Microbiology - PDF

MICROBIOLOGY  MICROBIOLOGY small living to study Microbiology is the branch of knowledge that deals with microorganisms/microbes → organisms that are too small to be seen with naked eye. The major groups are:...

MICROBIOLOGY  MICROBIOLOGY small living to study Microbiology is the branch of knowledge that deals with microorganisms/microbes → organisms that are too small to be seen with naked eye. The major groups are: Prokaryotes Eukaryotes Viruses Bacteria Fungi Parasites→Protozoa Microbes differ in shape, size, appearance and in genetic and metabolic characteristics.  MICROBES ARE SMALL resolution power = 0.25 nm resolution power = 0.25 m The light (or optical) microscope uses visible light and a system of lenses to generate magnified images of small objects Gram positive and negative bacteria at the light microscope VIRUSES -small size (18-600 nm) -structural organization of subcellular level -true parasite BACTERIA -prokaryotic cells -no nucleus -no intracellular compartmentalization -smaller than eukaryotes (typically 1-10 µm)  YEASTS, single cells FUNGI -eukaryotic cells -nucleus and intracellular compartmentalization MOULDS, filamentous -no motile -larger than bacteria (yeasts -unicellular organisms, typically 15-10 µm) (moulds-pluricellular organisms, typically up to 50 µm) AMOEBA PROTOZOA -eukaryotic cells -nucleus and intracellular compartmentalization PLASMODIUM -no motile or motile for pseudopodes, flagella or cilia -larger than bacteria (typically up to 100 µm) → Complex life cycle that involves biological vectors DISTINGUISHING FEATURES OF EUKARYOTES AND PROKARYOTES MICROBES ARE ABSOLUTELY EVERYWHERE MICROBES ARE UBIQUITOUS MICROBES ARE ABSOLUTELY EVERYWHERE  BENEFITS FROM MICROBIAL ACTIVITY Food production → microbial fermentation processes used to produce yogurt, buttermilk, cheeses, alcohol beverages, leavened breads, sauerkrauts, kimchi, and pickles. Energy production and cleaning up the environment → methane, or natural gas, is a product of methanogenic bacteria; some pollutants (pesticides, solvents, oil spills) can be cleaned up with the aid of microbes. Sustaining agriculture → nitrogen, carbon, and sulfur are converted into forms that can be used by plants in their growth. Production of gene products or products from bioengineering → enzymes, antibiotics, vaccines, and medications (human insulin, interferons, growth hormones).  MICROBES & HUMAN HEALTH Human microbiota refers to all microorganisms living in/on our body, and it is mainly established on the skin, oral and vaginal mucosa, as well as in the respiratory, urinary and gastrointestinal tracts. It has fundamental roles in health and disease. SKIN MICROFLORA  THE HUMAN MICROBIOTA OR NORMAL FLORA Colonization begins at birth and is of two types: - Permanent colonization by microbes that are part of the normal flora at all times. - Transient colonization by potential pathogens. WE ARE BORN 100% HUMAN AND WE DIE 90% MICROBIAL CELLS  THE INTESTINAL MICROBIOTA The microbes that colonize the relatively sterile gut of the infant are largely determined by the mode of delivery. The intestinal microbiota also develops differently determined by differences in infant feeding (breastfeeding vs formula feeding). Throughout infancy and early childhood, the microbiota changes with dietary alterations, infections and exposure to antibiotics. The intestinal microbiota of the infant is characterized by instability and low levels of diversity. By the toddler years (around 3 years of age), the intestinal microbiota is similar in diversity and stability to that of adults.  THE INTESTINAL MICROBIOTA Beyond bacteria, the human gastrointestinal tract is also home to a large number of fungi— 0,1% of the total gut microbes—which play crucial roles in human intestinal homoeostasis and disease pathogenesis  THE INTESTINAL MICROBIOTA FUNCTIONS o breaking down of food compounds o biosynthesis of vitamins (B12, K) and amino acids o activation of host innate and immune resposes o competition for nutrients with pathogenic microbes o competition to adhesion with pathogenic microbes o protection against epithelial injury o promotion of angiogenesis o fermentation of polysaccharides from vegetables o metabolism of therapeutics into active compounds DISRUPTION OF THE INTESTINAL MICROBIOTA Fecal microbiota transplantation (FMT) is a therapeutic intervention that involves the transfer of fecal microbiota from a healthy donor to a recipient with dysbiotic gut microbiota. FMT is thought to work by restoring gut microbial diversity and function, which leads to an improvement in gastrointestinal and nongastrointestinal symptoms.  