Basic microbiology

Introduction:

Microbiology is the study of organisms, called micro-organisms that are too small to be perceived clearly by the unaided eye. Antony van Leeuwenhoelk (1632 – 1723 ) first discovered the microbial world. Since then our knowledge in this field is increasing day by day especially after the introduction of high resolution microscopes like electron microscope. Microorganisms can be classified into major groups which are algae, protozoa, fungi, bacteria, viruses and number of organisms intermediate between bacteria and viruses (e.g. rickettsiae, chlamydiae). Most micro-organisms belong to Kingdom ‘Protista’ (protists).

  • Higher protists (eucaryotes – organized cell).
  • Lower protists (procaryotes – simple cell structure).

Viruses are unique, acellular, metabolically inert organism, and therefore can replicate only within living cells. 

Eukaryotes prokaryotes and viruses:

One simpler way to classifying cellular organisms is to divide them into prokaryotes and eukaryotes. Fungi, protozoa and humans, for instance, are eukaryotic, whereas bacteria are prokaryotic. In prokaryotes, the bacterial genome or chromosome is a single, circular molecule of double-stranded DNA, lacking a nuclear membrane (smaller, single or multiple circular DNA molecule called plasmids may also be present in bacteria) whereas the eukaryotic cell has a true nucleus with multiple chromosomes surrounded by a nuclear membrane. Following are properties of eukaryotes and prokaryotes,

Eucaryotic cell:

A eucaryotic cell is about 20 µm in diameter or larger. The main units of cell organization are:

  1. Nucleus consisting of sub-units called chromosomes which are composed of deoxyribonucleic acid (DNA), contains the genetic information. The nucleus is contained in a membrane.
  2. Mitochondria and chloroplasts are sites of energy generation.
  3. Vacuoles and lysosomes are involved in ingestion and digestion of food.
  4. Cytoplasm contains a colloidal suspension of proteins, carbohydrates and important organelles such as endoplasmic reticulum, Golgi apparatus and ribosomes, which are involved in protein synthesis. Cytoplasm is also a means of locomotion in cells without cell walls by amoeboid motion.
  5. Flagella provide a means of locomotion for cells, which have a rigid cell wall.

Procaryotic cell:

These are usually smaller than 5 µm in diameter and have much simpler structure:

  1. Nucleus contains a single long molecule of DNA and is not separated from cytoplasm by a membrane.
  2. Cytoplasm occupies most of the space and is relatively uniform in structure.
  3. Enzymes for respiration and photosynthesis are housed in the cell membrane, which also regulates the flow of materials into and out of the cell.
  4. Most cells are surrounded by a rigid cell wall.
  5. Procaryotes move by the action of flagella.

Viruses:

Viruses have a simpler chemical structure. They consist only of a protein coat surrounding a single kind of nucleic acid, either DNA or ribonucleic acid (RNA). With the exception of enzymes, which aid in penetration of the host cell they are devoid of enzyme activity and consequently cannot be considered true cells. When a virus injects its nucleic acid into the host cell, it takes over the regulation of the cell and directs it towards the production of more viruses. The cell fills up with the newly formed viruses and then bursts spilling the viruses into the medium where each particle can infect other host cells.

Morphology of bacteria:

Shape and size

The shape of a bacterium is determined by its rigid cell wall. Bacteria are classified into three main groups:

  • Cocci (spherical)
  • Bacilli (rod-shaped)
  • Spirochaetes (helical).

Some bacterial with variable shapes appearing both as coccal and bacillary forms, arc called pleomorphic in appearance. The size of bacteria ranges from about 0.2 µm to 5 µm.

Arrangement of bacteria:

Bacteria, whichever shape they may be, arrange themselves (usually according to the plane of successive cell divi­sion) as pairs (diplococcal); chains (streptococci), grape-like clusters (staphylococci) or as angled pairs or palisades (corynebacteria).

 Classification of bacteria according to their shape and arrangement

Shape

Name

Size range

Sphere Cocci (coccus, singular), Pairs (diplococcal),Chains (streptococci),Grape-like clusters (staphylococci), Angled pairs or palisades (corynebacteria). 1-3 µm
Rod bacilli (bacillus, singuar) 0.3-1.5 µm (diameter)1-10 µm (length)
curved rod orspiral vibrio (curved), spirilla (spiral; spirillus, singular) 0.6-1 µm (diameter), 2-6 µm (length)up to 50 µm
Filament (chains of Individual  cells) variety of names 100 µm and longer

Gram-staining characteristics:

Bacteria can be classified into two major subgroups according to the staining characteristics of their cell walls. The stain used, called the Gram stain (first developed by a Danish physician, Christian Gram), divides the bacteria into Gram-positive (purple) and Gram-negative (pink) groups. The Gram-staining property of bacteria is useful both for their identification and in the therapy of bacterial infections because, in general, Gram-positive bacteria are more susceptible to penicillin than Gram-negative bacteria.

