Chapter 9: Pathogenesis of Bacterial Infection

The pathogenesis of bacterial infection includes initiation of the infectious process and the mechanisms that lead to the development of signs and symptoms of disease. The biochemical, structural, and genetic factors that play important roles in bacterial pathogenesis are introduced in this chapter and may be revisited in the organism-specific sections. Characteristics of bacteria that are pathogens include transmissibility, adherence to host cells, persistence, invasion of host cells and tissues, toxigenicity, and the ability to evade or survive the host’s immune system. Resistance to antimicrobials and disinfectants can also contribute to virulence, or an organism’s capacity to cause disease. Many infections caused by bacteria that are commonly considered to be pathogens are inapparent or asymptomatic. Disease occurs if the bacteria or immunologic reactions to their presence cause sufficient harm to the person.

Terms frequently used in describing aspects of pathogenesis are defined in the Glossary (see below). Refer to the Glossary in Chapter 8 for definitions of terms used in immunology and in describing aspects of the host’s response to infection.

Humans and animals have abundant normal microbiota that usually do not produce disease (see Chapter 10) but achieve a balance that ensures the survival, growth, and propagation of both the bacteria and the host. Some bacteria that are important causes of disease are cultured commonly with the normal microbiota (eg, Streptococcus pneumoniae, Staphylococcus aureus). Sometimes bacteria that are clearly pathogens (eg, Salmonella serotype Typhi) are present, but infection remains latent or subclinical, and the host is a “carrier” of the bacteria.

It can be difficult to show that a specific bacterial species is the cause of a particular disease. In 1884, Robert Koch proposed a series of postulates that have been applied broadly to link many specific bacterial species with particular diseases. Koch’s postulates are summarized in Table 9-1.

Koch’s postulates have remained a mainstay of microbiology; however, since the late 19th century, many microorganisms that do not meet the criteria of the postulates have been shown to cause disease. For example, Treponema pallidum (syphilis) and Mycobacterium leprae (leprosy) cannot be grown in vitro; however, there are animal models of infection with these agents. In another example, there is no animal model of Neisseria gonorrhoeae (gonorrhea) infection even though the bacteria can readily be cultured in vitro; experimental infection in humans has been produced that substitutes for an animal model.

In other instances, Koch’s postulates have been at least partially satisfied by showing bacterial pathogenicity in an in vitro model of infection rather than in an animal model. For example, some forms of Escherichia coli (E coli)–induced diarrhea (see Chapter 15) have been defined by the interaction of the E coli with host cells in tissue culture.

The host’s immune responses also should be considered when an organism is being investigated as the possible cause of a disease. Thus, development of a rise in specific antibody during recovery from disease is an important adjunct to Koch’s postulates.

Modern-day microbial genetics has opened new frontiers to study pathogenic bacteria and differentiate them from nonpathogens. Molecular cloning has allowed investigators to isolate and modify specific virulence genes and study them with models of infection. The ability to study genes associated with virulence has led to a proposed form of molecular Koch’s postulates. These postulates are summarized in Table 9-1.

Some pathogens are difficult or impossible to grow in culture, and for that reason, it is not possible with Koch’s postulates or the molecular Koch’s postulates to establish the cause of their associated diseases. The polymerase chain reaction is used to amplify microorganism-specific nucleic acid sequences from host tissues or fluids. The sequences are used to identify the infecting organisms. The molecular guidelines for establishing microbial disease causation are listed in Table 9-1. This approach has been used to establish the causes of several diseases, including Whipple disease (Tropheryma whipplei), bacillary angiomatosis (Bartonella henselae), human monocytic ehrlichiosis (Ehrlichia chaffeensis), hantavirus pulmonary syndrome (Sin Nombre virus), and Kaposi sarcoma (human herpesvirus 8).

Analysis of infection and disease through the application of principles such as Koch’s postulates leads to classification of bacteria as pathogens, opportunistic pathogens, or nonpathogens. Some bacterial species are always considered to be pathogens, and their presence is abnormal; examples include Mycobacterium tuberculosis (tuberculosis) and Yersinia pestis (plague). Such bacteria readily meet the criteria of Koch’s postulates. Other species are commonly part of the normal microbiota of humans (and animals) but also can frequently cause disease. For example, E coli is part of the gastrointestinal microbiota of normal humans but is also a common cause of urinary tract infections, traveler’s diarrhea, and other diseases. Strains of E coli that cause disease are differentiated from those that do not by determining (1) whether they are virulent in animals or in vitro models of infection and (2) whether they have a genetic makeup that is significantly associated with production of disease. Other bacteria (eg, Pseudomonas species, Stenotrophomonas maltophilia, and many yeasts and molds) only cause disease in immunosuppressed and debilitated persons and are opportunistic pathogens.

Bacteria (and other microorganisms) can adapt to a variety of environments that include external sources such as soil, water, and organic matter or internal milieu as found within insect vectors, animals, and humans, where they normally reside and subsist. In doing so, the bacteria ensure their survival and enhance the possibility of transmission. By producing asymptomatic infection or mild disease rather than death of the host, microorganisms that normally live in people enhance the possibility of transmission from one person to another.

Some bacteria that commonly cause disease in humans exist primarily in animals and incidentally infect humans. For example, Salmonella and Campylobacter species typically infect animals and are transmitted in food products to humans. Other bacteria produce infection of humans that is inadvertent, a mistake in the normal life cycle of the organism; the organisms have not adapted to humans, and the disease they produce may be severe. For example, Y pestis (plague) has a well-established life cycle in rodents and rodent fleas, and transmission by the fleas to humans is inadvertent; Bacillus anthracis (anthrax) lives in the environment, occasionally infects animals, and is transmitted to humans by products such as raw hair from infected animals. The Clostridium species are ubiquitous in the environment and are transmitted to humans by ingestion (eg, Clostridium perfringens gastroenteritis and Clostridium botulinum [botulism]) or when wounds are contaminated by soil (eg, C perfringens [gas gangrene] and Clostridium tetani [tetanus]). Both B anthracis and the Clostridium species elaborate spores to protect the organisms’ nucleic acid from harsh environmental factors such as ultraviolet light, desiccation, chemical detergents, and pH extremes. These spores ensure survival in external environments including foods ingested by humans. After being ingested or inoculated, the spores germinate into the vegetative, metabolically active form of the pathogen.

