Lecture 1 Bacteria (I and II) PDF

11/3/2023 Clinically Relevant Microorganisms: Bacteria (I and II) Tomás Maira Litrán Contact Information Research Building, Office number: 3460 Extension number: 806408 email: [email protected] 1 11/3/2023 Microbiology and Microorganisms • Microorganisms (microbes) are life forms too small...

11/3/2023 Clinically Relevant Microorganisms: Bacteria (I and II) Tomás Maira Litrán Contact Information Research Building, Office number: 3460 Extension number: 806408 email: [email protected] 1 11/3/2023 Microbiology and Microorganisms • Microorganisms (microbes) are life forms too small to be seen by the human eye (require the use of microscopes) • Most microorganisms are harmless to humans and, in fact, many are helpful • Some are harmful causing various types of infections • Diverse in form/function • Inhabit every environment that supports life • Many single-celled, some form complex structures, some multicellular • Live in microbial communities Organisms and Biological Entities Acellular Cellular Prokaryotes (Unicellular) Eukaryotes Viruses Viroids/ Virusoids Bacteria Fungi Yeast (Unicellular) Parasites Mold (Multicellular) Protozoa (Unicellular) Worms Helminths (multicellular) Bugs Arthropods (vectors !!!) Dimorphic (Uni and Multicellular) 2 11/3/2023 Prokaryotes (Bacteria) • • • • • Genomic material:  Not surrounded by a nuclear membrane  Only DNA (no proteins)  Plasmids No specialized membrane-surrounded organelles 70S ribosomes Flagella with simple structure and cilia Complex cell wall with peptidoglycan and other components Eukaryotes • • • • • Genomic material  DNA and proteins isolated from cytoplasm by nuclear membrane Different organelles separated by internal membranes. Organelles specialized in different functions 80S ribosomes Complex flagella and cilia Classical cellular membranes: lipid bilayer and proteins Downloaded from: StudentConsult (on 10 April 2013 05:45 PM) © 2005 Elsevier Organisms and Biological Entities Acellular Cellular Prokaryotes (Unicellular) Eukaryotes Viruses Viroids/ Virusoids Bacteria Fungi Yeast (Unicellular) Parasites Mold (Multicellular) Protozoa (Unicellular) Worms Helminths (multicellular) Bugs Arthropods (vectors !!!) Dimorphic (Uni and Multicellular) 3 11/3/2023 Bacteria-Prokaryotes BACTERIA: Classification Bacteria can be classified by: • Shape • Cell wall structure  Staininig property • Formation of spores • Additional structures: capsule, flagella, etc • Growth characteristcis (e.g. +/- oxygen) • Metabolic properties, biochemical properties • Extracellular or Intracellular • Genotype 4 11/3/2023 Common Cell Morphologies and Arrangements Bacterial Classification According to Cell Wall Structure and Gram Staining De Agostini Picture Library / Getty Images The Gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria 5 11/3/2023 Bacterial Classification According to Cell Wall Structure and Gram Staining Gram-Positive Gram-Negative Bacterial Classification According to Cell Wall Structure and Gram Staining Gram-Positive Gram-Negative PM: plasma membrane P: periplasmic space W/M: peptidoglycan (murein) layer OM: outer membrane (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th edition. McGraw-Hill, 2008) 6 11/3/2023 Peptidoglycan Subunit Composition Lysozyme Peptidoglycan: • Provides rigid support to bacterial cells and maintains the characteristic shape of the cell and allows bacterial cells to withstand media of low osmotic pressure, such as water • Target for antibacterial drugs (beta-lactams-penicillins, glycopeptide antibiotic, etc) • Cleaved by lysozyme enzymes (human tears, mucus, and saliva) thus providing a major line of defense against bacterial infection Peptidoglycan Subunit Composition N-acetylglucosamine (NAG) + N-acetylmuramic acid (NAM) • Contains both D and L amino acids (D amino acids are not normally used in nature) • Three amino acids not present in proteins:  D-glutamic acid  D-Alanine  Diaminopimelic acid (DAP) • D-amino acids protects against degradation by peptidases (recognize only the L-isomers) DIAMINOPIMELIC ACID (DAP) 