General Concepts in Bacteriology (Part 1) PDF

General Concepts in Bacteriology (Part 1) Module 01: Principles & Perspectives II Jhason John J. Cabigon, MD | Asynchronous TABLE OF CONTENTS I. BACTERIAL CELL STRUCTURE................................................................... 1 A. CYTOPLASMIC COMPONENTS OF PROKARYOTES.......................

General Concepts in Bacteriology (Part 1) Module 01: Principles & Perspectives II Jhason John J. Cabigon, MD | Asynchronous TABLE OF CONTENTS I. BACTERIAL CELL STRUCTURE................................................................... 1 A. CYTOPLASMIC COMPONENTS OF PROKARYOTES............................... 1 B. CELL ENVELOPE...................................................................................2 C. CELL ENVELOPE: CELL MEMBRANE/PLASMA MEMBRANE................. 2 D. CELL ENVELOPE: CELL WALL............................................................... 2 E. OUTER SURFACE COMPONENTS......................................................... 5 II. BASIS OF BACTERIAL CLASSIFICATION..................................................... 6 A. GROWTH CHARACTERISTICS...............................................................6 B. MORPHOLOGIC CHARACTERISTICS.....................................................7 C. BIOCHEMICAL CHARACTERISTICS....................................................... 9 III. BACTERIAL CLASSIFICATION AND SUBTYPING...................................... 11 A. MAJOR BACTERIAL CLASSIFICATIONS............................................... 11 B. BACTERIAL SUBTYPING..................................................................... 11 QUESTIONS.............................................................................................. 12 ANSWER KEY............................................................................................ 13 RATIONALE...............................................................................................13 LEARNING OBJECTIVES 1. To understand the characteristics of bacteria, the structure of bacteria, and the basis of Gram staining 2. To understand the methods of classification of bacteria I. BACTERIAL CELL STRUCTURE ● Bacterial microbes are made of prokaryotic cells ○ Prokaryotic cells are simpler than eukaryotic cells at every level ▸ Except for the cell envelope (i.e., cell envelope of prokaryotes are more complex) ○ Prokaryotic cells do not have a true nucleus ▸ Main difference of eukaryotes and prokaryotes ▸ Eukaryotes have a true nucleus – “Eu” means true; “karyo” means nucleus ○ Prokaryotes package their DNA in the nucleoid ○ Prokaryotes have no nuclear membrane and mitotic apparatus ○ Prokaryotes do not contain other membrane-bound organelles Figure 2. Prokaryotic and Eukaryotic Ribosome Subunits A. CYTOPLASMIC COMPONENTS OF PROKARYOTES ● Nucleoid ○ Contains circular DNA ○ Where DNA packaging occurs ● Ribosomes ○ Most numerous intracellular structure ○ Site of protein synthesis in all living organisms ○ All prokaryotes have 70S ribosomes ▸ Measured in Svedberg units (S) – Measures how fast molecules move in a centrifuge – It is not a mathematical formula ▸ Serves as the target of certain antibiotics ▸ Eukaryotes contain 80S ribosomes ▸ 70S ribosome subunits include: – 50S ▪ Contains 23S and 5S rRNA and other proteins ▪ Site of action of erythromycin and chloramphenicol – 30S ▪ Contains 16S rRNA ▪ Site of action of aminoglycosides (e.g. streptomycin, gentamycin) Figure 3. Prokaryotic and Eukaryotic Ribosome Subunits Figure 1. Color-Enhanced Transmission Electron Micrograph of E. coli (DNA Shown in Red) YL6:01.29 ● Plasmids ○ Extrachromosomal genetic component ○ Can replicate independently of the chromosomes ○ Typically, small, circular DNA strands that may encode genes to: TG05: Artillaga, Avila, Cheng, Escalante, Lim, Marcos, Palencia, Quimson, Remitio, Salvador, Sandoval CG14: Acuña, Antonio, Ballelos, Carreon, Diño, Go, Lim, Liwanag, Manuel, Meneses, Plurad, Ubaldo 1 ▸ Resist or degrade antibiotics ▸ Tolerate toxic metabolites ● Inclusions/Inclusion Bodies ○ Store reserve materials in the form of insoluble granules ○ Store energy or structural building blocks ● The tetrapeptide side chains and peptide cross bridges vary depending on the species B. CELL ENVELOPE ● Complex covering of prokaryotic cells that differ in composition among major groups ● Protect organism from hostile environments (e.g., extreme osmolarity, harsh chemicals, antibiotics) ● Cell is enveloped by (from