Nonspecific Responses.

Some nonspecific responses are biochemical; others are cellular. Biochemical factors remove essential nutrients, such as iron, from tissues so that it is unavailable for use by invading microorganisms. Cellular responses are central to tissue and organ defenses, and the cells involved are known as phagocytes.

Phagocytes.

Phagocytes are cells that ingest and destroy bacteria and other foreign particles. The two major types of phagocytes are polymorphonuclear leukocytes, also known as PMNs or neutrophils, and macrophages. Phagocytes ingest bacteria by a process known as endocytosis and engulf them in a membrane-lined structure called a phagosome (Figure 3-6). The phagosome is then fused with a second structure, the lysosome. When the lysosome, which contains toxic chemicals and destructive enzymes, combines with the phagosome, the bacteria trapped within the structure, referred to as a phagolysosome, are neutralized and destroyed. This destructive process must be carried out inside membrane-lined structures; otherwise the noxious substances contained within the phagolysosome would destroy the phagocyte itself. This is evident during the course of rampant infections when thousands of phagocytes exhibit “sloppy” ingestion of the microorganisms and toxic substances spill from the cells, damaging the surrounding host tissue. This process is referred to as phagocytosis.

Although both PMNs and macrophages are phagocytes, these cell types differ. PMNs develop in the bone marrow and spend their short lives (usually a day or less) circulating in blood and tissues. Widely dispersed in the body, PMNs usually are the first cells on the scene of bacterial invasion. Macrophages also develop in the bone marrow but first go through a cellular phase in which they are called monocytes. Macrophages circulating in the bloodstream are called monocytes. When deposited in tissue or at a site of infection, monocytes transform into mature macrophages. In the absence of infection, macrophages usually reside in specific organs, such as the spleen, lymph nodes, liver, or lungs, where they live for days to several weeks, awaiting encounters with invading bacteria. In addition to the ingestion and destruction of bacteria, macrophages play an important role in mediating immune system defenses (see Specific Responses—The Immune System later in this chapter).

In addition to the inhibition of microbial proliferation by phagocytes and by biochemical substances such as lysozyme, microorganisms are “washed” from tissues during the flow of lymph fluid. The fluid carries infectious agents through the lymphatic system, where they are deposited in tissues and organs (e.g., lymph nodes and spleen) heavily populated with phagocytes. This process functions as an efficient filtration system.

Two Branches of the Immune System.

The immune system provides immunity that generally can be divided into two branches:

Antibody-mediated immunity is centered on the activities of B cells and the production of antibodies. When B cells encounter a microbial antigen, they become activated and a series of events is initiated. These events are mediated by the activities of helper T cells and the release of cytokines. Cytokines mediate clonal expansion, and the number of B cells capable of recognizing the antigen increases. Cytokines also activate the maturation of B cells into plasma cells that produce antibodies specific for the antigen. The process results in the production of B-memory cells (Figure 3-9). B-memory cells remain quiescent in the body until a second or subsequent exposure to the original antigen occurs. With secondary exposure, the B-memory cells are preprogrammed to produce specific antibodies immediately upon encountering the original antigen.

Antibodies protect the host in a number of ways:

Because a population of activated specific B cells is a developmental process as a result of the exposure to microbial antigens, antibody production is delayed when the host is first exposed to an infectious agent. This delay in the primary antibody response underscores the importance of nonspecific response defenses, such as inflammation, that work to hold the invading organisms in check while antibody production begins. This also emphasizes the importance of B-memory cell production. By virtue of this memory, any subsequent exposure or secondary (anamnestic) response to the same microorganism results in rapid production of protective antibodies so that the body is spared the delays characteristic of the primary exposure.

Some antigens, such as bacterial capsules and outer membranes, activate B cells to produce antibodies without the intervention of helper T cells. However, this activation does not result in the production of B-memory cells, and subsequent exposure to the same bacterial antigens does not result in a rapid host memory response.

The primary cells involved in cell-mediated immunity are T lymphocytes (cytotoxic T cells) that recognize and destroy human host cells infected with microorganisms. This function is extremely important for the destruction and elimination of infecting microorganisms. Some pathogens (e.g., viruses, tuberculosis, some parasites, and fungi) are able to survive in host cells, protected from antibody interaction. Antibody-mediated immunity targets microorganisms outside human cells, whereas cell-mediated immunity targets microorganisms inside human cells. However, in many instances these two branches of the immune system overlap and work together.

Like B cells, T cells must be activated. Activation is accomplished by T-cell interactions with other cells that process microbial antigens and present them on their surface (e.g., macrophages, dendritic cells, and B cells). The responses of activated T cells are very different and depend on the subtype of T cell (Figure 3-10). Activated helper T cells work with B cells for antibody production (see Figure 3-9) and facilitate inflammation by releasing cytokines. Cytotoxic T cells directly interact with and destroy host cells containing microorganisms or other infectious agents, such as viruses. The activated T-cell subset, helper or cytotoxic cells, is controlled by an extremely complex series of biochemical pathways and genetic diversity within the major histocompatibility complex (MHC). MHC molecules are present on cells and form a complex with the antigen to present them to the T cells. The two primary classes of major histocompatibility molecules are MHC I and MHC II. MHC I molecules are located on every nucleated cell in the body and are predominantly responsible for the recognition of endogenous proteins expressed from within the cell. MHC II molecules are located on specialized cell types, including macrophages, dendritic cells, and B cells, for the presentation of extracellular molecules or exogenous proteins.

