Test bank For Bailey and Scotts Diagnostic Microbiology 12th Edition by Betty A. Forbes

Front Matter

Dedication

CONTRIBUTORS

FOREWORD

PREFACE

ACKNOWLEDGMENTS

PART I Basic Medical Microbiology

CHAPTER 1 Microbial Taxonomy

CLASSIFICATION

SPECIES

GENUS

NOMENCLATURE

BOX 1-1 Role of Taxonomy in Diagnostic Microbiology and Infectious Diseases

IDENTIFICATION

IDENTIFICATION METHODS

Table 1-1 Identification Criteria and Characteristics for Microbial Classification

ADDITIONAL READING

CHAPTER 2 Bacterial Genetics, Metabolism, and Structure

BACTERIAL GENETICS

NUCLEIC ACID STRUCTURE AND ORGANIZATION

Nucleotide Structure and Sequence

DNA Molecular Structure

Figure 2-1 General overview of bacterial life processes.

Genes and the Genetic Code

Chromosomes

Nonchromosomal Elements of the Genome

Figure 2-2 A, Molecular structure of DNA showing nucleotide structure, phosphodiester bond connecting nucleotides, and complementary pairing of bases (A, adenine; T, thymine; G, guanine; C, cytosine) between antiparallel nucleic acid strands. B, 5 ′ and 3′ antiparallel polarity and helical (“twisted ladder”) configuration of DNA.

Figure 2-3 Molecular structure of nucleic acid bases. Pyrimidines: cytosine, thymine, and uracil. Purines: adenine and guanine.

REPLICATION AND EXPRESSION OF GENETIC INFORMATION

Replication

Figure 2-4 Bacterial DNA replication depicting bidirectional movement of two replication forks from origin of replication. Each parent strand serves as a template for production of a complementary daughter strand and, eventually, two identical chromosomes.

Expression of Genetic Information

Transcription.

Figure 2-5 Overview of gene expression components; transcription for production of mRNA and translation for production of polypeptide (protein).

Translation.

Table 2-1 The Genetic Code as Expressed by Triplet Base Sequences of mRNA*

Regulation and Control of Gene Expression

Figure 2-6 Overview of translation in which mRNA serves as the template for the assembly of amino acids into polypeptides. The three steps include initiation (A), elongation (B and C), and termination (not shown).

Figure 2-7 Transcriptional (i.e., genetic level) control of gene expression. Gene repression is depicted in A and B; induction is shown in C and D.

GENE EXCHANGE AND GENETIC DIVERSITY

Mutation

Genetic Recombination

Figure 2-8 Genetic recombination (A). The mechanisms of gene exchange between bacteria: transformation (B), transduction (C), and conjugational transfer of chromosomal (D) and plasmid (E) DNA.

Gene Exchange

Transformation.

Figure 2-9 Photomicrograph of Escherichia coli sex pilus between donor and recipient cell.

Transduction.

Conjugation.

Figure 2-10 Pathways for bacterial dissemination of plasmids and transposons, together and independently.

BACTERIAL METABOLISM

FUELING

Acquisition of Nutrients

Production of Precursor Metabolites

Energy Production

Figure 2-11 Overview of bacterial metabolism, which includes the processes of fueling, biosynthesis, polymerization, and assembly.

Figure 2-12 Overview diagram of the central metabolic pathways (Embden-Meyerhof-Parnas [EMP], the tricarboxylic acid [TCA] cycle, and the pentose phosphate shunt). Precursor metabolites (see also Figure 2-11) that are produced are highlighted in red; production of energy in the form of ATP (∼P) by substrate-level phosphorylation is highlighted in yellow, and reduced carrier molecules for transport of electrons used in oxidative phosphorylation are highlighted in green.

BIOSYNTHESIS

POLYMERIZATION AND ASSEMBLY

STRUCTURE AND FUNCTION OF THE BACTERIAL CELL

EUKARYOTIC AND PROKARYOTIC CELLS

BACTERIAL MORPHOLOGY

Figure 2-13 General structures of the gram-positive and gram-negative bacterial cell envelopes. The outer membrane and periplasmic space are only present in the gram-negative envelope. The murein layer is substantially more prominent in gram-positive envelopes.

BACTERIAL CELL COMPONENTS

Cell Envelope

Outer Membrane.

Cell Wall (Murein Layer).

Figure 2-14 Peptidoglycan sheet (A) and subunit (B) structure. Multiple peptidoglycan layers compose the murein structure, and different layers are extensively crosslinked by peptide bridges (NAG,N-acetylglucosamine; NAM, N-acetylmuramic acid). Note that amino acid chains only derive from NAM.

Periplasmic Space.

Cytoplasmic (Inner) Membrane.

Cellular Appendages.

Cell Interior

ADDITIONAL READING

CHAPTER 3 Host-Microorganism Interactions

THE ENCOUNTER BETWEEN HOST AND MICROORGANISM

THE HUMAN HOST’S PERSPECTIVE

Microbial Reservoirs and Transmission

Figure 3-1 General stages of microbial-host interaction.

Humans as Microbial Reservoirs

Animals as Microbial Reservoirs

Figure 3-2 Summary of microbial reservoirs and modes of transmission to humans.

Insects as Vectors

The Environment as a Microbial Reservoir

THE MICROORGANISM’S PERSPECTIVE

MICROORGANISM COLONIZATION OF HOST SURFACES

THE HOST’S PERSPECTIVE

Skin and Skin Structures

Figure 3-3 Skin and skin structures.

BOX 3-1 Protective Characteristics of the Skin and Skin Structures

Mucous Membranes

Figure 3-4 General features of mucous membranes highlighting protective features such as ciliated cells, mucus production, tight intercellular junctions, and cell sloughing.

BOX 3-2 Protective Characteristics of Mucous Membranes

General Protective Characteristics.

Specific Protective Characteristics

Figure 3-5 Protective characteristics associated with the mucosal linings of different internal body surfaces.

THE MICROORGANISM’S PERSPECTIVE

Microbial Colonization

BOX 3-3 Microbial Activities Contributing to Colonization of Host Surfaces

BOX 3-4 Factors Contributing to Disruption of Skin and Mucosal Surface

MICROORGANISM ENTRY, INVASION, AND DISSEMINATION

THE HOST’S PERSPECTIVE

Disruption Of Surface Barriers

Responses to Microbial Invasion of Deeper Tissues

Nonspecific Responses

Figure 3-6 Overview of phagocyte activity and possible outcomes of phagocyte-bacterial interactions.

Phagocytes

BOX 3-5 Components of Inflammation

Inflammation

Figure 3-7 Overview of the components, signs, and functions of inflammation.

SPECIFIC RESPONSES—THE IMMUNE SYSTEM

Figure 3-8 General structure of the IgG class antibody molecule.

Components of the Immune System

BOX 3-6 Cells of the Immune System

Two Arms of the Immune System.

Figure 3-9 Overview of B-cell activation that is central to antibody-mediated immunity.

Figure 3-10 Overview of T-cell activation that is central to cell-mediated immunity.

THE MICROORGANISM’S PERSPECTIVE

Colonization and Infection

Pathogens and Virulence

Microbial Virulence Factors

Attachment.

Invasion.

BOX 3-7 Microbial Strategies for Surviving Inflammation

Survival Against Inflammation.

Survival Against the Immune System.

BOX 3-8 Microbial Strategies for Surviving the Immune System

Microbial Toxins.

BOX 3-9 Summary of Bacterial Toxins

Genetics of Virulence: Pathogenicity Islands

Figure 3-11 Possible outcomes of infections and infectious diseases.

OUTCOME AND PREVENTION OF INFECTIOUS DISEASES

OUTCOME OF INFECTIOUS DISEASES

Figure 3-12 Host-microorganism interactions and stages of infection or disease.

