Introduction to Microbes - Lecture Slides PDF

INTRODUCTION TO MICROBES DR Y. MAHABEER LEARNING OBJECTIVES To Know: Classification & structure of microbes esp. bacteria Differences between eukaryotes & prokaryotes How bacteria are classified Differences between Gram negative and Gram positive bacteria DEFINITION...

INTRODUCTION TO MICROBES DR Y. MAHABEER LEARNING OBJECTIVES To Know: Classification & structure of microbes esp. bacteria Differences between eukaryotes & prokaryotes How bacteria are classified Differences between Gram negative and Gram positive bacteria DEFINITION Microbiology = Study of microscopic living forms BUT SOME ARE VERY LARGE! MEDICALLY IMPORTANT ORGANISMS Virus - 0.03um – 0.3um Bacteria - 0.2um – 2 um Fungi - 4- 40um (mushrooms!) Parasites eg worms – 0.3cm – 12m Naked eye : > 40 um TAXONOMIC HIERACHY  Binomial (Linnaeus) Genus (capitalised) Species – lowercase Both italicized or underlined WE ARE COVERED IN BACTERIA!! Symbiosis=living together  INDIGENOUS MICROBIOTA = NORMAL FLORA More bacterial cells on us than our own cells!!! Where?? Skin Mouth Upper airways Gastro-intestinal tract Genitourinary(vagina, urethra)  Prokaryotes – Bacteria Eukaryotes – fungi, parasites Virus – don’t fit in Differences between Eukaryotes and Prokaryotes Eukaryotic cells contain a nucleus with a nuclear membrane enclosing multiple chromosomes. Prokaryotic cells have a single chromosome that is not enclosed in a nuclear membrane. Characteristics Prokaryotes Eukaryotes Cell wall Contains Absent peptidoglycan, When present lipids and contains chitin proteins or cellulose Characteristic Prokaryotes Eukaryotes Nuclear structure: Nuclear Absent Present membrane Chromosomes Single,closed, Multiple, linear, circular, chromosomes dsDNA Ploidy Haploid Diploid, haploid Characteristics Prokaryotes Eukaryotes Cytoplasm Ribosomes Present, 70S Present, 80S Mitochondria Absent Present Golgi complex Absent Present Endoplasmic Absent Present reticulum Cytoplasmic Present; Present; membrane phospholipids, no phospholipids sterols (except…) and sterols Motility Flagella, simple Flagella, complex Characteristics Prokaryotes Eukaryotes Energy Cytoplasmic Mitochondria generation membrane associated Sexual Absent Present may reproduction alternate with asexual Recombination/ Chromosomal Diploid zygote gene exchange or plasmid formed from gene exchange haploid germ via cells; meiosis transformation, results in transduction or genetic conjugation recombination BACTERIA Smallest living cells(0,1-10um) Have a cell wall, cytoplasmic membrane Cytoplasm only contains ribosomes & single DS DNA NO organelles NO nucleus Free living Binary fission in artificial media-grow within a day THEREFORE… PROKARYOTIC CLASSIFICATION OF BACTERIA Phenotypic Gram stain & morphology Growth requirements Biochemical reactions Serological Genotypic Phylogenetic tree Ribosomal RNA Molecular (eg PFGE, sequencing) Gram stain & morphology Gram positive or Gram negative Shape Arrangement Size SHAPES Cocci- round Bacilli – rod-like Cocco-bacilli-very short rods Spirochaetes – spiral Curved ARRANGEMENT Clusters Chains Coryneform(chinese letters) GRAM STAIN Crystal violet (purple) Iodine (black) Decolouriser Safranin/carbol fucshin (pink) Method Crystal violet which serves as the primary stain A weak solution of iodine - bind dye. The cell is then treated with an organic decolouriser, such ethyl alcohol and acetone.  