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Introduction to Clinical Microbiology © Alex011973/Shutterstock, Inc

Introduction to Clinical Microbiology © Alex011973/Shutterstock, Inc

Chapter 1 Introduction to Clinical © Alex011973/Shutterstock, Inc.

Chapter Outline Classification and The Role of Clinical Microbiology Characteristics of and Prokaryotes The Infectious Process

Key Terms Acquired Infectious Inflammatory response carrier Innate immunity Cell-mediated immunity (CMI) Normal flora Colonization Nosocomial Endotoxin Pili Immunoglobulin Immunosuppressive

Learning Objectives 1. Discuss the purpose of clinical microbiology. 7. Define the following terms: 2. Describe the binomial system of taxonomy a. Infection and discuss how phenotypic and molecular b. Infectious disease characteristics are used to classify . c. True pathogen 3. Identify and give the of the bacterial cell d. Opportunistic pathogen components. e. Nosocomial infection 4. Differentiate the gram-positive cell wall from the f. Endogenous infection gram-negative cell wall. g. Exogenous infection 5. State the function of pili, fimbriae, flagella, and the h. Asymptomatic carriage (carriers) capsule. i. Colonization 6. Describe the important metabolic activities of the 8. Define and contrast: bacterial cell. a. and b. disease and

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9. Define normal flora and discuss its role in each of the d. Humoral immunity following sites: e. Cell-mediated immunity a. and oral cavity 12. Describe the function of B and T cells in the immune b. Nasopharynx response: c. Stomach and a. List and summarize the characteristics of the d. Colon immunoglobulin classes. 10. List and describe the major routes of infection. b. List and state the function of four populations of 11. Describe the following defense mechanisms: T cells. a. Innate (natural) immunity 13. Define and describe endotoxins and . b. Inflammatory response 14. List the signs of microbial infection. c. Acquired immunity 15. List the laboratory procedures that might be requested to identify infectious disease.

The purpose of clinical microbiology is to isolate and iden- Classification and Taxonomy tify pathogenic . Clinical work with clinicians and other personnel to assist in the The classification of into categories based on diagnosis, management, and treatment of infectious dis- genotypic and phenotypic characteristics is known as tax- ease. The microbiology laboratory can provide the phy- onomy. Historically, classification has been based mostly sician with information from direct smears and stains, on observable properties such as morphology, biochemical cultures, molecular analysis, serological testing, and an- characteristics, and antigenic relationships. Examples of tibiotic susceptibility testing. The also relies on phenotypical characteristics used to classify microorgan- the ’s medical ; ; and isms are shown in Box 1-1. results of X-rays, laboratory tests, and epidemiological in- This phenotypical classification is being replaced with formation (such as previous , travel, and illness systems based on genetic homology. Although these sys- in the family) to aid in the diagnosis of infectious disease. tems are more precise, at , they do not conform to This chapter provides an introduction to clinical mi- classification based on phenotypic characteristics. Genetic crobiology, including a review on taxonomy, bacterial homology includes classification based on DNA base com- structure, and . Also discussed are the con- position and ratio. The cytosine and guanine content (CG) cepts of and normal flora and the infectious to total base content is used as an indicator of relatedness. process, including symptoms and routes of infection. A Nucleic sequence analysis uses the order of bases along summary of the inflammatory process and immunity is the DNA or RNA sequence and determines similar se- discussed and important definitions provided. quences between two organisms.

Box 1-1 Phenotypical Classification Characteristics Macroscopic morphology: Size, texture, color, elevation Microscopic morphology: Size, shape (cocci, bacilli), arrangement (pairs, chains, clusters) characteristics: Gram-stain reaction (positive/negative), acid fastness Environmental requirements: Temperature optimum, oxygen needs, pH needs, carbon dioxide needs, need/able to withstand NaCl Nutritional requirements: Use carbon or nitrogen substrates Resistance profiles: Inherent resistance to , chemicals Antigenic properties: Serological or immunological methods (Lancefield groups of , properties of capsules)

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When identifying microorganisms, the key features are outlined based on genotypic characteristics, including Box 1-2 Example of Classification and nucleic and phenotypic characteristics, Family: Microcococceae which are observable. The hierarchy for classification is summarized below, beginning with the largest division, or : kingdom, and ending with the smallest division, or . Species: aureus Kingdom Accepted abbreviation: S. aureus Division Informal: staphylococci Class Order Family information on the is investigated, and the or- Genus ganism may or may not be reclassified or renamed. When Species a new name is accepted, the written format is “new name The species is the most basic taxonomic group and encom- (old name)” until sufficient has elapsed to recognize passes bacterial strains with common genetic, physiologic, the change. For example, faecalis was for- and phenotypic characteristics. There may be subgroups merly classified in the genus Streptococcus; when it was re- within the species, which are known as subspecies. Below classified, Enterococcus (Streptococcus) faecalis was written the subspecies level, there may be microorganisms that for clarification. Box 1-2 gives an example of nomenclature. share specific minor characteristics; these are known as biotypes, subtypes, or strains or . Strains or sub- types are genetic variants of the . Differ- Characteristics of Eukaryotes ent species with many important features in common are and Prokaryotes known as a genus (genera). Genera are based on genetic and phenotypic characteristics among several species. It Eukaryotic cells contain membrane-enclosed structures, is usually not practical in microbiology to classify similar which have specific functions. Fungi and parasites are cat- genera into higher taxonomic levels. However, at times, egorized as eukaryotes. Eukaryotic cells have a cytoskel- grouping into families may be helpful. eton that supports the cell and also various organelles, In the binomial system of nomenclature, two names, such as the nucleus, mitochondria, endoplasmic reticu- the genus and species, are used. These are generally derived lum, Golgi bodies, and lysosomes. Bacterial cells are pro- from the Latin or Greek language. Both the genus and species karyotic, which means that they do not contain organelles names should be italicized or underlined; the genus name is enclosed in membranes. Prokaryotes are unicellular or- always capitalized and the species name is never capitalized. ganisms without a nuclear membrane, mitochondria, en- Accepted abbreviations include the uppercase form of the doplasmic reticulum, or Golgi bodies. Bacteria multiply first letter of the genus with a period. Informal names are asexually, and all cellular functions occur in either the written in lower case without italics or underlining. cytoplasm or cytoplasmic membrane of the bacterial cell. Proposed changes in nomenclature are examined by Table 1-1 summarizes the characteristics of eukaryotic the International Journal for Systematic . New and prokaryotic organisms.