BOX 3-10 Signs and Symptoms of Infection and Infectious Diseases

PREVENTION OF INFECTIOUS DISEASES

BOX 3-11 Strategies for Prevention of Infectious Diseases

Immunization

Epidemiology

BOX 3-12 Definitions of Selected Epidemiologic Terms

ADDITIONAL READING

PART II GENERAL PRINCIPLES IN CLINICAL MICROBIOLOGY

SECTION 1 SAFETY AND SPECIMEN MANAGEMENT

CHAPTER 4 Laboratory Safety

STERILIZATION AND DISINFECTION

Figure 4-1 Gravity displacement type autoclave.A, Typical Eagle Century Series sterilizer for laboratory applications. B, Typical Eagle 3000 sterilizer piping diagram. The arrows show the entry of steam into the chamber and the displacement of air.

METHODS OF DISINFECTION

Physical Methods of Disinfection

Chemical Methods of Disinfection

CHEMICAL SAFETY

Figure 4-2 National Fire Protection Association diamond stating chemical hazard. This information can be customized as shown here for isopropyl alcohol by applying the appropriate self-adhesive polyester numbers to the corresponding color-coded hazard area.

FIRE SAFETY

Figure 4-3 Fume hood. A, Model ChemGARD. B, Schematics. Arrows indicate air flow through cabinet to outside vent.

ELECTRICAL SAFETY

HANDLING OF COMPRESSED GASES

BIOSAFETY

Figure 4-4 Fire evacuation plan. Arrows indicate quickest fire exits.

Figure 4-5 A, Gas cylinders chained to the wall. B, Gas cylinder chained to dolly during transportation.

EXPOSURE CONTROL PLAN

EMPLOYEE EDUCATION AND ORIENTATION

DISPOSAL OF HAZARDOUS WASTE

Figure 4-6 Autoclave bags.

STANDARD PRECAUTIONS

Figure 4-7 A, Various bench-top pipette discard containers. B, Bench-top serologic pipette discard container.

Figure 4-8 Cartons for broken glass.

Figure 4-9 Sharps containers.

ENGINEERING CONTROLS

LABORATORY ENVIRONMENT

Figure 4-10 Class I biological safety cabinet. A, Model BSC-100. B, Schematics showing airflow.

BIOLOGICAL SAFETY CABINET

Figure 4-11 Class II biological safety cabinet.A, Model Steril GARD II. B, Schematics showing airflow.

Figure 4-12 Class III biological safety cabinet. A, Custom-built class III system. B, Schematics. Arrows show airflow through cabinet.

Figure 4-13 Personal protective equipment. A, Microbiologist wearing a laboratory gown, gloves, goggles, and face mask.

PERSONAL PROTECTIVE EQUIPMENT

Figure 4-14 A, The Bio-Pouch (lower right-hand corner) is made of laminated, low-density polyethylene, which is virtually unbreakable. The label for shipping a diagnostic specimen is shown (UN 3373). B, The Bio-Bottle is made of high-density polyethylene and is used as the secondary container. This packaging is used for both infectious substances (with the Class 6 label shown) or for clinical and diagnostic specimens with the UN 3373 label.

POSTEXPOSURE CONTROL

CLASSIFICATION OF BIOLOGIC AGENTS BASED ON HAZARD

MAILING BIOHAZARDOUS MATERIALS

Table 4-1 Examples of Infectious Substances Included in Category A*

REFERENCES

ADDITIONAL READING

CHAPTER 5 Specimen Management

GENERAL CONCEPTS FOR SPECIMEN COLLECTION AND HANDLING

APPROPRI ATE COLLECTION TECHNIQUES

SPECIMEN TRANSPORT

SPECIMEN PRESERVATION

Figure 5-1 Specimen bag with biohazard label, separate pouch for paperwork, and self-seal.

Specimen Storage

Table 5-1 Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory*

SPECIMEN LABELING

SPECIMEN REQUISITION

REJECTION OF UNACCEPTABLE SPECIMENS

SPECIMEN PROCESSING

GROSS EXAMINATION OF SPECIMEN

DIRECT MICROSCOPIC EXAMINATION

SELECTION OF CULTURE MEDIA

SPECIMEN PREPARATION

Figure 5-2 Examples of various types of hemolysis on blood agar. A,,Streptococcus pneumoniae showing alpha (α)-hemolysis (i.e., greening around colony). B,,Staphylococcus aureus showing beta (β)-hemolysis (i.e., clearing around colony). C,, Enterococcus faecalis showing gamma (γ)-hemolysis (i.e., no hemolysis around colony).

Figure 5-3 MacConkey agar. A,,Escherichia coli, a lactose fermenter. B,, Pseudomonas aeruginosa, a non–lactose fermenter.

INOCULATION OF SOLID MEDIA

Figure 5-4 Methods of inoculating solid media. A,, Streaking for quantitation. B,, Streaking semiquantitatively.

INCUBATION CONDITIONS

SPECIMEN WORKUP

EXTENT OF IDENTIFICATION REQUIRED

COMMUNICATION OF LABORATORY FINDINGS

CRITICAL (PANIC) VALUES

EXPEDITING RESULTS REPORTING—COMPUTERIZATION

REFERENCE

ADDITIONAL READING

SECTION 2 APPROACHES TO DIAGNOSIS OF INFECTIOUS DISEASES

CHAPTER 6 Role of Microscopy

BRIGHT-FIELD (LIGHT) MICROSCOPY

PRINCIPLES OF LIGHT MICROSCOPY

Magnification

Resolution

Table 6-1 Microscopy for Diagnostic Microbiology

BOX 6-1 Applications of Microscopy in Diagnostic Microbiology

Figure 6-1 Principles of bright-field (light) microscopy.

Contrast

STAINING TECHNIQUES FOR LIGHT MICROSCOPY

Smear Preparation

Figure 6-2 Smear preparations by swab roll (A) and pipette deposition (B) of patient specimen on glass microscope slide.

Gram Stain

Procedure.

Principle.

Gram Stain Examination.

Figure 6-3 Gram stain procedures and principles. A, Gram-positive bacteria observed under oil immersion appear purple. B, Gram-negative bacteria observed under oil immersion appear pink.

Gram Stain of Bacteria Grown in Culture.

Figure 6-4 Examples of common bacterial cellular morphologies, Gram staining reactions, and cellular arrangements.

Acid-Fast Stains

Principle.

Figure 6-5 Gram stains of direct smears showing squamous cells and bacteria (A), proteinaceous debris alone (B), and proteinaceous debris with polymorphonuclear leukocytes and bacteria (C).

Figure 6-6 Gram stain of direct smears showing polymorphonuclear leukocytes, proteinaceous debris, and bacterial morphologies (arrows), including gram-positive cocci in chains (A), gram-positive diplococci (B), gram-positive cocci in clusters (C), gram-negative coccobacilli (D), gram-negative bacilli (E), gram-negative diplococci (F), and mixed gram-positive and gram-negative morphologies (G).

Figure 6-7 Gram stains of direct smears can reveal infectious etiologies other than bacteria, such as the yeast Candida tropicalis.

Figure 6-8 Example of a slide map for staining several bacterial colony samples on a single slide.

Procedure.

PHASE CONTRAST MICROSCOPY

Figure 6-9 The Ziehl-Neelsen acid-fast stain procedures and principles. A, Acid-fast positive bacilli. B, Acid-fast negative bacilli.

FLUORESCENT MICROSCOPY

PRINCIPLE OF FLUORESCENT MICROSCOPY

Figure 6-10 Acid-fast stain of direct smear to show acid-fast bacilli staining deep red (arrow A) and non–acid-fast bacilli and host cells staining blue with the counterstain methylene blue(arrow B).

STAINING TECHNIQUES FOR FLUORESCENT MICROSCOPY

Fluorochroming

Figure 6-11 Principle of fluorescent microscopy. Microorganisms in a specimen are stained with a fluorescent dye. On exposure to excitation light, organisms are visually detected by the emission of fluorescent light by the dye with which they have been stained (i.e., fluorochroming) or “tagged” (i.e., immunofluorescence).