inner to outer covering) ○ Cell membrane/Plasma membrane ○ Cell wall ○ Capsule ▸ Secreted by certain bacteria ▸ Not always found in all bacteria C. CELL ENVELOPE: CELL MEMBRANE/PLASMA MEMBRANE ● Innermost covering ● Major functions: ○ Selective permeability and transport of solutes ○ Electron transport and oxidative phosphorylation in aerobic species ▸ A functional analog of the mitochondrial membrane for energy or ATP generation ○ Excretion of hydrolytic exoenzymes ▸ E.g., harmful bacterial toxins ○ Biosynthetic function ▸ Contains enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids ○ Chemotactic functions ▸ Bears receptors and other proteins of the chemotactic and other sensory transduction systems Figure 4. Parts of a peptidoglycan molecule Polypeptide Backbone ● Composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM1) ● Connected by β1→4 linkages that are disrupted by lysozymes ● E.g., Lysozyme found in tears, sweat, and saliva Nice to Know! ● Because saliva contains lysozymes, it is part of our non-specific immunity ○ It does not cure infections ○ Do not spit on infected wounds D. CELL ENVELOPE: CELL WALL ● Serves as the basis for differentiation between Gram-positive and Gram-negative bacteria ○ Gram-positive bacteria have a thicker peptidoglycan layer than Gram-negative bacteria (30x thicker) ○ Gram-negative bacteria have an outer membrane lying outside the peptidoglycan layer ● Major functions: ○ Osmotic protection ▸ The high tensile strength of the cell wall prevents the bursting of the bacterial cell – Internal osmotic pressure of most bacteria is relatively high ○ Shape ○ Antigenic properties ▸ As seen in the cell wall of Gram-negative cells ▸ The site for major antigenic determinants including endotoxins STRUCTURAL COMPONENTS: PEPTIDOGLYCANS ● Aka murein or mucopeptide ● Cell wall strength is attributed to the presence of peptidoglycan ● 3 parts of Peptidoglycan molecule: ○ Polypeptide backbone ○ Tetrapeptide side chains ○ Peptide cross bridge ● The polypeptide backbone is the same for all bacteria YL6:01.29 General Concepts in Bacteriology (Part 1) Figure 5. Polypeptide backbone Tetrapeptide Side Chains and Peptide Cross Bridges ● Extend from the NAM subunits ● Most bacteria have the same polypeptides in the positions of 1, 2, and 4, while position 3 depends on the bacteria species ○ Position 1: L-alanine ○ Position 2: D-glutamate ○ Position 3: L-lysine for Gram-positive while diaminopimelic acid (DAP) for Gram-negative ○ Position 4: D-alanine ● Tetrapeptides can be directly cross-linked to one another by the peptide cross bridges ○ Stabilizes structure ○ Target of penicillin ● The fact that all peptidoglycan chains are cross-linked means that each layer is a single giant molecule ○ Especially in Gram-positive organisms 2 GRAM-POSITIVE BACTERIAL CELL WALL Figure 6. Tetrapeptide side chains GRAM STAINING ● Gram-staining procedure was named after histologist Hans Christian Gram ○ Difference in cell wall structure will determine if the bacterium is Gram-positive or Gram-negative ○ Developed to identify bacteria in infected tissue ● 4 reagents involved in Gram-staining procedure: ○ Crystal violet (primary stain) ○ Iodine (mordant) ○ Ethanol or acetone (decolorizer) ○ Safranin (counterstain) Figure 8. Gram-positive bacterial cell wall ● In Gram-positives, each peptidoglycan layer is a single giant molecule of crosslinked peptidoglycan chains Teichoic Acid and Teichuronic Acid ● Most Gram-positive cell walls contain considerable amounts of teichoic and teichuronic acid ○ Compose 50% dry weight of cell wall ● Both are similar polymers to one another other than: ○ Teichoic Acids contain repeating units of phosphate ○ Teichuronic acids contain repeating units of sugar acids ● Teichuronic acids are synthesized in place of teichoic acids when phosphate is limited ● There are two types of teichoic acids: ○ Wall teichoic acid (WTA) ○ Membrane teichoic acid/lipoteichoic acid (LTA) Table 1. Two types of teichoic acids Type Figure 7. Gram-staining procedure (Spherical microbes: Gram-positive; Rod-shaped microbes: Gram-negatives) Gram Staining Process 1. Addition of Crystal Violet ○ All bacteria, whether Gram positive or Gram negative, will stain violet at this step 2. Addition of Iodine (mordant) ○ Forms crystal violet-iodine complexes to ensure that the dye will not be removed easily 3. Treatment with Alcohol or Acetone ○ Gram-positive cells contain highly cross-linked layers of peptidoglycan ▸ Crystal violet-iodine complexes remain in that layer despite addition of alcohol ○ Gram-negative cells have an outer membrane and only a single layer of peptidoglycan ▸ Addition of alcohol will dehydrate the outer membrane ▸ Crystal violet-iodine complexes are washed away and leave Gram-negatives colorless, and can now take up safranin (red) 4. Safranin (1.32, 2025) ○ Stains pink or red on Gram-negative cells ▸ Gram-negative cells stain pink because it can take up the counterstain after alcohol treatment ○ Peptidoglycan layer of Gram-positive cells remain violet YL6:01.29 General Concepts in Bacteriology (Part 1) Characteristics Wall Teichoic Acid (WTA) ● Covalently linked to peptidoglycan layer ● Limited to cell wall Membrane Teichoic Acid / Lipoteichoic Acid (LTA) ● Covalently linked to membrane glycolipids ● Extends to cell membrane ● Intimately associated with lipids Figure 9. Two types of teichoic acids ● Functions: ○ Cation exchange ○ Tensile strength ○ Elasticity 3 ○ Antigen determinant ▸ In Streptococcus pneumoniae: Teichoic Acids (WTA) bare the antigenic determinants called Forssman antigen ▸ In Streptococcus pyogenes: Lipoteichoic Acid (LTA) helps to facilitate microbial attachment to animal cells Table 2. Three main components of LPS Component Lipid A ● Glycolipid ● Embedded in outer membrane ● Associated with toxicity when LPS is split (i.e. fever, blood clot) Core polysaccharide ● Provides stability O antigen ● Covers bacterial surface ● Highly variable among species and strains ● Used for antigen specificity Polysaccharides ● Found in some Gram-positive cell walls ● Yielded from hydrolysis of Gram-positive walls Characteristics GRAM-NEGATIVE BACTERIAL CELL WALL Figure 10. Gram-negative bacterial wall ● Main difference with Gram-positive cell walls: the Gram-negative cell wall consists of a single peptidoglycan layer and an outer membrane Outer Membrane Figure 11. Liposaccharide components (LPS) ● Lies outside the peptidoglycan layer ● Presence of lipopolysaccharide component (LPS) in the outer membrane makes it asymmetrical ○ Unlike the symmetrical bilayer of the inner cell/plasma membrane ● Excludes hydrophilic molecules due to its lipid nature ● Can exclude hydrophobic molecules ○ Unusual for biologic membranes ○ Protects cell from hydrophobic molecules ▸ Important in protecting enteric bacteria (i.e., intestinal bacteria) from deleterious substances (i.e., bile salts) ● Contains porins ○ Allow passive diffusion of low molecular weight and hydrophilic compounds ● Large antibiotic molecules penetrate the outer membrane relatively slow ○ Accounts for relatively high resistance of Gram-negative bacteria to some antibiotics Lipoprotein ● Provides stability ● Anchors outer membrane to peptidoglycan layer ○ Crosslinks the outer membrane and peptidoglycan layers Periplasmic Space ● Contains peptidoglycan layer and gel-like solution of proteins ● Periplasmic proteins include: ○ Binding proteins ○ Hydrolytic enzymes ○ Detoxifying enzymes (e.g. B-lactamases that inactivate certain antibiotics) Lipopolysaccharide Component (LPS) ● Endotoxin of Gram-negative bacteria cell wall ● Extremely toxic to animals because it is firmly bound to cell surface ○ Released only when the cell is lysed ● LPS has 3 main components: ○ Lipid A ○ Core polysaccharide ○ O antigen YL6:01.29 General Concepts in Bacteriology (Part 1) Figure 12. Lipoprotein and periplasmic space in Gram-negative cell wall SUMMARY: GRAM-POSITIVE VS. GRAM-NEGATIVE Table 3. Gram-positive vs Gram-negative cell walls Gram-Positive Gram-Negative Color of Gram-Stained Cell Purple Reddish-pink Representative Genera Bacillus Staphylococcus Streptococcus Escherichia Neisseria Pseudomonas 4 ○ Causes tuberculosis ○ Mycolic acid is responsible for “acid-fastness” characteristic of M. tuberculosis Distinguishing Structures/Components Peptidoglycan Thick layer Thin layer Teichoic acids Present Absent Outer membrane Absent Present Lipopolysaccharide (endotoxin) Absent Present Porin proteins Absent (unnecessary because there is no outer membrane) Present; allow passage of molecules through outer membrane Periplasm Absent Present Sensitivity to penicillin* Generally more susceptible (with notable exceptions) Generally less susceptible (with notable exceptions) Sensitivity to lysozyme Yes No General Characteristics Mycoplasma ● Bacteria that lack a cell wall ● Clinical significance: Resistant to cell wall-inhibiting antibiotics ○ Some antibiotics (e.g., penicillin family) target the peptidoglycan layer of the cell wall E. OUTER SURFACE COMPONENTS GLYCOCALYX ● ● ● ● An extracellular polysaccharide Secretion of bacteria Secreted in the environment Aids in adherence to surfaces Forms ● Capsule ○ Condensed, well-defined, closely surrounds the cells, and excludes particles ○ Contributes to the invasiveness of the organism ○ Protects encapsulated cell from phagocytosis ● Slime layer (Biofilm) ○ Loosely associated with the cell and does not exclude particles *Examples of commonly used penicillin: Penicillin V, Amoxicillin, Ampicillin SPECIAL CONSIDERATIONS ● Some bacteria have special structures requiring special stains ○ Acid fast ○ Mycoplasma Figure 14. Two forms of the glycocalyx Acid Fast FLAGELLA ● ● ● ● Thread-like appendages composed entirely of protein Organs of locomotion Respond to changes in the environment Highly antigenic ○ Called H antigens ○ Trigger immune response that is directed against proteins Types of Arrangement ● Monotrichous: single polar flagellum ● Lophotrichous: multiple polar flagella ● Amphitrichous: single flagellum found on each of the two opposite poles ● Peritrichous: multiple flagella distributed over the entire cell Figure 13. Acid-fast cell wall structure ● Cell wall is made of large amounts of mycolic acid ○ Mycolic acid: waxes, complex-branched hydrocarbons ▸ Inner part is linked to arabinogalactan ▸ Outer part is linked to lipids ● Hydrophobic structure renders acid-fast bacteria resistant to harsh chemicals (e.g. detergents, strong acids) ○ When stained through the acid-fast procedure, the specimen resists decolorization with acid-alcohol ● Best example: Tubercle bacillus ○ Also known as Mycobacterium tuberculosis YL6:01.29 General Concepts in Bacteriology (Part 1) Figure 15. Types of arrangements of flagella 5 FIMBRIAE / PILI ● Rigid surface appendages ● Shorter and thinner than flagella ● Contain adhesins ○ Proteins located at the tip of the fimbriae ○ Responsible for attachment ● Designed for adherence and conjugation ○ Not designed for movement except in certain organisms (e.g., Pseudomonas) ▸ In Pseudomonas: twitching movement – The tips strongly adhere to surfaces – The pili retracts inside the cell – The bacteria is pulled towards the adherent tip, resulting in twitching ● Has antigenic properties ○ Elicits formation of antibodies by the host ● Inhibits phagocytic ability of leukocytes 4. Which flagellar arrangement is characterized by a single flagellum found on each of the two opposite poles? a. Peritrichous b. Amphitrichous c. Monotrichous d. Lophotrichous Answers: 1B, 2F, 3F, 4B II. BASIS OF BACTERIAL CLASSIFICATION A. GROWTH CHARACTERISTICS ● In a culture medium, will help in identifying the organism through colony characteristics and pigmentation ● Motile organisms can be differentiated from non-motile organisms ● Type of culture media used is vital in identification COMMONLY USED CULTURE MEDIA ● Chocolate Agar Plate (CAP) ○ Non-selective ○ H. influenzae can be grown in CAP ▸ Fastidious organism ▸ Appears as round, smooth, convex, gray and opaque colonies Figure 16. Fimbriae or pili ENDOSPORES ● “Resting” state: cannot reproduce ● Two most common Gram-positive rods that are spore formers are: ○ Bacillus ○ Clostridium ● Sporulation ○ Triggered by the depletion of key nutrients ▸ E.g., Carbon and nitrogen ○ Each cell forms a single internal spore ▸ Mother cell will undergo autolysis, releasing the spore ● Spore ○ Resting cell ○ Highly resistant to desiccation, heat, and chemical agents ○ When returned to favorable nutritional conditions, spores will germinate back into their vegetative state ▸ The vegetative state is usually killed at 70°C but spores are NOT Figure 17. Chocolate Agar Plate (CAP). Shows positive growth of H. influenzae in CAP ● MacConkey Agar ○ Differential ○ Can be used to differentiate: ▸ Gram positives vs. Gram negatives – Gram positive bacteria (e.g. Staphylococcus aureus) do not grow on MacConkey agar ▪ Bile salts inhibit the growth of Gram-positive bacteria (01.28a, 2026) ▸ Lactose fermenting colonies vs. non-lactose fermenting colonies among Gram negatives – Pink colonies will indicate growth of Gram-negative lactose fermenters – White colonies will indicate non-fermenters Active Recall Box 1. Prokaryotes have what kind of ribosomes? a. 80S b. 70S c. 50S d. 30S 2. T/F: Most bacteria have the same polypeptides in the positions of L-alanine, D-glutamate, and L-lysine. 