THE SKIN MICROBIOTA The skin microbiota is the second largest microbiota of the human body in mass, made up 106 microbes/cm2 [108-1010 skin microbes in total]; The relative abundance of skin microbial species is restructured during puberty and microbial communities stabilize in postpubescent individuals  THE SKIN MICROBIOTA FUNCTIONS o maintain an healthy cutaneous barrier o help the immune system o limit pathogenic microorganism growth IMBALANCE IN SKIN MICROBIOTA Microorganisms 2020, 8, 1752  DYSBIOSIS OF THE SKIN MICROBIOTA Environmental factors, cosmetic products, and poor diet could harm the skin microbiota  WE START FROM BACTERIA Prokaryotes Eukaryotes Viruses Bacteria Fungi Parasites→Protozoa How to observe bacteria? The observation is performed by an light microscope, with or without stain.  BACTERIAL STRUCTURES The main bacterial structures are: o cytoplasmic membrane occasionally with mesosome surrounded by a cell wall/outer membrane o fluid cytoplasm containing the DNA chromosome, mRNA, ribosomes, granular inclusion o external structures as capsule, flagella, and fimbriae (pili)  DNA CHROMOSOME OR NUCLEOID o Single, circular, double-stranded and supercoiled DNA molecule (>106 base pairs, around 3500 genes, 1000 µm long) o Haploid genome, weakly bound to acid proteins (no hystones) o Contained not in a nucleus but in a discrete area known as the nucleoid o It is anchored to membrane invaginations (mesosome) o Topological changes in DNA architecture are supported by topoisomerases that are different from their eukaryotic counterparts → unique biochemical target for antibiotic action To enable a macromolecule this large to fit within the bacterium, histone-like proteins bind to the DNA, segregating the DNA molecule into chromosomal domains and making it more compact  PLASMIDS o Circular, double-stranded extrachromosomal DNA molecule (103 – 105 base pairs) o Self replicating → many copies of a plasmid whithin one bacterial cell. o They encode genes for auxiliary functions → usually not essential for the bacterium’s day- to-day survival, but they can help the bacterium to overcome occasional stressful situations. virulence plasmids fertility plasmids degradative plasmids  Replication of DNA chromosome Replication requires: o replication origin (oriC) o DNA-dependent DNA polymerase that synthesizes a copy of the DNA o helycase to unwind the DNA at the origin to expose the DNA o primase to synthesize primers to start the process o topoisomerases oriC New DNA is synthesized semiconservatively, and proceeds bidirectionally  Replication of DNA chromosome New DNA is synthesized semiconservatively, using both strands of the parental DNA as templates  Bacterial topoisomerases The progression of the replicating forks is associated with DNA unrolling that requires the intervention of topoisomerases: o Topoisomerase I: causes a series of positive windings (right-handed) transforming the DNA molecule into a more relaxed (less superspiralized) form o Topoisomerase II or gyrase: causes a series of negative (sinister) windings causing superspiralization of the DNA molecule o Topoisomerase IV: involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication (the two newly formed chromosomes are separated from each other) Topoisomerases are essential in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA  Replication of plasmids DNA rolling circle replication requires: o replication origin (oriC) o nicking enzyme o DNA-dependent DNA polymerase III o DNA-dependent DNA polymerase I A typical DNA rolling circle replication has five steps: 1. Circular dsDNA will be "nicked". 2. The 3’-end is elongated using "unnicked" DNA as leading strand (template); 5’-end is displaced. 3. Displaced DNA is a lagging strand and is made double stranded via a series of Okazaki fragments 4. Replication of both "unnicked" and displaced ssDNA. 5. Displaced DNA circularizes. New DNA is synthesized semiconservatively, and proceeds unidirectionally  CYTOPLASMIC STRUCTURES The cytoplasm is a colloidal matrix made up of H2O for 85%. It is the site of replication, transcription of genetic material, and protein synthesis. It contains the ribosomes and occasionally the granular inclusions with reserve function (lipids, glycogen, polyphosphates). Ribosomes 70S consist of two subunits: 30S (16S RNA, 21 proteins) 50S (5S RNA, 23S RNA, 34 proteins) Ribosomes form functional units called polysomes and are free in the cytoplasm, or in contact with the cytoplasmic membrane. PROTEIN SYNTHESIS AND ANTIBACTERIAL DRUGS  CYTOPLASMIC STRUCTURES The cytoplasmic membrane has a lipid bilayer structure similar to the structure of the eukaryotic membranes, but it contains no sterols (e.g., cholesterol). o symmetrical bilayer of phospholipids o non-glycosylated proteins Cytoplasmic membrane is o external or integrated proteins in the membrane responsible for many of the functions attributable to organelles in eukaryotes  CYTOPLASMIC MEMBRANE