SLP 1.3 Introduction of Bacteriology PDF

FACULTY OF DENTISTRY UNIVERSITI SAINS ISLAM MALAYSIA DBB1018 APPLIED BASIC SCIENCE SEMESTER I: SESSION 2024/2025 SELF-LEARNING PACKAGE (SLP) SLP 1.3 Applied Microbiology Introduction of Bacteriology By Dr Nor Zaihana Abdul Rahman Lecturer, Facu...

FACULTY OF DENTISTRY UNIVERSITI SAINS ISLAM MALAYSIA DBB1018 APPLIED BASIC SCIENCE SEMESTER I: SESSION 2024/2025 SELF-LEARNING PACKAGE (SLP) SLP 1.3 Applied Microbiology Introduction of Bacteriology By Dr Nor Zaihana Abdul Rahman Lecturer, Faculty of Dentistry, USIM Page | 1 LEARNING OUTCOMES At the end of this topic, you should be able to: 1. Differentiate eukaryotic and prokaryotic cells and different shapes of bacteria. 2. Describe briefly the structure and the stages in biosynthesis of the bacterial cell wall. 3. Differentiate Gram-positive and Gram-negative cell walls. 4. Describe the mechanisms of pathogenicity and virulence factors that enable bacteria to invade and colonize. REFERENCES 1. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Introduction to Bacteriology. 2. Brock Biology of Microorganisms by Michael T. Madigan, John M. Martinko, and David P. Stahl. 3. Scheffers DJ, Pinho MG. Bacterial cell wall synthesis: new insights from localization studies. Microbiol Mol Biol Rev. (2005) Dec;69(4):585-607. doi: 10.1128/MMBR.69.4.585-607.2005. PMID: 16339737; PMCID: PMC1306805. 4. Salton MRJ, Kim KS. Structure. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 2. 5. Moagi Shaku, Christopher Ealand, Ofentse Matlhabe, Rushil Lala, Bavesh D. Kana, Chapter Two - Peptidoglycan biosynthesis and remodeling revisited, Editor(s): Geoffrey Michael Gadd, Sima Sariaslani, Advances in Applied Microbiology. 2020. (112): 67-103. https://doi.org/10.1016/bs.aambs.2020.04.001. Page | 2 INTRODUCTION Bacteria are single-celled microorganisms that lack a nuclear membrane, are metabolically active and divide by binary fission. Medically they are a major cause of disease. Superficially, bacteria appear to be relatively simple forms of life; in fact, they are sophisticated and highly adaptable. Many bacteria multiply rapidly, and different species can utilize various hydrocarbon substrates, including phenol, rubber, and petroleum. These organisms exist widely in both parasitic and free-living forms. Because they are ubiquitous and have a remarkable capacity to adapt to changing environments by selection of spontaneous mutants, the importance of bacteria in every field of medicine cannot be overstated. The discipline of bacteriology evolved from the need of physicians to test and apply the germ theory of disease and economic concerns relating to the spoilage of foods and wine. The initial advances in pathogenic bacteriology were derived from identifying and characterising bacteria associated with specific diseases. During this period, great emphasis was placed on applying Koch's postulates to test proposed cause-and-effect relationships between bacteria and specific diseases. Today, most bacterial diseases of humans and their etiologic agents have been identified, although important variants continue to evolve and sometimes emerge, e.g., Legionnaire's Disease, tuberculosis and toxic shock syndrome. Significant advances in bacteriology over the last century resulted in the development of many effective vaccines (e.g., pneumococcal polysaccharide vaccine, diphtheria toxoid, and tetanus toxoid) as well as other vaccines (e.g., cholera, typhoid, and plague vaccines) that are less effective or have side effects. Another major advance was the discovery of antibiotics. These antimicrobial substances have not eradicated bacterial diseases but are powerful therapeutic tools. Their efficacy is reduced by the emergence of antibiotic-resistant bacteria (now a crucial medical management problem). In reality, improvements in sanitation and water purification have a greater effect on the incidence of bacterial infections in a community than the availability of antibiotics or bacterial vaccines. Nevertheless, many serious bacterial diseases remain. Page | 3 BASIC STRUCTURE OF BACTERIA Bacterial cells are between 0.3 and 5 lm in size. They have three primary forms: cocci, straight rods, and curved or spiral rods. The nucleoid consists of a very thin, long, circular DNA molecular double strand not surrounded by a membrane. Among the nonessential genetic structures are the plasmids. The cytoplasmic