The clinical manifestations of diseases (eg, diarrhea, cough, genital discharge) produced by microorganisms often promote transmission of the agents. Examples of clinical syndromes and how they enhance transmission of the causative bacteria are as follows: Vibrio cholerae can cause voluminous diarrhea, which may contaminate salt and fresh water; drinking water or seafood such as oysters and crabs may be contaminated; ingestion of contaminated water or seafood can produce infection and disease. Similarly, contamination of food products with sewage containing E coli that causes diarrhea results in transmission of the bacteria. M tuberculosis (tuberculosis) naturally infects only humans; it produces respiratory disease with cough and production of aerosols, resulting in transmission of the bacteria from one person to another.

Many bacteria are transmitted from one person to another on hands. A person with S aureus carriage in the anterior nares may rub his nose, pick up the staphylococci on the hands, and spread the bacteria to other parts of the body or to another person, where infection results. Many opportunistic pathogens that cause nosocomial infections are transmitted from one patient to another on the hands of hospital personnel. Handwashing is thus an important component of infection control.

The most frequent portals of entry of pathogenic bacteria into the body are the sites where mucous membranes meet with the skin, which are the respiratory (upper and lower airways), gastrointestinal (primarily mouth), genital, and urinary tracts. Abnormal areas of mucous membranes and skin (eg, cuts, burns, and other injuries) are also frequent sites of entry. Normal skin and mucous membranes provide the primary defense against infection. To cause disease, pathogens must overcome these barriers.

In the body, most bacteria that cause disease do so first by attaching or adhering to host cells, usually epithelial cells. After the bacteria have established a primary site of infection, they multiply and spread directly through tissues or via the lymphatic system to the bloodstream. This infection (bacteremia) can be transient or persistent. Bacteremia allows bacteria to spread widely in the body and permits them to reach tissues particularly suitable for their multiplication.

Pneumococcal pneumonia is an example of the infectious process. S pneumoniae can be cultured from the nasopharynx of 5–40% of healthy people. Occasionally, pneumococci from the nasopharynx are aspirated into the lungs; aspiration occurs most commonly in debilitated people and in settings such as coma when normal gag and cough reflexes are diminished. Infection develops in the terminal air spaces of the lungs in persons who do not have protective antibodies against that particular pneumococcal capsular polysaccharide type. Multiplication of the pneumococci and resultant inflammation lead to pneumonia. The pneumococci enter the lymphatics of the lung and move to the bloodstream. Between 10% and 20% of persons with pneumococcal pneumonia have bacteremia at the time the diagnosis of pneumonia is made. When bacteremia occurs, the pneumococci can spread to secondary sites of infection (eg, cerebrospinal fluid, heart valves, and joint spaces). The major complications of pneumococcal pneumonia are meningitis, septic arthritis, and rarely endocarditis.

The infectious process in cholera involves ingestion of V cholerae, chemotactic attraction of the bacteria to the gut epithelium, motility of the bacteria by a single polar flagellum, and penetration of the mucous layer on the intestinal surface. The V cholerae adherence to the epithelial cell surface is mediated by pili and possibly other adhesins. Production of cholera toxin results in flow of chloride and water into the lumen of the gut, causing diarrhea and electrolyte imbalance.

Bacteria are haploid (see Chapter 7) and limit genetic interactions that might change their chromosomes and potentially disrupt their adaptation and survival in specific environmental niches. One important result of the conservation of chromosomal genes in bacteria is that the organisms are clonal. For many pathogens, there are only one or a few clonal types that are spread in the world during a period of time. For example, epidemic serogroup A meningococcal meningitis occurs in Asia, the Middle East, and Africa and occasionally spreads into Northern Europe and the Americas. On several occasions, over a period of decades, single clonal types of serogroup A Neisseria meningitidis have been observed to appear in one geographic area and subsequently spread to others with resultant epidemic disease. There are two clonal types of Bordetella pertussis, both associated with disease. There are, however, mechanisms that bacteria use, or have used a long time in the past, to transmit virulence genes from one to another.

Mobile Genetic Elements

Primary mechanisms for exchange of genetic information between bacteria include natural transformation and transmissible mobile genetic elements such as plasmids, transposons, and bacteriophages (often referred to as “phages”). Transformation occurs when DNA from one organism is released into the environment and is taken up by a different organism that is capable of recognizing and binding DNA. In other cases, the genes that encode many bacterial virulence factors are carried on plasmids, transposons, or phages. Plasmids are extrachromosomal pieces of DNA and are capable of replicating. Transposons are highly mobile segments of DNA that can move from one part of the DNA to another. This can result in recombination between extrachromosomal DNA and the chromosome (illegitimate or nonhomologous recombination; Chapter 7). If this recombination occurs, the genes coding for virulence factors may become chromosomal. Finally, bacterial viruses or phages are another mechanism by which DNA can be moved from one organism to another. Transfer of these mobile genetic elements between members of one species or, less commonly, between species can result in transfer of virulence factors, including antimicrobial resistance genes. A few examples of plasmid- and phage-encoded virulence factors are given in Table 9-2.

Pathogenicity Islands

Large groups of genes that are associated with pathogenicity and are located on the bacterial chromosome are termed pathogenicity islands (PAIs). They are large organized groups of genes, usually 10–200 kb in size. The major properties of PAIs are as follows: they have one or more virulence genes; they are present in the genome of pathogenic members of a species but absent in the nonpathogenic members; they are large; they typically have a different guanine plus cytosine (G + C) content than the rest of the bacterial genome; they are commonly associated with tRNA genes; they are often found with parts of the genome associated with mobile genetic elements; they often have genetic instability; and they often represent mosaic structures with components acquired at different times. Collectively, the properties of PAIs suggest that they originate from gene transfer from foreign species. A few examples of PAI virulence factors are provided in Table 9-3.