Figure 6-12 Principles of fluorochroming and immunofluorescence. Fluorochroming (A) involves nonspecific staining of any bacterial cell with a fluorescent dye. Immunofluorescence (B) uses antibodies labeled with fluorescent dye (i.e., a conjugate) to specifically stain a particular bacterial species.

Procedure 6-1 Acridine Orange Stain

PRINCIPLE

METHOD

EXPECTED RESULTS

Acridine Orange.

Auramine-Rhodamine.

Figure 6-13 Comparison of acridine orange fluorochroming and Gram stain. Gram stain of mycoplasma demonstrates inability to distinguish these cell wall–deficient organisms from amorphous gram-negative debris (A). Staining the same specimen with acridine orange confirms the presence of nucleic acid–containing organisms (B). Gram stain distinguishes between gram-positive and gram-negative bacteria (C), but all bacteria stain the same with the nonspecific acridine orange dye (D).

Figure 6-14 Comparison of the Ziehl-Neelsen–stained (A) and auramine-rhodamine–stained (B) Mycobacterium spp.(arrows).

Calcofluor White.

Figure 6-15 Immunofluoresence stains of Legionella spp. (A) and Bordetella pertussis (B) used for identification.

Figure 6-16 Dark-field microscopy. Principle (A) and dark-field photomicrograph showing the tightly coiled characteristics of the spirochete Treponema pallidum (B).

Immunofluorescence

DARK-FIELD MICROSCOPY

Figure 6-17 A, Transmission electron micrograph showing Escherichia coli cells internalized by a human mast cell (arrows). B, Scanning electron micrograph of E. coli interacting with the surface of human mast cell (arrows).

ELECTRON MICROSCOPY

ADDITIONAL READING

CHAPTER 7 Traditional Cultivation and Identification

PRINCIPLES OF BACTERIAL CULTIVATION

NUTRITIONAL REQUIREMENTS

General Concepts of Culture Media

Phases of Growth Media

Figure 7-1 A, Clear broth indicating no bacterial growth (left) and turbid broth indicating bacterial growth (right). B, Individual bacterial colonies growing on the agar surface following incubation.

Media Classifications and Functions

Figure 7-2 Growth of Legionella pneumophila on the enrichment medium buffered charcoal-yeast extract (BCYE) agar, used specifically to grow this bacterial genus.

Figure 7-3 A, Heavy mixed growth of the gram-negative bacillus Escherichia coli (arrow A) and the gram-positive coccus Enterococcus spp. (arrow B) on the nonselective medium sheep blood agar (SBA). B, The selective medium phenylethyl-alcohol agar (PEA) only allows the enterococci to grow (arrow).

Figure 7-4 Differential capabilities of MacConkey agar as gram-negative bacilli capable of fermenting lactose appear deep purple (arrow A), whereas those not able to ferment lactose appear light pink or relatively colorless (arrow B).

Figure 7-5 Different colony morphologies exhibited on sheep blood agar by various bacteria, including alpha-hemolytic streptococci (arrow A), gram-negative bacilli (arrow B), beta-hemolytic streptococci (arrow C), and Staphylococcus aureus (arrow D).

Summary of Artificial Media for Routine Bacteriology

Table 7-1 Plating Media for Routine Bacteriology

Brain-Heart Infusion.

Chocolate Agar.

Columbia CNA with Blood.

Gram-Negative (GN) Broth.

Hektoen Enteric (HE) Agar.

MacConkey Agar.

Phenylethyl Alcohol (PEA) Agar.

Sheep Blood Agar.

Figure 7-6 Differential capabilities of HE agar for lactose-fermenting, gram-negative bacilli (e.g., Escherichia coli, arrow A), non–lactose-fermenters (e.g., Shigella spp., arrow B), and H2S producers (e.g., Salmonella spp., arrow C).

Thayer-Martin Agar.

Thioglycollate Broth.

Figure 7-7 Growth characteristics of various bacteria in thioglycollate broth. A, Facultatively anaerobic gram-negative bacilli (i.e., those that grow in the presence or absence of oxygen) grow throughout broth. B, Gram-positive cocci grow as “puff balls.” C, Strictly aerobic organisms (i.e., those that require oxygen for growth), such as Pseudomonas aeruginosa, grow toward the top of the broth. D, Strictly anaerobic organisms (i.e., those that do not grow in the presence of oxygen) grow in the bottom of the broth.

Xylose-Lysine-Desoxycholate (XLD) Agar.

Preparation of Artificial Media

Media Sterilization.

Figure 7-8 Differential capabilities of xylose-lysine-desoxycholate (XLD) agar for lactose-fermenting, gram-negative bacilli (e.g., Escherichia. coli, arrow A), non–lactose-fermenters (e.g., Shigella spp., arrow B), and H2S producers (e.g., Salmonella spp., arrow C).

Cell Cultures.

ENVIRONMENTAL REQUIREMENTS

Oxygen and Carbon Dioxide Availability

Temperature

pH

Moisture

Methods for Providing Optimum Incubation Conditions

BACTERIAL CULTIVATION

ISOLATION OF BACTERIA FROM SPECIMENS

Evaluation of Colony Morphologies

Type of Media Supporting Bacterial Growth.

Relative Quantities of Each Colony Type.

Colony Characteristics.

Figure 7-9 A, Dilution streak technique for isolation and semiquantitation of bacterial colonies. B, Actual plates show sparse, or 1+ bacterial growth that is limited to the first quadrant. C, Moderate, or 2+ bacterial growth that extends to the second quadrant. D, Heavy, or 3+ to 4+ bacterial growth that extends to the fourth quadrant.

Figure 7-10 A, Streaking pattern using a calibrated loop for enumeration of bacterial colonies grown from a liquid specimen such as urine. B, Actual plate shows well-isolated and dispersed bacterial colonies for enumeration obtained with the calibrated loop streaking technique.

Gram Stain and Subcultures.

PRINCIPLES OF IDENTIFICATION

Figure 7-11 Colony morphologic features and descriptive terms for commonly encountered bacterial colonies.

ORGANISM IDENTIFICATION USING GENOTYPIC CRITERIA

ORGANISM IDENTIFICATION USING PHENOTYPIC CRITERIA

Figure 7-12 Mixed bacterial culture on sheep blood agar (A) requires subculture of individually distinct colonies (arrows) to obtain pure cultures of Staphylococcus aureus (B) and Streptococcus pneumoniae (C).

Microscopic Morphology and Staining Characteristics

Figure 7-13 Flowchart example of a bacterial identification scheme.

Macroscopic (Colony) Morphology

Figure 7-14 Microscopic examination of a wet preparation demonstrates the size difference between most yeast cells, such as those of Candida albicans (arrow A), and bacteria, such as Staphylococcus aureus (arrow B).

Environmental Requirements for Growth

Resistance or Susceptibility to Antimicrobial Agents

Figure 7-15 A, Zone of growth inhibition around the 5-mg vancomycin disk is indicative of a gram-positive bacterium. B, The gram-negative organism is not inhibited by this antibiotic, and growth extends to the edge of the disk.

Nutritional Requirements and Metabolic Capabilities

Establishing Enzymatic Capabilities.

Types of Enzyme-Based Tests.

Single Enzyme Tests.

Catalase Test.

Oxidase Test.

Indole Test.

Urease Test.

PYR Test.

Hippurate Hydrolysis.

Tests for Presence of Metabolic Pathways.

Oxidation and Fermentation Tests.

Amino Acid Degradation.

Figure 7-16 Principle of glucose oxidative-fermentation (O-F) test. Fermentation patterns shown in O-F tubes are examples of oxidative, fermentative, and nonutilizing bacteria.

Single Substrate Utilization.

Establishing Inhibitor Profiles.

Figure 7-17 Four basic components of bacterial identification schemes and systems.