FUNCTIONS: o selective permeability and transport of ions and metabolites o electron transport and oxidative phosphorylation (energy production) o biosynthesis of the cell wall o excretion of proteins (enzymes, toxins) in the external environment o functioning in DNA and cell wall synthesis, cell division and spore formation o bearing the receptors of the chemotactic and other transduction systems  CYTOPLASMIC STRUCTURES Mesosomes are folded invaginations in the cytoplasmic membrane of bacteria; o they are prominent in Gram positive bacteria o they are smaller, harder to see in Gram negative bacteria FUNCTIONS: o cell wall synthesis (biosynthetic mesosomes) o cell division and spore formation (septal mesosomes) o helps in respiration and secretion processes (respiratory mesosomes)  BACTERIAL CELL WALL Gram positive Gram negative Gram stain is a rapid, powerful test that allows clinicians to distinguish between the two major classes of bacteria. CELL SHAPE AND STRUCTURE, RESISTANCE TO THE OSMOTIC PRESSURE, PROTECTION FROM THE ENVIRONMENT  BACTERIAL CELL WALL Gram positive Gram negative Thick, multilayered cell wall, ≥40 layers of Thin cell wall, two layers of peptidoglycan peptidoglycan (20 – 80 nm) (8 – 10 nm)  Peptidoglycan (mucopeptide, murein) The peptidoglycan is a rigid mesh made up of linear polisaccharide chains cross-linked by peptides. The polysaccharide NAG NAM is made up of repeating The tetrapeptide is disaccharides of attached to NAM, NAG and NAM and it is unsual because it contains both D and L The amino acid in the third amino acids position (LYSINE or DIAMINO PIMELIC ACID) is essential for the cross-linking of the peptidoglycan chain The Peptidoglycan Cell Wall - Bing video Peptidoglycan (mucopeptide, murein) Peptidoglycan in Gram positive AMINO ACID BRIDGE → GLYCINE5 PEPTIDE Peptidoglycan in Gram negative DIRECT BRIDGE BIOSYNTHESIS PATHWAY OF THE PETIDOGLYCAN  Peptidoglycan synthesis INSIDE THE CELL o NAG is enzymatically converted into NAM, and enegetically activated with UTP to form UDP-NAM. o the pentapeptide is assembled in a series of enzymatic reactions and attached to UDP-NAM to form the UDP-NAM pentapeptide Peptidoglycan synthesis AT MEMBRANE LEVEL o the UDP-NAM pentapeptide is attached to the bactoprenol “conveyor belt” (dolichol) o NAG is added and the disaccharide building block is made up o the pentaglycine in Gram positive bacteria is added to the third amino acid o the bactoprenol molecule translocates the disaccharide:peptide precursor to the outside of the cell.  Peptidoglycan synthesis OUTSIDE THE CELL o the disaccharide building block (NAG-NAM pentapeptide) is attached to a peptidoglycan chain by using the energy released by the pyrophosphate of the bactoprenol o the bactoprenol is recycled at membrane level o the peptide chains from adjacent glycan chains are cross-linked to each other by a peptide bond exchange 3rd 4th TRANSGLYCOSYLASES TRANSPEPTIDASES enzymes that attach the enzymes that catalyze the disaccharide building block transpeptidation reaction to to the existing crosslink adjacent peptidoglycan chain peptidoglycan chains  Peptidoglycan synthesis CARBOXYPEPTIDASES are enzymes that remove unreacted terminal D-Ala to limit the extent of cross-linking TRANSPEPTIDASES and CARBOXYPEPTIDASES are called penicillin-binding proteins (PBPs), they are used for extending the peptidoglycan, creating a septum for cell division and curving the peptidoglycan mesh (cell shape). Peptidoglycan extension and cross-linking is necessary for cell growth and division. AUTOLYSINS are important for determining bacterial shape  Cell wall – Gram positive bacteria In Gram positive bacteria, the cell wall is constituted by a thick layer of peptidoglycan, with quantities of other polymers intersected, essentially represented by the teichoic and lipoteichoic acids → water-soluble polymers of chemically modified ribose and glycerol connected by phosphates STRENGHTENS CELL WALL  Teichoic and lipoteichoic acids FUNCTIONS: o they give the cell surface an overall negative charge, thus promoting the intake of cations (K+, Ca2+, Mg2+) from the external environment (ions sequestration) o they stabilize the cell wall because they are anchored to the membrane (strengthens cell wall) o they are highly antigenic and show a remarkable diversity of composition in the different bacterial species o they promote attachment to other bacteria and surfaces (virulence Polyribitol phosphate (A) or glycerol factors) phosphate (B) cross-linked to peptidoglycan Cell wall – Gram positive bacteria  Cell wall – Gram negative bacteria In Gram negative bacteria, the cell wall consists of a thin layer of peptidoglycan surrounded by an outer membrane → asymmetric bilayered structure, the inner leaflet contains phospholipids, the outer leaflet is