3. T/F: A Gram-negative cell wall has no outer membrane. Figure 18. MacConkey Agar. Shows differential results per bacterial species YL6:01.29 General Concepts in Bacteriology (Part 1) 6 ● Blood Agar Plate (BAP) ○ Usually non-selective ○ Can be used to differentiate Streptococcus spp. ▸ Coccobacilli: bacilli that is oval ○ Spiral: comes in three forms ▸ Vibrio: curved or comma shaped ▸ Spirilla: thick, rigid spiral ▸ Spirochetes: thin, flexible spirals Figure 19. Blood Agar Plate (BAP) for Hemolysis of Streptococcus spp.: α-hemolysis (S. mitis, left); ß-hemolysis (S. pyogenes, middle); γ-hemolysis or non-hemolytic (S. salivarius, right) MOTILITY Figure 22. Shapes of bacteria ● Can be demonstrated using semisolid culture media ○ Sample is inoculated in a straight line stab with a needle and incubated ○ After incubation, turbidity away from the line of the stab indicates bacterial motility through the medium ARRANGEMENT Figure 23. Types of arrangement for bacteria Table 4. Types of arrangement for bacteria Figure 20. Straight line swab with a needle. B: positive for motility test. After incubation there is turbidity or cloudiness due to bacterial growth (bacteria can swim through the medium) B. MORPHOLOGIC CHARACTERISTICS SHAPE Prefix/Type Description Diplo- Exists in pairs Strepto- Twisted or easily bent (like a chain) Staphylo- Seen in clusters (“bunch of grapes”) Tetra- Seen as groups of four Sarcina Cube-like and in groups of 8 STAINING Figure 21. Shapes of unicellular bacteria: (A) Cocci, (B) Bacilli, (C) Spiral ● Basic cell shapes that occur among unicellular bacteria include the following: ○ Cocci: spherical ▸ “Coccus”: from the Greek word ‘cocco,’ meaning berry ▸ E.g., Staphylococcus, Streptococcus ○ Bacilli: rod-shaped ▸ E.g., Yakult contains Lactobacilli shirota strain ▸ Streptobacilli: bacilli in chains or strips YL6:01.29 General Concepts in Bacteriology (Part 1) ● Differential staining is a technique used to differentiate bacterial structures (01.32, 2025) ● Special/structural staining techniques can reveal important structures, such as: ○ Flagellar structure ○ Spores ○ Capsules ○ Inclusion bodies 7 Differential Staining: Gram Staining Figure 24. Gram-negative bacteria (left) and Gram-positive bacteria (right) ● Gram-staining: most important ○ Gram-positive bacteria: stain blue ○ Gram-negative bacteria: stain red or pink Figure 27. Haemophilus spp. Figure 27 ● Figure 27: Gram (-) coccobacilli ○ Oval shaped: coccobacilli ○ Another staining technique might have been used, hence the purple stain ▸ Haemophilus is Gram-negative Figure 25. Neisseria spp. Figure 25 ● Recall: Bacteria are prokaryotic organisms and very small (in comparison to eukaryotic cells) ● Figure 25: Gram (-) coccus ○ Red stain: Gram-negative ○ Spherical: coccus ● Neisseria spp: can cause meningitis and gonorrhea Figure 28. Vibrio spp. Figure 28 ● Figure 28: Gram (-) curved ○ Comma/curved shape: spiral, vibrio ● Vibrio spp.: can cause cholera Figure 26. Escherichia coli (E. coli) Figure 26 ● Figure 26: Gram (-) rods ○ Red stain: Gram-negative ○ Rod-shaped: bacilli YL6:01.29 General Concepts in Bacteriology (Part 1) 8 Figure 29. Neisseria gonorrhoeae (black circle) Figure 28 ● Figure 29: Gram (-) cocci ○ Red stain: Gram-negative ○ Spherical: coccus ○ In pairs Figure 31. Example of Gram (-) Bacilli (black arrow) Figure 31 ● Figure 31: Gram (-) bacilli ○ Red stain: Gram-negative Figure 32. Example of a smear using AFS Figure 30. Streptococcus pneumoniae Figure 30 ● Figure 30: Gram (+) cocci ○ Blue stain: Gram-positive ○ Spherical, in chains: streptococcus ○ In pairs Figure 32 ● Used Acid Fast Stain (AFS) ● Red organisms indicate bacterium C. BIOCHEMICAL CHARACTERISTICS ● Certain tests can demonstrate biochemical characteristics ● Examples ○ Oxidase test for G- rods: will determine whether the organism produces the enzyme oxidase ○ Catalase test for G+ cocci: can determine if the organism produces catalase ● Importance: not all bacteria will produce oxidase or catalase so we can use this as another basis for classification OXIDASE TEST ● Rapid test to determine whether an organism produces oxidase ● Process: ○ Dropping a reagent in an inoculated swab ● Result ○ Positive (color change to purple): means that the organism produces oxidase YL6:01.29 General Concepts in Bacteriology (Part 1) 9 Optochin Test ● Optochin test: differentiates the alpha hemolytic Streptococcus spp. ● Further differentiates the alpha hemolytic Streptococcus spp. ● Can