PRINCIPLES OF PHENOTYPE-BASED IDENTIFICATION SCHEMES

SELECTION AND INOCULATION OF IDENTIFICATION TEST BATTERY

Type of Bacteria to Be Identified

Clinical Significance of the Bacterial Isolate

Availability of Reliable Testing Methods

INCUBATION FOR SUBSTRATE UTILIZATION

Conventional Identification

Rapid Identification

DETECTION OF METABOLIC ACTIVITY

Colorimetry

Fluorescence

Turbidity

ANALYSIS OF METABOLIC PROFILES

Identification Databases

Figure 7-18 Example of converting a metabolic profile to an octal profile for bacterial identification.

Table 7-2 Generation and Use of Genus-Identification Database Probability: Percent Positive Reactions for 100 Known Strains

Use of the Database to Identify Unknown Isolates

Confidence in Identification.

Table 7-3 Generation and Use of Genus-Identification Database Probability: Probability That Unknown Strain × is Member of Known Genus Based on Results of Each Individual Parameter Tested

COMMERCIAL IDENTIFICATION SYSTEMS

ADVANTAGES AND EXAMPLES OF COMMERCIAL SYSTEM DESIGNS

Figure 7-19 Biochemical test panel (API; bioMérieux, Inc., Hazelwood, Mo). The test results obtained with the substrates in each cupule are recorded, and an organism identification code is calculated by octal code conversion on the form provided. The octal profile obtained then is matched with an extensive database to establish organism identification.

Figure 7-20 Plastic cards composed of multiple wells containing dried substrates that are reconstituted by inoculation with a bacterial suspension (bioMérieux, Inc., Hazelwood, Mo). Test results in the card wells are automatically read by the manufacturer’s reading device.

Overview of Commercial Systems

CHROMATOGRAPHY

PRINCIPLES

APPLICATIONS

ADDITIONAL READING

CHAPTER 8 Nucleic Acid–Based Analytic Methods for Microbial Identification and Characterization

OVERVIEW OF MOLECULAR METHODS

NUCLEIC ACID HYBRIDIZATION METHODS

Figure 8-1 Principles of nucleic acid hybridization. Identification of unknown organism is established by positive hybridization (i.e., duplex formation) between a nucleic acid strand from the known organism (i.e., the probe), and a target nucleic acid strand from the organism to be identified. Failure to hybridize indicates lack of homology between probe and target nucleic acid.

Hybridization Steps and Components

Production and Labeling of Probe Nucleic Acid

Figure 8-2 Reporter molecule labeling of nucleic acid probes and principles of hybridization detection. Use of probes labeled with a radioactive reporter, with hybridization detected by autoradiography (A); probes labeled with biotin-avidin reporter, with hybridization detected by a colorimetric assay (B); probes labeled with chemiluminescent reporter (i.e., acridinium), with hybridization detected by a luminometer to detect emitted light (C).

Preparation of Target Nucleic Acid.

Mixture and Hybridization of Target and Probe.

Detection of Hybridization.

Hybridization Formats

Solution Format.

Solid Support Format.

Figure 8-3 Principle of the solution hybridization format.

Figure 8-4 Principle of solid support hybridization formats. A, Filter hybridization. B, Southern hybridization. C, Sandwich hybridization.

Figure 8-5 Peptide nucleic acid (PNA) probes. Structure of DNA compared to the structure of a synthetic PNA probe; the chemical modification of DNA allows for greater sensitivity and specificity of the PNA probes compared to the DNA probes.

In Situ Hybridization.

Peptide Nucleic Acid (PNA) Probes.

Hybridization with Signal Amplification.

AMPLIFICATION METHODS—PCR-BASED

Figure 8-6 Using a fluorescent-tagged peptide nucleic acid (PNA) probe in conjunction with fluorescent in situ hybridization (FISH), Staphylococcus aureus (A) or Candida albicans (B) was directly identified in blood cultures within 2½ hours. A drop from the positive blood culture bottle is added to a slide containing a drop of fixative solution, which keeps the cells intact. After fixation, the appropriate fluorescent-labeled PNA probe is added. The PNA probe penetrates the microbial cell wall and hybridizes to the rRNA. Slides are examined under a fluorescent microscope. If the specific target is present, bright green, fluorescent-staining organisms will be present. Blood cultures negative for either S. aureus (C) or C. albicans (D) by PNA FISH technology are shown.

Overview of PCR and Derivations

Figure 8-7 Overview of polymerase chain reaction. The target sequence is denatured to single strands, primers specific for each target strand sequence are added, and DNA polymerase catalyzes the addition of deoxynucleotides to extend and produce new strands complementary to each of the target sequence strands (cycle 1). The second cycle begins by both double-stranded products of cycle 1 being denatured and subsequently serving as targets for more primer annealing and extension by DNA polymerase. After 25 to 30 cycles, at least 107 copies of target DNA may be produced.

Extraction and Denaturation of Target Nucleic Acid.

Primer Annealing.

Extension of Primer-Target Duplex.

Detection of PCR Products.

Derivations of the PCR Method.

Figure 8-8 Use of ethidium bromide–stained agarose gels to determine the size of PCR amplicons for identification. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B, C, and D contain PCR amplicons typical of the enterococcal vancomycin-resistance genes vanA (783 kb), vanB (297 kb), and vanC1 (822 kb), respectively.

Figure 8-9 Ethidium bromide–stained gels containing amplicons produced by multiplex PCR. Lane A shows molecular-size markers, with the marker sizes indicated in base pairs. Lanes B and C show amplicons obtained with multiplex PCR consisting of control primers and primers specific for the staphylococcal methicillin-resistance gene mecA. The presence of only the control amplicon (370 bp) in Lane B indicates that PCR was successful, but the strain on which the reaction was performed did not contain mecA. Lane C shows both the control and the mecA (310 bp) amplicons, indicating that the reaction was successful and that the strain tested carries the mecA resistance gene.

Real-Time PCR

Figure 8-10 Examples of real-time PCR instruments. A, Applied Biosystems. B, iCycler. C, Light Cycler. D, SmartCycler.

Table 8-1 Examples of Commercially Available Real-Time Polymerase Chain Reaction (PCR) Instruments

Figure 8-11 Fluorogenic probes (probe with an attached fluorophore, a fluorescent molecule that can absorb light energy and then is elevated to an excited state that is released as fluorescence in the absence of a quencher) commonly used for detection of amplified product in real-time PCR assays. A, Hydrolysis probe. In addition to the specific primers for amplification, an oligonucleotide probe with a reporter fluorescent dye (R) and a quencher dye (Q) at its 5′ and 3′ ends, respectively, is added to the reaction mix. During the extension phase, the quencher (molecule that can accept energy from a fluorophore and then dissipate the energy so that no fluorescence results) can only quench the reporter fluorescence when the two dyes are close to each other, which is only the case with an intact probe (a). Once amplification occurs and the fluorogenic probe binds to amplified product, bound probe is degraded by the 5′-3′ exonuclease activity of Taq polymerase; thus quenching is no longer possible and fluorescence is emitted and then measured (b). B, Molecular beacon. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore that fluoresces once the beacon probe binds to the amplified target and the quencher is no longer in close proximity to the fluorophore. These probes are designed such that the loop portion of the molecule is a sequence complementary to the target of interest (a). The “stem” portion of the beacon probe is formed by the annealing of complementary arm sequences on the respective ends of the probe sequence. In addition, a fluorescent moiety (R) and a quencher moiety (Q) at opposing ends of the probe are attached (a). The stem portion of the probe keeps the fluorescent and quencher moieties in close proximity to one another such that the fluorescence of the fluorophore remains quenched. When it encounters a target molecule with a complementary sequence, the molecular beacon undergoes a spontaneous conformational change that forces the stem apart, thereby causing the fluorophore and quencher to move away from each other and leading to restoration of fluorescence (b). C, Fluorescent resonant energy transfer (FRET) or hybridization probes. Two different hybridization probes are used, one carrying a fluorescent reporter moiety at its 3′ end (designated R1) and the other probe carrying a fluorescent dye at its 5′ end (designated R2) (a). These two oligonucleotide probes are designed such that they hybridize to amplified DNA target in a head-to-tail arrangement in very close proximity to one another. The first dye (R1) is excited by a filtered light source and emits a fluorescent light at a slightly longer wavelength. Because the two dyes are in close proximity to each other, the energy emitted from R1 excites R2 attached to the second hybridization probe, which emits fluorescent light at an even longer wavelength (b). This energy transfer is referred to as FRET. By selecting an appropriate detection channel on the instrument, the intensity of light emitted from R2 is filtered and measured.

