Bacteria

Question 1

Features of Bacteria

Answer 1

• Unicellular, haploid
• Usually 0.3 to 5μm
• Nucleoid (no true nucleus)
• Circular chromosome
• Ribosomes
• Outer and cytoplasmic membranes
• Unique surface
  structures (capsule, LPS, pilli, etc.)

Peptidoglycan Cell Wall
*Present in nearly all bacterial species
*Made up of glycan chains of N-acetyl glucosamine and N-
acetyl muramic acid
*Glycan chains are crosslinked by peptides
*Targeted by many clinically relevant antibiotics

Question 2

Virulence Factors

Answer 2

• Attachment to the mucosal surface: Pili, adhesins, and fimbriae
• Fight for resources:
  metal-binding transporters, siderophores
• Evade complements: capsule, C5a peptidases
• Evade antibodies: IgA proteases
• Kill phagocytes: Toxins-like

Question 3

Biospores

Answer 3

• Heat-resistant, desiccation resistant,
  survive for decades and many
  antiseptics, disperse rapidly
• Common endospore formers that are possible bioweapons
• Bacillus anthracis
• Clostridium tetani
• Visualized by endospore staining
  (malachite green and safranin)

Question 4

Gram Staining

Answer 4

1. Fixing (heat fix)
2. Crystal violet (primary stain)
3. Iodine treatment (mordant)
4. Ethanol and acetone (decolorization)
5. Counter stain with Safranin

peptidoglycan layer is 10x thicker in gram-positive

Question 5

Acid-fast staining to visualise mycobacteria

Answer 5

Stain cells with high lipid and wax-like surface (mycolic acids)

1. Fixing (heat fix)
2. Carbol fuchsin (primary stain, red)
3. Heat treatment
4. Ethanol and acetone (decolorization)
5. Counter stain with Methylene blue

Question 6

Other Staining

Answer 6

Flagella staining
* Chemicals (pararosaniline, tannic acid, or potassium alum) that bind to flagella to increase their diameter and change their color

Capsule staining
* Negative staining (exclusion of dye, dark background)
* Commonly used dyes (India Ink, nigrosin, or fluorescently labeled dextran)
* Immunostaining of capsule

FISH : Fluorescence in situ hybridization

Question 7

Other Methods

Answer 7

Examples included the Analytical profile index
(API) test, 16S rRNA sequencing, whole genome sequencing, and MAIDI-TOF

MALDI TOF -> antigen susceptibility, microorganism identification, subtyping

Question 8

MacConkey’s Agar

Answer 8

MacConkey’s agar: Gram positive bacteria failed to grow due to bile salts (selective).
Lactose fermentation will result in a color change of the pH indicator (differentiating)

Question 9

Carbon

Answer 9

• Need to synthesize biomolecules and provide energy
• Auxotroph (=more requirements) and prototroph (≈wild-type)
• Sugars: lactose/glucose/arabinose/
  fucose….
• Non-sugars: acetate, glycerol, alcohols, amino acids, fatty acids
• Coliforms: lactose-fermenters at high temp

Question 10

Nitrogen

Answer 10

Not all bacteria can synthesize all amino acids required
* Common requirements: arginine, aspartic acid, cysteine, isoleucine, leucine, proline, threonine, and valine.
* E. coli can synthesize all amino acids from nitrate.
* Some Neisseria gonorrhoeae strains require proline
* Common nitrogen source in nature
* Amino acids
* Salts like nitrate, nitrile, etc.
* Urea and ammonia (Is pee sterile?)

Question 11

Phosphate

Answer 11

• Absolutely required for life: synthesis of nucleotide phosphates, DNA, RNA, cofactors for various
  biochemical reactions, polyphosphate, etc. Can be
  found in many host niches.