be used to determine which species is susceptible to the antibiotic and which is resistant ○ Zone of inhibition (area of no growth): bacteria is susceptible to antibiotic ● Examples: ○ S. pneumoniae: optochin sensitive strain ○ S. viridans: optochin resistant strain Figure 33. Oxidase test CATALASE TEST ● Rapid test to determine whether an organism produces catalase ● Process: ○ Place hydrogen peroxide in the sample ○ If the sample produces catalase, it will trigger a reaction liberating oxygen which produces bubbles ▸ Positive: bubbling ▸ Negative: no bubbling ● Limitations: ○ If blood is present in the sample, it may produce a false positive catalase test ▸ Hydrogen peroxide causes wounds to bubble ▸ Means that a sample should not be taken from a blood agar plate when testing for catalase Figure 35. Optochin test Optochin Test Mnemonic: 1ORVSP 1: Alpha Optochin Resistant strain = S. viridans Sensitive strain = S. pneumoniae Bacitracin Test ● Differentiates the beta hemolytic Streptococcus spp. ● Can be used to determine which species is susceptible to the bacitracin and which is resistant. ○ Zone of inhibition (area of no growth): bacteria is susceptible to antibiotic ● Example: ○ S. pyogenes: bacitracin sensitive strain ○ S. agalactiae: bacitracin resistant strain Bacitracin Test Mnemonic: 2BRASPy 2: Beta Bacitracin Resistant strain = S. agalactiae Sensitive strain = S. pyogenes Figure 34. Catalase test OPTOCHIN TEST VS. BACITRACIN TEST ● Optochin test: differentiates the alpha hemolytic Streptococcus spp. ● Bacitracin test: differentiates the beta hemolytic Streptococcus spp. ● Recall: BAP is used to differentiate Streptococcus spp. based on their hemolytic reaction ○ Alpha hemolytic reaction: partial hemolysis producing greenish colonies ○ Beta hemolytic reaction completes hemolysis producing clearing of BAP ● Problem: still need to differentiate the streptococcal species under the alpha hemolytic group and beta hemolytic group using optochin and bacitracin test YL6:01.29 General Concepts in Bacteriology (Part 1) Figure 36. Bacitracin sensitivity test 10 SAMPLE FLOW OF STREPTOCOCCUS SPP. IDENTIFICATION ○ Example: Vibrio cholerae ▸ 130 serogroups have been identified based on the O antigens in the LPS – Only O1 and O139 are related to epidemics and pandemics – Others are nontoxigenic but found in food and environment ● How is this done? ○ Biotyping ▸ Uses biochemical test ○ Antimicrobial susceptibility testing ○ Antigenic Susceptibility ▸ Use of antibodies ○ Phage typing ▸ Uses bacteriophages ● Antimicrobial susceptibility testing Figure 37. Sample flow of Streptococcus spp. 1. Gram stain: Gram positive 2. Catalase test to differentiate Streptococcus spp. and Staphylococcus spp. a. Negative catalase test: Streptococcus spp. b. Positive catalase test: Staphylococcus spp. 3. BAP culture to determine the hemolytic reaction of Streptococcus spp. a. Greenish colored colonies: Alpha-hemolytic Streptococcus b. Clearing of BAP: Beta-hemolytic Streptococcus 4. Bacitracin and Optochin sensitivity test a. Optochin test i. Zone of inhibition: sensitive S. pneumoniae ii. No zone of inhibition: resistant S. viridans b. Bacitracin test: i. Zone of inhibition: sensitive S. pyogenes ii. No zone of inhibition: resistant S. agalactiae III. BACTERIAL CLASSIFICATION AND SUBTYPING A. MAJOR BACTERIAL CLASSIFICATIONS ● Bergey’s Manual of Systematic Bacteriology ○ Gram Negative Eubacteria ○ Gram Positive Eubacteria ○ Eubacteria Lacking Cell Walls ▸ Used to differentiate it with Archaebacteria ▸ E.g., Mycoplasma sp. ○ Archaebacteria ▸ Predominantly inhabit extreme terrestrial and aquatic environments such as those with high salts and high temperatures ▸ Often referred to as extremophiles ▸ Do not cause disease ▸ Can be distinguished by their lack of peptidoglycan cell wall and the presence of characteristic rRNA sequences B. BACTERIAL SUBTYPING ● Used to distinguish between strains of the same species ● Different strains of the species should be differentiated as well ○ May be done through subtyping ● Importance ○ Some species may be pathogenic and may cause epidemics ○ Some are normal flora YL6:01.29 General Concepts in Bacteriology (Part 1) Figure 38. Antimicrobial susceptibility testing with the use of impregnated discs (left) and e-strips (right) ● Antigen susceptibility ○ Differentiates serotype, serovar, serogroup ○ “Sero” ▸ antibodies ○ Species and subspecies ▸ Salmonella spp. 4400 types detected because of this