ability to evade phagosome-lysosome fusion. The persistence of chlamydia-specific IgA antibodies has been proposed as a better marker of chronic C. pneumoniae infections, and of chronic C. trachomatis infections as well. Perhaps studies that look at IgA levels in patients with chronic disease in conjunction with immuno-electron microscopy and molecular biological investigations of the patients' tissues would help to confirm both methodologies.

Helicobacter pylori is extraordinary among bacteria in its ability to colonize the stomachs of more than half of all people worldwide and often to persist for years or decades once it has become established. H. pylori can be detected and analyzed by a variety of different methods. Seroepidemiological studies performed in the United States and abroad have demonstrated the worldwide presence of H. pylori in a large majority of individuals from low socioeconomic groups and also in many people of higher socioeconomic status. The recent observation that H. pylori can be isolated from cats and that cats can be colonized by H. pylori following inoculation suggests that transmission from pets to humans (or humans to pets) is also possible. The presence of H. pylori can be diagnosed from gastric-biopsy specimens harvested at endoscopy. Long-term gastric colonization is associated with intense humoral and cellular immune responses. However, these responses are usually unable to eradicate H. pylori and they may, in fact, play a role in the persistence of the bacterium and/or in the production of disease. Interestingly, vaccination can reduce the severity of antral gastritis in mice, ferrets, and monkeys. However, immunization can eliminate colonization by H. pylori in mice and ferrets but not in monkeys or humans. The immune response to colonization by H. pylori has been proposed as being responsible in part for the production of peptic ulcer disease (PUD).

Lyme borreliosis (Lyme disease) is unequivocally a persistent infectious disease. Borrelia burgdorferi, the agent of Lyme disease, persists in both humans and animals, despite a vigorous and effective immune response by the infected host. The agent of Lyme disease also owes its survival to its propensity to infect a wide variety of mammalian and avian hosts, thereby allowing geographic spread and adaptation to new niches. The majority (60 to 80%) of patients with Lyme disease develop an expanding annular skin lesion (erythema migrans) at the site of the tick bite. Borrelia plasmids are fundamentally different from plasmids of other bacteria in that they contain essential, rather than nonessential, extrachromosomal elements of the genome. The phenomenon of lateral gene transfer and recombination may have selective advantages in mixed populations of spirochetes but does not provide an explanation for the immune evasion of clonal populations of spirochetes during persistent infection. The proof of the pudding for antigenic variation is whether antigenic changes in spirochetes provide selective advantages for the variant subpopulation in evading immune clearance. No evidence for this phenomenon has been observed with B. burgdorferi. Analysis of the outer and inner membranes of spirochetes indicates that the majority of protein is associated with the inner membrane and that proteins associated with the outer membrane, including OspC, may have only limited surface exposure.

This chapter discusses the spectrum of infections caused by Pseudomonas aeruginosa. Hospital-acquired pneumonias, urinary tract infections, surgical-site infections, and bacteremias are among the nosocomial infections frequently caused by P. aeruginosa. The major cause of high morbidity and mortality in cystic fibrosis (CF) is chronic respiratory infection with P. aeruginosa. The lack of nitric oxide synthase (iNOS) production in CF may have two important repercussions. First, the reduced NO levels have been linked to the hyperabsorption of sodium in CF. Second, nitric oxide has also been directly implicated as a bactericidal and bacteristatic agent. The major causes of high morbidity and mortality presently associated with CF are chronic inflammation and the resulting respiratory tissue destruction. The first phase is an insidious infection, with intermittent isolation of P. aeruginosa from the lungs of the patient with CF. The mucoid phenotype of P. aeruginosa is rarely seen in infections other than CF, although mucoid strains can be isolated during chronic urinary tract infections, but all mucoid isolates produce chemically similar polymers based on the polyuronic acid exopolysaccharide alginate. The majority of the support for such a role of alginate in allowing P. aeruginosa to persist in the CF lung comes from in vitro studies that have previously been extensively reviewed. P. aeruginosa causes life-threatening infections in patients with compromised innate immune defenses, such as burn victims, neutropenic individuals undergoing chemotherapy, and persons with CF.