membrane harbors numerous proteins such as permeases, cell wall synthesis enzymes, sensor proteins, secretion system proteins, and, in aerobic bacteria, respiratory chain enzymes. The membrane is surrounded by the cell wall, the most important element of which is the supporting murein skeleton. Because of the strength and rigidity of its wall, the bacterial cell has a characteristic size and shape. As long as the wall remains intact, large changes in the osmotic pressure of the environment have little effect on cell shape. SPHERICAL FORM OR "COCCUS" a) In general, a diameter of 0.8 to 1.0 μm b) Some organisms exhibit characteristic small variations from the sphere: e.g., the Pneumococcus (causes pneumonia) and the Gonococcus (causes Gonorrhoea) are elongated cocci. In contrast, the Staphylococcus (causes abscesses) is a perfect sphere. ROD SHAPED OR "BACILLUS" (CYLINDRICAL SHAPE OF CELL) The shape of individual rods is different for different species and may be useful for identification. For instance, the rod may be long and slender or short and thick. The ends of the rod may appear square or rounded or tapered. Some rods, such as Corynebacterium diphtheriae are characteristically pleomorphic. (Note: With some organisms, the line of demarcation between a coccus and a rod is difficult to draw, with the result that we call some forms "coccobacilli"). This is a problem for gram-negative rods, as you will find in the laboratory. CURVED ROD OR SPIRAL SHAPE The vibrios (e.g. the cholera vibrio) are curved rods. Page | 4 The spirochetes are bacteria with a spiral shape. However, they do not have a rigid wall and are therefore classified differently. (Treponema pallidum the causative agent of syphilis, is a spirochete as is Borrelia burgdorferi, the cause of Lyme Disease.) Compared to bacterial cells, mammalian cells lack a cell wall. The shape of mammalian cells will change depending on the ionic strength of the surrounding medium. In distilled water, mammalian cells will swell and burst, but bacterial cells remain stable. Figure 1: Different shapes of bacteria. EUKARYOTIC AND PROKARYOTIC CELLS All cells are classified as either prokaryotic or eukaryotic based primarily on the organization of their internal structures (Figure 2). Bacteria and Archaea exhibit a prokaryotic cell structure characterized by a comparatively simple, non- compartmentalized internal organization that lacks membrane-enclosed organelles (Figure 2a). Page | 5 In contrast, eukaryotic cells are typically larger and structurally more complex, having a compartmentalized cytoplasm containing membrane-bound organelles, such as a defined nucleus, mitochondria, and chloroplasts (Figure 2b). In addition to microbial eukaryotic cells (the algae, protozoa and fungi), plants and animals are also composed of eukaryotic cells. The cell wall of Gram-negative bacteria features a porous outer membrane into the outer surface of which the lipopolysaccharide responsible for the pathogenesis of Gram-negative infections is integrated. The cell wall of Gram-positive bacteria does not possess such an outer membrane. Its murein layer is thicker and contains teichoic acids and wall-associated proteins that contribute to the pathogenic process in Gram- positive infections. Many bacteria have capsules made of polysaccharides that protect them from phagocytosis. Attachment pili or fimbriae facilitate adhesion to host cells. Motile bacteria possess flagella. Foreign body infections are caused by bacteria that form a biofilm on inert surfaces. Figure 2: Cell structure of (a) bacteria and the archaeans and (b) eukaryotic cells. The membrane-enclosed organelles (nucleus, mitochondria, and chloroplasts) present in eukaryotic cells are absent from cells of Bacteria and Archaea. Chloroplasts are present only in photosynthetic eukaryotic cells, including algae and plant cells. mechanisms of pathogenicity and virulence factors Page | 6 DIFFERENCES OF GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA The cell wall and membrane structure of Gram-positive bacteria are much different in composition than those of gram negatives (early observations showed that Gram- positive bacteria were more susceptible to disinfectants and antibiotics). In addition to a thick layer of peptidoglycan, Gram-positive (Staphylococcus, Streptococcus, etc.) walls are composed mostly of different carbohydrate polymers. The key aspect is that the