Pathogenic bacteria (and other pathogens) have adapted both to saprophytic or free-living states, possibly environments outside the body, and to the human host. They have evolved complex signal transduction systems to regulate the genes important for virulence. Environmental signals often control the expression of the virulence genes. Common signals include temperature, iron availability, osmolality, growth phase, pH, and specific ions (eg, Ca²⁺) or nutrient factors. A few examples are presented in the following paragraphs.

The gene for diphtheria toxin from Corynebacterium diphtheriae is carried on temperate bacteriophages. Toxin is produced only by strains lysogenized by the phages. Toxin production is greatly enhanced when C diphtheriae is grown in a medium with low iron.

Expression of virulence genes of B pertussis is enhanced when the bacteria are grown at 37°C and suppressed when they are grown at lower temperatures or in the presence of high concentrations of magnesium sulfate or nicotinic acid.

The virulence factors of V cholerae are regulated on multiple levels and by many environmental factors. Expression of the cholera toxin is higher at a pH of 6.0 than at a pH of 8.5 and higher also at 30°C than at 37°C.

Osmolality and amino acid composition also are important. As many as 20 other genes of V cholerae are similarly regulated.

Y pestis produces a series of virulence plasmid-encoded proteins. One of these is an antiphagocytic fraction 1 capsular protein that results in antiphagocytic function. This protein is expressed maximally at 35–37°C, the host temperature, and minimally at 20–28°C, the flea temperature at which antiphagocytic activity is not needed. The regulation of other virulence factors in Yersinia species also is influenced by environmental factors.

Motility of bacteria enables them to spread and multiply in their environmental niches or in patients. Yersinia enterocolitica and Listeria monocytogenes are common in the environment where motility is important to them. Presumably, motility is not important in the pathogenesis of the diseases caused by these bacteria. Y enterocolitica is motile when grown at 25°C but not when grown at 37°C. Similarly, Listeria is motile when grown at 25°C and not motile or minimally motile when grown at 37°C.

Many factors determine bacterial virulence or the ability to cause infection and disease.

Adherence Factors

When bacteria enter the body of the host, they must adhere to cells of a tissue surface. If they did not adhere, they would be swept away by mucus and other fluids that bathe the tissue surface. Adherence, which is only one step in the infectious process, is followed by development of microcolonies and subsequent steps in the pathogenesis of infection.

The interactions between bacteria and tissue cell surfaces in the adhesion process are complex. Several factors play important roles, including surface hydrophobicity and net surface charge, binding molecules on bacteria (ligands), and host cell receptor interactions. Bacteria and host cells commonly have net negative surface charges and therefore repulsive electrostatic forces. These forces are overcome by hydrophobic and other more specific interactions between bacteria and host cells. In general, the more hydrophobic the bacterial cell surface, the greater the adherence to the host cell. Different strains of bacteria within a species may vary widely in their hydrophobic surface properties and ability to adhere to host cells.

Bacteria also have specific surface molecules that interact with host cells. Many bacteria have pili, thick rodlike appendages or fimbriae, shorter “hairlike” structures that extend from the bacterial cell surface and help mediate adherence of the bacteria to host cell surfaces. For example, some E coli strains have type 1 pili, which adhere to epithelial cell receptors; adherence can be blocked in vitro by addition of d-mannose to the medium. E coli organisms that cause urinary tract infections commonly do not have d-mannose–mediated adherence but have P-pili, which attach to a portion of the P blood group antigen; the minimal recognition structure is the disaccharide a-d-galactopyranosyl-(1–4)-β-d-galactopyranoside (GAL–GAL binding adhesin). The E coli that causes diarrheal diseases (see Chapter 15) have pilus (fimbriae)-mediated adherence to intestinal epithelial cells. The type of pili and specific molecular mechanisms of adherence appear to be different depending on the form of the E coli that induce the diarrhea.

Other specific ligand-receptor mechanisms have evolved to promote bacterial adherence to host cells, illustrating the diverse mechanisms used by bacteria. Group A streptococci (Streptococcus pyogenes) (see Chapter 14) also have hairlike appendages, termed fimbriae that extend from the cell surface. Lipoteichoic acid, protein F, and M protein are found on the fimbriae. The lipoteichoic acid and protein F cause adherence of the streptococci to buccal epithelial cells; this adherence is mediated by fibronectin, which acts as the host cell receptor molecule. M protein acts as an antiphagocytic molecule and is a major virulence factor.

Antibodies that act against the specific bacterial ligands that promote adherence (eg, pili and lipoteichoic acid) can block adherence to host cells and protect the host from infection.

After adherence occurs, conformational changes in the host cell ensue that can lead to cytoskeletal changes allowing organism uptake by the cell. Sometimes, changes in the adhesin molecule after attachment may trigger activation of virulence genes that promote invasion or that result in other pathogenic changes as described in the following pages.

Invasion of Host Cells and Tissues

Invasion is the term commonly used to describe the entry of bacteria into host cells and for many disease-causing bacteria, invasion of the host’s epithelium is central to the infectious process. Some bacteria (eg, Salmonella species) invade tissues through the junctions between epithelial cells. Other bacteria (eg, Yersinia species, N gonorrhoeae, and Chlamydia trachomatis) invade specific types of the host’s epithelial cells and may subsequently enter the tissue. In many infections, the bacteria produce virulence factors that cause the host cells to engulf (ingest) the bacteria. The host cells play a very active role in the process.

When inside the host cell, bacteria may remain enclosed in a vacuole composed of the host cell membrane, or the vacuole membrane may be dissolved and bacteria may be dispersed in the cytoplasm. Some bacteria (eg, Shigella species) multiply within host cells, but other bacteria do not.

Toxin production and other virulence properties are generally independent of the ability of bacteria to invade cells and tissues. For example, C diphtheriae is able to invade the epithelium of the nasopharynx and cause symptomatic sore throat even when the C diphtheriae strains are nontoxigenic.