Figure 8-12 Melting curve analyses performed using the Light Cycler HSV1/2 Detection Kit. DNA was extracted and subjected to real-time PCR using the Light Cycler to detect the presence of HSV DNA. Following amplification, melting curve analysis was performed in which amplified product was cooled to below 55° C and then the temperature slowly raised. The Tm is the temperature at which half of the DNA is single-stranded and is specific for the sequence of the particular DNA product. The specific melting temperature is determined at 640 nm (channel F2 on the cycler) for the clinical samples and the positive and negative controls. For illustration purposes, melting curve analyses are “overlaid” relative to one another in this figure for three clinical samples and the HSV-1 and HSV-2 positive or “template” control. The clinical specimens containing HSV-1 DNA (red line) or HSV-2 (green line) result in a melting peak at 54° C (the Tm) or 67° C (the Tm), respectively. The Light Cycler positive or template control containing HSV-1 and HSV-2 DNA, displayed as a purple line, shows two peaks at 54° C and 67° C, respectively. The clinical sample that is negative (brown line) for both HSV-1 and HSV-2 shows no peaks.

Figure 8-13 Quantitation using real-time PCR. A, In the example, four samples containing known amounts of target are amplified by real-time PCR. The inverse log of their fluorescence is plotted against the cycle number and their respective CT is determined; the fewer the number of targets, the greater the CT value. B, Similarly, the clinical specimen is also run by real-time PCR and its CT value is determined. C, The log of the nucleic concentration and the respective CT value for each specimen run containing a known amount of target or nucleic acid are plotted to generate a standard curve. By knowing the CT value of the clinical specimen, the concentration of target in the original sample can then be determined.

AMPLIFICATION METHODS: NON–PCR-BASED

Table 8-2 Examples of Commercially Available Signal Amplification Methods

SEQUENCING AND ENZYMATIC DIGESTION OF NUCLEIC ACIDS

Nucleic Acid Sequencing

High-Density DNA Probes

Enzymatic Digestion and Electrophoresis of Nucleic Acids

Table 8-3 Examples of Non–Polymerase Chain Reaction–Based Nucleic Amplification Tests

Figure 8-14 Overview of high-density DNA probes. High-density oligonucleotide arrays are created using light-directed chemical synthesis that combines photolithography and solid-phase chemical synthesis. Because of this sophisticated process, more than 500 to as many as a million different oligonucleotide probes may be formed on a chip; an array is shown in A. Nucleic acid is extracted from a sample and then hybridized within seconds to the probe array in a GeneChip Fluidics Station. The hybridized array (B) is scanned using a laser confocal fluorescent microscope that looks at each site (i.e., probe) on the chip and the intensity of hybridization is analyzed using imaging processing software. The Affymetrix system is shown in part C. From left to right is the GeneChip Fluidics Station, scanner, and computer system for data.

Figure 8-15 DNA enzymatic digestion and gel electrophoresis to separate DNA fragments resulting from the digestion. An example of a nucleic acid recognition site and enzymatic cut produced by Eco R1, a commonly used endonuclease, is shown in the inset.

Figure 8-16 Restriction fragment length polymorphisms of vancomycin-resistant Enterococcus faecalis isolates in lanes A through G as determined by pulsed-field gel electrophoresis. All isolates appear to be the same strain.

Figure 8-17 Although antimicrobial susceptibility profiles indicated that several methicillin-resistant S. aureus isolates were the same strain, restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis (Lanes A through F) demonstrates that only isolates B and C were the same.

Figure 8-18 Restriction patterns generated by pulsed-field gel electrophoresis for two Streptococcus pneumoniae isolates, one that was susceptible to penicillin (Lane B) and one that was resistant (Lane C), from the same patient. Restriction fragment length polymorphism analysis indicates that the patient was infected with different strains. Molecular-size markers are shown in Lane A.

APPLICATIONS OF NUCLEIC ACID–BASED METHODS

DIRECT DETECTION OF MICROORGANISMS

Advantages and Disadvantages

Specificity.

Sensitivity.

Amplification Techniques Enhance Sensitivity.

Applications for Direct Molecular Detection of Microorganisms

IDENTIFICATION OF MICROORGANISMS GROWN IN CULTURE

CHARACTERIZATION OF MICROORGANISMS BEYOND IDENTIFICATION

Detection of Antimicrobial Resistance

Table 8-4 Examples of Methods to Determine Strain Relatedness

Investigation of Strain Relatedness/Pulsed-Field Gel Electrophoresis

Figure 8-19 Procedural steps for pulsed-field gel electrophoresis (PFGE).

ADDITIONAL READING

CHAPTER 9 Immunochemical Methods Used for Organism Detection

PRODUCTION OF ANTIBODIES FOR USE IN LABORATORY TESTING

POLYCLONAL ANTIBODIES

MONOCLONAL ANTIBODIES

Figure 9-1 Group A Streptococcus (Streptococcus pyogenes) contains many antigenic structural components and produces various antigenic enzymes, each of which may elicit a specific antibody response from the infected host.

PRINCIPLES OF IMMUNOCHEMICAL METHODS USED FOR ORGANISM DETECTION

PRECIPITIN TESTS

Double Immunodiffusion

Figure 9-2 Production of a monoclonal antibody.

Counterimmunoelectrophoresis

Figure 9-3 Exo-Antigen Identification System, Immuno-Mycologics, Inc., Norman, Okla. The center well is filled with a 50s× concentrate of an unknown mold. The arrow identifies well 1; wells 2 to 6 are shown clockwise. Wells 1, 3, and 5 are filled with anti-Histoplasma, anti-Blastomyces, and anti-Coccidioides reference antisera, respectively. Wells 2, 4, and 6 are filled with Histoplasma antigen, Blastomyces antigen, and Coccidioides antigen, respectively. The unknown organism can be identified as Histoplasma capsulatum based on the formation of line(s) of identity (arc) linking the control band(s) with one or more bands formed between the unknown extract (center well) and the reference antiserum well (well 1).

Figure 9-4 Apparatus for performing counterimmunoelectrophoresis.

Figure 9-5 Alignment of antibody molecules bound to the surface of a latex particle and latex agglutination reaction.

PARTICLE AGGLUTINATION

Latex Agglutination

Figure 9-6 Cryptococcal Antigen Latex Agglutination System (CALAS), Meridian Diagnostics, Inc., Cincinnati, Ohio. Patient 1 shows positive agglutination; patient 2 is negative.

Figure 9-7 Streptex, Remel, Inc., Lenexa, Kan. Colony of beta-hemolytic Streptococcus agglutinates with group B Streptococcus (Streptococcus agalactiae) latex suspension.

Coagglutination

Figure 9-8 Coagglutination.

Liposome-Enhanced Latex Agglutination

IMMUNOFLUORESCENT ASSAYS

Figure 9-9 Diagram of liposome-latex agglutination reaction.

Figure 9-10 Legionella (Direct) Fluorescent Test System, Scimedx Corp., Denville, NJ. Legionella pneumophila serogroup 1 in sputum.

Figure 9-11 Direct and indirect fluorescent antibody tests for antigen detection.

ENZYME IMMUNOASSAYS

Figure 9-12 ProSpecT Giardia/Cryptosporidium Microplate Assay. A, Breakaway microwell cupules and kit components. B, Positive (yellow) and negative (blue) reactions.

Solid-Phase Immunoassay

Figure 9-13 Principle of direct solid-phase enzyme immunosorbent assay (SPIA). A, Solid phase is microtiter well. B, Solid phase is bead.