test ○ Antigens ▸ To classify organisms, we can differentiate these from each other: – Cell wall (O) antigen – Flagellar (H) antigen – Capsular (K) antigen ▸ These are used to aid in classifying certain organisms at the species level – To serotype strains of medically important species for epidemiologic purposes – To identify serotypes of public health importance – To distinguish strains of exceptional virulence or public health importance ▸ Ex: V. cholerae (O1 is the pandemic strain) and E. coli (enterotoxigenic, enteroinvasive, enterohemorrhagic, and enteropathogenic serotypes) ● Bacteriophage typing ○ Phages are virus that infect the bacteria ○ Similar to antimicrobial susceptibility testing, phage typing determines the susceptibility pattern of an isolate to a set of specific bacteriophages ▸ Used as an aid in epidemiologic surveillance of certain diseases 11 Figure 39. Bacteriophage typing ○ Phage susceptibility ▸ Phage typing – Determining the susceptibility pattern of an isolate to a set of specific bacteriophages – Has been used primarily as an aid in epidemiologic surveillance of diseases caused by Staphylococcus aureus, mycobacteria, P. aeruginosa, V. cholerae, and S. typhi – Susceptibility to bacteriocins has also been used as an epidemiologic strain marker Figure 40. Phage typing. Outcome is the same as antimicrobial susceptibility testing Active Recall Box 5. T/F. Inclusion bodies cannot be seen using differential staining. 6. What is the resistant strain in the Optochin test? 7. T/F. Antigen susceptibility helps differentiate serotype, serovar, and serogroup. Answers: 5F, 6 S. viridans, 7T QUICK REVIEW QUESTIONS 1. T/F. The cytoplasmic components of prokaryotes include the nucleus, ribosomes, plasmids, and inclusion bodies. 2. What is the correct arrangement of cellular envelope coverings from outermost to innermost? A. Cell membrane, Cell wall, Capsule B. Capsule, Cell wall, Cell membrane C. Cell wall, Cell membrane, Capsule D. Capsule, Cell membrane, Cell wall 3. T/F. In Gram-staining, both Gram-positive and Gram-negative bacteria can be stained with Crystal Violet. YL6:01.29 General Concepts in Bacteriology (Part 1) 4. Archaebacteria do not cause disease. They are distinguished from Eubacteria by their lack of rRNA sequences. A. Only statement 1 is true B. Only statement 2 is true C. Both statements are true D. Both statements are false 5. What is the primary difference between Teichoic Acid and Teichuronic Acid? A. Teichoic Acids contain repeating units of sugar acids. B. Teichuronic Acids contain repeating units of phosphate. C. Teichoic Acids contain repeating units of phosphate. D. Teichuronic Acids contain repeating units of sugar acids. 6. How do porins in the outer membrane contribute to bacterial function? A. They enhance the synthesis of lipopolysaccharides. B. They allow passive diffusion of hydrophobic molecules. C. They promote the formation of a symmetrical bilayer. D. They protect the cell from hydrophilic molecules. 7. Which paired statement is incorrectly paired? A. Flagella: antigenic appendages composed entirely of protein B. Glycocalyx: aids in adherence to surfaces C. Endospore: “resting” state D. Fimbriae: contributes to invasiveness of organisms 8. Which of the following is a characteristic of Gram-positive bacteria? A. Gram-stained cell stains reddish-pink B. Porins are present C. Teichoic acids are present D. Not sensitive to lysozyme 9. Explain the concept of phage typing and how it is similar to antimicrobial susceptibility testing. A. Phage typing involves determining the color of bacteria colonies. B. Phage typing is a method to identify viral infections in bacteria. C. Phage typing determines the susceptibility of bacteria to specific viruses. D. Phage typing helps in studying the size of bacterial colonies. 10. Which of the following factors, when observed in a culture medium, can aid in the identification of an organism? A. The presence of antibiotics in the medium. B. The concentration of glucose in the medium. C. Colony characteristics and pigmentation. D. The number of agar plates used. 11. Which is correctly paired in terms of morphological classification of bacteria? A. Strepto: bacteria existing in clusters B. Vibrio: a subclassification of bacilli C. Red stain: Gram-positive D. Spherical: coccus 12. Classify the following bacteria through the given morphological descriptions: blue staining, rod-shaped seen in clusters A. Gram (-) Streptobacilli B. Gram (-) Staphylobacilli C. Gram (+) Streptobacilli D. Gram (+) Staphylobacilli 13. Which of the following statements are true? A. Alpha-hemolytic Streptococcus appear as complete hemolysis with greenish colonies. B. Beta-hemolytic Streptococcus appear as partial hemolysis with clearing of BAP 12 C. Catalase test can be used to differentiate between Streptococcus spp. and Staphylococcus spp. D. Bubbling of a sample from BAP indicates the presence of catalase. 