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Microbiology

7.5 Using Biochemistry to Identify Microorganisms

Microbiology 7.5 Using Biochemistry to Identify Microorganisms
  1. Preface
  2. 1 An Invisible World
    1. Introduction
    2. 1.1 What Our Ancestors Knew
    3. 1.2 A Systematic Approach
    4. 1.3 Types of Microorganisms
    5. Summary
    6. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  3. 2 How We See the Invisible World
    1. Introduction
    2. 2.1 The Properties of Light
    3. 2.2 Peering Into the Invisible World
    4. 2.3 Instruments of Microscopy
    5. 2.4 Staining Microscopic Specimens
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  4. 3 The Cell
    1. Introduction
    2. 3.1 Spontaneous Generation
    3. 3.2 Foundations of Modern Cell Theory
    4. 3.3 Unique Characteristics of Prokaryotic Cells
    5. 3.4 Unique Characteristics of Eukaryotic Cells
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  5. 4 Prokaryotic Diversity
    1. Introduction
    2. 4.1 Prokaryote Habitats, Relationships, and Microbiomes
    3. 4.2 Proteobacteria
    4. 4.3 Nonproteobacteria Gram-Negative Bacteria and Phototrophic Bacteria
    5. 4.4 Gram-Positive Bacteria
    6. 4.5 Deeply Branching Bacteria
    7. 4.6 Archaea
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  6. 5 The Eukaryotes of Microbiology
    1. Introduction
    2. 5.1 Unicellular Eukaryotic Parasites
    3. 5.2 Parasitic Helminths
    4. 5.3 Fungi
    5. 5.4 Algae
    6. 5.5 Lichens
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  7. 6 Acellular Pathogens
    1. Introduction
    2. 6.1 Viruses
    3. 6.2 The Viral Life Cycle
    4. 6.3 Isolation, Culture, and Identification of Viruses
    5. 6.4 Viroids, Virusoids, and Prions
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  8. 7 Microbial Biochemistry
    1. Introduction
    2. 7.1 Organic Molecules
    3. 7.2 Carbohydrates
    4. 7.3 Lipids
    5. 7.4 Proteins
    6. 7.5 Using Biochemistry to Identify Microorganisms
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. True/False
      3. Matching
      4. Fill in the Blank
      5. Short Answer
      6. Critical Thinking
  9. 8 Microbial Metabolism
    1. Introduction
    2. 8.1 Energy, Matter, and Enzymes
    3. 8.2 Catabolism of Carbohydrates
    4. 8.3 Cellular Respiration
    5. 8.4 Fermentation
    6. 8.5 Catabolism of Lipids and Proteins
    7. 8.6 Photosynthesis
    8. 8.7 Biogeochemical Cycles
    9. Summary
    10. Review Questions
      1. Multiple Choice
      2. True/False
      3. Matching
      4. Fill in the Blank
      5. Short Answer
      6. Critical Thinking
  10. 9 Microbial Growth
    1. Introduction
    2. 9.1 How Microbes Grow
    3. 9.2 Oxygen Requirements for Microbial Growth
    4. 9.3 The Effects of pH on Microbial Growth
    5. 9.4 Temperature and Microbial Growth
    6. 9.5 Other Environmental Conditions that Affect Growth
    7. 9.6 Media Used for Bacterial Growth
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  11. 10 Biochemistry of the Genome
    1. Introduction
    2. 10.1 Using Microbiology to Discover the Secrets of Life
    3. 10.2 Structure and Function of DNA
    4. 10.3 Structure and Function of RNA
    5. 10.4 Structure and Function of Cellular Genomes
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Matching
      4. Fill in the Blank
      5. Short Answer
      6. Critical Thinking