In vitro studies with cells in tissue culture have helped characterize the mechanisms of invasion for some pathogens; however, the in vitro models have not necessarily provided a complete picture of the invasion process. Full understanding of the invasion process, as it occurs in naturally acquired infection, has required study of genetically engineered mutants and their ability to infect susceptible animals and humans. Thus, understanding of eukaryotic cell invasion by bacteria requires satisfying much of Koch’s postulates and the molecular Koch’s postulates. The following paragraphs contain examples of bacterial invasion of host cells as part of the infectious process.

Shigella species adhere to host cells in vitro. Commonly, HeLa cells are used; these undifferentiated unpolarized cells were derived from a cervical carcinoma. The adherence causes actin polymerization in the nearby portion of the HeLa cell, which induces the formation of pseudopods by the HeLa cells and engulfment of the bacteria. Adherence and invasion are mediated at least in part by products of genes located on a large plasmid common to many shigellae. There are multiple proteins, including the invasion plasmid antigens (IpA-D) that contribute to the process. Inside the HeLa cells, the shigellae either are released or escape from the phagocytic vacuole, where they multiply in the cytoplasm. Actin polymerization propels the shigellae within a HeLa cell and from one cell into another. In vivo the shigellae adhere to integrins on the surface of M cells in Peyer’s patches and not to the polarized absorptive cells of the mucosa. M cells normally sample antigens and present them to macrophages in the submucosa. The shigellae are phagocytosed by the M cells and pass through the M cells into the underlying collection of macrophages. Shigellae inside the M cells and macrophages can cause these cells to die by activating the normal cell death process (apoptosis). The shigellae spread to adjacent mucosal cells in a manner similar to the in vitro model by actin polymerization that propels the bacteria.

From studies using cells in vitro, it appears that the adherence-invasion process with Y enterocolitica is similar to that of Shigella. Yersiniae adhere to the host cell membrane and cause it to extrude protoplasmic projections. The bacteria are then engulfed by the host cell with vacuole formation. Invasion is enhanced when the bacteria are grown at 22°C rather than at 37°C. When yersiniae have entered the cell, the vacuolar membrane dissolves and the bacteria are released into the cytoplasm. In vivo, the yersiniae are thought to adhere to and invade the M cells of Peyer’s patches rather than the polarized absorptive mucosal cells, much like shigellae.

L monocytogenes from the environment is ingested in food. Presumably, the bacteria adhere to and invade the intestinal mucosa, reach the bloodstream, and disseminate. The pathogenesis of this process has been studied in vitro. L monocytogenes adheres to and readily invades macrophages and cultured undifferentiated intestinal cells. The listeriae induce engulfment by the host cells. Proteins, called internalins, have a primary role in this process. The engulfment process, movement within a cell and movement between cells, requires actin polymerization to propel the bacteria, as with shigellae.

Legionella pneumophila infects pulmonary macrophages and causes pneumonia. Adherence of the legionellae to the macrophage induces formation of a long, thin pseudopod that then coils around the bacteria, forming a vesicle (coiling phagocytosis). The vesicle remains intact, phagolysosome fusion is inhibited, and the bacteria multiply within the vesicle.

N gonorrhoeae uses pili as primary adhesins and opacity-associated proteins (Opa) as secondary adhesins to host cells. Certain Opa proteins mediate adherence to polymorphonuclear cells. Some gonococci survive after phagocytosis by these cells. Pili and Opa together enhance the invasion of cells cultured in vitro. In uterine (fallopian) tube organ cultures, the gonococci adhere to the microvilli of nonciliated cells and appear to induce engulfment by these cells. The gonococci multiply intracellularly and migrate to the subepithelial space by an unknown mechanism.

Toxins

Toxins produced by bacteria are generally classified into two groups: endotoxin, which is present in the outer membrane of gram-negative rods, and toxins that are secreted, such as enterotoxins and exotoxins. Enterotoxins and exotoxins are often classified by mechanisms of action and the impact on host cells and they are discussed in more detail below. The primary features of the two groups are listed in Table 9-4.

A. Exotoxins

Many gram-positive and gram-negative bacteria produce exotoxins of considerable medical importance. Some of these toxins have had major roles in world history. For example, tetanus caused by the toxin of C tetani killed as many as 50,000 soldiers of the Axis powers in World War II; the Allied forces, however, immunized military personnel against tetanus, and very few died of that disease. Vaccines have been developed for some of the exotoxin-mediated diseases and continue to be important in the prevention of disease. These vaccines—called toxoids—are made from exotoxins, which are modified so that they are no longer toxic. Many exotoxins consist of A and B subunits (often referred to as binary toxins or type III toxins). The B subunit generally mediates adherence of the toxin complex to a host cell and aids entrance of the exotoxin into the host cell. The A subunit provides the toxic activity. Examples of some pathogenetic mechanisms associated with exotoxins are given below. Other toxins of specific bacteria are discussed in the chapters covering those bacteria.

C diphtheriae is a gram-positive rod that can grow on the mucous membranes of the upper respiratory tract or in minor skin wounds (see Chapter 12). Strains of C diphtheriae that carry a lysogenic, temperate corynebacteriophage (β-phage or ω-phage) with the structural gene for the toxin are toxigenic and produce diphtheria toxin and cause diphtheria. Many factors regulate toxin production; when the availability of inorganic iron is the factor limiting the growth rate, then maximal toxin production occurs. The toxin molecule is secreted as a single polypeptide molecule (molecular weight [MW], 62,000). This native toxin is enzymatically degraded into two fragments, A and B, linked together by a disulfide bond. Fragment B (MW 40,700) binds to specific host cell receptors and facilitates the entry of fragment A (MW 21,150) into the cytoplasm. Fragment A inhibits peptide chain elongation factor EF-2 by catalyzing a reaction that attaches an adenosine diphosphate–ribosyl group to EF-2, yielding an inactive adenosine diphosphate–ribose–EF-2 complex. Arrest of protein synthesis disrupts normal cellular physiologic functions. Diphtheria toxin is very potent.