Membrane-Bound SPIA

OTHER IMMUNOASSAYS

Figure 9-14 Principle of indirect solid-phase enzyme immunosorbent assay (SPIA).

Figure 9-15 Directigen respiratory syncytial virus (RSV) membrane-bound cassette. A, Positive reaction. B, Negative reaction.

Figure 9-16 Optical ImmunoAssay (OIA) for group A Streptococcus. A, Positive reaction.B, Negative reaction. C, Invalid reaction.

ADDITIONAL READING

CHAPTER 10 Serologic Diagnosis of Infectious Diseases

FEATURES OF THE IMMUNE RESPONSE

CHARACTERISTICS OF ANTIBODIES

Figure 10-1 Structure of immunoglobulin G.

Figure 10-2 Structure of immunoglobulin M.

Features of the Humoral Immune Response Useful in Diagnostic Testing

Figure 10-3 Relative humoral response to antigen stimulation over time.

Interpretation of Serologic Tests

SERODIAGNOSIS OF INFECTIOUS DISEASES

Table 10-1 Tests Available for Serodiagnosis of Infectious Diseases

PRINCIPLES OF SEROLOGIC TEST METHODS

SEPARATING IGM FROM IGG FOR SEROLOGIC TESTING

METHODS OF ANTIBODY DETECTION

Direct Whole Pathogen Agglutination Assays

Particle Agglutination Tests

Flocculation Tests

Figure 10-4 MACRO-VUE RPR card test. R, Reactive (positive) test; N, nonreactive (negative) test.

Counterimmunoelectrophoresis

Immunodiffusion Assays

Hemagglutination Inhibition Assays

Neutralization Assays

Complement Fixation Assays

Figure 10-5 Complement fixation test.

Enzyme-Linked Immunosorbent Assays

Figure 10-6 Indirect fluorescent antibody tests for Toxoplasma gondii, IgG antibodies. A, Positive reaction. B, Negative reaction.

Indirect Fluorescent Antibody Tests and Other Immunomicroscopic Methods

Figure 10-7 Diagram of Western blot immunoassay system.

Radioimmunoassays

Fluorescent Immunoassays

Western Blot Immunoassays

Figure 10-8 Human immunodeficiency virus type 1 (HIV-1) Western blot. Samples are characterized as positive, indeterminate, or negative based on the bands found to be present in significant intensity. A positive blot has any two or more of the following bands: p24, gp41, and/or gp120/160. An indeterminate blot contains some bands but not the definitive ones. A negative blot has no bands present. Lane 16 shows antibodies from a control serum binding to the virus-specific proteins (p) and glycoproteins (gp) transferred onto the nitrocellulose paper.

ADDITIONAL READING

SECTION 3 EVALUATION OF ANTIMICROBIAL ACTIVITY

CHAPTER 11 Principles of Antimicrobial Action and Resistance

ANTIMICROBIAL ACTION

PRINCIPLES

Figure 11-1 The basic steps required for antimicrobial activity and strategic points for bacterial circumvention or interference (marked by X) of antimicrobial action leading to resistance.

Table 11-1 Anatomic Distribution of Some Common Antibacterial Agents

BOX 11-1 Bacteriostatic and Bactericidal Antibacterial Agents*

GENERALLY BACTERIOSTATIC

GENERALLY BACTERICIDAL

MODE OF ACTION OF ANTIBACTERIAL AGENTS

Table 11-2 Summary of Mechanisms of Action for Commonly Used Antibacterial Agents

Inhibitors of Cell Wall Synthesis

Beta-Lactam Antimicrobial Agents.

Figure 11-2 Basic structures and examples of commonly used beta-lactam antibiotics. The core beta-lactam ring is highlighted in yellow in each structure.

Glycopeptides.

Figure 11-3 Structure of vancomycin, a non–beta-lactam antibiotic that inhibits cell wall synthesis.

Inhibitors of Cell Membrane Function

Inhibitors of Protein Synthesis

Aminoglycosides.

Macrolide-Lincosamide-Streptogramin (MLS) Group.

Figure 11-4 Structure of the commonly used aminoglycoside gentamicin. Potential sites of modification by adenylylating, phosphorylating, and acetylating enzymes produced by bacteria are highlighted.

Ketolides.

Oxazolidinones.

Chloramphenicol.

Tetracyclines.

Glycylglycines.

Inhibitors of DNA and RNA Synthesis

Fluoroquinolones.

Metronidazole.

Figure 11-5 Structures of the fluoroquinolones ciprofloxacin and ofloxacin.

Rifampin.

Inhibitors of Other Metabolic Processes

Sulfonamides.

Trimethoprim.

Nitrofurantoin.

MECHANISMS OF ANTIBIOTIC RESISTANCE

PRINCIPLES

BIOLOGIC VS. CLINICAL RESISTANCE

Figure 11-6 Bacterial folic acid pathway indicating the target enzymes for sulfonamide and trimethoprim activity.

ENVIRONMENTALLY MEDIATED ANTIMICROBIAL RESISTANCE

Figure 11-7 Cations (Mg++ and Ca++) and aminoglycosides (AG++) compete for the negatively charged binding sites on the outer membrane surface of Pseudomonas aeruginosa. Such competition is an example of the impact that environmental factors (e.g., cation concentrations) can have on the antibacterial activity of aminoglycosides.

MICROORGANISM-MEDIATED ANTIMICROBIAL RESISTANCE

Intrinsic Resistance

Acquired Resistance

BOX 11-2 Examples of Intrinsic Resistance to Antibacterial Agents

COMMON PATHWAYS FOR ANTIMICROBIAL RESISTANCE

Resistance to Beta-Lactam Antibiotics

Figure 11-8 Overview of common pathways bacteria use to effect antimicrobial resistance.

Table 11-3 Summary of Resistance Mechanisms for Beta-Lactams, Vancomycin, Aminoglycosides, and Fluoroquinolones

Figure 11-9 Mode of beta-lactamase enzyme activity. By cleaving the beta-lactam ring, the molecule can no longer bind to penicillin-binding proteins (PBPs) and is no longer able to inhibit cell wall synthesis.

Resistance to Glycopeptides

Resistance to Aminoglycosides

Figure 11-10 Diagrammatic summary of beta-lactam resistance mechanisms for gram-positive and gram-negative bacteria. A, Among gram-positive bacteria, resistance is mediated by beta-lactamase production and altered PBP targets. B, In gram-negative bacteria, resistance can also be mediated by decreased uptake through the outer membrane porins.

BOX 11-3 Bacterial Resistance Mechanisms for Miscellaneous Antimicrobial Agents

CHLORAMPHENICOL

TETRACYCLINES

MACROLIDES (I.E., ERYTHROMYCIN) AND CLINDAMYCIN

SULFONAMIDES AND TRIMETHOPRIM

RIFAMPIN

Resistance to Quinolones

Resistance to Other Antimicrobial Agents

EMERGENCE AND DISSEMINATION OF ANTIMICROBIAL RESISTANCE

Figure 11-11 Factors contributing to the emergence and dissemination of antimicrobial resistance among bacteria.

ADDITIONAL READING

CHAPTER 12 Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing

GOAL AND LIMITATIONS

STANDARDIZATION

LIMITATIONS OF STANDARDIZATION

TESTING METHODS

PRINCIPLES

METHODS THAT DIRECTLY MEASURE ANTIMICROBIAL ACTIVITY

Conventional Testing Methods: General Considerations

Inoculum Preparation.

Figure 12-1 Bacterial suspension prepared to match the turbidity of the 0.5 McFarland turbidity standard. Matching this turbidity provides a bacterial inoculum concentration of 1 to 2× 108 CFU/mL.

Selection of Antimicrobial Agents for Testing.

BOX 12-1 Criteria for Antimicrobial Battery Content and Use

ORGANISM IDENTIFICATION OR GROUP:

ACQUIRED RESISTANCE PATTERNS COMMON TO LOCAL MICROBIAL FLORA:

ANTIMICROBIAL SUSCEPTIBILITY TESTING METHOD USED:

SITE OF INFECTION:

AVAI LABILITY OF ANTIMICROBIAL AGENTS IN FORMULARY:

Table 12-1 Summary of Broth Dilution Susceptibility Testing Conditions

Conventional Testing Methods: Broth Dilution

Procedures.