Table 1-1 Comparing the Characteristics of Eukaryotic and Prokaryotic Cells

Eukaryotes Prokaryotes Microorganisms included Algae, fungi, Bacteria Nucleus Enclosed in nuclear membrane No nuclear membrane Mitochondria Present Absent Golgi bodies Present Absent Endoplasmic reticulum Present Absent Ribosomes 80S (60S + 40S) 70S (50S + 30S)

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Bacterial cells range from 1 μm to 3 μm in length and ribosomes, the DNA , mRNA, proteins and thus are not visible to the human eye without the aid of a metabolites, nucleoid, and . The cytosol contains . Bacteria that are round are known as cocci; many and is the site of most metabolic processes cocci may arrange in pairs, chains, or clusters. Bacteria for the bacterial cell. The bacterial chromosome is one that have a rod shape are known as bacilli, and those with double-stranded circle contained in a discrete area of the a spiral form are known as spirochetes. cytoplasm, known as the nucleoid. The DNA is not con- Typical morphologies of bacteria are shown in tained within a nucleus, and there are no histones. Plas- Figure 1-1. mids, which are small, circular extrachromosomal DNA may also be found in the cytoplasm. Plasmids play a role Bacterial Components in the development of resistance. Bacterial cells contain a number of components that are The cytosol has a granular appearance as a result of the located within the interior of the cell, known as the cyto- presence of polysomes and inclusions. The polysomes con- plasm or cytosol. These cytoplasmic structures include tain messenger RNA bound to ribosomes, serving as the site of protein formation. Transcription and occur Coccus or spheres as a coupled reaction in the cytoplasm; ribosomes bind to Diplococci - arranged in pairs mRNA, and protein is made at the same time that the mRNA is being synthesized and is still bound to the DNA. The Sved- berg unit (S) expresses the sedimentation coefficient for mac- Streptococcus Neisseria pneumoniae romolecules and serves as a measure of particle size. It is used to describe the complex subunits of ribosomes. The bacterial ribosome consists of 30S and 50S subunits that form a 70S ribosome. By contrast, the eukaryotic ribosome contains 40S Streptococci Cocci arranged in chains and 60S subunits that form an 80S ribosome. The inclusions contain glycogen and inorganic phos- phates. Other inclusions may be present depending on the specific bacterial species. The bacterial cell may convert to an endospore form under poor environmental conditions. The spore state is Staphylococci Cocci arranged in clusters inactive but is able to resist the adverse conditions. When the situation becomes favorable, the spore can germinate into the bacterial cell. or rods The cytosol is enclosed by the cellular envelope, which Various sizes includes the cytoplasmic membrane and cell wall. There is also periplasmic space and an outer membrane in gram- Filamentous negative bacteria. The cytoplasmic, or inner membrane, is found in both gram-positive and gram-negative bacteria. It consists of a lipid bilayer intermixed with protein mole- Bacillus - large bacilli arranged in chains cules including enzymes. The functions of the cytoplasmic membrane include generation of ATP, of solutes Curved in and out of the cell, cell motility, chromosomal segrega- tion, and sensors for detecting environmental changes. Coccobacilli Enzymes involved in cell-wall and outer-wall formation

Fusiform and the synthesis and secretion of various compounds are also located here. Cellular structures or appendages also are located in Spirochete the cellular envelope. These include pili or fimbriae, which Figure 1-1 Bacterial shapes and morphology are hair-like extensions that extend into the environment.

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There are common pili, which permit the organism to at- Gram-negative bacteria also have an outer membrane, tach to the host cells, and sex pili, which are involved in which acts as a barrier for the cell against the external en- conjugation. Flagella are connected to the cellular enve- vironment and contains important enzymes and proteins. lope and found in those bacteria that are motile. Monot- This outer membrane is external to the cytoplasmic mem- richous flagella are located at one end of the cell, while brane and is made up of lipopolysaccharide (LPS). LPS, lophotrichous flagella are located on both ends of the cell. also known as endotoxin, has several biological functions Peritrichous flagella cover the entire bacterial surface. The important in the disease process. When LPS is released presence or absence and location of flagella are important upon cell lysis into the environment of the host, B cells are identification characteristics. activated, which stimulates macrophage and other cells to Other bacteria have capsules, which are composed of release interleukin-1, interleukin-2, tumor factor, or protein layers. When these materials are and other cytokines. LPS can also induce and more loosely arranged, it is known as a slime layer. Cap- shock; disseminated intravascular coagulation (DIC) is sules are poor and are antiphagocytic and impor- one severe consequence of large amounts of LPS released tant factors for bacteria. Capsules also may serve into the blood. DIC is characterized by systemic activation as barriers to hydrophobic compounds such as detergents of blood coagulation with the formation of fibrin clots, and can enable the bacteria to adhere to other bacteria or which may result in thrombi, or clots, within the blood and to host surfaces. organs of the body. This may ultimately result in multiple The periplasmic space contains the murein layer and a organ failure and bleeding from consumption of the co- gel-like structure that assists the bacteria in obtaining nu- agulation proteins and platelets. There are several causes trients. There are also enzymes that can break down large of DIC that include severe infection and , trauma, molecules in this area. It is located between the inner part , hepatic failure, and transfusion reactions. of the outer membrane and the outer external membrane LPS consists of three sections: lipid A, core polysaccha- and is found only in gram-negative bacteria. ride, and O antigen. Lipid A is associated with endotoxin The cell wall, or murein layer, is more commonly activity; its structure is similar for closely related bacteria. known as peptidoglycan; it serves as the external wall of Core polysaccharide is important structurally and for the most bacteria. This layer provides stability and strength viability of the bacterial cell. The O antigen is important to the bacterial cell and blocks the passage of some mac- in differentiating serotypes or strains of a bacterial spe- romolecules. It is composed of the disaccharides N- cies. For example, there are over 150 types of O antigen for acetylglucosamine and N-acetylmuramic acid, which are E. coli; one particularly important type is O157. Porins cross-linked to form peptidoglycan sheets when they are present in the outer membrane contain water and help bound by peptide molecules. These peptidoglycan sheets regulate the passage of nutrients, antibiotics, and other further cross-link to form a multilayered structure. The hydrophilic compounds into the cell. peptidoglycan is much thicker in the gram-positive cell The cell walls of gram-positive and gram-negative bac- wall when compared to that of gram-negative bacteria. In teria are compared in Figure 1-2. addition, there are also techoic acids linked to the cellular membrane in the gram-positive cell wall. Techoic acids Cellular Metabolism are water-soluble polymers of polyol phosphates bound to peptidoglycan and essential for the viability of the cell. Cellular metabolism of bacteria is important when deter- Lipoteichoic acids contain a fatty acid and are important mining how bacteria cause disease and also for the biochem- as surface antigens to differentiate bacterial serotypes. ical identification of bacteria. Bacteria must obtain nutrients Lipoteichoic acids also aid the attachment of the organism from the environment through the cell envelope; water, oxy- to host receptors. Techoic acids also play a role in viru- gen, and carbon dioxide diffuse across the cell membrane. lence. The mycobacteria contain a waxy substance, mycolic Active transport is needed to move other molecules such acid, in their cell walls, which enables them to resist the as organic acids, amino acids, and inorganic ions into the actions of acid. Other compounds that contribute to the acid- bacterial cell. These processes are facilitated through carrier resistance and waxy character of the mycobacterial cell molecules. Other compounds such as sugars, fatty acids, and wall are cord factor, wax D, and sulfolipids. nucleotide bases are chemically modified through group