Gram-positive bacteria has no phospholipid outer membrane. For example, a polymer of ribitol (a sugar alcohol) phosphate (a teichoic acid) in Staphylococcus or a polymer of other sugars in Streptococcus. The teichoic acids linked to the peptigoglycan are important antigens. The host’s antibody response seems mainly directed against these polymers rather than against the peptidoglycan. Thus, these polymers probably are on the outermost surface of the cell, with the peptidoglycan underneath closest to the cytoplasmic membrane. Teichoic acids, for instance, may be slightly modified by the addition of an amino acid, and this will impart immunological specificity to the cell surface. Gram-negative: the peptidoglycan (mucopeptide) layer is thinner. Gram-negative organisms also have an outer membrane (OM). The outer surface of the OM bilayer contains lipopolysaccharide (LPS). As in Gram positives, the peptidoglycan layer of the gram-negative cell wall is closest to the cytoplasmic membrane. Outside of the peptidoglycan, and attached to it by lipoprotein, is the important outer membrane containing lipopolysaccharide. In summary, gram negatives have the inner cytoplasmic membrane surrounded by a thin layer of peptidoglycan, surrounded by the unique outer membrane. Thus, Gram-negative bacteria have unique, 3-layered envelopes (Figure 3), with the outer membrane being unique to Gram-negative bacteria. A periplasmic space containing some binding proteins, and some digestive or hydrolytic enzymes occurs in gram-negative but not gram-positive bacteria. This periplasmic space is located between the inner and the outer membrane. Page | 7 Figure 3: Differences in structure of Gram-positive and Gram-negative bacteria. Table 1: Example of Gram-positive and Gram-negative bacteria. The Gram staining results explain the fundamental differences in surface structures of Gram-positive and Gram-negative bacteria. Gram-positive and Gram-negative bacteria take up the identical amounts of crystal violet (CV) and iodine (I). The CV-I complex, however, is trapped inside the Gram-positive cell by the dehydration and reduced porosity of the thick cell wall due to the differential washing step with 95 per Page | 8 cent ethanol or other solvent mixture. In contrast, the thin peptidoglycan layer and probable discontinuities at the membrane adhesion sites do not impede solvent extraction of the CV-I complex from the Gram-negative cell. Moreover, mechanical disruption of the cell wall of Gram-positive organisms or its enzymatic removal with lysozyme results in complete extraction of the CV-I complex and conversion to a Gram-negative reaction. Therefore, autolytic wall-degrading enzymes that cause cell wall breakage may account for Gram-negative or variable reactions in cultures of Gram-positive organisms (such as Staphylococcus aureus, Clostridium perfringens, Corynebacterium diphtheriae, and some Bacillus spp.). BIOSYNTHESIS OF CELL WALL Unique features of almost all prokaryotic cells (except for Halobacterium halobium and mycoplasmas) are cell wall peptidoglycan and the specific enzymes involved in its biosynthesis. These enzymes are target sites for inhibition of peptidoglycan synthesis by specific antibiotics. The primary chemical structures of peptidoglycans of both Gram-positive and Gram-negative bacteria have been established; they consist of a glycan backbone of repeating groups of β1, 4-linked disaccharides of β1,4-N- acetylmuramyl-N-acetylglucosamine. Tetrapeptides of L-alanine-D-isoglutamic acid-L- lysine (or diaminopimelic acid)-n-alanine are linked through the carboxyl group by amide linkage of muramic acid residues of the glycan chains; the D-alanine residues are directly cross-linked to the 𝛆-amino group of lysine or diaminopimelic acid on a neighbouring tetrapeptide, or a peptide bridge links them. In S. aureus peptidoglycan, a glycine pentapeptide bridge links the two adjacent peptide structures. The extent of direct or peptide-bridge cross-linking varies from one peptidoglycan to another. The staphylococcal peptidoglycan is highly cross-linked, whereas that of E. coli is much less so, and has a more open peptidoglycan mesh. The diamino acid providing the 𝛆- amino group for cross-linking is lysine or diaminopimelic acid, which is uniformly present in Gram-negative peptidoglycans. The structure of the peptidoglycan is illustrated in Figure 4. Page | 9 Figure 4: Schematic structure