Microbiology Fundamentals

• Pharmaceutical Microbiology is covered in Microbiology-I (PHBTt-401).

Recommended Textbooks

• Hugo & Russell's Pharmaceutical Microbiology, 8th edition, Wiley-Blackwell, Edited by S.P. Denyer et al., ISBN 9781444330632.
• Essential Microbiology for Pharmacy and Pharmaceutical Science, Wiley-Blackwell, By Geoffrey Hanlon and Norman Hodges, ISBN 978-0-470-66534-3 (pbk.).
• Pharmaceutical microbiology : essentials for quality assurance and quality control / Tim Sandle, By: Sandle, Tim, Publisher: Amsterdam: ElSevier, 2016 - ISBN: 0081000227; 9780081000229.

Role of Pharmacists in Microbiology

• Pharmacists must: understand sterilization and aseptic manufacturing to limit contamination, and produce antibiotics, steroids, insulin and other recombinant proteins, amino acids, organic acids, enzymes and polysaccharides.
• Pharmacists must: understand the use of antibiotics, including their capabilities and limitations.
• Pharmacists must: have a clear understanding of viral, fungal, and bacterial diseases.
• Pharmaceutical microbiology directly impacts medicine production and usage.

Aims of Pharmaceutical Microbiology (PM)

• Reduce microorganism numbers and exclude microorganisms and microbial byproducts from water and starting materials.
• Ensure the finished pharmaceutical product is sterile.
• Detect mutagenic and carcinogenic activity in prospective drugs.
• Apply biotechnological approaches using microorganisms to manufacture pharmaceutical products like insulin.
• Pharmaceutical microbiologists work within quality assurance and quality control.
• Pharmaceutical microbiologists are involved in: laboratory testing, environmental monitoring and act as subject matter experts in sanitization, autoclave operation, de-pyrogenation, process validation, cleanroom gowns, and raw materials.

Key Facts About Microorganisms

• Microorganisms are abundant and live both in and on our bodies.
• Some microorganisms are beneficial or commercially important, most are harmless, and some are pathogens.
• Fungi and protozoa are more complex than bacteria.
• Most fungi are contaminants/spoilage organisms, and few are pathogenic.
• Protozoa are of limited pharmaceutical concern as pathogens, and not as spoilage organisms.
• Infectious diseases arise from not living prions (rogue protein molecules), and viruses (nucleic acid and protein).
• Bacteria are the simplest living cells.
• Most bacteria of pharmaceutical interest are easily grown in the lab.

Benefits, Uses, and Problems with Microorganisms

• Microorganisms play an essential role in carbon and nitrogen cycles.
• Microorganisms are important in brewing, dairy, and food industries.
• Microorganisms are used in manufacturing industrial solvents and other chemicals.
• Microorganisms can be used as insecticides.
• Microorganisms are used for chemical detoxification.
• Microorganisms are used in oil extraction.
• Microorganisms can be used in the production of 'biological' detergents.
• Microorganisms are used in manufacturing antibiotics, steroids, vaccines, and biotechnology.
• Microorganisms are used in assays to measure antibiotic concentrations.
• Microorganisms are used as biological indicators during sterilization.
• Microorganisms are used in tests to detect metabolic disorders and mutagenicity.
• Microorganisms can cause infections.
• Microorganisms that cause infections are called pathogens.
• Harmless microorganisms may transmit antibiotic resistance.
• Dead bacteria can cause fever by releasing endotoxins.
• Microorganisms can contaminate/spoil nonsterile medicines.
• Microorganisms can cause noninfectious diseases (e.g., gastric ulcers, some cancers).

General Characteristics of Microbial Cells

• Acellular microbes include viroids (single-stranded RNA without protein), prions, and viruses.
• Cellular microbes include prokaryotes (archaea and bacteria) and eukaryotes (some algae, all protozoa, and some fungi).
• Prokaryotes lack a true nucleus and membrane-bound organelles where eukaryotes have a true nucleus and membrane-bound organelles.
• Prokaryotes reproduce asexually and eukaryotes reproduce sexually or asexually.
• Prokaryotes genetic variation is from mutations and eukaryotes genetic variation is from mutations.
• Mitochondria and Chloroplasts are absent in prokaryotes but may be present in eukaryotes.
• A prokaryote's ribosome size is 70s and a eukaryote's ribosome size is 80s.
• Prokaryotes lack sterols but have peptidoglycan in the cell wall where eukaryotes possess sterols but lack peptidoglycan in the cell walls.