  12. 11 Mechanisms of Microbial Genetics
    1. Introduction
    2. 11.1 The Functions of Genetic Material
    3. 11.2 DNA Replication
    4. 11.3 RNA Transcription
    5. 11.4 Protein Synthesis (Translation)
    6. 11.5 Mutations
    7. 11.6 How Asexual Prokaryotes Achieve Genetic Diversity
    8. 11.7 Gene Regulation: Operon Theory
    9. Summary
    10. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  13. 12 Modern Applications of Microbial Genetics
    1. Introduction
    2. 12.1 Microbes and the Tools of Genetic Engineering
    3. 12.2 Visualizing and Characterizing DNA, RNA, and Protein
    4. 12.3 Whole Genome Methods and Pharmaceutical Applications of Genetic Engineering
    5. 12.4 Gene Therapy
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  14. 13 Control of Microbial Growth
    1. Introduction
    2. 13.1 Controlling Microbial Growth
    3. 13.2 Using Physical Methods to Control Microorganisms
    4. 13.3 Using Chemicals to Control Microorganisms
    5. 13.4 Testing the Effectiveness of Antiseptics and Disinfectants
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  15. 14 Antimicrobial Drugs
    1. Introduction
    2. 14.1 History of Chemotherapy and Antimicrobial Discovery
    3. 14.2 Fundamentals of Antimicrobial Chemotherapy
    4. 14.3 Mechanisms of Antibacterial Drugs
    5. 14.4 Mechanisms of Other Antimicrobial Drugs
    6. 14.5 Drug Resistance
    7. 14.6 Testing the Effectiveness of Antimicrobials
    8. 14.7 Current Strategies for Antimicrobial Discovery
    9. Summary
    10. Review Questions
      1. Multiple Choice
      2. True/False
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  16. 15 Microbial Mechanisms of Pathogenicity
    1. Introduction
    2. 15.1 Characteristics of Infectious Disease
    3. 15.2 How Pathogens Cause Disease
    4. 15.3 Virulence Factors of Bacterial and Viral Pathogens
    5. 15.4 Virulence Factors of Eukaryotic Pathogens
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  17. 16 Disease and Epidemiology
    1. Introduction
    2. 16.1 The Language of Epidemiologists
    3. 16.2 Tracking Infectious Diseases
    4. 16.3 Modes of Disease Transmission
    5. 16.4 Global Public Health
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  18. 17 Innate Nonspecific Host Defenses
    1. Introduction
    2. 17.1 Physical Defenses
    3. 17.2 Chemical Defenses
    4. 17.3 Cellular Defenses
    5. 17.4 Pathogen Recognition and Phagocytosis
    6. 17.5 Inflammation and Fever
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  19. 18 Adaptive Specific Host Defenses
    1. Introduction
    2. 18.1 Overview of Specific Adaptive Immunity
    3. 18.2 Major Histocompatibility Complexes and Antigen-Presenting Cells
    4. 18.3 T Lymphocytes and Cellular Immunity
    5. 18.4 B Lymphocytes and Humoral Immunity
    6. 18.5 Vaccines
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  20. 19 Diseases of the Immune System
    1. Introduction
    2. 19.1 Hypersensitivities
    3. 19.2 Autoimmune Disorders
    4. 19.3 Organ Transplantation and Rejection
    5. 19.4 Immunodeficiency
    6. 19.5 Cancer Immunobiology and Immunotherapy
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  21. 20 Laboratory Analysis of the Immune Response
    1. Introduction
    2. 20.1 Polyclonal and Monoclonal Antibody Production
    3. 20.2 Detecting Antigen-Antibody Complexes
    4. 20.3 Agglutination Assays