C tetani is an anaerobic gram-positive rod that causes tetanus (see Chapter 11). C tetani from the environment contaminates wounds, and the spores germinate in the anaerobic environment of the devitalized tissue. Infection often is minor and not clinically apparent. The vegetative forms of C tetani produce the toxin tetanospasmin (MW 150,000) that is cleaved by a bacterial protease into two peptides (MW 50,000 and 100,000) linked by a disulfide bond. The toxin initially binds to receptors on the presynaptic membranes of motor neurons. It then migrates by the retrograde axonal transport system to the cell bodies of these neurons to the spinal cord and brainstem. The toxin diffuses to terminals of inhibitory cells, including both glycinergic interneurons and γ-aminobutyric acid (GABA)–secreting neurons from the brainstem. The toxin degrades synaptobrevin, a protein required for docking of neurotransmitter vesicles on the presynaptic membrane. Release of the inhibitory glycine and GABA is blocked, and the motor neurons are not inhibited. Spastic paralysis results. Extremely small amounts of toxin can be lethal for humans. Tetanus is totally preventable in immunologically normal people by immunization with tetanus toxoid.

C botulinum causes botulism. This anaerobic, gram-positive spore-forming organism is found in soil or water and may grow in foods (eg, canned, vacuum packed) if the environment is appropriately anaerobic. An exceedingly potent toxin (the most potent toxin known) is produced. It is heat labile and is destroyed by sufficient heating. There are seven distinct serologic types of toxin. Types A, B, E, and F are most commonly associated with human disease. The toxin is very similar to tetanus toxin, with a 150,000 MW protein that is cleaved into 100,000-MW and 50,000-MW proteins linked by a disulfide bond. Botulinum toxin is absorbed from the gut and binds to receptors of presynaptic membranes of motor neurons of the peripheral nervous system and cranial nerves. Proteolysis, by the light chain of botulinum toxin, of target proteins in the neurons inhibits the release of acetylcholine at the synapse, resulting in lack of muscle contraction and flaccid paralysis.

Spores of C perfringens are introduced into wounds by contamination with soil or feces. In the presence of necrotic tissue (an anaerobic environment), spores germinate and vegetative cells can produce several different toxins. Many of these are necrotizing and hemolytic and—together with distention of tissue by gas formed from carbohydrates and interference with blood supply—favor the spread of gas gangrene. The alpha toxin of C perfringens is a lecithinase that damages cell membranes by splitting lecithin to phosphorylcholine and diglyceride. Theta toxin also has a necrotizing effect. Collagenases and DNAses are produced by clostridiae as well.

Some S aureus strains growing on mucous membranes (eg, the vagina in association with menstruation) or in wounds, elaborate toxic shock syndrome toxin-1 (TSST-1), which causes toxic shock syndrome (see Chapter 13). The illness is characterized by shock, high fever, and a diffuse red rash that later desquamates; multiple other organ systems are involved as well. TSST-1 is a superantigen (also referred to as a type I toxin), and superantigens do not need to enter cells to cause their potent cellular disruption. TSST-1 stimulates most T-cells by binding directly to MHC-II and T-cell receptors. The net result is production of large amounts of the cytokines interleukin-2 (IL-2), interferon γ, and tumor necrosis factor (TNF) (see Chapter 8). The major clinical manifestations of the disease appear to be secondary to the effects of the cytokines. The systemic effects of TSST-1 are due to the massive cytokine stimulation. Some strains of group A β-hemolytic streptococci produce pyrogenic exotoxins A and C. Rapidly progressive soft tissue infection by streptococci that produce the pyrogenic exotoxin A has many clinical manifestations similar to those of staphylococcal toxic shock syndrome. The pyrogenic exotoxins A and C are also superantigens that act in a manner similar to TSST-1.

Type II toxins are proteins that typically affect cell membranes facilitating invasion by the pathogen secreting them (see also Tissue-Degrading Enzymes later in the chapter). Examples include hemolysins and phospholipases which are also discussed in the appropriate organism chapters.

B. Exotoxins Associated With Diarrheal Diseases and Food Poisoning

Exotoxins associated with diarrheal diseases are frequently called enterotoxins and many belong to the type III toxin family. (See also Table 48-3.) Characteristics of some important enterotoxins are discussed as follows.

V cholerae has produced epidemic diarrheal disease (cholera) in many parts of the world (see Chapter 17) and is another toxin-produced disease of historical and current importance. After entering the host via contaminated food or drink, V cholerae penetrates the intestinal mucosa and attaches to microvilli of the brush border of gut epithelial cells. V cholerae, usually of the serotype O1 (and O139), can produce an enterotoxin with a MW of 84,000. The toxin consists of two subunits—A, which is split into two peptides, A₁ and A₂, linked by a disulfide bond, and B. Subunit B has five identical peptides and rapidly binds the toxin to cell membrane ganglioside molecules. Subunit A enters the cell membrane and causes a large increase in adenylate cyclase activity and in the concentration of cAMP. The net effect is rapid secretion of electrolytes into the small bowel lumen, with impairment of sodium and chloride absorption and loss of bicarbonate. Life-threatening massive diarrhea (eg, 20–30 L/day) can occur, and acidosis develops. The deleterious effects of cholera are due to fluid loss and acid–base imbalance; treatment, therefore, is by electrolyte and fluid replacement.

Some strains of S aureus produce enterotoxins while growing in meat, dairy products, or other foods. In typical cases, the food has been recently prepared but not properly refrigerated. There are at least seven distinct types of the staphylococcal enterotoxin. After the preformed toxin is ingested, it is absorbed in the gut, where it stimulates vagus nerve receptors. The stimulus is transmitted to the vomiting center in the central nervous system. Vomiting, often projectile, results within hours. Diarrhea is less frequent. Staphylococcal food poisoning is the most common form of food poisoning. S aureus enterotoxins are superantigens.

Enterotoxins are also produced by some strains of Y enterocolitica (see Chapter 19), Vibrio parahaemolyticus (see Chapter 17), Aeromonas species (see Chapter 17), and other bacteria, but the role of these toxins in pathogenesis is not as well defined. The enterotoxin produced by C perfringens is discussed in Chapter 11.