The Microbial World - Classification

• The former classification was: Eukaryotes, Prokaryotes.
• The new classification is: Bacteria (Eubacteria), Archaea, Eukarya.

Types of Microorganisms

• Living organisms are made of prokaryotic and eukaryotic cells.
• Prokaryotic cells include Bacteria and Archaea but only Bacteria will be considered further.
• Eukaryotic cells include other microorganisms such as fungi, protozoa and algae, and also parasitic worms, mites, animals, plants including humans.
• Viruses do not have a cellular structure and some do not consider them living, but they are indisputably agents of infection and are part of the microbial world.

Evolutionary Relationships Using Ribosomal RNA Gene Phylogenies

• DNA is extracted from cells.
• Copies of the gene encoding rRNA are made by the polymerase chain reaction (PCR).
• The gene is sequenced and compared with sequences from other organisms with a computer algorithm.
• This generates a phylogenetic tree that shows evolutionary relationships.
• The phylogenetic tree of life shows three domains of organisms with representative groups in each.

Viruses

• Viruses are parasites infecting animals, plants, protozoa, and bacteria.
• Viruses contain nucleic acid and protein with a protein coat to protect the nucleic acid core (DNA or RNA).
• Viruses need a host cell to grow because the range of hosts are narrow.
• Viruses can be contaminants in pharmaceutical raw materials of animal origin.
• Viruses are susceptible to heat and organic solvents so they are unlikely to arise in gelatin.
• Viruses are unaffected by antibiotics that treat bacterial and fungal infections.

Prions

• Prions are the simplest infectious agents and considered microorganisms because they transmit disease from one person to another.
• Similar to viruses for their lack of cellular structure, but prions have no nucleic acids.
• Prions are incorrectly folded mammalian proteins interacting with normal proteins and induce structural changes.
• Prions cause transmissible spongiform encephalopathies, like bovine spongiform encephalopathy (mad cow disease) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans.
• Prions are stable and difficult to inactivate by disinfectants, gamma-radiation, and steam-sterilization.

Bacteria

• Bacteria are the most widely studied type of microorganism and cause more diseases than fungi and protozoa.
• Bacteria are easily grown in the lab.
• Bacteria have a robust cell wall for resisting osmotic pressure and bacteria cells can reproduce asexually.
• Bacteria can reproduce (double) in 20 minutes and mutants can be quickly selected and become dominant.
• Chlamydia and rickettsia are pathogenic bacteria and intracellular parasites.
• Mycoplasmas do not have a cell wall which makes them resistant to penicillins.

Fungi: Yeasts and Moulds

• Yeasts are unicellular and larger than bacteria (5-10 μm).
• Yeasts reproduce sexually and reproduce by binary fission or budding.
• Yeasts are contaminants of medicines and spoilage organisms and they can cause infections.
• Moulds are multicellular filaments collectively forming a mycelium colony with tubes called hyphae and moulds are contaminants of medicines instead of pathogens.

Protozoa

• Protozoa are single-celled animals in water and soil
• Some protozoa are grown in bacterial cultures in the lab but cannot arise as contaminants of raw materials/medicines.
• Protozoa that can cause malaria and amoebic dysentery are of pharmaceutical interest.

Distinguishing Characteristics of the Major Groups of Infectious Agents

• Prions don't have a cellular structure, and no nucleic acids. The laboratory cultivation is only within living organisms and all prion diseases are untreatable/fatal.
• Viruses don't have a cellular structure and contain DNA or RNA. The laboratory cultivation is only within living organisms and most cause active disease.
• Chlamydia and rickettsia contain DNA in a single chromosome. The laboratory cultivation is only within living organisms and many are human pathogens.
• Bacteria are prokaryotes using DNA in a single chromosome without a nucleus. Most that cause human infection can be grown easily and despite being pathogens, most are harmless.
• Fungi are eukaryotes with DNA in multiple chromosomes in a nucleus. They are grown easily and few are pathogens, and the majority are harmless.
• Protozoa are eukaryotes with DNA in multiple chromosomes in a nucleus. The majority are difficult to grow in the lab and few are pathogens, but the majority are harmless.

Bacterial Cell Structure

• External bacterial structures include: slime layers, capsules from Glycocalyx, cell walls and Cell membranes (different than archaea).
• Gram staining differentiates Gram positive and Gram negative bacteria, and Flagella is for motility.
• Cytoplasm contains ribosomes, nucleoid, and gas vesicles, inside of a cell membrane.