    5. 20.4 EIAs and ELISAs
    6. 20.5 Fluorescent Antibody Techniques
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  22. 21 Skin and Eye Infections
    1. Introduction
    2. 21.1 Anatomy and Normal Microbiota of the Skin and Eyes
    3. 21.2 Bacterial Infections of the Skin and Eyes
    4. 21.3 Viral Infections of the Skin and Eyes
    5. 21.4 Mycoses of the Skin
    6. 21.5 Protozoan and Helminthic Infections of the Skin and Eyes
    7. Summary
    8. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  23. 22 Respiratory System Infections
    1. Introduction
    2. 22.1 Anatomy and Normal Microbiota of the Respiratory Tract
    3. 22.2 Bacterial Infections of the Respiratory Tract
    4. 22.3 Viral Infections of the Respiratory Tract
    5. 22.4 Respiratory Mycoses
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  24. 23 Urogenital System Infections
    1. Introduction
    2. 23.1 Anatomy and Normal Microbiota of the Urogenital Tract
    3. 23.2 Bacterial Infections of the Urinary System
    4. 23.3 Bacterial Infections of the Reproductive System
    5. 23.4 Viral Infections of the Reproductive System
    6. 23.5 Fungal Infections of the Reproductive System
    7. 23.6 Protozoan Infections of the Urogenital System
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  25. 24 Digestive System Infections
    1. Introduction
    2. 24.1 Anatomy and Normal Microbiota of the Digestive System
    3. 24.2 Microbial Diseases of the Mouth and Oral Cavity
    4. 24.3 Bacterial Infections of the Gastrointestinal Tract
    5. 24.4 Viral Infections of the Gastrointestinal Tract
    6. 24.5 Protozoan Infections of the Gastrointestinal Tract
    7. 24.6 Helminthic Infections of the Gastrointestinal Tract
    8. Summary
    9. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  26. 25 Circulatory and Lymphatic System Infections
    1. Introduction
    2. 25.1 Anatomy of the Circulatory and Lymphatic Systems
    3. 25.2 Bacterial Infections of the Circulatory and Lymphatic Systems
    4. 25.3 Viral Infections of the Circulatory and Lymphatic Systems
    5. 25.4 Parasitic Infections of the Circulatory and Lymphatic Systems
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Fill in the Blank
      3. Short Answer
      4. Critical Thinking
  27. 26 Nervous System Infections
    1. Introduction
    2. 26.1 Anatomy of the Nervous System
    3. 26.2 Bacterial Diseases of the Nervous System
    4. 26.3 Acellular Diseases of the Nervous System
    5. 26.4 Fungal and Parasitic Diseases of the Nervous System
    6. Summary
    7. Review Questions
      1. Multiple Choice
      2. Matching
      3. Fill in the Blank
      4. Short Answer
      5. Critical Thinking
  28. A | Fundamentals of Physics and Chemistry Important to Microbiology
  29. B | Mathematical Basics
  30. C | Metabolic Pathways
  31. D | Taxonomy of Clinically Relevant Microorganisms
  32. E | Glossary
  33. Answer Key
    1. Chapter 1
    2. Chapter 2
    3. Chapter 3
    4. Chapter 4
    5. Chapter 5
    6. Chapter 6
    7. Chapter 7
    8. Chapter 8
    9. Chapter 9
    10. Chapter 10
    11. Chapter 11
    12. Chapter 12
    13. Chapter 13
    14. Chapter 14
    15. Chapter 15
    16. Chapter 16
    17. Chapter 17
    18. Chapter 18
    19. Chapter 19
    20. Chapter 20
    21. Chapter 21
    22. Chapter 22
    23. Chapter 23
    24. Chapter 24
    25. Chapter 25
    26. Chapter 26
  34. Index