C. Lipopolysaccharides of Gram-Negative Bacteria

The LPS (endotoxin) of gram-negative bacteria are bacterial cell wall components that are often liberated when the bacteria lyse. The substances are heat stable, have MWs between 3000 and 5000 (lipooligosaccharides, LOS), and several million (lipopolysaccharides) can be extracted (eg, with phenol-water). They have three main regions (see Figure 2-19). The lipid A domain is the region recognized by the immune system and is the component that is responsible for cytokine stimulation (see below). The other two components are an oligosaccharide core and an outermost O-antigen polysaccharide.

The pathophysiologic effects of LPS are similar regardless of their bacterial origin except for those of Bacteroides species, which have a different structure and are less toxic (see Chapter 21). LPS in the bloodstream is initially bound to circulating proteins, which then interact with receptors on macrophages, neutrophils, and other cells of the reticuloendothelial system. Proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-a, and other cytokines are released, and the complement and coagulation cascades are activated. The following can be observed clinically or experimentally: fever, leukopenia, and hypoglycemia; hypotension and shock resulting in impaired perfusion of essential organs (eg, brain, heart, kidney); intravascular coagulation; and death from massive organ dysfunction.

Injection of LPS produces fever after 60–90 minutes, the time needed for the body to release IL-1. Injection of IL-1 produces fever within 30 minutes. Repeated injection of IL-1 produces the same fever response each time, but repeated injection of LPS causes a steadily diminishing fever response because of tolerance partly caused by reticuloendothelial blockade and partly caused by IgM antibodies to LPS.

Injection of LPS produces early leukopenia, as does bacteremia with gram-negative organisms. Secondary leukocytosis occurs later. The early leukopenia coincides with the onset of fever caused by liberation of IL-1. LPS enhances glycolysis in many cell types and can lead to hypoglycemia.

Hypotension occurs early in gram-negative bacteremia or after injection of LPS. There may be widespread arteriolar and venular constriction followed by peripheral vascular dilation, increased vascular permeability, decrease in venous return, lowered cardiac output, stagnation in the microcirculation, peripheral vasoconstriction, shock, and impaired organ perfusion and its consequences. Disseminated intravascular coagulation (DIC) also contributes to these vascular changes.

LPS is among the many different agents that can activate the alternative pathway of the complement cascade, precipitating a variety of complement-mediated reactions (eg, anaphylatoxins, chemotactic responses, membrane damage) and a drop in serum levels of complement components (C3, C5–C9).

Disseminated intravascular coagulation is a frequent complication of gram-negative bacteremia and can also occur in other infections. LPS activates factor XII (Hageman factor)—the first step of the intrinsic clotting system—and sets into motion the coagulation cascade, which culminates in the conversion of fibrinogen to fibrin. At the same time, plasminogen can be activated by LPS to plasmin (a proteolytic enzyme), which can attack fibrin with the formation of fibrin split products. Reduction in platelet and fibrinogen levels and detection of fibrin split products are evidence of DIC. Heparin can sometimes prevent the lesions associated with DIC.

LPS causes platelets to adhere to vascular endothelium and occlusion of small blood vessels, causing ischemic or hemorrhagic necrosis in various organs.

Endotoxin levels can be assayed by the limulus test: A lysate of amebocytes from the horseshoe crab (limulus) gels or coagulates in the presence of 0.0001 μg/mL of endotoxin. This test is rarely used in clinical laboratories because it is difficult to perform accurately.

D. Peptidoglycan of Gram-Positive Bacteria

The peptidoglycan of gram-positive bacteria is made up of cross-linked macromolecules that surround the bacterial cells (see Chapter 2 and Figure 2-15). Vascular changes leading to shock may also occur in infections caused by gram-positive bacteria that contain no LPS. Gram-positive bacteria have considerably more cell wall–associated peptidoglycan than do gram-negative bacteria. Peptidoglycan released during infection may yield many of the same biologic activities as LPS, although peptidoglycan is invariably much less potent than LPS.

Enzymes

Many species of bacteria produce enzymes that are not intrinsically toxic but do play important roles in the infectious process. Some of these enzymes are discussed as follows.

A. Tissue-Degrading Enzymes

Many bacteria produce tissue-degrading enzymes. The best-characterized are enzymes from C perfringens (see Chapter 11), and, to a lesser extent, anaerobic bacteria (see Chapter 21), S aureus (see Chapter 13), and group A streptococci (see Chapter 14). The roles of tissue-degrading enzymes in the pathogenesis of infections appear obvious but have been difficult to prove, especially those of individual enzymes. For example, antibodies against the tissue-degrading enzymes of streptococci do not modify the features of streptococcal disease.

In addition to lecithinase, C perfringens produces the proteolytic enzyme collagenase, which degrades collagen, the major protein of fibrous connective tissue, and promotes spread of infection in tissue.

S aureus produces coagulase, which works in conjunction with blood factors to coagulate plasma. Coagulase contributes to the formation of fibrin walls around staphylococcal lesions, which helps them persist in tissues. Coagulase also causes deposition of fibrin on the surfaces of individual staphylococci, which may help protect them from phagocytosis or from destruction within phagocytic cells.

Hyaluronidases are enzymes that hydrolyze hyaluronic acid, a constituent of the ground substance of connective tissue. They are produced by many bacteria (eg, staphylococci, streptococci, and anaerobes) and aid in their spread through tissues.

Many hemolytic streptococci produce streptokinase (fibrinolysin), a substance that activates a proteolytic enzyme of plasma. This enzyme is then able to dissolve coagulated plasma and probably aids in the rapid spread of streptococci through tissues. Streptokinase has been used in treatment of acute myocardial infarction to dissolve fibrin clots.

Many bacteria produce substances that are cytolysins—that is, they dissolve red blood cells (hemolysins) or kill tissue cells or leukocytes (leukocidins). Streptolysin O, for example, is produced by group A streptococci and is lethal for mice and hemolytic for red blood cells from many animals. Streptolysin O is oxygen labile and can therefore be oxidized and inactivated, but it is reactivated by reducing agents. It is antigenic. The same streptococci also produce oxygen-stable, serum-inducible streptolysin S, which is not antigenic. Clostridia produce various hemolysins, including the lecithinase described earlier. Hemolysins are produced by most strains of S aureus; staphylococci also produce leukocidins. Most gram-negative rods isolated from sites of disease produce hemolysins. For example, whereas E coli strains that cause urinary tract infections typically produce hemolysins, strains that are part of the normal gastrointestinal flora may or may not produce hemolysins.