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Fermentative processes vary with each bacterial species GRAM-NEGATIVE GRAM-POSITIVE BACTERIA BACTERIA and can be used to identify bacteria. For example, all mem- bers of the Enterobacteriaceae family possess the necessary enzymes to ferment the glucose. Identifying CAPSULECAPSULE CAPSULE the production of acid end products from a specific sub-

Lipopoly- strate is useful in the identification of bacteria. saccharide Bacteria can also use oxidative phosphorylation to

OUTEROUTOOUUUTTER Protein CELLLLLLLW WWAALLAALLLLL generate energy. In this process, carrier molecules trans- MEMBRANMEMMMEEEMMBBRRRAAANNNEE (PEPTIDOGL(P( DOGLDODOGOGLGLYCAN)YCYCACAANN)N) electrons from a reduced carrier molecule to a ter- CELLCCECELELELLLW WAWALLLL (PEPTIDOGLGLLYYCAN)YCYCANNN)) Teichoic minal electron acceptor. In the process, ATP is generated PERIPLASMICIICC acids from ADP. In aerobic respiration, oxygen is used as the SPACE CYTOPLASMICOPOPLAASMIASSSMMMIIICC terminal electron acceptor. In anaerobic respiration, an CYTOPLASMICASMIAASSMMIC ProteinsProteins MEMBRANRARAANNENE MEMBRANEMBMBBRRRANERAANNEE electron acceptor other than oxygen is used. CYTOPLASMLASM CYTOPLASMLASM Bacteria that totally rely on aerobic respiration and cannot grow in the absence of oxygen are known as obli- Figure 1-2 Cell wall comparisons gate (strict) aerobes; those that cannot tolerate any amount of oxygen are known as obligate (strict) anaerobes. Fac- ultative anaerobic bacteria can use aerobic respiration or translocation to enter the cell. Nutrients also include nitrate, fermentative processes. phosphate, hydrogen sulfide, sulfate, potassium, magne- sium, calcium, sodium, iron, and ammonia. Exotoxins and Endotoxins Precursor molecules are formed from the nutrients once Bacteria produce systemic effects of infection through the they are within the bacterial cell. These compounds are production of . Exotoxins are associated with gram- produced through the pentose phosphate shunt, Embden- positive organisms and are secreted by the living bacterial Meyerhof-Parnas (EMP) pathway, and the tricarboxylic acid cell. Exotoxins are usually found in high concentration in (TCA) cycle. These metabolites include acetyl CoA, glucose- fluid media and are not associated with the production of 6-phosphate, pyruvate, oxaloacetate, and other compounds. fever. Examples include leukocidin, produced by S. aureus, Through biosynthesis, these molecules form fatty acids, sug- which inhibits white blood cells (WBCs); the toxic shock ars, amino acids, and nucleotides, which then polymerize to of S. aureus; toxin of Coryne- form larger macromolecules. Macromolecules include lipid, bacterium diphtheriae; and theta toxin, a necrotizing toxin lipopolysaccharide, glycogen, murein, protein, DNA, and produced by perfringens. Other exotoxins are RNA. The final step is assembly whereby inclusions; cyto- extracellular enzymes such as DNAse, which inhibits the sol; polyribosomes; pili; flagella; and the cellular envelope, host deoxyribonucleic acid (DNA); , which con- nucleoid, and capsule are formed from the macromolecules. verts fibrinogen into fibrin; , which lyse red Energy is required for all bacterial metabolism and oc- blood cells (RBCs); proteases, which break down protein; curs through the breakdown of chemical substrates. The and fibrinolysins, which lyse fibrin clots. Endotoxins are substrate is oxidized and donates electrons to an electron- usually associated with gram-negative bacteria and con- acceptor molecule, which is reduced. Carrier molecules sist of lipopolysaccharide (LPS), the component of the cell such as nicotinamide-adenine-dinucleotide (NAD+) and wall. Endotoxins are released at cell lysis or and are nicotinamide-adenine-dinucleotide-phosphate (NADP+) capable of inducing fever in the host. mediate this process. The energy released is transferred to phosphate-containing compounds such as ATP, where high-energy phosphate bonds are found. This provides the The Role of Clinical Microbiology needed energy for the biochemical reactions for the cell. Fermentative processes within the cell do not require Clinical microbiology begins when the patient presents oxygen; typical end products produced from fermenta- signs of infection to the physician. An initial diagnosis tion include , acids, hydrogen, and carbon dioxide. is made, and the physician then orders diagnostic medical