The host's cellular immune response appears to determine the fate of the organisms escaping the dormant state. It is the depletion of CD4 cells which leads to the increased rate of reactivation of latent tuberculosis infection in patients dually infected with human immunodeficiency virus (HIV) and tuberculosis. This chapter discusses mechanisms responsible for these phenomena and describes the methods of studying dormancy. Tuberculosis is a chronic infection caused by Mycobacterium tuberculosis. M. tuberculosis predominantly causes disease in the lungs, although it can affect any organ in the body. The infected macrophages spread through the lymphatic channels to regional lymph nodes and then metastasize throughout the body. The interactions of macrophages, lymphocytes, and theM. tuberculosis organism result in regulation of the immune response in tuberculosis. One study of human alveolar macrophages failed to demonstrate either production of nitric oxide synthase mRNA or nitrous oxide following stimulation of the macrophages by mycobacteria. The human immune response to mycobacteria is decidedly a TH1-like response and is characteristic of the cellular immune response to intracellular pathogens. The adaptive process may then involve a switchover to a reduced metabolic state or to a spore-like state that becomes insusceptible to drug action. Investigation of mycobacterial cells which pass from an actively multiplying state to a low-metabolic nonmultiplying state identified a switch in metabolism to the glyoxalate cycle, which permits adaptation to the usually lethal effects of anaerobiosis.

This chapter provides an introduction to the rapidly expanding but still very limited knowledge about the interactions of these Bartonella species and their reservoirs and vectors and the adaptations these bacteria have developed to maintain persistent infection in the host mammalian species. There are now at least 13 named species and probably an equal number of unnamed species. Of these, only four have been definitively associated with human disease: B. henselae, B. quintana, B. bacilliformis, and B. elizabethae. B. quintana infection may be asymptomatic or may be characterized by high fever, severe shin pain, and relapsing symptoms over weeks to months. The species most commonly associated with endocarditis in humans is B. quintana, and most patients with B. endocarditis require replacement of the infected cardiac valve. Granulomatous inflammatory disease (CSD), most commonly of the lymph nodes, occurs in immunocompetent humans infected with B. henselae. In humans and cats, B. quintana and B. henselae bacteremia, respectively, persist despite the development of a humoral antibody response. The study of Bartonella pathogenesis is still in its infancy, and although there is a tractable system for genetic manipulation (via conjugation), it will be essential to identify and characterize virulence factors. The availability of the B. henselae genome sequence in the near future will greatly enhance these studies, and the next phase of comparative genomics and microarrays will provide further insight into the mechanisms of pathogenesis and persistence of B. bacilli.

Infective endocarditis describes a family of persistent microbial infections of the heart valves. This chapter explores their ability to behave as endogenous pathogens and is instructive as we try to understand persistence of the larger constellation of endocarditis-associated pathogens. Infective endocarditis generally occurs in individuals with previously diseased or damaged heart valves, most frequently after bacteremia containing viridans streptococci or Staphylococcus aureus. Many of the most common microorganisms associated with infective endocarditis are considered to be of low virulence, often causing no known disease in their native niches. Patients with infective endocarditis show elevated anti-phospholipid antibodies associated with endothelial-cell activation, thrombin generation, and impairment of fibrinolysis. Adhesion to platelets and preformed platelet-fibrin clots would appear intuitively to be associated with the ability of microbes to infect platelet vegetations and cause infective endocarditis. Dextran synthesis by viridans streptococci has been suggested to be a virulence factor in infective endocarditis, promoting adhesion and persistence. The ability of microbes to induce platelet aggregation in vitro may be associated with the pathogenicity of those strains in infective endocarditis. The host distinguishes the commensals from exogenous pathogens. When commensals breach the mucosa and infect the heart valves in infective endocarditis, the host immune repertoire against these endogenous pathogens is programmed for systemic tolerance.