B. IgA1 Proteases

Immunoglobulin A is the secretory antibody on mucosal surfaces. It has two primary forms, IgA1 and IgA2 that differ near the center, or hinge region of the heavy chains of the molecules (see Chapter 8). IgA1 has a series of amino acids in the hinge region that are not present in IgA2. Some bacteria that cause disease produce enzymes, IgA1 proteases that split IgA1 at specific proline–threonine or proline–serine bonds in the hinge region and inactivate its antibody activity. IgA1 protease is an important virulence factor of the pathogens N gonorrhoeae, N meningitidis, Haemophilus influenzae, and S pneumoniae. The enzymes are also produced by some strains of Prevotella melaninogenica, some streptococci associated with dental disease, and a few strains of other species that occasionally cause disease. Nonpathogenic species of the same genera do not have genes coding for the enzyme and do not produce it. Production of IgA1 protease allows pathogens to inactivate the primary antibody found on mucosal surfaces and thereby eliminate protection of the host by the antibody.

Antiphagocytic Factors

Many bacterial pathogens are rapidly killed after they are ingested by polymorphonuclear cells or macrophages. Some pathogens evade phagocytosis or leukocyte microbicidal mechanisms by adsorbing normal host components to their surfaces. For example, S aureus has surface protein A, which binds to the Fc portion of IgG. Other pathogens have surface factors that impede phagocytosis (eg, S pneumoniae, N meningitidis); many other bacteria have polysaccharide capsules. S pyogenes (group A streptococci) has M protein. N gonorrhoeae (gonococci) has pili. Most of these antiphagocytic surface structures show much antigenic heterogeneity. For example, there are more than 90 pneumococcal capsular polysaccharide types and more than 150 M protein types of group A streptococci. Antibodies against one type of the antiphagocytic factor (eg, capsular polysaccharide, M protein) protect the host from disease caused by bacteria of that type but not from those with other antigenic types of the same factor.

A few bacteria (eg, Capnocytophaga and Bordetella species) produce soluble factors or toxins that inhibit chemotaxis by leukocytes and thus evade phagocytosis by a different mechanism.

Intracellular Pathogenicity

Some bacteria (eg, M tuberculosis, L monocytogenes, Brucella species, and Legionella species) live and grow in the hostile environment within polymorphonuclear cells, macrophages, or monocytes. The bacteria accomplish this feat by several mechanisms: they may avoid entry into phagolysosomes and live within the cytosol of the phagocyte; they may prevent phagosome–lysosome fusion and live within the phagosome; or they may be resistant to lysosomal enzymes and survive within the phagolysosome.

Many bacteria can live within nonphagocytic cells (see previous section, Invasion of Host Cells and Tissues).

Antigenic Heterogeneity

The surface structures of bacteria (and of many other microorganisms) have considerable antigenic heterogeneity. Often these antigens are used as part of a serologic classification system for the bacteria. The classification of the 2000 or so different salmonellae is based principally on the types of the O (LPS side chain) and H (flagellar) antigens. Similarly, there are more than 150 E coli O types and more than 100 E coli K (capsule) types. The antigenic type of the bacteria may be a marker for virulence, related to the clonal nature of pathogens, although it may not actually be the virulence factor (or factors). V cholerae O antigen type 1 and O antigen type 139 typically produce cholera toxin, but very few of the many other O types produce the toxin. Only some of the group A streptococcal M protein types are associated with a high incidence of poststreptococcal glomerulonephritis. N meningitidis capsular polysaccharide types A and C are associated with epidemic meningitis. In the examples cited earlier and in other typing systems that use surface antigens in serologic classification, antigenic types for a given isolate of the species remain constant during infection and on subculture of the bacteria.

Some bacteria and other microorganisms have the ability to make frequent shifts in the antigenic form of their surface structures in vitro and presumably in vivo. One well-known example is Borrelia recurrentis, which causes relapsing fever. A second widely studied example is N gonorrhoeae (see Chapter 20). The gonococcus has three surface-exposed antigens that switch forms at very high rates of about one in every 1000; lipooligosaccharide, 6–8 types; pili, innumerable types; and Opa, 10–12 types for each strain. The number of antigenic forms is so large that each strain of N gonorrhoeae appears to be antigenically distinct from every other strain. Switching of forms for each of the three antigens appears to be under the control of different genetic mechanisms. It is presumed that frequent switching of antigenic forms allows gonococci to evade the host’s immune system; gonococci that are not attacked by the immune system survive and cause disease.

Bacterial Secretion Systems

Bacterial secretion systems are important in the pathogenesis of infection and are essential for the interaction of bacteria with the eukaryotic cells of the host. The gram-negative bacteria have cell walls with cytoplasmic membranes and outer membranes; a thin layer of peptidoglycan is present. Gram-positive bacteria have a cytoplasmic membrane and a very thick layer of peptidoglycan (see Chapter 2). Some gram-negative bacteria and some gram-positive bacteria have capsules as well. The complexity and rigidity of the cell wall structures necessitate mechanisms for the translocation of proteins across the membranes. These secretion systems are involved in cellular functions such as the transport of proteins that make pili or flagella and in the secretion of enzymes or toxins into the extracellular environment. The differences in cell wall structure between gram-negative and gram-positive bacteria result in some differences in the secretion systems. The basic mechanisms of the different bacterial secretion systems are discussed in Chapter 2. (Note: The specific bacterial secretion systems were named in the order of their discovery and not by their mechanisms of action.)