Question 12

Oxygen Requirement

Answer 12

Oxygen requirement
* In E. coli, aerobic respiration generates 38 molecules of ATP per glucose
* By contrast, 2 ATP per glucose without oxygen
* Oxygen generates toxic products: ROS

Question 13

Temperature

Answer 13

• Mesophilic – grows best between 25ºC and 40ºC.
• Psychrophilic (cold loving) – grows best below 20ºC
• Thermophilic – grows best at high temp, 55- 80ºC

Question 14

Other Requirements for Growth

Answer 14

Other requirements for growth
* pH range/ optimal pH
* Neutral pH: Most pathogenic bacteria
* Acidic: Lactobacilli; Helicobacter pylori
* Alkaline: Vibrio cholerae
* Metal ions
* Biologically available Fe2+ is scarce. Competition between host and pathogens
* Make siderophores and chelators. Pyoverdine and pyochelin from Pseudomonas aeruginosa. Lactoferrin from the host.
* Growth factors (probably the reason why there are many viable but not culturable (VBNC) bacteria)

Question 15

VBNC

Answer 15

• About 80% of human gut bacteria and 90% of soil bacteria are VBNC.
• Microchip in the soil allow grow
  ≈80% of the bacterial species

Question 16

Plasmids

Answer 16

Small, circular double-stranded DNA, also vary widely in size (1.8Mb to 846bp)
* Origins of replication determine the copy number
* Genes that lead to better survival: antibiotic-resistant, virulence factors
* Horizontal gene transfer

Question 17

Mutations

Answer 17

Spontaneous mutations
* Random, undirected alteration of the nucleotide sequence
* Frequency of point mutation in E. coli?
* Three modes of mutation
* Insertion
* Deletion
* Substitution

Question 18

Horizontal Gene Transfer

Answer 18

Horizontal gene transfer
* Three mechanisms
* Transformation: genes transferred from one bacterium to another as “naked” DNA
* Transduction: DNA transferred from one bacteria to another through a virus
* Conjugation: plasmids transferred 1 bacteria to another via a pilus

Question 19

Targeting Bacteria

Answer 19

• Understanding the structural features of a bacterial cell is fundamentally important because they inform drug and vaccine development. For example, most clinically relevant antibiotics target cell wall peptidoglycan synthesis. Features that are unique to bacteria are also pathogen-associated molecular patterns that are detected by the host. Some of these features are important for pathogenesis. They are called virulence factors. Examples include capsules, adhesins, pili, and toxins, to name a few.

Question 20

Identification

Answer 20

• Identifying and classifying bacterial pathogens are of utmost importance because they decide treatment options. The most simplistic way is to name them according to the diseases they caused. Alternatively, they are named and classified based on their sizes, shapes, cellular organization (clustering or chains), and staining properties. Recent advancements in the clinical microbiology labs include MALDI-TOF. These approaches help diagnostics in a timely fashion, such that the medical doctors are well-informed about the pathogens they are dealing with and the possible treatments that are available at their disposal.

Question 21

Bacterial Processes

Answer 21

• Nutritional and growth requirements assist identification and classification of bacterial pathogens. For example, many gut pathogens are gram-negative, rod-shaped, and lactose fermenting. These properties are useful for preventing food-borne diseases. Before the time when antibiotics became widely available, people developed alternative treatment options, such as inducing an artificial fever. By increasing the body temperature, pathogens are clear. They are unimaginable in the current standard. Yet, it illustrates our long-term fight against infectious diseases, as well as the importance of securing suitable antimicrobials.

Question 22

Resistance

Answer 22

• There are many ways that bacterial pathogens gain resistance to antibiotics. One of them is spontaneous mutations of the drug target. As the drug’s binding affinity reduces, we will need more to treat the disease. Another more dangerous form of gaining antibiotic resistance is horizontal gene transfer. This can be done by genetic transformation, transduction, and conjugation. It is very difficult to stop these processes from happening except for the prudent use of antibiotics. Once the resistance is in place, it may take a long time for the bacteria to become sensitive again, if it is even possible.

Question 23

Developing Antibiotics

Answer 23

• Finding new antibiotics is easier said than done. Traditionally, antibiotics are discovered from soil bacteria and fungi. While this pipeline has been fruitful for a few decades, the antibiotic discovery process has had a diminishing return. A larger screening is required, which partly leads to reduced economic returns for the pharmaceutical industry. Moving forward, the biomedical sector may partner with academia to buffer the cost of antibiotic development. For a selected subset of pathogens in which treatment options are scanty, government support may be required to expedite antibiotic discovery. Alternative treatments such as phage therapy and anti-virulence molecules should be explored to fill the void.