14. Which of the following are correctly matched? A. S. viridans: optochin sensitive B. S. pneumoniae: bacitracin sensitive C. S. pyogenes: optochin resistant D. S. agalactiae: bacitracin resistant ANSWER KEY 1F, 2B, 3T, 4A, 5C, 6B, 7D, 8C, 9C, 10C, 11D, 12D, 13C, 14D Vibrio is a subclassification of bacilli. Red staining indicates the presence of Gram-negative bacteria. 12. D. Gram (+) Staphylobacilli. Blue staining indicates the presence of Gram-positive bacteria. Staphylo is used to classify bacteria found in clusters. Bacilli are rod-shaped bacteria. 13. C. Catalase test can be used to differentiate between Streptococcus spp. and Staphylococcus spp. D. is incorrect as bubbling of a BAP sample may be due to the effect of H2O2. 14. D. S. agalactiae: bacitracin resistant. S. agalactiae is a bacitracin resistant strain. RATIONALE False. Prokaryotes do not have a true nucleus. They package their DNA in the nucleoid instead. Thus, the cytoplasmic components of prokaryotes include the nucleoid, ribosomes, plasmids, and inclusions. 2. B. Capsule, Cell wall, Cell membrane. The capsule is the outermost envelope of the prokaryotic cell. The cell wall is the intermediate envelope. The cell membrane is the innermost envelope. 3. True. In the first step of Gram-staining, both Gram-positive and Gram-negative bacteria are stained with Crystal Violet. 4. A. Only statement 1 is true. Archaebacteria can be distinguished from Eubacteria by the presence of characteristic rRNA sequences. 5. C. Teichoic Acids contain repeating units of phosphate. Teichoic Acids are characterized by their repeating phosphate units, which are an essential component of their structure. This is different from Teichuronic Acids, which contain repeating units of sugar acids instead. 6. B. They allow passive diffusion of hydrophilic compounds. Porins are protein channels present in the outer membrane of Gram-negative bacteria. They allow for the passive diffusion of low molecular weight and hydrophilic compounds, such as nutrients and ions. This mechanism plays a role in nutrient uptake and waste elimination for the bacterial cell. 7. D. Fimbriae - contributes to invasiveness of organisms. The capsule form of glycocalyx contributes to invasiveness of organisms. Fimbriae or pili are rigid surface appendages that have antigenic properties and inhibit phagocytosis. 8. C. Teichoic acids are present. The other choices are characteristics of Gram-negative bacteria. 9. C. Phage typing determines the susceptibility of bacteria to specific viruses. Phage typing is a laboratory technique used to determine the susceptibility pattern of a bacterial isolate to a specific set of bacteriophages. This technique involves exposing the bacterial isolate to different phages and observing whether the bacteria get infected and undergo lysis or not. 10. C. Colony characteristics and pigmentation. Colony characteristics and pigmentation refer to the visual appearance of bacterial colonies grown on culture media. Different bacterial species can exhibit unique colony morphologies, such as size, shape, texture, and color. Additionally, some bacteria produce distinct pigments when they grow on specific media. 11. D. Spherical: coccus. The prefix strepto- is used to classify bacteria seen in chains or strips that twist or bend easily. REFERENCES 1. YL6:01.29 General Concepts in Bacteriology (Part 1) REQUIRED ● 💻 Cabigon, J. J. J., MD (2023, August 15) General Concepts in Bacteriology [Lecture slides]. SUPPLEMENTARY 📄 ASMPH 2025. 01.32: General Concepts in Bacteriology by Cabigon, J. J. J., MD. ● 📄 ASMPH 2026. 01.28a: General Concepts in Bacteriology by Cabigon, J. J. J., MD. ● Concerns and Feedback form: http://bit.ly/YL6CFF2027 How’s My Transing? form: https://bit.ly/2027YL6HMT Mid-Semester Evaluation form: https://bit.ly/2027YL6MidSem End-of-Semester Evaluation form: https://bit.ly/2027YL6EndofSem Errata Points Trackers: https://bit.ly/YL62027EPT YL6 TransMap: https://bit.ly/2027YL6TransMap FREEDOM SPACE 13