Learning Objectives

  • Describe examples of biosynthesis products within a cell that can be detected to identify bacteria

Accurate identification of bacterial isolates is essential in a clinical microbiology laboratory because the results often inform decisions about treatment that directly affect patient outcomes. For example, cases of food poisoning require accurate identification of the causative agent so that physicians can prescribe appropriate treatment. Likewise, it is important to accurately identify the causative pathogen during an outbreak of disease so that appropriate strategies can be employed to contain the epidemic.

There are many ways to detect, characterize, and identify microorganisms. Some methods rely on phenotypic biochemical characteristics, while others use genotypic identification. The biochemical characteristics of a bacterium provide many traits that are useful for classification and identification. Analyzing the nutritional and metabolic capabilities of the bacterial isolate is a common approach for determining the genus and the species of the bacterium. Some of the most important metabolic pathways that bacteria use to survive will be discussed in Microbial Metabolism. In this section, we will discuss a few methods that use biochemical characteristics to identify microorganisms.

Some microorganisms store certain compounds as granules within their cytoplasm, and the contents of these granules can be used for identification purposes. For example, poly-β-hydroxybutyrate (PHB) is a carbon- and energy-storage compound found in some nonfluorescent bacteria of the genus Pseudomonas. Different species within this genus can be classified by the presence or the absence of PHB and fluorescent pigments. The human pathogen P. aeruginosa and the plant pathogen P. syringae are two examples of fluorescent Pseudomonas species that do not accumulate PHB granules.

Other systems rely on biochemical characteristics to identify microorganisms by their biochemical reactions, such as carbon utilization and other metabolic tests. In small laboratory settings or in teaching laboratories, those assays are carried out using a limited number of test tubes. However, more modern systems, such as the one developed by Biolog, Inc., are based on panels of biochemical reactions performed simultaneously and analyzed by software. Biolog’s system identifies cells based on their ability to metabolize certain biochemicals and on their physiological properties, including pH and chemical sensitivity. It uses all major classes of biochemicals in its analysis. Identifications can be performed manually or with the semi- or fully automated instruments.

Another automated system identifies microorganisms by determining the specimen’s mass spectrum and then comparing it to a database that contains known mass spectra for thousands of microorganisms. This method is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and uses disposable MALDI plates on which the microorganism is mixed with a specialized matrix reagent (Figure 7.26). The sample/reagent mixture is irradiated with a high-intensity pulsed ultraviolet laser, resulting in the ejection of gaseous ions generated from the various chemical constituents of the microorganism. These gaseous ions are collected and accelerated through the mass spectrometer, with ions traveling at a velocity determined by their mass-to-charge ratio (m/z), thus, reaching the detector at different times. A plot of detector signal versus m/z yields a mass spectrum for the organism that is uniquely related to its biochemical composition. Comparison of the mass spectrum to a library of reference spectra obtained from identical analyses of known microorganisms permits identification of the unknown microbe.

A flowchart. A square plate with a grid of circles is labeled grow microorganisms on MALDI-plate. Then irradiate sample. Then use mass spectrometer to measure gaseous ions released. Then compare mass spectrum of sample to reference spectra; two small graphs are shown here. Then identify specied.
Figure 7.26 MALDI-TOF methods are now routinely used for diagnostic procedures in clinical microbiology laboratories. This technology is able to rapidly identify some microorganisms that cannot be readily identified by more traditional methods. (credit “MALDI plate photo”: modification of work by Chen Q, Liu T, Chen G; credit “graphs”: modification of work by Bailes J, Vidal L, Ivanov DA, Soloviev M)

Microbes can also be identified by measuring their unique lipid profiles. As we have learned, fatty acids of lipids can vary in chain length, presence or absence of double bonds, and number of double bonds, hydroxyl groups, branches, and rings. To identify a microbe by its lipid composition, the fatty acids present in their membranes are analyzed. A common biochemical analysis used for this purpose is a technique used in clinical, public health, and food laboratories. It relies on detecting unique differences in fatty acids and is called fatty acid methyl ester (FAME) analysis. In a FAME analysis, fatty acids are extracted from the membranes of microorganisms, chemically altered to form volatile methyl esters, and analyzed by gas chromatography (GC). The resulting GC chromatogram is compared with reference chromatograms in a database containing data for thousands of bacterial isolates to identify the unknown microorganism (Figure 7.27).

