Microbiology for the Health Sciences

Chapter 4. Diversity of Microorganisms

Part 1: Acellular and Procaryotic Microbes

Chapter 4 Outline
Categories of Microorganisms Acellular Infectious Agents

Viruses
Viroids and Prions The Domain Bacteria

Characteristics
Unique Bacteria Photosynthetic Bacteria The Domain Archaea

Categories of Microorganisms
Microbiology is the study of microorganisms. Microorganisms can be divided into those that are truly cellular (bacteria, archaeans, algae, protozoa and fungi) and those that are acellular (viruses, viroids and prions). Cellular microorganisms can be divided into those that are procaryotic (bacteria and archaeans) and those that are eucaryotic (algae, protozoa, and fungi).

Viruses, viroids and prions are often referred to as infectious agents or infectious particles.

Acellular Infectious Agents

Viruses Complete virus particles are called virions.

Most viruses are from 10 to 300 nm in diameter.

Viruses infect humans, animals, plants, fungi, protozoa, algae and bacterial cells.

Some viruses, called oncogenic viruses or oncoviruses, cause specific types of cancer.
A typical virion consists of a genome of either DNA or RNA, surrounded by a capsid (protein coat) which is composed of protein units called capsomeres. Some viruses (enveloped viruses) have an outer envelope composed of lipids and polysaccharides.

Acellular Infectious Agents (continued)

Viruses have 5 properties that distinguish them from living cells: 1. They possess either DNA or RNA living cells possess both. 2. They are unable to replicate on their own.

3. Unlike cells, they do not divide by binary fission, mitosis or meiosis.

4. They lack the genes and enzymes necessary for energy production.

5. They depend on the ribosomes, enzymes and metabolites of the host cell for protein and nucleic acid production.

Model of an icosahedral virus: adenovirus

Acellular Infectious Agents (continued)

Viruses are classified by: Type of genetic material (either DNA or RNA) Shape and size of capsid Number of capsomeres Presence or absence of an envelope Type of host it infects Disease it produces Target cell Immunologic/antigenic properties

Acellular Infectious Agents (continued)

Viruses
There are 4 categories of viruses, based on the type of nucleic acid they possess. Most viral genomes are of the first two types. Double-stranded DNA viruses Single-stranded RNA Single-stranded DNA Double-stranded RNA Most viral genomes are circular molecules, but some are linear.

Herpesviruses acquiring their envelopes as they leave a host cells nucleus by budding.

Comparative sizes of virions, their nucleic acids and bacteria.

Acellular Infectious Agents (continued)

Viruses
Origin of Viruses

One theory states that viruses represent ancient derivatives of degenerate cells or cell fragments.
Most scientists agree that viruses are nonliving entities. Bacteriophages Viruses that infect bacteria are known as bacteriophages or simply, phages. Virulent bacteriophages always cause what is known as the lytic cycle, which ends with the destruction of the bacterial cell.

A partially lysed cell of Vibrio cholerae with attached virions of phage CP-T1.

The bacteriophage T4 is an assembly of protein components.

Viral DNA enters the cell through the core.

20 facets, filled with DNA

Acellular Infectious Agents (continued)

Animal Viruses The steps in multiplication of animal viruses are: Attachment Penetration Uncoating Biosynthesis Assembly Inclusion bodies are remnants or collections of viruses; often seen in infected cells and used as a diagnostic tool to identify particular viral diseases.

Multiplication of Herpes Simplex on HeLa Cells

Adsorption

Penetration

Penetration

Penetration

Uncoating

Uncoating

Uncoating

Acellular Infectious Agents (continued)

Latent Virus Infections Viral infections in which the virus is able to hide from a hosts immune system by entering cells and remaining dormant.

Herpes viral infections are examples.

Once acquired, herpes virus infections (e.g., those that cause cold sores, genital herpes, and chickenpox/shingles) never completely go away; for example, chickenpox may be followed, years later, by shingles - both the result of the same virus.

Acellular Infectious Agents (continued)

Antiviral Agents Antibiotics are not effective against viral infections.