In summary, the host presents a spectrum of challenges to invading microorganisms, from physical barriers, including the skin and mucous membranes, to the interactive cellular and biochemical components of inflammation and the immune system. All these systems work together to minimize microbial invasion and prevent damage to vital tissues and organs resulting from the presence of infectious agents.

Attachment.

Whether humans encounter microorganisms in the air, through ingestion, or by direct contact, the first step of infection and disease development, a process referred to as pathogenesis, is microbial attachment to a surface (exceptions being instances in which the organisms are directly introduced by trauma or other means into deeper tissues).

Many of the microbial factors that facilitate attachment of pathogens are the same as those used by nonpathogenic colonizers (see Box 3-2). Most pathogenic organisms are not part of the normal microbial flora, and attachment to the host requires that they outcompete colonizers for a place on the body’s surface. Medical interventions, such as the overuse of antimicrobial agents, result in the destruction of the normal flora, creating a competitive advantage for the invading pathogenic organism.

Invasion.

Once surface attachment has been secured, microbial invasion into subsurface tissues and organs (i.e., infection) is accomplished by disruption of the skin and mucosal surfaces by several mechanisms (see Box 3-3) or by the direct action of an organism’s virulence factors. Some microorganisms produce factors that force mucosal surface phagocytes (M cells) to ingest them and then release them unharmed into the tissue below the surface. Other organisms, such as staphylococci and streptococci, are not so subtle. These organisms produce an array of enzymes (e.g., hyaluronidases, nucleases, collagenases) that hydrolyze host proteins and nucleic acids, destroying host cells and tissues. This destruction allows the pathogen to “burrow” through minor openings in the outer surface of the skin and into deeper tissues. Once a pathogen has penetrated the body, it uses a variety of strategies to survive attack by the host’s inflammatory and immune responses. Alternatively, some pathogens cause disease at the site of attachment without further penetration. For example, in diseases such as diphtheria and whooping cough, the bacteria produce toxic substances that destroy surrounding tissues. The organisms generally do not penetrate the mucosal surface they inhabit.

Survival Against Inflammation.

If a pathogen is to survive, the action of phagocytes and the complement components of inflammation must be avoided or controlled (Box 3-5). Some organisms, such as Streptococcus pneumoniae, a common cause of bacterial pneumonia and meningitis, avoid phagocytosis by producing a large capsule that inhibits the phagocytic process. Other pathogens may not be able to avoid phagocytosis but are not effectively destroyed once internalized and are able to survive within phagocytes. This is the case for Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Still other pathogens use toxins and enzymes to attack and destroy phagocytes before the phagocytes attack and destroy them.

The defenses offered by the complement system depend on a series of biochemical reactions triggered by specific microorganism molecular structures. Therefore, microbial avoidance of complement activation requires that the infecting agent either mask its activating molecules (e.g., via production of a capsule that covers bacterial surface antigens) or produce substances (e.g., enzymes) that disrupt critical biochemical components of the complement pathway.

Any single microorganism may possess numerous virulence factors, and several may be expressed simultaneously. For example, while trying to avoid phagocytosis, an organism may also excrete other enzymes and toxins that destroy and penetrate tissue and produce other factors designed to interfere with the immune response. Microorganisms may also use host systems to their own advantage. For example, the lymphatic and circulatory systems used to carry monocytes and lymphocytes to the site of infection may also serve to disperse the organism throughout the body.

Survival Against the Immune System.

Microbial strategies to avoid the defenses of the immune system are outlined in Box 3-6. Again, a pathogen can use more than one strategy to avoid immune-mediated defenses, and microbial survival does not necessarily require devastation of the immune system. The pathogen may merely need to “buy” time to reach a safe area in the body or to be transferred to the next susceptible host. Also, microorganisms can avoid much of the immune response if they do not penetrate the surface layers of the body. This strategy is the hallmark of diseases caused by microbial toxins.

Microbial Toxins.

Toxins are biochemically active substances released by microorganisms that have a particular effect on host cells. Microorganisms use toxins to establish infections and multiply within the host. Alternatively, a pathogen may be restricted to a particular body site from which toxins are released to cause systemic damage throughout the body. Toxins also can cause human disease in the absence of the pathogens that produced them. This common mechanism of food poisoning involves ingestion of preformed bacterial toxins (present in the food at the time of ingestion) and is referred to as intoxication, a notable example of which is botulism.

Endotoxin and exotoxin are the two general types of bacterial toxins (Box 3-7). Endotoxin is a component of the cellular structure of gram-negative bacteria and can have devastating effects on the body’s metabolism, the most serious being endotoxic shock, which often results in death. The effects of exotoxins produced by gram-positive bacteria tend to be more limited and specific than the effects of gram-negative endotoxin. The activities of exotoxins range from enzymes produced by many staphylococci and streptococci that augment bacterial invasion by damaging host tissues and cells to highly specific activities (e.g., diphtheria toxin inhibits protein synthesis, and cholera toxin interferes with host cell signals). Examples of other highly active and specific toxins are those that cause botulism and tetanus by interfering with neuromuscular functions.