A flowchart. A bacterial culture is grown (image is of an agar plate). Then fatty acids are extracted and converted to methyl esters (image is of a test tube). Then gas chromatography analyzes methyl ester fingerprints (image is of a chromatography machine). Then bacteria are identified (image is of a graph). The X axis of the graph is column retention time. The Y axis is of detector signal. The line has various peaks.
Figure 7.27 Fatty acid methyl ester (FAME) analysis in bacterial identification results in a chromatogram unique to each bacterium. Each peak in the gas chromatogram corresponds to a particular fatty acid methyl ester and its height is proportional to the amount present in the cell. (credit “culture”: modification of work by the Centers for Disease Control and Prevention; credit “graph”: modification of work by Zhang P. and Liu P.)

A related method for microorganism identification is called phospholipid-derived fatty acids (PLFA) analysis. Membranes are mostly composed of phospholipids, which can be saponified (hydrolyzed with alkali) to release the fatty acids. The resulting fatty acid mixture is then subjected to FAME analysis, and the measured lipid profiles can be compared with those of known microorganisms to identify the unknown microorganism.

Bacterial identification can also be based on the proteins produced under specific growth conditions within the human body. These types of identification procedures are called proteomic analysis. To perform proteomic analysis, proteins from the pathogen are first separated by high-pressure liquid chromatography (HPLC), and the collected fractions are then digested to yield smaller peptide fragments. These peptides are identified by mass spectrometry and compared with those of known microorganisms to identify the unknown microorganism in the original specimen.

Microorganisms can also be identified by the carbohydrates attached to proteins (glycoproteins) in the plasma membrane or cell wall. Antibodies and other carbohydrate-binding proteins can attach to specific carbohydrates on cell surfaces, causing the cells to clump together. Serological tests (e.g., the Lancefield groups tests, which are used for identification of Streptococcus species) are performed to detect the unique carbohydrates located on the surface of the cell.

Clinical Focus

Resolution

Penny stopped using her new sunscreen and applied the corticosteroid cream to her rash as directed. However, after several days, her rash had not improved and actually seemed to be getting worse. She made a follow-up appointment with her doctor, who observed a bumpy red rash and pus-filled blisters around hair follicles (Figure 7.28). The rash was especially concentrated in areas that would have been covered by a swimsuit. After some questioning, Penny told the physician that she had recently attended a pool party and spent some time in a hot tub. In light of this new information, the doctor suspected a case of hot tub rash, an infection frequently caused by the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that can thrive in hot tubs and swimming pools, especially when the water is not sufficiently chlorinated. P. aeruginosa is the same bacterium that is associated with infections in the lungs of patients with cystic fibrosis.

The doctor collected a specimen from Penny’s rash to be sent to the clinical microbiology lab. Confirmatory tests were carried out to distinguish P. aeruginosa from enteric pathogens that can also be present in pool and hot-tub water. The test included the production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42 °C. Cetrimide is a selective agent that inhibits the growth of other species of microbial flora and also enhances the production of P. aeruginosa pigments pyocyanin and fluorescein, which are a characteristic blue-green and yellow-green, respectively.

Tests confirmed the presence of P. aeruginosa in Penny’s skin sample, but the doctor decided not to prescribe an antibiotic. Even though P. aeruginosa is a bacterium, Pseudomonas species are generally resistant to many antibiotics. Luckily, skin infections like Penny’s are usually self-limiting; the rash typically lasts about 2 weeks and resolves on its own, with or without medical treatment. The doctor advised Penny to wait it out and keep using the corticosteroid cream. The cream will not kill the P. aeruginosa on Penny’s skin, but it should calm her rash and minimize the itching by suppressing her body’s inflammatory response to the bacteria.

Skin with red raised bumps.
Figure 7.28 Exposure to Pseudomonas aeruginosa in the water of a pool or hot tub can sometimes cause a skin infection that manifests as “hot tub rash.” (credit: modification of work by “Lsupellmel”/Wikimedia Commons)

Go back to the previous Clinical Focus box.

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