Some bacterial species because of thicker cell walls will retain the crystal violet and appear purple under the microscope and are called Gram-positive Other bacteria are decolourised and pick up the safranin counter stain and appear pink- red microscopically and are called Gram- negative Gram stain The Gram-stained reaction in conjunction with the types, e.g. cocci and bacilli, as well as the arrangement of the bacterial cells is then used to make a presumptive identification. Gram positive cocci In clusters: Staphylococcus aureus In chains: Streptococcus pneumoniae Streptococcus pyogenes Gram positive bacilli Spore-forming Bacillus spp Clostridium spp Chinese letters/club-shaped Corynebacterium (diphtheriae) Short rods/coccobacilli Listeria (monocytogenes) Branching Nocardia spp Actinomyces spp Gram negative Cocci Neiserria (gonorrhoeae or meningititdis) Bacilli Enterobacterales (E.coli, Klebsiella pneumonia, Proteus mirabilis, Salmonella spp, Shigella spp) Pseudomonas aeruginosa Curved bacilli Vibrio (cholerae) Campylobacter spp Growth requirements eg O2  based on the types of reactions to generate energy for growth ◦ Aerobic - oxygen is terminal electron acceptor and cannot grow in its absence eg Pseudomonas aeruginosa ◦ Anaerobic - depend on other substances (organic acids/alcohols etc) as electron acceptors eg Clostridium spp, Bacteroides spp ◦ facultative anaerobes- preferentially utilize oxygen as a terminal electron acceptor, but also can metabolize in the absence of oxygen by reducing other compounds eg E.coli STRUCTURE OF BACTERIA SLIME extracellular polysaccharide loosely associated with the bacteria Not all bacteria facilitates colonization of smooth, prosthetic surfaces such as intravascular catheters EG Staph aureus & Staph spp CAPSULE Hydrophilic gel Almost all bacteria Not readily stained Capsule- discreet layer Smooth,mucoid colonies on agar Polysaccharide Mucoid colonies due to thick capsule (Klebsiella spp) CELL WALL Internal to capsule ALL bacteria (except cell wall-less Chlamydia, mycoplasma) Function Rigidity & shape Protection from bursting & mechanical disruption Barrier from toxins Anti-phagocytosis  main structural component of the cell wall is a ‘peptidoglycan’, a mixed polymer of hexose sugars and amino acids. PG (aka murein) – only in prokaryotes NAGA (N-acetyl-glucosamine) NAMA (N-acetyl-muramic acid) Alternating units 1:4 linkages Lysozyme can break this bond  Tetrapeptide side chains –alternating L- & D- amino acids Cross bridges – sheets –peptide bonds 3rd aa to 4th(terminal) Structure of peptidoglycan Structure of peptidoglycan  Penicillin binding proteins(PBP) – transpeptidases/co- peptidases are enzymes - export new polymers from cell & link them to inner aspect of cell wall(ANTIBIOTICS!!) Autolysins remove old PG Bacteria are classified according to their cell wall as Gram positive or Gram negative. Draughtsman colonies due to autolysin in Strep pneumoniae Gram positive peptidoglycan forms a thick layer external to the cell membrane May have techoic acid – polymers of ribitol/glycerol from PG or cell membrane Stabilise cell wall, antigenic, adherence Other cell wall structures eg M protein, S layer, C polysaccharide Gram negatives peptidoglycan layer is thin(single layer) overlaid by an outer membrane, anchored to lipoprotein molecules in the peptidoglycan layer Outer membrane proteins – attachment Porins- pores thro which small hydrophilic solutes can pass Periplasmic space bet. CM & OM  Lipopolysaccharide core polysaccharide lipid A O-sp polysaccharide side chains - major surface antigenic determinants in GNB endotoxin activity- extremely toxic to man Even minute amt endotoxic shock(fever & shock)/Gram negative shock- high mortality & ICU admission Structure of LPS Cell membrane Encloses cytoplasm Phospholipid bilayer Rich in proteins No sterols Contains enzymes for respiration, PG & lipid synthesis etc Bacterial chromosome attached to it Cytoplasm amorphous gel with enzymes, ions, granules, plasmids, ribosomes Many ribosomes- faster