Bacterial Cell Structure: Gram Stain

• Apply heat to fix bacterial smear on a glass slide..
• Stain with crystal violet solution, the cells should appear blue/violet.
• Add Gram's iodine (I/KI solution), the cells remain the same colour.
• Apply alcohol or acetone to decolorize; some cells turn blue/violet, and others are colourless.
• Stain with safranin solution (red).
• Wash with water.
• Gram-positive cells appear blue/violet and Gram-negative cells appear pink/red for viewing.

Bacterial Cell Walls

• Gram-positive cell walls contain a cytoplasmic membrane, peptidoglycan, phospholipid, protein, and lipoteichoic acid.
• Gram-negative cell walls contain an inner membrane, periplasmic space, outer membrane, phospholipid, peptidoglycan, lipoprotein, protein, LPS, and porins.

Bacterial Cell Wall: Peptidoglycan(murein)

• Peptidoglycan in bacteria contain the glycan backbone consisting of N-Acetylglucosamine(NAG) and N-Acetylmuramic acid (NAM).
• The peptide crosslinks contain L-Alanine, D-Glutamic acid , Diaminopimelic acid (DAP)/ Lysine and D-Alanine.
• Lysozyme acts on the glycan backbone and beta lactam antibiotics prevent cross-linking.

Bacterial Cell Wall: Peptidoglycan (murein) Continued

• Acetylated sugar backbone: almost invariable and the tetrapeptide is variable with DAP OR lysine and has D-aa being protease resistance.

Bacterial Cell Wall: Peptidoglycan

• Gram-negative Bacteria has direct no interbridge and Gram-positive bacteria has Glycine interbridge.
• Three-dimensional structure consists of peptidoglycan ribbons cross-linked to one another.

Archaeal Cell Wall

• Archaeal Cells contain an S-layer pseudocrystalline protein/glycoprot, extreme halophils, methanogens and hyperthermophiles.
• Archaeal Cells pseudopeptidoglycan contains Glycan backbone, NAG, Glu,NAT (N-Acetyltalosaminuronic acid) for Peptide cross-links.
• The pseudopeptidoglycan contains Methanobacterium spp.

Gram-positive Cell Wall

• The gram-positive cell wall has teichoic acids, which form the outermost part of peptidoglycan-shell, contain glycerophosphate/ribitol phosphate, connected by phosphodiesters with often modifications: sugars, D-Ala, L-Ser, L-Gly in the outer membrane.
• The gram-positive wall has also contains: localization (membrane bound), glycerol-containing acids linked to a lipid and during infection, lipoteichoic acid molecules trigger an inflammatory response.

Gram-negative Cell Wall

• It includes lipopolysaccharide, lipid A, phospholipid, porins, and lipoprotein with a periplasm in the outer membrane.

Gram-Negative Cell Wall: LPS

• Contains O-specific polysaccharide, Core polysaccharide KDO,HEp,Gal and Lipid A.
• Has Much heterogeneity.

Gram-Negative Cell Wall: Lipid A

• Also known as endotoxin and is recognized by the innate immune response, triggering septic shock.
• The lipid A, hydrophobic anchor of LPS is similar to common glycerophospholipids.

Limulus Amoebocyte Lysate (LAL) Test

• LAL is used for the detection of endotoxins.
• Inactive protease enzyme becomes an active enzyme, from endotoxin.
• Clotting protein (coagulogen) forms a clot as a response.

Gram-Negative Cell Wall

• Contains an "inner" core polysaccharide which is phosphorylated and is non-repeating oligosaccharide, and required for the barrier function of the outer membrane.

The Bacterial Cell Wall Comparison

• Peptidoglycan level is 90% for the gram-positive cell and 10% for the gram-negative cell with the number of layers being up to 25 (20-50nm) for gram-positive and 1 (3nm) for gram-negative cell, and there is a different crosslink; gram-positive is peptide-interbridge while gram-negative cell is direct.
• N-acetylmuramic acida is present for both gram-positive and gram-negative bacteria while special aa is Lys > DAP (gram-positive cell) and DAP (gram-negative cell) .
• There is no pseudomurein in the gram-positive and gram-negative cell where archaea has it.
• Teichoic acid is present (+) in the gram-positive and lipoteichoic aceid is present (+) in the gram-positive and archaea where the gram-negative doesn't contain it.