7 11/3/2023 Peptidoglycan Cross-Link Gram negative Gram positive Tetrapeptide residues (depends on the bacterial species) Most Gram-negative bacteria: (L-ala)(D-glu)-(DAP)-(D-ala), where DAP is diaminopimelic acid Gram-positive bacteria may have DAP or L-lys or another amino acid at position 3 Peptidoglycan thickness: Gram-positive: ≈50–100 molecules thick Gram-negative: ≈1–2 molecules think 8 11/3/2023 Teichoic and Lipoteichoic Acids-(Gram Positive) Lipoteichoic acid has a fatty acid and is anchored in the membrane Teichoic acid is attached to the peptide of peptidoglycan • Polymers of glycerol or ribitol joined by phosphate groups • Aa and sugars attached to glycerol/ribitol • Negatively charged • Maintain structure of cell envelope • Defence against: - Antibiotics - Host defences • Binding to host tissue Bacterial Lipopolysaccharide (Gram-Negative) 9 11/3/2023 Lipopolysaccharide (LPS) Structure Consists of three parts 1. Lipid A 2. Core polysaccharide 3. O side chain (O antigen) Lipopolysaccharide (LPS) Structure O chain: major surface antigen • (O-polysaccharide = O-antigen) • Sugars chain linear or branched • Variation • Immunogenic (serotypes) Core polysaccharide: outer and inner core • KDO* (specific sugar), heptoses and hexoses which can be modified with P groups, phophoethanolamine, etc Lipid A (Endotoxin): conserved among related bacteria (similar in all enterobacteriaceae) • Glucosamines + P groups • Fatty acids • Toxic. Endotoxic shock. Death *KDO: 3-deoxy-D-mannooctulosonic acid ketodeoxyoctonate 10 11/3/2023 Lipopolysaccharide (LPS) Biological Role • Amphipathic molecule; helps to create a permeability barrier • Stabilize outer membrane • Protects Gram-negative bacteria from host defenses like TEICHOIC and LipaCHOIC ACIDS • Pathogenesis • Adhesion • Biofilm formation Smooth and Rough (LPS) Structures of a Gram-Negative Bacterium LPS may be present in the smooth or rough form • Smooth LPS (S-LPS) contains the complete core oligosaccharide and OMore virulent antigen regions => • Rough LPS (R-LPS) does not contain the O-antigen region but have either complete or truncated core oligosaccharide regions LESS VIRULENT = • Presence of smooth vs rough LPS will generally impact bacterial virulence = linked to severity Colony morphology of Yersinia enterocolitica with S-LPS and R-LPS 11 11/3/2023 Periplasmic Space Proteins involved in: • Transport  inside the cell: nutrientes  outside the cell: waste products • Electron transport • Peptidoglycan and LPS synthesis • Flagella structure • Secretion systems Other Bacteria–Other Cell Walls (Mycobacterias) Mycobacterial cell walls • Waxy, hydrophobic coat, with a high percentage of lipids (≈60%) • Contain long chain C60-90 fatty acids called mycolic acids (thick layer) linked to peptidoglycan by arabinogalactan polysaccharide (thing layers) • Lipids: lipoarabinomannan and other glycolipids • Impenetrable by many antibiotics, desinfectants etc 12 11/3/2023 Other Bacteria–Other Cell Envelopes (Mycoplasma) The Mycoplasma • Lack cell walls (only plasma membrane) and are pleomorphic • Sterols may stabilize plasma membrane • Smallest bacteria size: capable of self-reproduction (0.2–0.3 μm vs 1.3 x 4 μm for E. coli) • Smallest genome size: < 1,000 genes and one of the smallest found in prokaryotes • Resist the action of β-lactams acting on cellular wall • Grow as fried egg appearance on agar surface Bacterial Secretion in Prokaryotes DOI:10.3390/toxins11060332 13 11/3/2023 Bacterial Secretion Systems The secretion systems are molecular complexes inserted in the bacterium envelope, which specifically export to the outside environment or to another cell Eukaryotic cell Effectors OUTSIDE T3SS T4SS T6SS Bacteria Type III secretion system Salmonella INSIDE Cell Wall Vs Cell Envelope Gram-Positive Gram-Negative Cell Envelope Cell wall: rigid structure that surrounds a microorganism and has a role in cell shape and homeostasis Cell envelope: membrane(s) and other structures that surround and protect the cytoplasm 14 11/3/2023 Components Outside of the Cell Wall • The cell envelope often