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and laboratory procedures. A direct stain, a culture, and through the blood to other organs. The pathogen is met an antibiotic susceptibility test are typical tests that may by the host’s inflammatory and , which involve the microbiology laboratory. The appropriate lab- have been stimulated by the microbe and its products. oratory specimens are collected, labeled, and sent to the Depending on the immune status of the host and severity laboratory with a requisition or laboratory order form. The of the infection, there may be various outcomes. The host laboratory performs direct stains, plates the specimen on ap- may destroy the pathogen, or the pathogen may destroy propriate culture media, and incubates the plates at the suit- the host; in some cases, the pathogen may remain latent able temperature and atmosphere. The plates are examined within the host. If the host recovers, the signs of infection and interpreted for the presence of pathogens, most often at decline, and the host eventually enters the convalescent 24 and 48 hours. Subcultures are performed as needed, and phase of infection. Consequences of infection may re- any biochemical, serological, molecular, antibiotic suscepti- main as a result of toxins or other compounds produced bility, and automated procedures are performed. These tests by the microbe. If the host continues to deteriorate, the are interpreted and the organisms are identified. The final symptoms may worsen, eventually resulting in perma- report of the identification and antibiotic susceptibility tests nent damage or even death. is sent to the physician. The physician interprets the report All pathogens in a specimen must be identified and and treats the patient appropriately. reported to the physician. A pathogen is a microorganism, Early diagnosis is associated with early treatment and including bacteria, , fungi, and parasites, that is ca- a better for the patient. Testing must be per- pable of causing infectious disease. The identification of formed in a timely, yet accurate, manner. Often, a pre- pathogens may be difficult because many microorganisms sumptive identification can be sent to the physician so are present in the normal microbial flora. Normal flora re- that antibiotic can be initiated. A final, definitive fers to those microorganisms normally residing in a partic- identification is then sent to update the report. ular body site or niche that do not generally cause infection. Normal flora may also be known as indigenous or resident flora or microbiota; these bacteria may perform important The Infectious Process functions and compete with pathogenic microorganisms. An infection is the entrance and multiplication of a mi- Normal Flora croorganism in or on a host. The microorganism may enter the host through many routes including the respiratory A limited number of organisms can be categorized as nor- tract, , or breaks in the . The mi- mal flora. The slightly acidic pH of the skin (5.5 to 6.0) re- croorganism next establishes itself and multiplies. Infec- sults from the presence of acids produced by a number of tions can spread directly through tissues or the lymphatic bacteria. For example, Propionibacterium acnes produces system into the blood. Infectious disease refers to an large amounts of propionic acid. Other normal skin flora infection with functional and structural harm to the host include Staphylococcus epidermidis, viridans streptococ- that usually is accompanied by . cus, and enterococcus. In addition, a number of contami- In this process, the pathogen first must come in con- nating organisms may be found transiently on the skin, tact with and colonize the host. Colonization refers to the such as intestinal tract and soil contaminants. state when the microbe has established itself in a par- In the mouth and oral cavity, the major normal flora ticular niche or body site. This generally corresponds is the viridans streptococcus, a collection of streptococcal with the , and the host has no signs of species exhibiting alpha or greening of sheep infection. Next, the pathogen breaks the host’s protec- blood . In addition, S. epidermidis, nonpathogenic tive barrier and multiplies. This coincides with the first Neisseria species, , lactobacilli, and signs of clinical infection, known as the prodromal stage, diphtheroids may be present. Members of the anaerobic during which time the host is particularly infective. The normal flora in this area include Actinomyces, Veillonella, clinical symptoms peak during the clinical sage of in- and Bacteroides species. fection. At this time the pathogen continues to multiply, The nasopharynx may serve as a site for asympto- attacking deeper tissues and eventually disseminating matic carriage of several microorganisms. Asymptomatic

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carriers maintain a reservoir for the microorganism but do Congenital contact may occur across the not have an infectious disease. The carrier state may be tran- or as the baby passes through the vaginal canal during sient or permanent, and these individuals may serve as an delivery. and may be acquired dur- infectious source to transmit the pathogen to others. Bacteria ing , whereas and Neis- that may be carried asymptomatically in the nasopharynx seria gonorrhoeae are examples of bacteria that may be include and Neisseria meningitidis. transmitted to the during delivery. The stomach and upper small intestine are usually Sexual contact may be the route of infection for sev- sterile, containing less than 1,000 organisms per milliliter. eral sexually transmittable , including gonorrhea Organisms entering the stomach are usually killed by the (N. gonorrhoeae); syphilis (); chla- hydrochloric acid and resulting low pH of the stomach, as mydia ( trachomatis); acquired immunodefi- well as by gastric enzymes. Other organisms are passed to ciency syndrome, or AIDS (human the small intestine, where they may be destroyed by bile virus, or HIV); and herpes ( virus). and pancreatic enzymes. When the gastric pH increases Hand-to-hand is the mode of direct over 5.0, colonization from bacteria of oral, nasopharyn- contact seen with the spread of the from geal, or colon origin may occur. . This route is also involved in the transmission The colon is heavily colonized and serves as a reser- of various GI infections when the hands are not properly voir for infection for numerous body sites, including the washed and are fecally contaminated. urinary tract and peritoneal cavity. Major components of Infectious respiratory secretion or droplet infection the normal bowel flora include Bacteroides, , serves as a route for several respiratory viruses as well Clostridium, Eubacterium, coliforms such as Escherichia as for bacterial pathogens including Streptococcus pyo- coli, aerobic and anaerobic streptococci, and yeast. genes (the agent in streptococcal pharyngitis throat) and The normal flora of the distal urethra in both males N. meningitidis. Infectious secretions include coughing, and females may contain diphtheroids, alpha and non- sneezing, kissing, and nasal drainage. Respiratory secre- hemolytic streptococci, Peptococcus, S. epidermidis, and tions can become dried on clothing, bedding, or floors Bacteroides. and converted to dust, which may serve as a route of in- Those sites considered to be sterile in the body, con- direct transmission. taining no normal flora, include the blood, cerebrospi- Indirect routes of infection include , nal fluid, and urinary bladder. Normal flora can become of contaminated food and water, airborne routes, and ani- pathogenic if they are moved to another site. Thus normal mal or vectors. flora E. coli of the colon is an important cause of urinary Fomites are inanimate objects such as uten- tract infection (UTI) once it enters the bladder or kidneys. sils, drinking devices (water bottles and cups), hospital Likewise, although viridans streptococcus is considered instruments, clothing, money, doorknobs, and . to be normal flora of the oral cavity, the organism is a Sometimes, these inanimate objects may serve as a route significant cause of subacute bacterial when of nosocomial (hospital-acquired) infection. Fomites may established in the heart. also be referred to as vehicles of infection, which refers to nonliving objects that have been contaminted with the infectious agent. Modes of Transmission—Routes Water may be contaminated as a result of improper of Infection sanitary measures or after it has been treated. Microor- Infectious disease can be transmitted by several routes, ganisms that may be associated with contaminated water which can be categorized as direct or indirect modes of include Shigella, , enteropathogenic E. coli, and transmission. The etiologic agent is the microorganism that A virus (HAV). Improperly prepared, processed, has caused the infection. The reservoir of infection refers preserved, or stored food may become contaminated with to the origin of the etiologic agent or its location such as various microorganisms, including Salmonella, , contaminated food, another human, or an infected animal. E. coli O157:H7, and S. aureus. Milk and milk products may Types of direct transmission include congenital contact, be contaminated through improper or lack of pasteuriza- sexual contact, hand-to-hand contact, and droplet infection. tion, while undercooked may serve as sources of

