microbio 3

Page 1: Title and Author

  • From normal flora to infectious disease

  • Associate Professor Li Zhang

  • School of Biotechnology and Biomolecular Sciences

  • University of New South Wales

  • Email: L.Zhang@unsw.edu.au

Page 2: Learning Outcomes

At the successful completion of this teaching activity, students will be able to:

  1. Appreciate the diversity of microorganisms associated with the skin and mucous membranes of the human body and their impact on pathogen isolation.

  2. Explain the fundamental principles behind collecting patient samples.

  3. Describe laboratory tests commonly used in diagnostic laboratories for the identification and characterization of infectious agents.

Page 3: Normal Flora

  • The human body is naturally colonized by various microorganisms known as normal flora, which typically do not cause disease.

  • Commensal microorganisms help prevent the colonization of pathogenic microorganisms.

  • Some normal flora microorganisms can become opportunistic pathogens under certain conditions.

  • They play a crucial role in pathogen isolation and detection.

    Normal flora are tiny living things, like bacteria, that naturally live on our bodies. They usually don’t make us sick and can actually help keep us healthy! They work like friendly helpers, preventing bad germs (the ones that can make us sick) from taking over. Sometimes, if someone is sick or their body's defenses are weak, these friendly bacteria can turn into 'bad' bacteria and cause problems. So, normal flora play an important role in keeping us safe from diseases and figuring out if we are sick.

Page 4: Bacterial Species on Human Body

  • Different body sites harbor distinct bacterial species:

    • Oral cavity: Various Streptococcus species (Gram-positive cocci).

    • Skin: Predominantly Staphylococcus epidermidis and Staphylococcus aureus (Gram-positive cocci).

    • Large intestine: Mainly Gram-negative rods such as Bacteroides species and Escherichia coli.

Page 5: Infectious Diseases

  • Infectious diseases are caused by infectious agents, mostly microorganisms including bacteria, viruses, and fungi.

Page 6: Diagnosis of Infectious Disease

  • Diagnosis involves:

    • Evaluating symptoms and signs.

    • Assessing patient history and conducting physical examinations.

    • Laboratory tests may be performed for confirmation in some cases.

Page 7: Laboratory Diagnosis of Infection

  • Clinicians must provide specimens to diagnostic laboratories.

  • Microbiologists are responsible for:

    • Identifying causative agents.

    • Testing the antibiotic susceptibilities of pathogens if necessary.

Page 8: Types of Samples for Microbial Tests

  • Types of specimens vary according to clinical situations:

    • Pneumonia: Sputum, blood sample.

    • COVID-19: Oropharyngeal swab or bilateral deep nasal swab.

    • Urinary tract infection: Mid-stream urine sample.

    • Gastroenteritis: Fecal samples.

    • Bacteremia: Blood sample.

    • Meningitis: Cerebrospinal fluid (CSF).

    • Wound infection: Exudates, pus.

Page 9: General Principles of Specimen Collection

  • Collect specimens ideally during the acute phase of the disease and before antibiotic administration, unless life-threatening conditions like meningitis are present.

  • Ensure appropriate type of specimen is collected: sputum or saliva?

  • Avoid contamination by following correct procedures, especially for blood samples.

  • Proper storage and transport of samples is crucial, e.g., urine samples.

Page 10: Proper Techniques for Specimen Collection

  • Clean the skin with an antibacterial swab prior to needle insertion to collect specimens, preventing contamination.

  • Do not touch:

    • The swabbed area.

    • The needle.

    • The top of the specimen container.

  • Use different types of growth mediums for various microorganisms.

Page 11: Diagnostic Laboratories

  • Upon arrival at the diagnostic lab, specimens and patient information are recorded, and the specimen is processed.

Page 12: Commonly Used Microbiological Laboratory Tests

  • Direct microscopy: Using stained or non-stained samples; Gram stain is a common method.

  • Bacterial cultivation and identification: Gram stain can also be performed on cultured bacteria, along with biochemical tests for identification.

  • Polymerase chain reaction (PCR).

  • Serology: Detection of antibodies in blood.

Page 13:

Gram Stain Process

The Gram stain is a laboratory technique used to identify and classify bacteria into two main groups: Gram-positive and Gram-negative. This classification is important because it helps in determining how bacteria respond to antibiotics and how they may cause disease. Here's how the process works, broken down step by step:

  1. Fixation: This is the first step where the bacterial sample is prepared. This usually involves applying heat to the slide, which kills the bacteria and helps them stick to the glass, making it ready for staining.

  2. Crystal Violet: This is the primary stain used in the Gram stain process. When the crystal violet dye is applied to the fixed bacterial sample, it penetrates the cells and colors them purple. All bacteria take up this dye initially.