includes layers and structures outside the cell wall 1. Capsule 2. Pili 3. Flagella Components Outside of the Cell Wall: Capsules • Usually composed of polysaccharides (sometimes peptides/proteins) • Well organized and not easily removed from cell Bacterial capsules Capsule • Not considered part of cell wall because these do not confer significant structural strength Biological function Capsule • Assist in attachment to surfaces • Role in development and maintenance of biofilms • Resistant to phagocytosis • Protect from desiccation • Exclude viruses and detergents 15 11/3/2023 Components Outside of the Cell Wall: Capsules Capsule in Gram Positive and Gram-Negative Bacteria Capsule Components Outside of the Cell Wall: Capsules Polysaccharide Neisseria meningitidis Haemophilus influenzae Streptococcus pneumoniae Escherichia coli Protein Bacillus anthracis Yersinia pestis • Capsular polysaccharides are a key component of numerous vaccines against encapsulated pathogens  Example of a polysaccharide (capsule)-based vaccine: Neisseria meningitidis serotypes A, C, W, Y polysaccharide vaccine 16 11/3/2023 Components Outside of the Cell Wall: Pili Fimbriae (s., fimbria); pili (s., pilus) • Short, thin, hairlike, protein appendages (up to 1,000/cell) • Can mediate attachment to surfaces, motility, DNA uptake Sex pili (s., pilus) • Longer, thicker, less numerous (1-10/cell) • Genes for formation on plasmids • Required for genetic exchange between bacteria (during conjugation) Components Outside of the Cell Wall: Flagella • Threadlike, locomotor appendages extending outward from plasma membrane and cell wall • Found in both Gram-positive and Gram-negative bacteria • Functions  Motility  Attachment to surfaces  May be virulence factors 17 11/3/2023 Pathogenic Bacteria Display Two Basic Types of Metabolism Many Types of Organic Matter (Partial oxidation through very many different pathways) Alcohols Acids Other compounds Air (O2) is not necessary in these metabolic types CO2 RESPIRATION FERMENTATION NADH2 FADH2 Krebs cycle Respiratory electron chain coupled to: O2 H2O Aerobic respiration NO3NH4++H2O SO42- Others SH2+H2O Anaerobic respiration Oxygen Requirements of Microorganisms Obligate aerobe: completely dependent on atmospheric O2 for growth Facultative anaerobe: do not require O2 but grow better in its presence Aerobes In the O2 of oxygen, they can respire nitrates, nitrites, sulfates, etc. or ferment Microaerophile: requires low O2 tensions (2-10%) Damaged by atmospheric O2 levels (21%) Obligate anaerobe: O2 is toxic for them Anaerobes Some respire nitrates, sulfate or other oxidized inorganic compounds Some ferment Aerotolerant anaerobe: can tolerate O2 Always ferment 18 11/3/2023 Detoxification of Reactive Oxygen Species • Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified:  superoxide radical O2 hydrogen peroxide H2O2  hydroxyl radical OH● • Aerobes produce protective enzymes that break down some of the ROS: • Three main enzymes break down those toxic byproducts:  Catalase  Peroxidase  Superoxide dismutase Protective Enzymes in Various Aerotolerant Groups Aerobes and aerotolerant anaerobes • All organisms which can live in the presence of O2 (whether or not they utilize it in their metabolism) contain superoxide dismutase (SOD) • Nearly all organisms contain the enzyme catalase, which decomposes H2O2 Obligate/Strict anaerobes • Lack/have very low quantities of superoxide dismutase and catalase 19 11/3/2023 Binary Fission • Growth: increase in the number of cells • Generally, bacteria multiply by a type of asexual reproduction called binary fission • Step of binary fission 1 1. DNA replication 2. Cell elongation 3. Septum formation 2 4. Cell separation • Generation time 3  Depends on several factors: nutritional, genetic factors, temperature etc  Example: Escherichia coli ~20 min 4 • Daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell Microbial Growth Cycle Phases of Bacterial Growth 1. Lag phase > NCULATION • No growth • Time between inoculation of a culture and beginning of growth • Biosynthesis of new enzymes; production