composed primarily of lipopolysaccharide (LPS) CELL STRUCTURE AND PROTECTION FROM THE ENVIRONMENT  Cell wall – Gram negative bacteria The outer membrane contains also: o outer membrane porins (Omp, trimeric transmembrane proteins): allow the passage of small hydrophilic substances o lipoproteins: anchor the outer membrane to the peptidoglycan and to the membrane o secretion devices (types I, II, III, IV and V): transport systems for the release and uptake across membranes of metabolites, proteins including virulence factors  Cell wall – Gram negative bacteria The type III secretion device (injectisome) is a major virulence factor for some bacteria, with a complex structure that traverses both the inner and outer membranes and can act as a syringe to inject proteins (toxins) into eukaryotic host cells.  Cell wall – Gram negative bacteria Periplasmic space → compartment between the external surface of the cytoplasmic membrane and the internal surface of the outer membrane. It is a protein gel containing : o transport systems for iron, proteins, sugars and other metabolites o a variety of hydrolytic enzymes (include proteases, phosphatases, lipases, nucleases, and carbohydrate-degrading enzymes) o different virulence factors (collagenases, hyaluronidases, proteases, and β-lactamases) Collagenases are enzymes that break the peptide bonds in the collagen, a key component of the animal extracellular matrix. Hyaluronidases are a family of enzymes that catalyze the degradation of the hyaluronic acid. They are mainly produce in bacteria that initiate their infection at the skin and mucosa surfaces. They both allow the bacteria to spread through subcutaneus tissue.  Lipopolysaccharide (LPS) O antigen Core polysaccharide Lipid A 2-keto-3-deoxy-octanoate LIPID A has a phosphorylated glucosamine disaccharide backbone with fatty acids attached to anchor the structure in the outer membrane. It is responsible for the endotoxin activity of LPS; it is released following cell lysis. CORE POLYSACCHARIDE is a branched phosphorylated polysaccharide of 9 to 12 sugars (unusual sugars, C6-8). O ANTIGEN is a long, linear or branched polysaccharides. It varies from one species to another, constituting the somatic antigenic component of the cell.  Cell wall - GRAM positive Vs GRAM negative o ~40 layers of peptidoglycan (50% of the cell wall) o 2-3 layers of peptidoglycan (5-10% of the cell o thickness: 20 – 80 nm wall) o L-LYS in the tetrapeptide o thickness: 8 – 10 nm o pentaglycine bridge o DAP in the tetrapeptide o direct bridge  Bacteria with alternative cell wall structure - Mycobacteria lipid coat wax-like Mycobacteria possess a complex, lipid-rich cell wall that is responsible for: o acid-fastness (resist to decoloration by acid solution) o slow growth (requiring incubation of 1 month or longer) o resistance to detergents and to common antibacterial drugs o resistance to phagocytosis  Bacteria cell wall and Gram stain The structural features of the cell wall allow to classify bacteria in two main groups, Gram positive and Gram negative, based on their characteristics. The classification of the bacteria in Gram groups was carried out by the histologist Hans Christian Joachin Gram (1884), who developed this differential staining technique. This technique involves the use of 2 dyes: o crystal violet o safranin  Gram stain is a differential stain The crystal violet of Gram stain is precipitated by Gram iodine and is trapped in the thick peptidoglycan layer in Gram positive bacteria. The decolorizer disperses the gram-negative outer membrane and washes the crystal violet from the thin layer of peptidoglycan. Gram negative bacteria are visualized by the red counterstain  Gram stain is a differential stain Streptococci Staphylococci Gram positive bacteria are visualized purple (Positive→P→Purple) Neisseria gonhorreae E. coli Gram negative bacteria are visualized red  The Bacterial Cell: Shapes and Arrangements COCCUS BACILLUS - ROD SPIRAL Types of bacteria  Importance of Gram stain o rapid semi-presuntive diagnosis of the infectious agent o classification of the bacteria: Gram positive or Gram negative o appreciation of the morphology: cocci, rods, spirals o appreciation of the arrangement of the bacteria: chains, clusters o identification of polymicrobial organisms However: o not suitable on some clinical specimens as feces or sputum o not appropriate for the detection of acid-fast bacteria (Mycobacteria)  Ziehl-Neelsen stain Ziehl-Neelsen staining is a type of bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. It is named for two German doctors who improved the stain, the bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen. “Acid-fast” refers to bacteria whose cell wall has a high lipid content of mycolic acids, which causes them to bind and retain the complex basic dye carbolfuchsin even after strong decoloration with acid-alcohol (thus “resistant to acid”). This differential staining technique involves the use of 2 dyes: o carbolfuchsin, a mixture of phenol and basic fuchsin o methylene blue  Ziehl-Neelsen stain 3% HCl in 70% isopropyl alcohol (Gram stain: ethanol-acetone 1:1) Ziehl-Neelsen stain The acid-fast bacteria (B) appear red (carbolfuchsin) while the other bacteria (A) take on the blue staining (methylene blue).  