Antiviral agents are drugs that are used to treat viral infections.
These agents interfere with virus-specific enzymes and virus production by disrupting critical phases in viral multiplication or inhibiting synthesis of viral DNA, RNA, or proteins.

Acellular Infectious Agents (continued)

Oncogenic Viruses or Oncoviruses Viruses that cause cancer.

Examples include Epstein-Barr virus, human papillomaviruses and HTLV-1.

Human Immunodeficiency Virus (HIV)

The cause of acquired immunodeficiency syndrome (AIDS).

It is an enveloped, double-stranded RNA virus. The primary targets for HIV are CD4+ cells.

Human Immunodeficiency Virus (HIV)

Acellular Infectious Agents (continued)

Viroids and Prions (smaller and less complex infectious agents than viruses) Viroids

Viroids are short, naked fragments of singlestranded RNA, which can interfere with the metabolism of plant cells.
Viroids are transmitted between plants in the same manner as viruses. Examples of plant diseases caused by viroids: potato spindle tuber, and citrus exocortis.

Acellular Infectious Agents (continued)

Prions Prions are small infectious proteins that cause fatal neurologic diseases in animals; examples: Scrapie, Bovine Spongiform Encephalopathy (Mad Cow Disease) and Creutzfeldt-Jacob disease. Of all pathogens, prions are the most resistant to disinfectants.

The mechanism by which prions cause disease remains a mystery.

The Domain Bacteria Characteristics

Bacteria are divided into 3 major phenotypic categories: Those that are Gram-negative and have a cell wall. Those that are Gram-positive and have a cell wall. Those that lack a cell wall. Characteristics of bacteria used in classification and identification include: cell morphology, staining reactions, motility, colony morphology, atmospheric requirements, nutritional requirements, biochemical and metabolic activities, enzymes that the organism produces, pathogenicity and genetic composition.

The Domain Bacteria Cell Morphology

There are 3 basic shapes of bacteria: Cocci (round bacteria) Bacilli (rod-shaped bacteria) Curved and spiral-shaped bacteria Cocci may be seen singly or in pairs (diplococci), chains (streptococci), clusters (staphylococci), packets of 4 (tetrads), or packets of 8 (octads). The average coccus is about 1 m in diameter. Some cocci have coccus in their name.

Morphologic Arrangements of Cocci

Gram-positive Staphylococcus aureus in clusters.

SEM of Streptococcus mutans illustrating cocci in chains.

Diagram Showing Various Forms of Bacteria

The Domain Bacteria Cell Morphology (continued)

Bacilli
Often referred to as rods; may be short or long, thick or thin, and pointed or with curved or blunt ends. They may occur singly, in pairs (diplobacilli), in chains (streptobacilli), in long filaments, or branched. An average sized bacillus is 1 x 3 m. Extremely short bacilli are called coccobacilli. Examples of medically important bacilli: Escherichia, Klebsiella, and Proteus spp. Pseudomonas, Haemophilus, and Bacillus spp.

The Domain Bacteria Cell Morphology (continued)

Curved and Spiral-Shaped Bacteria Examples of curved bacteria: Vibrio spp. Campylobacter spp.

Helicobacter spp.
Examples of spiral-shaped bacteria: Treponema spp.

Borrelia spp.

Spiral-Shaped Bacteria

Borrelia hermsii in stained blood smear; a cause of relapsing fever.

Domain Bacteria Staining Procedures

Three Categories of Staining Procedures 1. Simple stains 2. Structural staining procedures Capsule stains Spore stains Flagella stains 3. Differential staining procedures Gram and acid-fast staining procedures.

Simple Bacterial Staining Technique

Domain Bacteria The Gram Staining Procedure

Divides bacteria into 2 major groups: Gram-positive (blue-to-purple) Gram-negative (pink-to-red) The final Gram reaction (positive or negative) depends upon the organisms cell wall structure. The cell walls of Gram-positive organisms have a thick layer of peptidoglycan, making it difficult to remove the crystal violet-iodine complex. Gram-negative organisms have a thin layer of peptidoglycan, making it easier to remove the crystal violet; the cells are subsequently stained with safranin.