required metabolites before growth can begin 2. Exponential phase /LOG in a culture * INACULATION in organism b INTRODUCING MICROORGANISM INTO CULTURE PHASE Most rapid growth Constant growth rate Cells are typically in the healthiest state 3. Stationary phase • Growth rate of is zero. Some cells grow, others die • Essential nutrient is used up, waste products accumulate, or culture runs out of O2 • Metabolism continues at greatly reduced rate 4. Death phase • If incubation continues the cells will eventually die 20 11/3/2023 Planktonic Growth vs Biofilm Growth • Planktonic growth: growth as suspension • Biofilms: adherent bacterial communities embedded in a polymer matrix • Protected from environmental stresses, such as desiccation, antimicrobials attack by the immune system or antibiotics→ Recalcitrant infections Scanning Electron Micrographs of Bacterial Biofilms Staphylococcus aureus Staphylococcus aureus Pseudomonas aeruginosa Bacillus cereus 21 11/3/2023 Types of Tissue and Device Related Biofilm Infections Caused by Bacterial Pathogens • Red: biofilms developing on tissues in our body • Purple: biofilms developing on implanted material Endospores • Some Gram-positive, members of the genera Bacillus and Clostridium are spore formers • Formed in response to unfavorable growth conditions e.g. limitation of nutrients (vegetative state → dormant state, or spore) • Allow cells to survive long periods without food or water, as well as exposure to chemicals, extreme temperatures, and even radiation • The location/shape/size of the spore within a cell is a characteristic of the bacteria and can assist in identification of the bacterium • Resistance to:  Heat  Dessication  UV light  Radiations  Chemical agents  Enzymes 22 11/3/2023 Sporulation • Process of endospore formation • Occurs in a hours (generally 6-8 hours) • Normally begins when growth ceases because of lack of nutrients • Complex multistage process Endospores • Exosporium: thin protein covering, is the outermost layer of a bacterial endospore • Protein coat: keratin-like proteins • Outer membrane • Cortex: thick layer of loosely cross-linked peptidoglycan • Spore wall: normal peptidoglycan • Inner membrane • Core Cortex = Modified peptidoglycan - NAcGM --- Nac MuR - NAcGM --- Nac MuR L- Ala L- Ala - NAcGM --- Nac MuR Lactic D- Glu • Few crosslinking • Alanine subunits • Lactic subunits Meso-DAP D- Ala 15 % 30 % 55 % 23 11/3/2023 Endospores Core Characteristics • Complete copy of the chromosome • Bare minimum concentrations of essential proteins and ribosomes • Small acid-soluble DNA-binding proteins (SASPs) Nucleic acids Enzymes Ribosomes Ca-DPA SASPs • High concentration of dipicolinic acid (DPA) complexed with Ca2+ ions (Ca-DPA) • Very low water content • Thermostable enzymes • Impermeable layers Clinically Relevant Bacteria that Produce Spores • Spore-forming bacteria mainly belong to two GramPositive genera: Bacilli and Clostridia Anthrax bioterrorist attack 2001 • Bacillus anthracis: anthrax; bioterrorism • Clostridium tetani: tetanus Bacillus anthracis • Clostridium difficile: antibiotic associated diarrhea • Clostridium perfringens: gas gangrene and food poisoning • Clostridium botulinum: botulism, food spoilage • These pathogens are difficult to combat because their endospores are so hard to kill Clostridium tetani • Autoclaves should be used to sterilize material that contains spores 24 11/3/2023 Antibiotics and Chemotherapeutic Agents Antibiotics and Chemotherapeutic Agents Antibiotic • Microbial products or their derivatives that kill susceptible microbes or inhibit their growth • Today the term has a broader meaning, in one sense to include designed molecules (chemotherapeutic agent) Selective Toxicity • Ability of drug to kill or inhibit pathogen while damaging host as little as possible Eukaryote ANTIBIOTIC Prokaryote 25 11/3/2023 Antibiotics and Chemotherapeutic Agents Bacteriostatic antibiotic • Antibiotic that inhibits the growth of bacteria but does not kill Bactericidal antibiotic • Antibiotic that kills bacteria Antibacterial spectrum • Range of activity of an antimicrobial against bacteria  Broad-spectrum drugs can inhibit a wide variety of bacteria (e.g Grampositive and Gram-negative bacteria)  Narrow-spectrum drug is active against a limited variety of bacteria  Advantages and disadvantages of both broad and narrow-spectrum drugs Measuring Effectiveness of Antimicrobial Drugs Effectiveness expressed in two ways • Minimal inhibitory concentration (MIC)  lowest concentration of drug that inhibits growth of pathogen • Minimal lethal concentration (MLC)  lowest concentration of drug that kills pathogen MICs are considered the gold standard' for determining the susceptibility of organisms to antimicrobials A variety of laboratory methods can be used to evaluate or screen the in vitro antimicrobial activity of an antibiotic/antimicrobial agent The most common techniques to determine MICs are: 1. Classical method: broth dilution method 2. Agar disk-diffusion method (Kirby-Bauer test) 3. Antimicrobial gradient method (Etest) 26 11/3/2023 Measuring Effectiveness of Antimicrobial Drugs: Broth Dilution Method Antibiotic Concentration Decreases MBC 1. Prepare two-fold dilutions of the antimicrobial in a liquid growth medium dispensed in tubes 2. Inoculated each tube with a microbial inoculum prepared in the same medium after dilution of standardized microbial suspension MIC + Bacteria 3. Mix and incubate under suitable conditions depending upon the test microorganism 4. MIC is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in tubes (or microdilution) as detected by the naked eye (MIC=8 µg/ml) 5. MBC is determined after broth macrodilution by sub-culturing a sample on non-selective agar plates after 24 h of incubation. MBC is the lowest antimicrobial concentration yielding a negative microbial growth after incubation. (MBC=32 µg/ml) 6. Interpret results (Susceptible, Intermediate, Resistant?) MIC What is the MIC of Vancomycin against Staphylococcus aureus? 27 11/3/2023 Measuring Effectiveness of Antimicrobial Drugs: Agar Disk-Diffusion Method (Kirby-Bauer Test) 1. Inoculate agar plates with a standardized inoculum of the test microorganism 2. Place filter paper disc(s) containing antibiotics at a desired concentration (single concentration) on the agar surface 3. Incubate plates under suitable conditions; Generally, antimicrobial agent diffuses into the agar and inhibits growth of the test microorganism 4. Measure diameters of inhibition growth zones and interpret results (Susceptible, Intermediate, Resistant?) • Halo diameter is a function of diffusion, activity and bacterial growth rates • Different antibiotics have different minimal diameters that define resistance/sensitivity Measuring Effectiveness of Antimicrobial Drugs: (Etest) Antimicrobial Gradient Method • Alternative method used to determine MIC; combination of the Kirby-Bauer disk diffusion test and dilution methods 1. Inoculate agar plate with standardized inoculum of the test bacteria 2. Deposit strip impregnated with an increasing concentration gradient of the antimicrobial agent from one end to the other on the agar surface 3. The point where the border of the zone of inhibition touches the strip defines the MIC (arrow) 4. Interpret results (Susceptible, Intermediate, Resistant?) Vancomycin Tetracycline Ciprofloxacin Ampicillin MIC MIC MIC MIC (1 μg/ml) Escherichia coli Staphylococcus aureus 28 11/3/2023 MIC Interpretation • Convert inhibition zone diameters or MIC values (from broth dilution and Etest methods) into SIR categories (Susceptible, Intermediate, Resistant), cut-off values (breakpoints) • Such breakpoints are published for clinically relevant organisms by the Clinical and Laboratory Standards Institute (CLSI) Staphylococcus aureus Mechanisms of Action of Major Antibacterial Agents • Antibiotics inhibit essential bacterial processes such as cell wall synthesis, plasma membrane integrity, nucleic acid synthesis, ribosomal function folate synthesis etc • Antibiotics are classified by their mechanism of action, as each group targets different parts of bacterial anatomy or physiology 4 29