  3. Iodine Treatment: After the crystal violet, iodine is added. Iodine acts like a fixer or a helper that locks the crystal violet dye inside the cells. This step is crucial because it forms a complex with the crystal violet, making it harder for the dye to wash away later.

  4. Decolorization: Next, a decolorizing agent (often alcohol or acetone) is applied. This step is key in differentiating between Gram-positive and Gram-negative bacteria:

    • Gram-positive bacteria have thick cell walls that hold onto the dye, so they remain purple.

    • Gram-negative bacteria, on the other hand, have thinner cell walls and lose the purple dye, becoming colorless after this step.

  5. Counterstain with Safranin: Finally, a secondary stain called safranin is added. This stains the now colorless Gram-negative bacteria pink. Gram-positive bacteria, which are still purple, do not take up the pink color because they already have the darker crystal violet stain.

In the end, under a microscope, you'll see purple (Gram-positive) and pink (Gram-negative) bacteria, which allows scientists and doctors to identify the type of bacteria present. This is a crucial step in diagnosing bacterial infections and deciding on the best treatment.

Page 14: Information Obtained from Gram Stain

  • Identification of bacteria as Gram-positive or Gram-negative.

  • Type of bacteria shape (coccus or rod).

    • Classified into four groups:

      • Gram-positive cocci

      • Gram-positive rods

      • Gram-negative cocci

      • Gram-negative rods

  • Immune cells may be visible when directly using clinical samples for Gram staining.

  • Note: Gram stain is not applicable for viruses.

Page 15: Bacterial Cell Wall

  • Reference for bacterial cell wall structure is cited from relevant literature on extracellular vesicles in Gram-positive bacteria and fungi.

Gram-positive and Gram-negative bacteria have distinct differences in their cell wall structures, which significantly impact their characteristics and response to antibiotics:

  1. Gram-positive Cell Walls

    • Structure: The cell wall is thick and primarily composed of peptidoglycan (up to 90%) which retains the crystal violet stain during the Gram staining process, making them appear purple.

    • Additional Components: They may also contain teichoic acids, which are essential for cell wall maintenance and integrity.

    • Function: The thick peptidoglycan layer provides structural support and protection from physical stresses. It also serves in retaining the dye during staining, crucial for identification.

  2. Gram-negative Cell Walls

    • Structure: The cell wall is thinner (approximately 10-20% peptidoglycan) and is located between an inner and outer membrane. The outer membrane contains lipopolysaccharides (LPS), which contribute to the bacterial virulence and can trigger strong immune responses.

    • Function: The thin peptidoglycan layer does not retain the crystal violet stain and instead takes up the counterstain (safranin), appearing pink. The outer membrane acts as a barrier to many substances, including antibiotics, contributing to antibiotic resistance.

In summary, the main differences lie in the thickness of the peptidoglycan layer and the presence of an outer membrane in Gram-negative bacteria, which affects their staining properties and susceptibility to antibiotics.

Page 16: Microscopic Examination of CSF

  • Examination of Gram-stained cerebrospinal fluid (CSF) reveals Gram-negative cocci and immune cells, as well as Gram-positive cocci.

  • Note: Gram stain is not typically used for fecal samples.

    Fecal samples are collected during diagnostic assessments to identify gastrointestinal infections, pathogens, and the presence of microorganisms. They are important for diagnosing conditions such as gastroenteritis and other digestive disorders.

Page 17: Bacterial Cultivation

  • Cultivated bacterial species help identify causative agents.

  • They can also be used for antibiotic sensitivity testing.

Page 18: Selective and Differential Media

  • MacConkey Agar: Designed for isolating enteric bacterial pathogens.

    • Contains basic nutrients, crystal violet (selective), bile salts (selective), lactose (differential), and neutral red (pH indicator).

    • Inhibits growth of Gram-positive bacteria and differentiates lactose fermenting from non-fermenting bacteria.

      • Lactose fermenter (LF): Escherichia coli

      • Non-lactose fermenter (NLF): Salmonella species

  • Clinical samples often come from sites with normal flora.

    Selective and differential media are specialized growth mediums used in microbiology to isolate and identify specific types of microorganisms. **Selective Media**: These media contain substances that inhibit the growth of certain bacteria while allowing others to grow. For instance, MacConkey Agar is selective for Gram-negative bacteria and inhibits Gram-positive bacteria through the inclusion of crystal violet and bile salts. **Differential Media**: These media contain specific ingredients that allow for the differentiation between various types of bacteria based on their biochemical characteristics. In MacConkey Agar, lactose serves as a differential component, permitting the identification of lactose fermenters (such as Escherichia coli) that produce acid and can change the pH indicator color, versus non-lactose fermenters (like Salmonella species) that do not acidify the medium and remain colorless. This combination helps isolate enteric bacterial pathogens effectively.