Different types of osteomyelitis can be classified according to the source of the infection (i.e., hematogenous or contiguous focus) and the vascular capability of the host (i.e., with or without generalized vascular insufficiency). Due to the abundant vascular supply, the most common sites of hematogenous osteomyelitis are the metaphyses of tubular bones. The major types of hematogenous osteomyelitis seen clinically include long-bone and vertebral osteomyelitis. Chronic vertebral osteomyelitis arises most often in those patients that have predisposing factors, such as the use of nonsterile injection techniques by intravenous drug abusers, previous spinal surgery, or implanted hardware. All three mechanisms may cause skin ulceration with subsequent skin infection, which may lead to contiguous-focus osteomyelitis. Staphylococcal products that have a role in osteomyelitis may be classified as virulence factors responsible for adherence, direct host damage, or immune avoidance. Clinical strains of Staphylococcus spp. are able to persist through a number glycocalyx properties. First, this layer has been shown to protect the embedded pathogens from the action of antimicrobial agents and the host immune system by forming a mechanical barrier. Second, local immune deficiency often occurs through frustrated phagocytosis, since the normal phagocytic processes are devoted to the removal of the glycocalyx and the implant, if present. Different types of chronic osteomyelitis can be classified according to the source of the infection (i.e., hematogenous or contiguous focus) and the vascular capability of the host (i.e., with or without generalized vascular insufficiency).

This chapter examines (i) the predisposing conditions that lead to abscess formation, (ii) the bacteria that predominate in various abscesses and their contribution to abscess formation, and (iii) the host response to the invading organisms and why that response is usually ineffective in eradicating the infection. The formation of intra-abdominal abscesses is used throughout the chapter as a model. Due to the prevalence of intra-abdominal abscesses, much research has been conducted to examine the molecular interactions between the invading microorganisms and the host that lead to abscess formation. The chapter discusses other examples of abscesses such as renal, brain, lung and skin. Intra-abdominal infection is caused by the leakage of gastrointestinal contents laden with bacteria. Following bacterial spillage, the majority of organisms are removed by the diaphragmatic lymphatics. The best-studied system for understanding the molecular interactions between host and microorganism that lead to abscess formation is the induction of intra-abdominal abscesses by the capsular polysaccharide complex (CPC) of Bacteroides fragilis. Studies in which the purified capsular polysaccharide from B. fragilis induced abscesses in the absence of viable organisms demonstrated the importance of this virulence factor in abscess formation. The authors propose that abscessogenic bacteria produce the structural components that induce the formation of abscesses because these structures serve an important function in the organisms' normal niche rather than serving solely as a mechanism to enhance their persistence as pathogens.

The salivary pellicle is an organic film derived from the saliva and deposited on the tooth surface. It is, however, rapidly colonized by bacteria which make up the dental plaque. Based on the dental plaque's relationship to the gingival margin, it has been separated for microbiological studies into two different communities: supragingival and subgingival plaque. Saliva plays two roles: it limits colonization of most surfaces in the oral cavity by a variety of mechanisms, and it provides these surfaces with colonizing microorganisms shed primarily from the tongue, dental plaque, and other mucosal surfaces. The presence of the normal supragingival oral flora generally results in oral health. Dental caries and periodontal diseases are conditional diseases, requiring both the presence of critical numbers of certain indigenous species and the response of the host. Dental plaque is a structurally organized community, and the response in planktonic or monospecies cultures is not necessarily applicable to their behavior within a biofilm. The role of glucans in human plaque formation is only now beginning to be understood through in vivo studies. Changes in species numbers and metabolic activities of these microbes are influenced by the environment, which affects the expression of virulence genes.

The study of bacterial biofilms is more advanced in the engineering field than in the medical field, but the simple realization that biofilms are involved in chronic infections opens the way for a massive transfer of valuable information from the engineering realm to the medical realm and for its application to the treatment of infectious diseases. Pseudomonas aeruginosa first came to the attention of biofilm microbiologists because it predominates in cold alpine streams and grows predominantly (99.99%) in biofilms in this natural habitat. The Center for Biofilm Engineering (CBE) has established the fact that most biofilms assume this microcolony and water channel structure, including all biofilms formed by the few grampositive species examined to date, and the most significant consequence of this new observation is that we must now explain how these elaborate structures are established and maintained. If we try to imagine the bacterial survival strategies that would have been effective in the earliest stages of the development of life on this planet, growth in stationary biofilms that were protected from unfavorable conditions would prevent bacteria from being swept into acid or boiling downstream pools and from surges of threatening water from upstream sources. The role of host defenses in controlling biofilm infections is discussed in the chapter. There is a growing conviction that antibiotics are losing their ability to control bacterial infections because the bacteria have mobilized all of their survival strategies in the face of this frontal attack.