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contamination. Ingestion of the microorganisms or of pre- disease prevalence describes the percentage of infected formed toxins plays a role in infections acquired through individuals in a given population at a given time. An contaminated foods. endemic is when a disease consistently occurs at a con- Infections may be incidentally transmitted to stant rate in a given location. An epidemic occurs when through infected animals or insect or arthropod vectors. there are larger numbers of cases of a disease in a given , (Pasteurella multocida), and tulare- location. are that occur around the mia (Francisella tularensis) are examples of infections that world. Outbreaks generally refer to instances where there can be acquired through the bite or scratch of an infected is a disproportionately larger number of infected individu- animal. Arthropod vectors such as , , lice, , als in a fairly short amount of time. and mosquitoes may transmit microorganisms from an infected animal to a human host. () is transmitted by mosquitoes, whereas (Borrelia True versus Opportunistic Pathogens burgdorferi) is transmitted by the bite of an infected . A true pathogen has the ability to cause an infection under Airborne routes of infection include the inhalation all conditions in all types of individuals. By contrast, an op- of infectious particles that may be suspended in the air. portunistic pathogen refers to an organism that is patho- Infectious aerosols can be formed in the laboratory that genic only under particular, favorable conditions. Under can serve as a route for laboratory-acquired infection. My- other conditions, opportunisms are nonpathogenic and cobacterium , the agent in tuberculosis, can generally harmless and are often members of the human remain suspended in droplets that serve as infectious aero- normal flora. Opportunistic infections are infections that sols. Dimorphic fungi, which cause systemic infections occur when there are preexisting conditions that increase such as Coccidioides immitis, can cause airborne infections the susceptibility of the host to infection. Generally, oppor- through the inhalation of infectious spores. tunistic pathogens do not cause disease in individuals with Figure 1-3 summarizes routes of infection for a normal immune system. These infections are increasing microorganisms. as a result of a number of factors including the widespread Once established in a particular area, the microorgan- use of broad-spectrum antibiotics that can alter the normal ism may multiply and spread from its original site of entry flora, the increased use of immunosuppressive to pre- to contiguous or disseminate through the blood to vent organ transplant rejection, the use of chemotherapeutic other, distant sites. Microorganisms spread and multiply agents to treat , and the increased and prolonged use of when host defense mechanisms are overcome. urethral catheters. Thus an opportunistic pathogen is one The incidence of a disease refers to the number of that attacks an already debilitated host but usually presents those individuals infected in a population, whereas the no danger to an individual with an intact immune system.

DISEASE TRANSMISSION

can occur by (A) Direct contact (B) Indirect contact

involves person-to-person by involving

Horizontal Vertical Fomites Vehicle transmission transmission transmission transmission through from via which can involve a Human or animal Sexual Respiratory Mother to Mother to contact intercourse droplets which can newborn harbor with diseases with diseases Foods Water Aerosols Mechanical Biological such as that can carry such as vector vector Gonorrhea Pertussis Neonatal carrying disease Syphilis Common cold Congenital carrying disease agents for Chlamydia Inuenza agents for Rabies syphilis (gonorrhea, Ringworm AIDS AIDS chlamydia) Gastroenteritis TB Lyme disease Leptospirosis Trachoma West Nile Athlete’s Amoebiasis Valley fever Shigellosis Malaria

Figure 1-3 Routes of infection

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In immunosuppressive conditions the host immune membranes of the . Those bacteria that system is unable to effectively battle those microorgan- are phagocytized are transported through the lymphatic isms considered to be normal flora for the general popula- system and eventually removed from the body. tion. Other individuals at for opportunistic pathogens The respiratory tract is a common portal of entry for include dialysis , those with HIV-AIDS, several microorganisms, including Streptococcus pneu- victims, diabetics, and any individual who has chronic moniae, M. tuberculosis, , and medical problems or is undergoing invasive medical tech- several respiratory viruses. Those microorganisms pos- niques. Those individuals with implants in- sessing pili, appendages for attachment, are able to attach cluding heart valves, prosthetic devices, and indwelling to the mucous membranes. intravenous catheters; alcoholics; and intravenous Saliva in the oral cavity possesses various hydrolytic users are also at increased risk of opportunistic pathogens. enzymes that can break down bacteria. The low pH of the By contrast, a true pathogen has the ability to infect stomach and the presence of gastric enzymes result in a those individuals with a healthy immune system as well limited amount of microorganisms that can sustain in as those with an immunosuppressed state. the stomach. The rapid peristalsis in the small intestine, together with the presence of enzymes and bile, keeps the Host Defense Mechanisms microbial population in this site at very low levels. The has a large amount of normal flora that Host defense mechanisms include innate, or natural, im- competes with pathogens, thus decreasing the chance for munity; acquired immunity; and phagocytosis. Immu- establishment of infection. The anaerobic normal flora of nity refers to host properties that confer resistance of the the large intestine produces fatty acids, while facultative host to foreign substances. It is the sum of all mechanisms anaerobes produce bacteriocins, which also hinder the used by the body as protection against environmental multiplication of pathogens. agents that are not normally present in the body. The constant action and low pH of urine prevent pathogens from establishing in the urinary system. Females, Innate or Natural Immunity who have shorter urethras, are more prone to UTIs than are Innate, or natural, immunity is inborn, mainly genetically males, who have longer urethras. Bacteria have easier access determined, and nonspecific. It is not acquired through to the female bladder from the GI and vaginal tracts. previous contact with an infectious agent. The skin acts as Lactobacilli, a major normal flora in the female vaginal a protective barrier, and a limited number of microorgan- tract, produce large amounts of , which results in isms can penetrate the intact skin barrier. Many microor- a low pH. This acidic environment decreases the amount of ganisms, however, can enter the body through breaks in other microorganisms that can cause infection in this area. the skin resulting from bites, , cuts, or needles. In The eyes are protected through the constant flushing addition, microorganisms can enter through the sweat and provided by as well as the presence of lysozyme in tears. fat glands. The normal flora bacteria of the skin produce Other elements of innate immunity include fever, in- free fatty acids from oil, which will produce low pH on terferon, phagocytosis, and various proteins. These the skin and thus prevent the establishment of infection. components are described with the inflammatory response. Mucus covers the surfaces of the respiratory tract. Natural immunity can be affected by a number of fac- The mucus can trap bacteria and, with the assistance of tors. For example, very young and very old persons are cilia, which are also present in the nasal cavity, can trap more susceptible to infection than are other age groups. many microorganisms. The bacteria are either swallowed, Hormonal changes such as those occurring during preg- entering the stomach where they are destroyed by the nancy, mellitus, and Addison’s and Cushing’s acidic environment, or removed through external open- diseases can alter the metabolism and immune response, ings such as the nose and mouth by sneezing or coughing. which results in increased susceptibility to infection. The Lysozyme, an that lyses bacterial cell walls, is also use of broad-spectrum antibiotics, which alter the host’s present in respiratory secretions. Some types of immu- normal flora, can also promote the growth of pathogens. noglobulin A (IgA) have a secretory component that can The normal flora is no longer present in large amounts to prevent attachment of the microorganisms to the mucous compete with the pathogenic organisms. Such infections