of the peptidoglycan. The glycan strands (consisting of GlcNAc and MurNAc), the stem peptides (with 4–3 or 3–3 cross-links) and targets bonds cleaved by the different PG hydrolases. A peptidoglycan with a chemical structure substantially different from that of all eubacteria has been discovered in certain archaebacteria. Instead of muramic acid, this peptidoglycan contains talosaminuronic acid and lacks the D-amino acids found in the eubacterial peptidoglycans. Interestingly, organisms containing this wall polymer (pseudomurein) are insensitive to penicillin, an inhibitor of the transpeptidases involved in peptidoglycan biosynthesis in eubacteria. The ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine is cleaved explicitly by the bacteriolytic enzyme lysozyme. Widely distributed in nature, this enzyme is present in human tissues and secretions and can cause complete digestion of the peptidoglycan walls of sensitive organisms. When lysozyme is allowed to digest the cell wall of Gram-positive bacteria suspended in an osmotic stabilizer (such as sucrose), protoplasts are formed. These protoplasts are able to survive and continue to grow on suitable media in the wall-less state. Gram-negative bacteria treated similarly produce spheroplasts, which retain much of the outer membrane structure. The dependence of bacterial shape on the peptidoglycan is shown by the Page | 10 transformation of rod-shaped bacteria to spherical protoplasts (spheroplasts) after enzymatic breakdown of the peptidoglycan. The mechanical protection the wall peptidoglycan layer provides is evident in the osmotic fragility of both protoplasts and spheroplasts. Two groups of bacteria lack the protective cell wall peptidoglycan structure, the Mycoplasma species, one of which causes atypical pneumonia and some genitourinary tract infections and the L-forms, which originate from Gram- positive or Gram-negative bacteria and are so designated because of their discovery. The mycoplasmas and L-forms are Gram-negative, insensitive to penicillin, and bounded by a surface membrane structure. L-forms arising "spontaneously" in cultures or isolated from infections are structurally related to protoplasts and spheroplasts; all three forms (protoplasts, spheroplasts, and L-forms) revert infrequently and only under particular conditions. BACTERIAL INFECTIONS Infection is the invasion of the host by microorganisms, which then multiply in close association with the host's tissues. Infection is distinguished from disease, a morbid process that does not necessarily involve infection (diabetes, for example, is a disease with no known causative agent). Bacteria can cause many infections, ranging in severity from inapparent to fulminating. Table 2 lists these types of infections. TYPES OF BACTERIAL INFECTIONS. The capacity of a bacterium to cause disease reflects its relative pathogenicity. On this basis, bacteria can be organized into three major groups. When isolated from a patient, primary pathogens are considered to be probable agents of disease (e.g., when the cause of the diarrheal disease is identified by the laboratory isolation of Salmonella spp. from faeces). Opportunistic pathogens are those isolated from patients whose host defense mechanisms have been compromised. They may be disease agents (e.g., in patients predisposed to urinary tract infections with Escherichia coli by catheterization). Finally, some bacteria, such as Lactobacillus acidophilus, are considered to be nonpathogens because they rarely or never cause human disease. However, their categorization as nonpathogens may change because of the adaptability of bacteria and the detrimental effect of modern radiation therapy, Page | 11 chemotherapy, and immunotherapy on resistance mechanisms. In fact, some bacteria previously considered to be nonpathogens are now known to cause disease. Serratia marcescens, for example, is a common soil bacterium that causes pneumonia, urinary tract infections, and bacteremia in compromised hosts. Table 2: Selected major infectious diseases of humans caused by bacteria. Type of Infection Description Inapparent No detectable clinical symptoms of infection Dormant Carrier state Accidental Zoonosis or environment or inadvertent exposures Opportunistic Infection is caused by normal flora or transient bacteria when normal host defences are compromised Primary Clinically apparent (e;g; invasion and multiplication