Various Gram-Positive Bacteria

Chains of streptococci in smear from broth culture.

Streptococcus pneumoniae in blood culture.

Various Gram-Positive Bacteria

Spores

A bacillus, Clostridium perfringens, in a smear from a broth culture.

Clostridium tetani in a smear from a broth culture (note terminal spores on some cells).

Various Gram-Positive Bacteria

Epithelial cells

Many Gram-positive bacteria

Gram-Negative Bacteria

Gram-negative bacilli in a smear from a bacterial colony.

Loosely coiled Gram-negative spirochetes, Borrelia burgdorferi, the cause of Lyme disease.

Domain Bacteria Staining Procedures (continued)

Some bacteria are neither consistently purple nor pink after Gram staining; they are known as Gram-variable bacteria; example Mycobacterium spp.

Mycobacterium spp. are often identified using the acidfast stain.

The acid-fast stain

Carbol fuchsin is the red dye that is driven through the bacterial cell wall.
Heat is used to soften the waxes in the cell wall Because mycobacteria are not decolorized by the acid-alcohol mixture, they are said to be acid-fast.

Domain Bacteria Acid-Fast Mycobacteria

Many acid-fast mycobacteria in a liver biopsy.

Acid-fast bacilli in a digested sputum specimen.

Domain Bacteria Motility

If a bacterium is able to swim, it is said to be motile. Bacterial motility is most often associated with flagella or axial filaments.

Most spiral-shaped bacteria and about 50% of bacilli are motile; cocci are generally nonmotile.
Motility can be demonstrated by stabbing the bacteria into a tube of semisolid medium or by using the hangingdrop technique.

Semisolid Agar Method for Determining Motility

Nonmotile Motile

Hanging-Drop Prep for Study of Living Bacteria

Depression slide

Depression slide with coverglass

Side view of hanging-drop prep.

Domain Bacteria Colony Morphology

A bacterial colony contains millions of organisms. Colony morphology (appearance of the colony) varies from one species to another. Colony morphology includes: size, color, overall shape, elevation and the appearance of the edge or margin of the colony.

Colony morphology also includes the results of enzymatic activity on various types of media.
As is true for cell morphology and staining characteristics, colony morphology is an important clue to the identification of bacteria.

Size of colonies is determined by the organisms generation time and is another important characteristic of a particular bacterial species.

Formation of a bacterial colony on solid growth medium.

Domain Bacteria Atmospheric Requirements

Bacteria can be classified on the basis of their atmospheric requirements, including their relationship to O2 and CO2 With respect to O2, bacterial isolates can be classified as: Obligate aerobes Microaerophilic aerobes Facultative anaerobes Aerotolerant anaerobes Obligate anaerobes Capnophilic organisms grow best in the presence of increased concentrations of CO2 (5 to 10%).

Domain Bacteria Nutritional Requirements

All bacteria need some form of the elements carbon, hydrogen, oxygen, sulfur, phosphorus and nitrogen for growth. Some bacteria require special elements (e.g., calcium, iron or zinc). Organisms with especially demanding nutritional requirements are said to be fastidious (fussy).

The nutritional needs of a particular organism are usually characteristic for that species and are sometimes important clues to its identity.

Domain Bacteria Biochemical and Metabolic Activities

As bacteria grow, they produce many waste products and secretions, some of which are enzymes. Pathogenic strains of many bacteria, like staphylococci and streptococci, can be tentatively identified by the enzymes they secrete. In particular environments, some bacteria produce gases such as carbon dioxide or hydrogen sulfide.

To identify bacteria in the lab, they are inoculated into various substrates (i.e., carbohydrates and amino acids) to determine whether they possess the enzymes necessary to break down those substrates.

Domain Bacteria Pathogenicity

Many pathogens are able to cause disease because they possess capsules, pili, or endotoxins, or because they secrete exotoxins and exoenzymes that damage cells and tissues.