Both gram-negative and gram-positive bacteria have a general secretion pathway (Sec) as the major mechanism for protein secretion. This pathway is involved in the insertion of most of the bacterial membrane proteins and provides the major pathway for proteins crossing the bacterial cytoplasmic membrane. Gram-negative organisms have an additional six mechanisms, secretion systems (SS) 1–6 (sometimes denoted I–VI), for protein secretion. These can be further characterized as Sec dependent (types 2 and 5) and Sec independent (types 1, 3, 4, 6). Type 2 SS uses the general Sec to transport the proteins to the periplasm and then create an outer membrane channel made by a special pore-forming protein complex. This type 2 SS is used to secrete portions of bacterial A B type toxins, such as cholera toxin. Similarly, the type 5 SS uses the general Sec to export an autotransporter to the periplasm; from there it transports itself across the outer membrane. An example of this type of SS includes the IgA proteases secreted by H influenzae. The sec-independent pathways include the type 1 secretion system or ABC secretion system (ATP-binding cassette) and the type 3 secretion system. The type 1 and 3 pathways do not interact with proteins that have been transported across the cytoplasmic membrane by the Sec system. Instead, these systems translocate proteins across both the cytoplasmic and outer membranes. The type 3, which is activated upon contact with a eukaryotic host cell, promotes transport of proteins directly from inside the bacterium to the inside of the host cell using a needlelike structure called an injectosome; when in the host cell cytoplasm, the transported proteins can manipulate host cell function. Pseudomonas aeruginosa possesses a type 3 secretion system that when expressed, may be associated with more serious disease. The type 4 secretion pathway consists of a protein complex that forms a “tunnel” that is able to directly transport proteins or DNA. The most recent SS to be discovered is the type 6 SS. This SS plays a role in the secretion of virulence proteins in V cholerae and P aeruginosa among other gram-negative pathogens. A seventh SS has been described in M tuberculosis and is not well understood. Its function appears to be transport of proteins across both the inner and outer membranes. Some other examples of the secretion systems and their roles in pathogenesis are shown in Table 9-5. These examples are but a small sample designed to illustrate the roles of the large number of molecular secretion activities used by bacteria to provide nutrients and facilitate their pathogenesis.

The Requirement for Iron

Iron is an essential nutrient for the growth and metabolism of nearly all microorganisms and is an essential cofactor of numerous metabolic and enzymatic processes. The availability of iron in humans for microbial assimilation is limited because the iron is sequestered by the high-affinity iron-binding proteins transferrin in serum and lactoferrin on mucosal surfaces. The ability of a microbial pathogen to efficiently obtain iron from the host environment is critical to its ability to cause disease. The requirement for iron, how bacteria acquire iron, and bacterial iron metabolism are discussed in Chapter 5.

Iron availability affects the virulence of many pathogens. For example, iron is an essential virulence factor in P aeruginosa. The use of animal models in L monocytogenes infection has demonstrated that increased iron results in enhanced susceptibility to infection, but iron depletion results in prolonged survival; iron supplementation therapy yields an increase in lethal infections.

Decreased iron availability can also be important in pathogenesis. For example, the gene for diphtheria toxin resides on a lysogenic bacteriophage, and only strains of C diphtheriae that carry the lysogenic bacteriophage are toxigenic. In the presence of low iron availability, there is increased production of diphtheria toxin and potentially more severe disease. The virulence of N meningitidis for mice is increased 1000-fold or more when the bacteria are grown under iron-limited conditions.

Human iron deficiency also plays a role in the infectious process. Iron deficiency affects hundreds of millions of people worldwide. Iron deficiency can affect multiple organ systems, including the immune system, and can result in impaired cell-mediated immunity and decreased polymorphonuclear cell function. Providing iron therapy during an active infection probably should be delayed because many pathogenic microorganisms can use the small amounts of supplemental iron, resulting in an increase in virulence.

The Role of Bacterial Biofilms

A biofilm is an aggregate of interactive bacteria attached to a solid surface or to each other and encased in an exopolysaccharide matrix. This is distinct from planktonic or free-living bacteria, in which interactions of the microorganisms do not occur in the same way. Biofilms form a slimy coat on solid surfaces and occur throughout nature. A single species of bacteria may be involved or more than one species may coaggregate to form a biofilm. Fungi, including yeasts, are occasionally involved. After a biofilm is formed, quorum-sensing molecules produced by the bacteria in the biofilm accumulate, resulting in a modification of the metabolic activity of the bacteria. The basic biology of biofilm exopolysaccharide (glycocalyx) is discussed in Chapter 2; the quorum-sensing molecules are discussed in Chapter 1.

The bacteria in the exopolysaccharide matrix may be protected from the host’s immune mechanisms. This matrix also functions as a diffusion barrier for some antimicrobials, but other antimicrobials may bind to it. Some of the bacteria within the biofilm show marked resistance to antimicrobials compared with the same strain of bacteria grown free living in broth, which helps to explain why it is so difficult to treat infections associated with biofilms.

Biofilms are important in human infections that are persistent and difficult to treat. A few examples include Staphylococcus epidermidis and S aureus infections of central venous catheters, eye infections such as what occur with contact lenses and intraocular lenses, in dental plaque, and in prosthetic joint infections. Perhaps the most profound example of a biofilm in human infection is in P aeruginosa airway infections in cystic fibrosis patients.

• Animals and humans are colonized with abundant microbiota, normal commensals that do not cause disease and are protective to the host.

• Virulent bacteria cause disease through the elaboration of factors that facilitate adherence, persistence, invasion, and toxigenicity.

• Genes that encode virulence factors may be carried on mobile genetic elements such as plasmids or bacteriophages or are found on large pathogenicity islands on bacterial chromosomes.

• Pili and fimbriae are rodlike or hairlike structures, respectively, that facilitate attachment to host cells.

• Invasion of host cells is a complex mechanism that involves elaboration of proteins that facilitate entry.

• Bacterial toxins may be extracellular (exotoxins) or are a component of the bacterial cell membrane (endotoxin, LPS) and are among the most powerful toxins in nature (eg, botulinum toxin).

• Other mechanisms important to bacterial survival and virulence include tissue-degrading enzymes, antiphagocytic factors, IgA proteases, antigenic heterogeneity, and the ability to chelate iron.

• There are at least seven known bacterial secretion systems, protein complexes, or channels that ensure transport of structural and toxigenic proteins through the bacterial cell after translation.