Crystalline layer – S layer Gram positive Gram negative The crystalline layer is made up of protein subunits, some of which can act as adhesins. It contributes to cell shape and helps protect the cell from osmotic stress.  Capsule Capsule, sometimes referred to as a slime layer or a glycocalyx, is a viscous layer outside the cell of some bacteria (Gram positive or Gram negative). It is made up of capsular polysaccharides. It is unnecessary for the growth but is very important for bacterial cell. India ink stain IMPORTANT FEATURES: o prevention from desiccation and drying o protection from mechanical injury, temperature o adherence to other bacteria or host tissue surfaces o resistance to phagocytosis o resistance to biocides (antibiotics or disinfectants) and host immune response o role in biofilm formation  What are biofilms? Biofilms are well-structured population of bacterial cells enclosed in a self-produced extracellular polymer matrix that are attached to a surface (biotic and abiotic surfaces). The matrix contains also metal ions, cations, proteins, lipids and extracellular DNA (eDNA). Bacterial cells within the biofilm are in the sessile state→strictly bound and firmly attached to each other, and to a solid surface  planktonic state → independently floating in a liquid environment. planktonic cells sessile forms Biofilm  BIOFILMS AND PUBLIC HEALTH Biofilms have many potential consequences for public health. Positive impact on human health: o commensal Staphylococcus epidermidis can impede the colonisation of potentially pathogenic bacteria through the stimulation of host-cell immune defences and the prevention of adhesion Negative impact on human health because biofilms are association with many diseases: o pneumonia in cystic fibrosis patients o chronic infection of wounds o chronic otitis media o implant- and catheter-associated infection o dental plaque Biofilm infections have improved pathogenic efficiency compared to those associated with planktonic cells, as microbial cells within the biofilm are recalcitrant to clearance by antimicrobials and host defence molecules BIOFILMS AND DISEASES BIOFILMS AND MEDICAL DEVISES BIOFILMS AND MEDICAL DEVISES  BIOFILMS AND PROCESSING INDUSTRIES Pipes damages: o Corrosion (pitting, craking) o Choking o Decrease of heat exchange efficiency, decrease of the flow  BIOFILMS AND PROCESSING INDUSTRIES The growth of biofilms on industrial processing equipment can lead to microbial contamination of the end-product. Unfortunately, regular cleaning and sanitation aren’t enough to prevent biofilm formation, because bacteria are highly protected under the residues forming the biofilm, and they will continue to grow until the overlying film is removed. Biofilms are difficult to remove through standard cleaning and sanitizing procedures. A specific process is needed to remove biofilms effectively: peracetic acid BIOFILMS AND PROCESSING INDUSTRIES  Flagella Flagella are ropelike propellers that allow the movement of the bacterial cell. They are composed of helically coiled protein subunits (flagellin→H antigen), anchored in the cytoplasmic membrane. Flagella consist of three main parts: o basal body o hook o filament  Flagella The movement is due to the coordinated activity of the flagella and is induced and oriented by chemical stimuli (chemotaxis); it is affected by the concentration gradients of attractive or repellent substances. Counterclockwise rotation leads to a straight line movement; clockwise rotation leads to a change of direction. The alternation of these phases leads to a directional movement towards an attractive substance or far from a repellent substance  Flagella a single flagellum at one pole flagella at both end of the cell a tuft of flagella at one pole flagella around the whole surface  Fimbriae (pili) Fimbriae (pili) are hairlike structures on the outside of bacteria and they are composed of protein subunits (pilin). Some important characteristics: o smaller in diameter compared to flagella o usually not coiled in structure o generally, several hundred fimbriae are arranged uniformly over the entire surface of the cell Function: o adherence to other bacteria or to the host (adhesins) o colonization of tissues the tips of the fimbriae may contain lectins that bind to specific sugars (mannose) on the host cell