Medium and Antimicrobial Agents.

Figure 12-2 Microtitre tray for broth microdilution testing. Doubling dilutions of each antimicrobial agent in test broth occupies one vertical row of wells.

Inoculation and Incubation.

Figure 12-3 Bacterial growth profiles in a broth microdilution tray. The wells containing the lowest concentration of an antibiotic that completely inhibits visible growth (arrow) μ g/mL, as the minimal inhibitory concentration (MIC).

Reading and Interpretation of Results.

BOX 12-2 Definitions of Susceptibility Testing Interpretive Categories*

SUSCEPTIBLE:

INTERMEDIATE:

RESISTANT:

Advantages and Disadvantages.

Figure 12-4 Growth pattern on an agar dilution plate. Each plate contains a single concentration of antibiotic, and growth is indicated by a spot on the agar surface. No spot is seen for isolates inhibited by the concentration of antibiotic incorporated into the agar of that particular plate.

Conventional Testing Methods: Agar Dilution

Table 12-2 Summary of Agar Dilution Susceptibility Testing Conditions

Table 12-3 Summary of Disk Diffusion Susceptibility Testing Conditions

Table 12-4 Supplemental Methods for Detection of Antimicrobial Resistance

Conventional Testing Methods: Disk Diffusion

Figure 12-5 A, By the disk diffusion method, antibiotic disks are placed on the surface just after the agar surface was inoculated with the test organism. B, Zones of growth inhibition around various disks are apparent following 16 to 18 hours of incubation.

Figure 12-6 Example of a regression analysis plot to establish zone-size breakpoints for defining the susceptible, intermediate, and resistant categories for an antimicrobial agent. In this example, the maximum achievable serum concentration of the antibiotic is 8 μ g/mL. Disk inhibition zones less than or equal to 18 mm in diameter indicate resistance, zones greater than or equal to 26 mm indicate susceptibility, and the intermediate category is indicated by zones ranging from 19 to 25 mm.

Procedures.

Medium and Antimicrobial Agents.

Inoculation and Incubation.

Figure 12-7 Disk diffusion plate that was inoculated with a mixed culture as evidenced by different colony morphologies (arrows) appearing throughout the lawn of growth.

R eading and Interpretation of Results.

Figure 12-8 Examination of a disk diffusion plate by transmitted and reflected light.

Figure 12-9 Individual bacterial colonies within a more obvious zone of inhibition (arrows). This could indicate inoculation with a mixed culture. However, emergence of resistant mutants of the test isolate is a more likely reason for this growth pattern.

A dvantages and Disadvantages.

Commercial Susceptibility Testing Systems

Broth Microdilution Methods.

Agar Dilution Derivations.

Diffusion in Agar Derivations.

Figure 12-10 Growth patterns on a plate containing an antibiotic gradient (concentration decreases from center of the plate to the periphery) applied by the Spiral Gradient instrument. The distance from where growth is noted at the edge of the plate to where growth is inhibited toward the center of the plate is measured. This value is used in a formula to calculate the MIC of the antimicrobial agent against each of the bacterial isolates streaked on the plate.

A utomated Antimicrobial Susceptibility Test Systems.

Figure 12-11 Etest uses the principle of a predefined antibiotic gradient on a plastic strip to generate an MIC value. It is processed like the disk diffusion. A, Individual antibiotic strips are placed on an inoculated agar surface. B, After incubation, the MIC is read where the growth/inhibition edge intersects the strip graduated with an MIC scale across 15 dilutions (arrow). Several antibiotic strips can be tested on a plate.

Figure 12-12 The Vitek 2 antimicrobial susceptibility test card contains 64 wells with multiple concentrations of up to 22 antibiotics. The antibiotic is rehydrated when the organism suspension is introduced into the card during the automated filling process.

Figure 12-13 The components of the Vitek 2 System consist of the instrument housing; the sample processing and reader/incubator; the computer workstation, which provides data analysis, sto rage, and epidemiology reports; the Smart Carrier Station, which is the direct interface between the microbiologist on the bench and the instrument; and a bar code scanner to facilitate data entry.

Figure 12-14 Microdilution tray format (A) used with the MicroScan WalkAway instrument (B) for automated incubation, reading, and interpretation of antimicrobial susceptibility tests.

Alternative Approaches for Enhancing Resistance Detection

Supplemental Testing Methods.

Predictor Antimicrobial Agents.

METHODS THAT DIRECTLY DETECT SPECIFIC RESISTANCE MECHANISMS

Phenotypic Methods

Beta-Lactamase Detection.

Figure 12-15 The chromogenic cephalosporin test allows direct detection of beta-lactamase production. When the beta-lactam ring of the cephalosporin substrate in the disk is hydrolyzed by the bacterial inoculum, a deep pink color is produced (A). Lack of color production indicates the absence of beta-lactamase (B).

C hloramphenicol Acetyltransferase Detection.

Genotypic Methods

SPECIAL METHODS FOR COMPLEX ANTIMICROBIAL–ORGANISM INTERACTIONS

Bactericidal Tests

Minimal Bactericidal Concentration.

Time-Kill Studies.

Serum Bactericidal Test.

Tests for Activity of Antimicrobial Combinations

Figure 12-16 Goals of effective antimicrobial susceptibility testing strategies.

LABORATORY STRATEGIES FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING

RELEVANCE

Table 12-5 Categorization of Bacteria According to Need for Routine Performance of Antimicrobial Susceptibility Testing*

WHEN TO PERFORM A SUSCEPTIBILITY TEST

Determining Clinical Significance

Predictability of Antimicrobial Susceptibility

Availability of Reliable Susceptibility Testing Methods

SELECTION OF ANTIMICROBIAL AGENTS FOR TESTING

Table 12-6 Selection of Antimicrobial Agents for Testing Against Common Bacterial Groups*

ACCURACY

USE OF ACCURATE METHODOLOGIES

REVIEW OF RESULTS

Components of Results-Review Strategies

Table 12-7 Examples of Susceptibility Testing Profiles Requiring Further Evaluation

Data Review.

Resolution.

ACCURACY AND ANTIMICROBIAL RESISTANCE SURVEILLANCE

COMMUNICATION

ADDITIONAL READING

PART III BACTERIOLOGY

SECTION 1 PRINCIPLES OF IDENTIFICATION

CHAPTER 13 Overview of Bacterial Identification Methods and Strategies

RATIONALE FOR A METHOD OF ORGANISM IDENTIFICATION

HOW TO USE PART III: BACTERIOLOGY

Table 13-1 Examples of Commercial Identification Systems for Various Organisms

Procedure 13-1 Acetamide Utilization

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-1 Acetamideutilization. A, Positive. B, Negative.

Procedure 13-2 Acetate Utilization

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-2 Acetate utilization. A, Positive. B, Negative.

Procedure 13-3 Bacitracin Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-3 Anyzone of inhibitionis positive(Streptococcuspyogenes); growthup to the disk isnegative(Streptococcusalgalatiae).

Procedure 13-4 Bile Esculin Agar

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-4 Bileesculin agar. A, Positive. B, Negative.

Procedure 13-5 Bile Solubility Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-5 Bilesolubility test. A, Colony lysed. B, Intact colony.

Procedure 13-6 Butyrate Disk

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-6 Butyrate disk. A, Positive. B, Negative.

Procedure 13-7 CAMP Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-7 CAMP test. A, Positive, arrowhead zone ofbeta-hemolysis (atarrow) typical ofgroup Bstreptococci. B, Negative, noenhancement ofhemolysis.

Procedure 13-8 Catalase Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-8 Catalase test. A, Positive. B, Negative.

Procedure 13-9 Cetrimide

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-9 Cetrimide. A, Positive. B, Negative.