of microbes in body tissues, causing local tissue injury) Secondary Microbial invasion subsequent to primary infection Page | 12  Mixed Two or more microbes infecting the same tissue Acute Rapid onset (hours or days); onset duration (days or weeks) Chronic Prolonged duration (months or years) Localized Confined to an area or to an organ Generalized Disseminated to many body region Pyogenic Pus-forming Retrograde Microbes ascending in a duct against the flow of secretions or excretions Fulminant Infections that occur suddenly and intensely Table 3: Types of Bacterial Infections Virulence is the measure of the pathogenicity of an organism. The degree of virulence is related directly to the ability of the organism to cause disease despite host resistance mechanisms; it is affected by numerous variables such as the number of infecting bacteria, route of entry into the body, specific and nonspecific host defense mechanisms, and virulence factors of the bacterium. Virulence can be measured experimentally by determining the number of bacteria required to cause animal death, illness, or lesions in a defined period after a designated route administers the bacteria. Consequently, calculations of a lethal dose affecting 50 percent of a population of animals (LD50) or an effective dose causing a disease symptom in 50 percent of a population of animals (ED50) are useful in comparing the relative virulence of different bacteria. Pathogenesis refers both to the mechanism of infection and to the mechanism by which disease develops. Pathogenic mechanisms of many bacterial diseases are poorly understood, while those of others have been probed at the molecular level. The relative importance of an infectious disease to the health of humans and animals does not always coincide with the depth of our understanding of its pathogenesis. Page | 13 HOST SUSCEPTIBILITY Susceptibility to bacterial infections depends on the host's physiologic and immunologic condition and the bacteria's virulence. Before increased amounts of specific antibodies or T cells are formed in response to invading bacterial pathogens, the “nonspecific” mechanisms of host resistance (such as polymorphonuclear neutrophils and macrophage clearance) must defend the host against the microbes. Development of effective specific immunity may require several weeks. The normal bacterial flora of the skin and mucosal surfaces also serves to protect the host against colonization by bacterial pathogens. In most healthy individuals, bacteria from the normal flora that occasionally penetrate the body (e.g., during tooth extraction or routine teeth brushing) are cleared by the host's cellular and humoral mechanisms. In contrast, individuals with defective immune responses are prone to frequent, recurrent infections with even the least virulent bacteria. The best-known example of such susceptibility is acquired immune deficiency syndrome (AIDS), in which the CD4+ helper lymphocytes are progressively decimated by human immunodeficiency virus (HIV). However, resistance mechanisms can be altered by many other processes. For example, aging often weakens both nonspecific and specific defense systems so that they can no longer effectively combat the challenge of bacteria from the environment. Infants are also especially susceptible to certain pathogens (such as group B streptococci because their immune systems are not yet fully developed and cannot mount a protective immune response to important bacterial antigens. In addition, some individuals have genetic defects of the complement system or cellular defenses (e.g., inability of polymorphonuclear neutrophils to kill bacteria). Finally, a patient may develop granulocytopenia as a result of a predisposing disease, such as cancer, or immunosuppressive chemotherapy for organ transplants or cancer. Host resistance can be compromised by trauma and by some underlying diseases. An individual becomes susceptible to infection with a variety of bacteria if the skin or mucosa is breached, particularly in the case of severe wounds such as burns or contaminated surgical wounds. Cystic fibrosis patients, who have poor ciliary function and consequently cannot clear mucus efficiently from the respiratory tract, are abnormally susceptible to infection with mucoid strains of Pseudomonas aeruginosa, resulting in serious respiratory distress. Ascending urinary tract infections with Escherichia