Page 19: Selective Media Considerations

  • No selective media is 100% effective; various bacteria may be present on culture plates.

  • For pure culture, typically need to subculture the target bacteria from a single colony to a fresh culture plate.

Page 20: Identification of Pure Cultures

Once a pure culture is obtained, identification methods include:

  1. Gram stain

  2. Biochemical characteristics (usually multiple tests)

  3. PCR (direct detection of pathogens in clinical samples)

Page 21: Biochemical Tests for Bacterial Identification

  • Catalase Test: Tests ability to decompose hydrogen peroxide to oxygen gas and water.

  • Coagulase Test: Tests coagulation of plasma (converts fibrinogen to fibrin).

Page 22: Carbohydrate Fermentation

  • Fermentation of carbohydrates by facultative anaerobic and anaerobic bacteria breaks down carbohydrates for energy, producing acid or acid plus gas as byproducts.

    • Negative Control: No fermentation activity

    • Positive Control: Fermentation observed

    • Example: Glucose fermentation.

Page 23: Polymerase Chain Reaction (PCR)

  • PCR amplifies specific DNA fragments for pathogen detection.

  • It is very sensitive and allows qualitative or quantitative detection of pathogens.

    **Polymerase Chain Reaction (PCR)**: PCR is like a copying machine for DNA! Imagine you have a tiny piece of a very important book, but the book is too small to read. PCR can take that little piece and make many more copies of it, so it’s easier to read and study. This helps scientists find out if germs (like viruses or bacteria) are making someone sick. When they use PCR, they can see if that tiny piece of DNA belongs to any harmful germs. It’s very good at finding even a small amount of germs in a sample, like blood or spit, which makes it super useful for doctors and scientists who want to know if someone has an infection.

Page 24: PCR Applications

  • PCR is often used for viral detection, such as for SARS-CoV2, HSV, and HPV.

  • Additionally used for some bacterial pathogens including Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycobacterium tuberculosis.

  • Despite PCR’s advantages, bacterial culture remains essential for some diagnostics.

    PCR (Polymerase Chain Reaction) is often used for virus detection due to its high sensitivity and ability to amplify small amounts of viral DNA or RNA, making it easier to identify infections such as SARS-CoV2, HSV, and HPV. This method allows for quicker results compared to traditional cultures. However, bacterial culture remains necessary for certain diagnostics because it not only confirms the presence of bacteria but also allows for antibiotic susceptibility testing. This is crucial in guiding treatment decisions, as some bacterial pathogens may not be detected by PCR or may require specific identification methods that culture provides.

Page 25: Antibiotic Sensitivity Testing

  • Measures bacteria's susceptibility to antibiotics via various methods, including disk diffusion.

  • Utilizes cultured bacteria to guide treatment decisions.

  • Culture and sensitivity tests typically require 48-72 hours, but in some cases may take longer.

Page 26: Serology Testing

  • Checks for antibodies in blood, primarily for diagnosing viral infections.

  • It may also support the detection of difficult-to-culture bacterial species.

  • Difference between serology tests and other methods discussed; acute infection indicated by specific antibody classes.

Page 27: Enzyme-Linked Immunosorbent Assay (ELISA)

  • Commonly used to measure antibody levels through antigen-antibody interactions.

  • An enzyme conjugated to one antibody catalyzes a substrate, resulting in a colored product for qualitative or quantitative measurements.

  • ELISA can also be employed to detect antigens. ELISA stands for Enzyme-Linked Immunosorbent Assay and has different types based on the detection method and the target. Here's a breakdown: 1. **Direct ELISA**: Utilizes a single antibody attached to an enzyme that binds directly to the target antigen. After binding, a substrate is added, resulting in a color change indicating the presence of the antigen. 2. **Indirect ELISA**: Involves two antibodies; the first (primary) binds to the target antigen, and the second (secondary) which is enzyme-conjugated, binds to the primary antibody. This amplification allows for increased sensitivity in detecting the target. 3. **Sandwich ELISA**: Requires two antibodies that bind to different sites on the target antigen. The antigen is 'sandwiched' between the two antibodies. This method is highly specific and typically used for detecting proteins that may be present in low quantities. 4. **Competitive ELISA**: Both the sample antigen and a labeled antigen compete to bind to a specific antibody. The amount of color change or signal output is inversely proportional to the concentration of the antigen in the sample. More antigen present in the sample results in less signal output. Each ELISA type serves specific purposes depending on the required sensitivity and specificity for detecting either antigens or antibodies.