Biofilm Formation.

Microorganisms typically exist as a group or community of organisms capable of adhering to each other or to other surfaces. A variety of bacterial pathogens, along with other microorganisms, are capable of forming biofilms, including S. aureus, P. aeruginosa, and Candida albicans. A biofilm is an accumulation of microorganisms embedded in a polysaccharide matrix. Pathogenic microorganisms use the formation of biofilm to adhere to implants and prosthetic devices. For example, nosocomial infections with Staphylococci spp. associated with implants have become more prevalent. Interestingly, biofilm-forming strains have a much more complex antibiotic resistance profile, indicating failure of the antibiotic to penetrate the polysaccharide layer. In addition, some of the cells in the sessile or stationary biofilm may experience nutrient deprivation and therefore exist in a slow-growing or starved state, displaying reduced susceptibility to antimicrobial agents. These organisms also have demonstrated a differential gene expression, compared to their planktonic or free-floating counter parts. The biofilm-forming communities are able to adapt and respond to changes in their environment, similar to a multicellular organism.

Biofilms may form from the accumulation of a single microorganism (monomicrobic aggregation) or from the accumulation of numerous species (polymicrobic aggregation). It is widely accepted that the cells in a biofilm are physiologically unique from the planktonic cells and are referred to as persister cells. During biofilm accumulation, the cells reach a critical mass that results in alteration in metabolism and gene expression. This is accomplished through a mechanism of signaling between cells or organisms through chemical signals or inducer molecules, such as acyl homoserine lactone (AHL) in gram-negative bacteria or oligopeptides in gram-positive bacteria. These signals are capable of interspecies and intraspecies communication.

Microbial biofilm formation is important to many disciplines, including environmental science, industry, and public health. Biofilm formation affects the efficient treatment of wastewater; it is essential for the effective production of beer, which requires aggregation of yeast cells; and it affects bioremediation for toxic substances such as oil. It has been reported that approximately 65% of hospital-acquired infections are associated with biofilm formation. Box 3-8 provides an overview of pathogenic organisms associated with biofilm formation in human infections.

Immunization

Medical strategies exist for minimizing the risk of disease development when exposure to infectious agents occurs. One of the most effective methods is vaccination, also referred to as immunization. This practice takes advantage of the specificity and memory of the immune system. The two basic approaches to immunization are active immunization and passive immunization. With active immunization, modified antigens from pathogenic microorganisms are introduced into the body and cause an immune response. If or when the host encounters the pathogen in nature, the memory of the immune system ensures minimal delay in the immune response, thus affording strong protection. With passive immunization, antibodies against a particular pathogen that have been produced in one host are transferred to a second host, where they provide temporary protection. The passage of maternal antibodies to the newborn is a key example of natural passive immunization. Active immunity is generally longer lasting, because the immunized host’s own immune response has been activated. However, for complex reasons, naturally acquired active immunity has had limited success for relatively few infectious diseases, necessitating the development of vaccines. Successful immunization has proven effective against many infectious diseases, including diphtheria, whooping cough (pertussis), tetanus, influenza, polio, smallpox, measles, hepatitis, and certain Streptococcus pneumoniae and Haemophilus influenzae infections.

Prophylactic antimicrobial therapy, the administration of antibiotics when the risk of developing an infection is high, is another common medical intervention for preventing infection.

Epidemiology

To prevent infectious diseases, information is required regarding the sources of pathogens, the mode of transmission to and among humans, human risk factors for encountering the pathogen and developing infection, and factors that contribute to good and bad outcomes resulting from the exposure. Epidemiology is the science that characterizes these aspects of infectious diseases and monitors the effect diseases have on public health. Fully characterizing the circumstances associated with the acquisition and dissemination of infectious diseases gives researchers a better chance of preventing and eliminating these diseases. Additionally, many epidemiologic strategies developed for use in public health systems also apply in long-term care facilities (i.e., nursing homes, hospitals, assisted living centers) for the control of infections acquired within the facility (i.e., nosocomial infections; for more information on infection control, see Chapter 80).

The field of epidemiology is broad and complex. Diagnostic microbiology laboratory personnel and epidemiologists often work closely to investigate problems. Therefore, familiarity with certain epidemiologic terms and concepts is important (see Box 3-1).

Because the central focus of epidemiology is on tracking and characterizing infections and infectious diseases, this field heavily depends on diagnostic microbiology. Epidemiologic investigations cannot proceed unless researchers first know the etiologic or causative agents. Therefore, the procedures and protocols used in diagnostic microbiology to detect, isolate, and characterize human pathogens are essential for patient care and also play a central role in epidemiologic studies focused on disease prevention and the general improvement of public health. In fact, microbiologists who work in clinical laboratories are often the first to recognize patterns that suggest potential outbreaks or epidemics.