growth rate 70S(50 S + 30 S)  Nucleoid Single chromosome 4000 genes-ds DNA Coiled-1000 times length of cell! DNA gyrase & topoisomerase play a role in this Plasmids Extrachromosomal DNA Small circular 1 type/>1 type Genes code for enzymes that protect cell from toxins eg abt Flagella For motility Flagellin Filament,hook, basal body From cytoplasm Position Peritrichous/lophotrichous/monotrichous  Fimbria(pili) Non-flagellar hair-like projections on surface Adhesion – virulence eg neiserria Sex pili in Gram negatives –transfer genetic material Compose of fimbrillin  Spores Resting/dormant stage For survival-can survive adverse conditions Bacillus/Clostridia Spherical/oval in bacteria Location, size, shape Doesn’t stain Need sterilisation temp. to kill SPORES FUNGI  Mycology-study of fungi Free-living -Widely found in nature Slow-growing Yeast/mould/both (dimorphic fungi) Many fungi that infect man are dimorphic Yeast at body temperature - 370C Mould at room temperature - 250C Yeast on agar plate-Candida spp COLONY OF MOULD STRUCTURE Eukaryotic- nucleus, nuclear membrane, nucleolus, linear chromosomes Organelles Cytoplasmic membrane contains ergosterol & not cholesterol like human cells BUT have rigid cell wall – different structure from bacteria  Rigid external cell wall comprising glucan- form fibrils to strengthen Mannan – mannose-based polymers Chitin- poly-NAGA. Similar to crab shell STRUCTURE OF FUNGAL CELL WALL STRUCTURE Size varies (2-4um) yeast to visible with naked eye eg mushrooms Bud extends from yeast cell, constricts-new cell (blastoconidia) Hyphae – tube-like extensions - mould Aerial hyphae – “fluffy”. Bear reproductive structures Vegetative- portion grows into medium/soil  Mycelium- intertwined mass of hyphae Septa-cross-walls that divide hyphae into subunits Conidia arise from from hyphae/ from conidiophore (stalk-like) STRUCTURE OF YEAST CELL STRUCTURE OF MOULD Lactophenol cotton blue stain from a culture of mould REPRODUCTION Haploid usually Diploid during sexual reproduction Asexual - Multiply by budding- mitosis - conidia Sexual – produce spores - meiosis Fungi: Type of infection Three types of infection(mycoses) are recognized: 1. Superficial mycoses where the fungus grows at the body surface on skin or hair. 2. Cutaneous and subcutaneous mycoses where nails and deeper layers of the skin are involved. 3. Systemic or deep mycoses with involvement of internal organs. YEASTS Candida spp Candida albicans Candida parapsilosis Candida glabrata Cryptococcus neoformans MOULDS Aspergillus spp Rhizopus spp PARASITES  Protozoa Single celled Free-living/parasites Mostly tropical & sup-tropical countries Helminths Macroscopic Multi-cellular worms 100um Nucleus & cytoplasm Endoplasm – food vacuoles Ectoplasm – locomotion eg flagella/pseudopodia  4 classes Amoebae eg Entamoeba histolytica Ciliates eg Balantidium coli Flagellates eg Trichomonas spp Sporozoa eg malaria(Plasmodium falciparam) Protozoa infect tissue and body organs as: Intracellular parasites in a wide variety of cells (red cells, macrophages, epithelial cells, brain, muscle). Extracellular parasites in the blood, intestine or urogenital system. AMOEBA – Entamoeba histolytica HELMINTHS PARASITIC WORMS Tapeworms(Cestodes)- flat eg Taenia Flukes(Trematodes) – leaf-shape eg Schistosoma Roundworms(nematodes) eg Ascaris Tapeworms & flukes-flattened bodies with suckers/hooks Roundworms – long, cylindrical bodies  Complex body structure Nervous/reproductive/alimentary systems No circulatory system Suckers/hooks/teeth/plates for attachment at anterior Body has tough cuticle Larval stages(100-200um) Adults- cm - metres WORM BOLUS - Ascaris WHIPWORM HOOKWORM TAPEWORM Schistosoma –adults (causes bilharzia)