    ### Enzyme-Linked Immunosorbent Assay (ELISA) **Overview**: ELISA is a widely used laboratory technique employed to detect and quantify specific proteins, such as antibodies or antigens, in a sample. It exploits the specificity of antigen-antibody interactions and involves enzyme-linked antibodies and substrates to generate measurable signals. **Principle**: ELISA utilizes the interaction between antigens and antibodies. When an antigen is introduced into a sample, it binds to specific antibodies. One of these antibodies is coupled to an enzyme that will facilitate a detectable reaction. **Detailed Steps**: 1. **Coating**: The wells of a microplate are coated with an antigen (for antibody detection) or an antibody (for antigen detection). This process immobilizes the protein of interest on the surface. 2. **Blocking**: To prevent non-specific binding, a blocking solution is added to cover potential binding sites not occupied by the targeted antibodies or antigens. 3. **Sample Addition**: The sample containing the antibodies (or antigens) is added to the wells. If the target protein is present, it will bind to the immobilized antigen (or antibody). 4. **Detection**: A second enzyme-linked antibody is introduced which binds to the target protein. This conjugated antibody is directed towards a different epitope on the target than the immobilized one, ensuring specificity. 5. **Substrate Reaction**: A substrate specific to the conjugated enzyme is added. The enzyme catalyzes this substrate, leading to a color change or the formation of a detectable product. The intensity of the color change correlates with the amount of the target protein present in the original sample. 6. **Measurement**: The colored product is quantified using a spectrophotometer, measuring the absorbance, which gives a direct indication of the concentration of antibody or antigen in the sample. **Applications**: ELISA is versatile; it is not only used for measuring antibody levels but can also be applied to detect antigens directly, such as viral proteins in infectious diseases. This method is crucial in disease diagnosis and monitoring immune responses.

Page 28: Bacterial Growth Identification

  • Bacterial growth characteristics can assist in identifying species.

  • Hemolysis:

    • Streptococcus pyogenes (beta-hemolysis on horse blood agar)

    • Classification of hemolytic activity:

      • Alpha (greenish)

      • Beta (transparent)

      • Gamma (no hemolysis).

Hemolytic Activity:

  • Definition: Hemolytic activity refers to the ability of certain bacteria, particularly streptococci, to lyse red blood cells and produce changes in the appearance of blood agar. This property is often used to help identify bacterial species.

  • Classification of Hemolytic Activity:

    • Alpha Hemolysis: Produces a greenish discoloration around the colony due to partial lysis of red blood cells; an example is Streptococcus pneumoniae.

    • Beta Hemolysis: Results in a clear, transparent zone around the colony indicating complete lysis of red blood cells; an example is Streptococcus pyogenes.

    • Gamma Hemolysis: Indicates no hemolysis, meaning the bacteria do not lyse red blood cells; an example is Enterococcus faecalis.

  • Use in Identification: Evaluating hemolytic activity on blood agar helps microbiologists differentiate between species of bacteria, especially in clinical samples where specific identification is crucial for treatment decisions.

Page 29: Case Study - Ryan's Knee

  1. Why did Ryan’s wound get infected?

    • Damage to the skin barrier allowed skin microorganisms to enter tissues.

  2. Likely microorganisms causing the infection:

    • Skin bacterial flora: Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis.

Page 30: Identifying Pathogens in Ryan's Case

  1. How can you identify causative pathogens?

    • Collect wound exudates for bacterial culture.

    • Perform subculture to obtain a single colony.

    • Utilize Gram stain and biochemical tests:

    • Staphylococcus aureus: Catalase +, Coagulase +

    • Streptococcus pyogenes: Catalase -, Coagulase -

    • Staphylococcus epidermidis: Catalase +, Coagulase -

  2. Need for microorganism identification:

    • To guide antibiotic treatment through sensitivity testing.

  3. Timing of sample collection in relation to antibiotic administration:

    • Sample collected before antibiotics given.

Page 31: Summary of Key Points

  • Normal flora (oral cavity, skin, large intestine) and their impact on diagnostic microbiology.

  • Importance of selective and differential media, e.g., MacConkey agar.

  • General principles of specimen collection: blood, urine, and other samples.

  • Overview of several detection and isolation methods employed in diagnostic laboratories.

  • Specific case study: Ryan’s skin infection.

Facultative Anaerobic vs. Anaerobic Bacteria:

  1. Facultative Anaerobic Bacteria:

  • These bacteria can grow in both the presence and absence of oxygen.

  • When oxygen is available, they utilize aerobic respiration; in the absence of oxygen, they can switch to fermentation or anaerobic respiration for energy.

  • Common examples include Escherichia coli which can thrive in aerobic conditions but switch to fermentation when oxygen is lacking.

  1. Anaerobic Bacteria:

  • These bacteria can only grow in environments devoid of oxygen.

  • They rely exclusively on anaerobic respiration or fermentation for energy production, as oxygen is toxic to them.

  • Examples include Clostridium species, which thrive in anaerobic conditions and can be harmful in certain infections.