coli are common in women and are particularly troublesome in Page | 14 patients with urinary tract obstructions. A variety of routine medical procedures, such as tracheal intubation and catheterization of blood vessels and the urethra, increase the risk of bacterial infection. The plastic devices used in these procedures are readily colonized by bacteria from the skin, which migrate along the outside of the tube to infect deeper tissues or enter the bloodstream. Because of this problem, it is standard practice to change catheters frequently (e.g., every 72 hours for peripheral intravenous catheters). Many drugs have been developed to treat bacterial infections. Antimicrobial agents are most effective, however, when the infection is also being fought by healthy phagocytic and immune defenses. Some reasons for this situation are the poor diffusion of antibiotics into certain sites (such as the prostate gland), the ability of many bacteria to multiply or survive inside cells (where many antimicrobial agents have little or no effect), the bacteriostatic rather than bactericidal action of some drugs, and the capacity of some organisms to develop resistance to multiple antibiotics. Many bacterial pathogens are transmitted to the host by a vector, usually an arthropod. For example, Rocky Mountain spotted fever and Lyme disease are vectored by ticks, and fleas spread bubonic plague. Susceptibility to these diseases depends partly on the host's contact with the vector. PATHOGENIC MECHANISMS Bacterial Infectivity Factors produced by a microorganism that evoke disease are called virulence factors. Examples are toxins, surface coats that inhibit phagocytosis, and surface receptors that bind to host cells. Most frank (as opposed to opportunistic) bacterial pathogens have evolved specific virulence factors that allow them to multiply in their host or vector without being killed or expelled by the host's defenses. Many virulence factors are produced only by specific virulent strains of a microorganism. For example, only certain strains of E. coli secrete diarrhea-causing enterotoxins. Page | 15 Figure 5: Mechanisms of acquiring bacterial virulence gene. Virulence factors should never be considered independently of the host's defenses; the clinical course of a disease often depends on the interaction of virulence factors with the host's response. An infection begins when the balance between bacterial pathogenicity and host resistance is upset. In essence, we live in an environment that favors the microbe, simply because the growth rate of bacteria far exceeds that of most eukaryotic cells. Furthermore, bacteria are much more versatile than eukaryotic cells in substrate utilization and biosynthesis. The high mutation rate of bacteria combined with their short generation time results in rapid selection of the best-adapted strains and species. In general, bacteria are much more resistant to toxic components in the environment than eukaryotes, particularly when the major barriers of eukaryotes (skin and mucous membranes) are breached. From a practical standpoint, bacteria can be said to have a single objective: to multiply. Only a few of the vast number of bacterial species in the environment consistently Page | 16 cause disease in a given host. From a teleologic standpoint, it is not in the best interest of the pathogen to kill the host, because in most cases the death of the host means the death of the pathogen. The most highly evolved or adapted pathogens are the ones that acquire the necessary nutritional substances for growth and dissemination with the smallest expenditure of energy and the least damage to the host. For example, Rickettsia akari, the etiologic agent of rickettsialpox, causes a mild, self- limited infection consisting of headache, fever, and a papulovesicular rash. Other members of the rickettsial group, such as R. rickettsii, the agent of Rocky Mountain spotted fever, elicit more severe, life-threatening infections. Some bacteria that are poorly adapted to the host synthesize virulence factors (e.g., tetanus and diphtheria toxin) so potent that they threaten the host's life. Host Resistance Although easily damaged, the skin represents one of the most important barriers of the body to the microbial world, which contains a diverse array of bacteria in enormous numbers. Fortunately, most bacteria in the environment are relatively benign to individuals with