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Box 1-3 Hallmark Signs of Sign Latin Derivation Description Redness Rubor Dilation of small blood vessels in area of Heat Calor Increased blood flow through the inflamed area in peripheral parts of body; fever as a result of chemical mediators of inflammation Swelling Edema Fluid accumulates outside of blood vessels Dolar As a result of edema because tissues are inflamed; effects of serotonin, bradykinin, and prostaglandins

are termed secondary infections or be- are formed in the bone marrow and enter cause the infection results from the use of an antibiotic. the blood and tissue. Monocytes, also formed in the bone marrow, are known as macrophages once the cells have en- Inflammatory Response tered the tissue. Macrophages are present in tissues but are The inflammatory response can be activated by trauma also fixed to the blood vessels of the , , and lymph or tissue injury. Inflammation is the total of the changes nodes. Macrophages can become activated by microorgan- occurring in tissue factors upon injury. There are four isms, endotoxins, and antigen–antibody complexes. Once hallmark signs of inflammation: redness, heat, swelling, activated, macrophages produce an increased number of lyso- and pain. These are described in Box 1-3. Hemodynamic somes that produce interleukin-1. Interleukin-1 is associated changes, such as increased vascular permeability, dilation with the stimulation of fever and activation of phagocytosis. of arterioles and capillaries, and increased blood flow to Inflammation caused by microorganisms may be ini- the injured area, occur. Plasma proteins, such as comple- tiated through activation of the or ment, interferon, and , are also released. Edema the blood coagulation cascade. These systems initiate the may occur as a result of , while there is also an release of several chemical mediators of inflammation, influx of red blood cells to the area. which are summarized in Table 1-2. Phagocytic white blood cells, which include neutro- phils (PMNs) and macrophages, have four functions: mi- Acquired Immunity gration, chemotaxis, ingestion, and killing. These cells Acquired immunity can be either passive or active. Pas- may adhere to the vascular endothelium or migrate from sively acquired immunity refers to the temporary resis- the blood to the affected tissues in a process known as dia- tance to infectious agents by administration of antibodies pedesis. Chemotaxis refers to the attraction of the phago- preformed in another host. Examples include gamma glob- cytes to the affected area by the microorganisms or its ulin and to Clostridium . Passive products from the blood to the injured site. Neutrophils acquisition is also seen in the fetus, who acquires antibod- and monocytes are attracted by bacterial products such as ies from the mother’s blood during pregnancy. These anti- endotoxins and enzymes. Attachment initiates the phago- bodies remain active in the newborn until 4 to 6 months of cytic process. The microorganism must be coated with age. Newborns may also acquire immunity passively from such as IgG or complement units for effective their mothers through antibodies carried in breast milk. phagocytosis. Those microbes that have a capsule are re- Actively acquired immunity is a state of resistance sistant to the effects of phagocytosis. The next step is in- built in an individual following contact with foreign an- gestion, which involves the formation of a phagosome that tigens such as microorganisms. The immunity may result undergoes a . The respiratory burst results from clinical or , through in the release of superoxide anion and peroxide, both of of live or inactivated microorganisms, their antigens, or which are toxic to microorganisms. Lysosomes within the nucleic acids, or through the absorption of bacterial prod- cell combine with phagosomes to form phagolysosomes, ucts such as toxins or . In active immunity the host which eventually release hydrolytic enzymes. actively produces antibody in reaction to a foreign antigen.

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Table 1-2 Chemical Mediators of Inflammation

Compound Effect Dilates blood vessels, increases permeability of blood vessels Kinins Increase vascular permeability and initiate or enhance release of other mediators from white blood cells; derived in the clotting cascade from activation of the precursor kininogen by kallikrein Leukotrienes white mobility and metabolism Prostaglandins Formed in the hypothalamus in the thermoregulation center; induce fever C-reactive protein, serum A, Liver proteins playing a role in the response antitrypsin Interleukin-1 Stimulates cells of immune response, increases fever by interaction with prostaglandins, increases adhesion of neutrophils to endothelium, promotes proliferation Interleukin-2 Causes proliferation of activated T and B cells Cytokines Stimulate white blood cells, promoting their growth and differentiation Gamma interferon Promotes growth of T and B cells

Acquired immunity involves contact with a foreign immunoglobulins: IgG, IgM, IgA, IgD, and IgE. Each im- agent or antigen. This process is known as , munoglobulin class has unique biological abilities. and it initiates a series of reactions that lead to the activa- IgG is able to cross the placenta and is most active at tion of lymphocytes and the synthesis of specialized serum 37°C. It is the major immunoglobulin in normal serum. proteins known as antibodies. Thus antigens are substances IgG is primarily involved in the secondary, or anamnestic, foreign to the body that initiate the production of antibodies. immune response. Three major cell types are involved in the immune re- IgM is involved mainly in the primary immune sponse: T lymphocytes, B lymphocytes, and macrophages. response and appears in the serum following initial expo- T and B lymphocytes arise from a common lymphoid sure to an antigen. IgM has its greatest activity at 20°C to precursor cell but mature in different areas of the body. 25°C. T cells mature in the thymus, while B cells, named for the IgA is unique in that it is the only immunoglobulin bursa of Fabricius (the area in the chicken where the cells found in secretions such as tears, saliva, and of the respira- were first isolated), mature in the bone marrow. Although tory tract. Important characteristics of the immunoglobu- B and T cells have different functions, both play a role in lins are summarized in Table 1-3, and a summary of their the recognition of antigen and reaction to it. The third primary functions is found in Box 1-4. type of cells, macrophages, are phagocytic cells that ingest, The complement system is another important aspect process, and present antigens to T cells. of humoral immunity. The complement system involves Humoral immunity is mediated by serum anti- more than 20 serum proteins and enzymes that can be acti- bodies secreted by B cells. These antibodies are known vated by immune (antigen–antibody) complexes or nonim- as immunoglobulins. There are five major classes of mune routes such as lipopolysaccharide. If the complement