Frequently, pathogenicity is tested by injecting the organism into mice or cell cultures.
Examples of some common pathogenic bacteria:

Neisseria meningitidis, Salmonella typhi, Shigella spp., Vibrio cholerae, Yersina pestis, Treponema pallidum.

Domain Bacteria Genetic Composition

Identification of bacteria is moving toward analyzing the organisms DNA or RNA techniques collectively referred to as molecular diagnostic procedures. The composition of the genetic material (DNA) of an organism is unique to each species. DNA probes make it possible to identify an isolate without relying on phenotypic characteristics.

Through the use of 16S rRNA sequencing, the degree of relatedness between 2 different bacteria can be determined.

Unique Bacteria
Rickettsias, chlamydias and mycoplasmas are bacteria, but do not possess all the attributes of typical bacterial cells. Rickettsias and chlamydias have a Gram-negative type of cell wall and are obligate intracellular pathogens (i.e., they must live within a host cell). Rickettsias do not grow on artificial culture media; they have leaky membranes. Chlamydias are energy parasites, meaning they prefer to use ATP molecules produced by their host cell.

Rickettsia prowazekii, the cause of epidemic louseborne typhus.

Unique Bacteria (continued)

Mycoplasmas Smallest of the cellular microbes. Lack a cell wall and therefore assume many shapes (they are pleomorphic).

In humans, pathogenic mycoplasmas cause primary atypical pneumonia and genitourinary infections.
Because they have no cell wall, they are resistant to drugs like penicillin that attack cell walls. They produce tiny fried egg colonies on artificial media.

SEM of Mycoplasma pneumoniae

Photosynthetic Bacteria
Photosynthetic bacteria include purple bacteria, green bacteria and cyanobacteria; they all use light as an energy source, but not in the same way. Purple and green bacteria do not produce oxygen, whereas cyanobacteria do. Photosynthesis that produces oxygen is called oxygenic photosynthesis.

Photosynthesis that does not produce oxygen is called anoxygenic photosynthesis.

The Domain Archaea

Archaea (meaning ancient) were discovered in 1977; they are procaryotic organisms. Genetically, archaeans are more closely related to eucaryotes than they are to bacteria. Archaeans vary widely in shape and live in extreme environments, such as extremely acidic, extremely hot and extremely salty environments.

Archaeans possess cell walls, but they do not have peptidoglycan (in contrast, all bacterial cell walls contain peptidoglycan).

Review of Key Points

Microbes are divided into those that are cellular and those that are acellular. Complete virus particles, called virions, may be distinguished from living cells because they possess either DNA or RNA never both. Viruses are classified by type of nucleic acid, shape of the capsid, size of the capsid, number of capsomeres, presence or absence of an envelope, type of hosts and host cells they infect, type of disease they cause, and antigenic properties.

Review of Key Points (continued)

Bacteriophages are viruses that infect bacteria. Viroids are infectious RNA molecules that interfere with the metabolism of plant cells.

Prions are infectious protein molecules that cause diseases in animals.

Characteristics used for identification and classification of bacteria include cell morphology, staining reaction, motility, colony morphology, atmospheric requirements, nutritional needs, biochemical and metabolic activities, pathogenicity, amino acid sequencing of proteins, and genetic composition.

Review of Key Points (continued)

The three basic shapes of bacteria are cocci, bacilli and curved and spiral-shaped bacteria Bacterial smears must be fixed before staining; using either heat-fixation or methanol-fixation. Most motile bacteria possess whip-like structures called flagella. A pile or mound of bacteria on the surface of a solid culture medium is referred to as a colony. Bacteria can be classified based on their oxygen requirements.

Review of Key Points (continued)

All bacteria need some form of the elements carbon, hydrogen, oxygen, sulfur, phosphorus, and nitrogen. Pathogenic bacteria may produce pili, capsules, endotoxin, extoxins, and exoenzymes that enable them to cause disease. Rickettsias, chlamydias, and mycoplasmas are rudimentary Gram-negative bacteria. Certain bacteria, including a group of bacteria referred to as cyanobacteria, are photosynthetic. Genetically, archaeans are more closely related to eucaryotic organisms than to bacteria.