normal immune systems. However, patients who are immunosuppressed, such as individuals receiving cancer chemotherapy or have AIDS, opportunistic microbial pathogens can establish life-threatening infections. Normally, microbes in the environment are prevented from entering the body by the skin and mucous membranes. The outermost surface of the skin consists of squamous cell epithelium, largely comprised of dead cells that are sloughed off as new cells are formed below them. In addition to the skin barrier, mucous membranes of the respiratory, gastrointestinal, and urogenital systems represent other portals through which bacteria can gain access to the body. Like the squamous epithelial cells of the skin, the mucosal epithelial cells divide rapidly, and as the cells mature, they are pushed laterally toward the intestinal lumen and shed. The entire process is reported to require only 36-48 hours for complete replacement of the epithelium, which diminishes the number of bacteria associated with the epithelium. The skin surface is a dry, acidic environment, and the temperature is less than 37° C. The pores and crevices of the skin also are colonized by the “normal bacterial flora”, which ensure competition for pathogens to which the skin is exposed. Similarly, the mucous layer that covers the epithelia contains hostile substances to microbial colonization. Page | 17 Protective levels of lysozyme, lactoferrin, and lactoperoxidase in the mucus either kill bacteria or restrict their growth. In addition, the mucus contains secretory immunoglobulins (predominantly sIgA) synthesized by plasma cells resident in the submucosal tissue. During the normal course of life, individuals develop local antibodies specific for various intestinal bacteria that colonize mucosal surfaces. Another mechanism of restricting the growth of bacteria that penetrate the skin and mucous membranes is competition for iron. Typically, the amount of free iron in tissues and blood available to bacteria is very low since plasma transferrin binds virtually all iron in the blood. Similarly, haemoglobin in the erythrocytes binds iron. Bacterial growth is restricted without free iron unless the bacteria synthesize siderophores or receptors for iron-containing molecules competing for transferrin-bound iron. Such siderophores strip iron from transferrin and present it to the bacteria, which enables them to grow. The phagocytic cells of the body patrol the blood and tissues for foreign substances, including bacteria. This task is assumed predominantly by polymorphonuclear neutrophils; however, monocytes, macrophages, and eosinophils also participate. After phagocytosis, these bacterial cells are usually killed unless their numbers are excessive or possess virulence factors that enable them to survive the lysosomal enzymes and acidic pH. In some instances, the bacteria kill the phagocyte or multiply within the macrophage, escaping the hostile extracellular environment. When inflammation occurs, phagocytic cells, along with lymphocytes, play an essential role in innate immunity to bacterial infections. During the interaction of bacterial cells with macrophages, T cells, and B cells, specific antibody responses and/or cell- mediated immunity develop to protect against reinfection. Genetic and Molecular Basis for Virulence Virulence factors in bacteria may be encoded on chromosomal DNA, bacteriophage DNA, plasmids, or transposons in either plasmids or the bacterial chromosome. For example, the capacity of the Shigella species to invade cells is a property encoded in part on a 140-mega-dalton plasmid. Similarly, the heat-labile enterotoxin (LTI) of E. coli is plasmid encoded, whereas the heat-labile toxin (LTII) is encoded on the chromosome. Other virulence factors are acquired by bacteria following infection by a particular bacteriophage, which integrates its genome into the bacterial chromosome by lysogeny. Temperate bacteriophages often serve as the basis of toxin production in Page | 18 pathogenic bacteria. Examples include diphtheria toxin production by Corynebacterium diphtheriae, erythrogenic toxin formation by Streptococcus pyogenes, Shiga-like toxin synthesis by E. coli, and production of botulinum toxin (types C and D) by Clostridium botulinum. Other virulence factors are encoded on the bacterial chromosome (e.g., cholera toxin, Salmonella enterotoxin, and Yersinia invasion factors). Page | 19