Procedure 13-10 Citrate Utilization

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-10 Citrate utilization. A, Positive. B, Negative.

Procedure 13-11 Coagulase Test

PRINCIPLE

METHOD

EXPECTED RESULTS

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-11 Coagulase test. A, Slide coagulase test forclumping factor. Left side is positive; right side is negative. B, Tube coagulase test for free coagulase. Tube on the left ispositive, exhibiting clot. Tube on the right is negative.

Procedure 13-12 Decarboxylase Tests (Moeller’s Method)

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-12 Decarboxylase tests(Moeller’ smethod). A, Positive. B, Negative. C, Uninoculatedtube.

Procedure 13-13 DNA Hydrolysis

PRINCIPLE

METHDO

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-13 DNA hydrolysis. A, Positive, Staphylococcusaureus. B, Positive, Serratia marcescens. C, Negative.

Procedure 13-14 Esculin Hydrolysis

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-14 Esculin hydrolysis. A, Positive, blackening of slant. B, Uninoculated tube.

Procedure 13-15 Fermentation Media

PRINCIPLE

METHOD

EXPECTED RESULTS

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-15 Fermentation media. A, Peptone medium with Andrade’s indicator. The tube on the left ferments glucose withthe production of gas (visible as a bubble [arrow] in the inverted[Durham] tube), the tube in the middle ferments glucose with nogas production, and the tube on the right does not fermentglucose. B, Heart infusion broth with bromcresol purpleindicator. The tube on the left is positive; the tube on the right is negative.

Procedure 13-16 Flagella Stain (Wet-Mount Technique)

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-16 Flagella stain (wet mounttechnique). A, Alcaligenesspp., peritrichousflagella (arrows). B, Pseudomonasaeruginosa, polarflagella (arrows).

Procedure 13-17 Gelatin Hydrolysis

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-17 Gelatin hydrolysis. A, Positive; note liquefactionat top of tube. B, Uninoculated tube.

Procedure 13-18 Growth at 42°C

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-18 Growth at 42°C. A, Positive, goodgrowth. B, Negative, nogrowth.

Procedure 13-19 Hippurate Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-19 Hippurate test. A, Positive. B, Negative.

Procedure 13-20 Indole Production

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-20 Indole production. A, Positive. B, Negative.

Procedure 13-21 LAP Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-21 LAP Test. A, Positive. B, Negative.

Procedure 13-22 Litmus Milk

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-22 Litmus milk. A, Acid reaction. B, Alkalinereaction. C, Nochange. D, Reduction ofindicator. E, Clot. (Note separation ofclear fluid from clotat arrow.) F, Peptonization.

Procedure 13-23 Lysin Iron Agar

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-23 Lysine iron agar. A, Alkalineslant/alkaline butt(K/K). B, Alkalineslant/alkaline butt, H2S positive (K/KH2S+). C, Alkalineslant/acid butt (K/A). D, Redslant/acid butt (R/A). E, Uninoculatedtube.

Procedure 13-24 Methyl Red/Voges-Proskauer (MRVP) Tests

PRINCIPLE

METHOD

EXPECTED RESULTS

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-24 Methyl Red/Voges-Proskauer (MRVP) tests. A, Positive methyl red. B, Negative methyl red. C, Positive Voges-Proskauer. D, Negative Voges-Proskauer.

Procedure 13-25 Microdase Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-25 Microdase Test. A, Positive. B, Negative.

Procedure 13-26 Motility Testing

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-26 Motility test. A, Positive. B, Negative.

Procedure 13-27 MRS Broth

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-27 MRS broth. A, Positive, gas production by Leuconostoc (arrow). B, Negative, no gas production.

Procedure 13-28 MUG Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-28 MUG Test. A, Positive. B, Negative.

Procedure 13-29 Nitrate Reduction

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-29 Nitrate reduction. A, Positive, no gas. B, Positive, gas(arrow). C, Positive, no color afteraddition of zinc (arrow). D, Uninoculatedtube.

Procedure 13-30 Nitrite Reduction

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-30 Nitrite reduction. A, Positive, nocolor change afteraddition of zincdust and gas in Durham tube(arrow). B, Negative.

Procedure 13-31 ONPG (o-Nitrophenyl- β – d-Galactopyranoside) Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-31 OPNG test. A, Positive. B, Negative.

Procedure 13-32 Optochin Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-32 Optochin test. A, Streptococcuspneumoniaeshowing zone ofinhibition >14 mm. B, Alpha-hemolytic Streptococcus growing up to thedisk.

Procedure 13-33 Oxidase Test (Kovac’s Method)

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-33 Oxidase test. A, Positive. B, Negative.

Procedure 13-34 Oxidation/Fermentation (OF) Medium (CDC Method)

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-34 Oxidation/fermentationmedium (CDCmethod). A, Fermenter. B, Oxidizer. C, Nonutilizer.

Procedure 13-35 Phenylalanine Deaminase

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-35 Phenylalaninedeaminase. A, Positive. B, Negative.

Procedure 13-36 PYR Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-36 PYR test. A, Positive. B, Negative.

Procedure 13-37 Pyruvate Broth

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-37 Pyruvate broth. A, Positive. B, Negative.

Procedure 13-38 Salt Tolerance Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Procedure 13-39 Spot Indole Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-38 Salt tolerance test. A, Positive. B, Negative.

Figure 13-39 Spot indole test. A, Positive. B, Negative.

Procedure 13-40 Triple Sugar Iron Agar (TSI)

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Procedure 13-41 Urea Hydrolysis (Christensen’s Method)

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-40 Triple sugar ironagar. A, Acidslant/acid butt withgas, no H2S (A/A). B, Alkalineslant/acid butt, nogas, H2S-positive(K/A H2S+). C, Alkalineslant/no changebutt, no gas, no H2S(K/NC). D, Uninoculatedtube.

Figure 13-41 Urea hydrolysis(Christensen’smethod). A, Positive. B, Negative.

Procedure 13-42 X and V Factor Test

PRINCIPLE

METHOD

EXPECTED RESULTS

QUALITY CONTROL

Figure 13-42 X and V factor test. A, Positive, growth aroundXV disk only. B, Positive, growth around V disk. C, Negative, growth over entire plate.

REFERENCES

ADDITIONAL READING

CHAPTER 14 General Considerations and Applications of Information Provided In Bacterial Sections of Part III

RATIONALE FOR APPROACHING ORGANISM IDENTIFICATION

Figure 14-1 Data demonstrating that more than 500 different bacterial species or taxa are reported from clinical laboratories across the United States. However, 95% of the isolates reported are distributed among only 27 different taxa and more than 90% are represented by 16 different taxa.

Figure 14-2 Relative frequency with which the most common bacterial species and taxa are reported by clinical laboratories.

FUTURE TRENDS OF ORGANISM IDENTIFICATION

CHAPTER 15 Bacterial Identification Flow Charts and Schemes: A Guide to Part III

Figure 15-1 Flow chart guide for anaerobic bacteria.

Figure 15-2 Flow chart guide for aerobic and facultatively anaerobic gram-positive bacteria.

Figure 15-3 Flow chart guide for aerobic and facultatively anaerobic gram-negative bacteria.

Figure 15-4 Flow chart guide for mycobacteria and other bacteria not characterized by the Gram reaction.

SECTION 2 CATALASE-POSITIVE, GRAM-POSITIVE COCCI

CHAPTER 16 Staphylococcus, Micrococcus, and Similar Organisms

Genera and Species to Be Considered

GENERAL CHARACTERISTICS

EPIDEMIOLOGY

PATHOGENESIS AND SPECTRUM OF DISEASE

Table 16-1 Epidemiology

Table 16-2 Pathogenesis and Spectrum of Diseases

Figure 16-1 Gram stain of Staphylococcus aureus from blood agar.

LABORATORY DIAGNOSIS

SPECIMEN COLLECTION AND TRANSPORT

SPECIMEN PROCESSING

DIRECT DETECTION METHODS

Table 16-3 Colonial Appearance and Characteristics on 5% Sheep Blood Agar