Table 1-3 Summary of Human Immunoglobulins

IgG IgM IgA IgD IgE Percentage in normal serum 75%–85% 5%–10% 5%–15% 0.001% 0.0003% Molecular weight (approximate) 150,000 900,000 160,000 (serum), 400,000 (secretory) 180,000 190,000 Heavy chain class gamma (γ) mu (μ) alpha (α) delta (δ) epsilon (ε)

Heavy chain subclasses γ1γ2γ3γ4 μ1μ2 α1α2 None None Cross placenta Yes No No No No Activate complement Yes for IgG-1, -2, -3 Yes No No No

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Box 1-4 Primary Functions of Human Immunoglobulins IgG: for newborns, neutralization of viruses and exotoxins; responds best to protein antigens, mainly involved in secondary (anamnestic) immune response IgM: Endotoxin neutralization, bacterial agglutination, complement-mediated bacteriolysis, strong opsonization abil- ity; responds best to polysaccharide antigens, mainly involved in primary immune response IgA: Prevention of bacterial and viral invasion of mucous membranes through interference with adherence of micro- organism to site; found in tears, milk, saliva, and respiratory and GI secretions IgD: Little is known; may serve as a B cell receptor or play a role in autoallergic diseases IgE: Major role in allergic response; found on surface of mast cells

cascade is activated, the target cell may be lysed, or phago- include a high-grade, spiking fever; chills; vasodilation cytic cells may be stimulated. with flushing; and an increased pulse rate. Chronic or subacute infections may be accompanied by the following Cell-Mediated Immunity systemic signs: intermittent, low-grade fever; ; Cell-mediated immunity (CMI) involves the T lympho- and . Local signs of infection include pain, heat, cytes, which circulate to the antigen to perform their func- redness, and swelling. The hallmark signs of inflamma- tion. There are several populations of T cells including: tion are found in Box 1-3. 77 T helper (inducer) cells In the laboratory, specific procedures are used to diag- 77 Enhance proliferation and differentiation of B cells nose infection. These include the leukocyte count, which and precursors to cytotoxic T cells is elevated for most infectious processes, and the differ- 77 Increase ability of macrophages to ingest and de- ential count, which enables the clinician stroy pathogens to determine the type of infection. In general, but not al- 77 Enhance the production of antibody by B cells ways, bacterial infections are associated with an elevated 77 Release lymphokines, including interleukin-1 (IL-1) white blood cell count and an increased percentage of neu- and interleukin-2 (IL-2) and B cell–stimulating fac- trophils. By contrast, lymphocytes are the predominant tor, which helps to activate B cells WBC in most viral infections. The erythrocyte sedimen- 77 Cytotoxic T cells—destroy targets on direct contact tation rate (ESR) is a nonspecific indicator of inflamma- through the recognition and destruction of antigen- tion and is frequently increased in infectious disease and bearing cells numerous other inflammatory states. C-reactive protein 77 T suppressor cells—suppress or regulate the response is another plasma protein that is present during infectious of T and B cells disease. Finally, the presence of type-specific antibodies 77 Null cells (natural killer [NK] and killer [K] cells)— in a patient’s serum can be used to identify the presence of kill tumor or viral-infected cells, although not with a particular pathogen. On exposure to a bacterial or viral the specificity of cytotoxic T cells pathogen, the patient produces antibodies against the anti- gens of the organism. The antibodies then can be detected Although presented as separate functions, the humoral through use of antigenic markers. and cell-mediated immune systems interact in the im- Radiographic signs of infectious disease that a clini- mune response. cian would note include pulmonary infiltrates, gas and swelling in the tissues, and the accumulation of fluid in Signs of Infection a body cavity. The need for clinical microbiology begins with a patient Gastrointestinal signs such as nausea, vomiting, and who is exhibiting one or more signs of infection. Some , as well as various neuromuscular and cardiopul- common general or systemic signs of acute infection monary signs, are also noted by the clinician.

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Nosocomial and Care–Associated Nosocomial urinary tract infections may be the result Infections of catheterization or the presence of indwelling catheters or other urological techniques such as . The organ- A nosocomial, or –associated, infection is ism is frequently of endogenous origin, as with E. coli, which acquired in a hospital or other health care setting. The is a member of the normal flora of the large intestine. Ex- organism is not present and not incubating in the pa- ogenous sources include the contaminated hands of health tient on entry or admission into the health care facility. care providers or contaminated equipment or solutions. A community-acquired infection is present or incubat- Nosocomial surgical infections usually involve ing at the time of admission into the health care facil- S. aureus, Enterococcus, or gram-negative bacilli. These ity. Community-acquired infections also are those that infections may be endogenous or exogenous. are acquired within an individual’s community such as Nosocomial may result from aspiration her or his school, workplace, or athletic or social setting. of the organisms from the stomach or upper respiratory As with other infections, nosocomial and community- tract. The airways or stomach may become colonized with acquired infections can be categorized as endogenous or bacteria including S. aureus, P. aeruginosa, and Klebsiella exogenous. Endogenous infections result from organisms pneumoniae. Respiratory care procedures, such as endo- that are a part of the patient’s normal flora, whereas ex- tracheal suctioning and inhalation therapy, also present a ogenous infections result from organisms from external greater risk for nosocomial pneumonia. A high mortality sources. These sources may include contaminated medi- rate is associated with nosocomial infections of the lower cal instruments or equipment or inanimate objects in the respiratory tract. health care setting or from contact with health care per- Bacteremia may result from the patient’s own flora as sonnel. Individuals, including health care providers, may well as that of the health care provider. In addition, intra- be colonized with an organism. Colonization is defined venous devices or solutions may be contaminated. as the presence and multiplication of a microorganism in Host factors that lead to increased susceptibility to a host, with no clinical signs of infection. Such individu- nosocomial infections include a compromised immune als may serve as a reservoir of infection and transmit the system, underlying medical disease or diseases, age, organism to susceptible individuals. trauma, , poor nutritional status, anatomical abnor- The most common types of nosocomial infections are malities, use of medical instrumentation, and diagnostic urinary tract infections (35% to 40%), surgical wound in- procedures. fections (20%), lower respiratory tract infections (15%), and Some, but not all, nosocomial infections can be pre- bacteremia (5%–10%). These percentages may vary with each vented. The universal use of gloves and practice of aseptic health care setting. Those bacteria most often associated with techniques, including thorough , can de- nosocomial infections include S. aureus, E. coli, Enterococ- crease the incidence of nosocomial infections. The rou- cus, and . Many nosocomial patho- tine disinfection of inanimate surfaces and prevention of gens are resistant to multiple agents. aerosols are also important factors.

Review Questions Matching b. Infection in an immunocompromised host that Match the following terms with the correct definition: does not cause infection in an immunocompetent 1. Infection individual 2. Infectious disease c. Infection acquired in a health care setting 3. d. Presence and multiplication of a microorganism in a 4. Nosocomial infection host with no clinical signs of infection 5. Colonization e. Entrance and multiplication of a microorganism in a a. Condition associated with functional and structural host harm to the host, accompanied by signs and symptoms

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Multiple Choice c. T lymphocytes d. B lymphocytes 6. All the following sites contain normal flora except: 14. Which of the following cells play a major role in cell- a. Oral cavity mediated immunity? b. Skin a. Macrophages c. Colon b. Neutrophils d. Cerebrospinal fluid c. T lymphocytes 7. Which of the following is not classified as a direct route d. B lymphocytes of infection? 15. The immunoglobulin found in the highest a. Ingestion of contaminated food or water concentration in normal serum is: b. Sexual contact a. IgA c. Hand-to-hand contact b. IgD d. Congenital contact c. IgE 8. Droplet infection through contact with infectious d. IgG respiratory secretions may be described as: e. IgM a. Inhalation of infectious aerosols during laboratory 16. Which of the following immunoglobulins is involved procedures mainly in the primary immune response? b. Transmission of rhinovirus through failing to wash a. IgA hands b. IgD c. Spread of respiratory viruses and Streptococcus c. IgE pyogenes through coughing or sneezing d. IgG d. Inhalation of bacteria or viruses that have dried on e. IgM bedding or clothing 17. Gram-negative bacteria contain , 9. Which of the following organisms are typically spread which are not found in gram-positive bacteria. through the ingestion of contaminated food or water? a. Capsules a. Neisseria meningitidis and S. pyogenes b. Periplasmic space and outer membrane b. Salmonella and Shigella c. Teichoic acids c. and Treponema pallidum d. Cross-linked peptidoglycan d. Plasmodium and Borrelia 18. Which of the following is true for bacterial cells? 10. Which of the following organisms are spread through a. The DNA is contained within a nuclear membrane. arthropod vectors? b. Their mitochondria, Golgi bodies, and endoplasmic a. N. meningitidis and S. pyogenes reticulum are present in the cytoplasm. b. Salmonella and Shigella c. The DNA is found in the nucleoid. c. Herpes simplex virus and T. pallidum d. The ribosomes are 80S. d. Plasmodium and Borrelia 19. The are important for motlity of the 11. Innate, or natural, immunity involves which of the bacterial cell. following mechanisms? a. pili a. Mucus and cilia in the respiratory tract that help to b. capsules trap and clear microorganisms c. flagella b. Humoral immunity d. LPS c. Cell-mediated immunity 20. Phenotypic properties used to classify bacteria include d. Immunity resulting from all of the following except: 12. The movement of neutrophils and monocytes from the a. DNA relatedness blood to injured tissue is known as: b. a. Diapedesis c. Biochemical properties b. Chemotaxis d. Antibiotic resistance patterns c. Ingestion d. Hematopoiesis 13. Antibody-producing white blood cells are: a. Macrophages b. Neutrophils

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Bibliography Bower, S., & Rosenthal, K. S. (2006). Bacterial cell walls: The Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2009). Mecha- armor, artillery and Achilles heel. Infectious Diseases in nisms of bacterial . In , Clinical Practice, 15, 309–316. 6th ed. (pp. 179–187). Philadelphia, PA: Mosby. Casadevall, A., & Pirofski, L. (2001). Host-pathogen interac- Parham, P. (2009). Antibody structure and the generation of tions: The attributes of virulence. The Journal of Infectious B-cell diversity. In The immune system, 3rd ed (pp. 1–31). Diseases, 184, 337–344. New York, NY: Garland Taylor & Francis Group. Diekema, D. J., & Pfaller, M. A. (2007). Infection control epi- Parham, P. (2009). Elements of the immune system and their demiology and clinical microbiology. In P. R. Murray, E. roles in defense. In The immune system, 3rd ed (pp. 1–31). J. Baron, J. H. Jorgensen, M. L. Landry, & M. A. Pfaller New York, NY: Garland Science Taylor & Francis Group. (Eds.), Manual of clinical microbiology, 9th ed. (pp. 118– Schaechter, M. (2012). Biology of infectious diseases. In N. C. 128). Washington, DC: American Society for Microbiology. Engleberg, T. Dermody, & V. DiRita (Eds.), Schaechter’s Finlay, B. B., & Falkow, S. (1989). Common themes in microbial mechanisms of microbial disease, 5th ed. (pp. 18–37). Phila- pathogenicity. Microbiology and Molecular Biology Reviews, delphia, PA: Lippincott Williams & Wilkins. 53, 210. Soule, B. M., & LaRocco, M. T. (1993). Nosocomial infections: Forbes, B. A., Sahm, D. F., & Weissfeld, A. S. (2007). Bacterial An overview. In B. J. Howard, J. F. Keiser, A. S. Weissfeld, genetics, metabolism and structure. In Bailey and Scott’s & F. Thomas (Eds.), Clinical and pathogenic microbiology, , 12th ed. St. Louis, MO: Mosby. 2nd ed. (pp. 83–99). St. Louis, MO: Mosby. Klevins, R. M., Edwards, J. R., Richards Jr., C. L., Horan T. C., Turgeon ML (2003). Antigens and antibodies. In Gaynes, R. P., Pollock, D. A., & Cardo, D. M. (2007). Es- and in laboratory medicine, 3rd ed. (pp. 15–35). St. timating health care-associated infections and in Louis, MO: Mosby. U.S. hospitals, 2002. Reports, 122, 160–166.

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