Structure of the Bacterial Cell

Although bacterial cells are much smaller and simpler in structure than eukaryotic cells, the bacteria are an exceedingly diverse group of organisms that differ in size, shape, habitat and metabolism. All bacterial cells are surrounded by at least one membrane, the cytoplasmic membrane enclosing the cytoplasm, but most cells are surrounded in addition by a thick cell wall (the Gram-positives) and another group by a thin cell wall followed by a second membrane, the outer membrane (the Gram-negatives), where both membranes are separated by the periplasm. So, Gram-positive bacteria consist of three compartments, the cytoplasm, the cytoplasmic membrane and the extracytoplasm, while Gram-negatives contain two additional compartments, the periplasm and the outer membrane. Following structures can be found in bacterial cells:

  1. Capsule – Found in some bacterial cells, this additional outer covering protects the cell when it is engulfed by other organisms and helps the cell adhere to surfaces and nutrients. It is visible with negative (background) staining.  it protects against chemicals and desiccation, stores waste products and protects the bacterium from attack by phagocytic cells it helps bacteria to form colonies
  2. Cell Wall – In both Gram-negative and –positive cells, the cell wall is located on the outside of the inner membrane, but is further surrounded by the outer membrane in Gram-negative bacteria. The major components of the bacterial cell wall are long glycan strands that are cross-linked by short peptides containing amino acids in both the D- and L-isoform and the whole ensemble is called peptidoglycan or murein, forming the murein sacculus.
  3. Cytoplasm – A gel-like substance composed mainly of water that also contains enzymes, salts, cell components and various organic molecules.
  4. Cell Membrane or Plasma Membrane – Surrounds the cell’s cytoplasm and regulates the flow of substances in and out of the cell. It has a typical unit membrane structure composed of proteins and lipids, basically similar to the membrane that surrounds all eukaryotic cells. In electron micrographs it appears as a triple-layered structure of lipids and proteins that completely surround the cytoplasm.
  5. Pili – Pili (singular: pilus), also called fimbriae, are hair-like appendages built by protein subunits called pilin or fimbrin and usually extend 1±2 μm from the surface of Gram-negative bacteria, with a diameter ranging from 2 nm to 8 nm. They function in bacterial cell-to-cell interactions, adhesion to specific receptors of host cells, either uptake or transfer of genetic material and twitching motility, a form of locomotion that is powered by extension and retraction of the pilus filament, and they provide receptors for bacteriophages.
  6. Flagella – Used in the locomotion of many motile bacteria; a rigid, hollow cylinder of protein, the base of which rotates propelling the cell along.
  7. Ribosomes – Cell structures responsible for translation (protein synthesis).
  8. Plasmids – plasmids are small circular pieces of DNA which are present in some bacteria, containing genes additional to those in the chromosome; some bacteria contain more than one plasmid. Plasmids are known to carry genes which may help the bacterium to survive in adverse conditions and carry genes for antibiotic resistance. Plasmids can be transferred to another bacterium in conjugation, transformation or transduction.
  9. Nucleiod Region – Area of the cytoplasm that contains the single bacterial DNA molecule.
  10. Endospore- A hard outer covering of some bacteria forms a resistant endospore which ensures survival in severe conditions of drought, toxic chemicals and extremes of temperature, e.g. Bacillus anthracis spores, which cause the disease anthrax, are known to be viable after 50 years in the soil.
  11. Mesosomes- A tightly folded infoldings of the plasma membrane, that may be the site of respiration and involved in cell division and the uptake of DNA; they might be an artifact of preparation for electron microscopy.

Taxonomy:

Systematic classification and categorizations of organisms into ordered groups is called taxonomy. A working knowledge of taxonomy is useful for diagnostic microbiology and for studies in epidemiology and pathogenicity. Although higher organisms are classified according to their evolutionary pathways (i.e. phylogenetically ), bacteria cannot be similarly categorized because of the insufficiency of their morphological featured. Bacterial classification is somewhat artificial in that they are categorized according to phenotypic features, which facilitate their laboratory identification. These comprise

  • Morphology (Cocci, Bacilli, spirochetes etc.)
  • Staining properties (Gram-positive, Gram-negative. etc.)
  • Spore formers or non spore formers.
  • Cultural requirements (aerobic. facultative anaerobic, anaerobic etc.)
  • Biochemical reactions (saccharolytic and asacchantlytic. according to sugar fermentation reactions. etc.)
  • Antigenic structure (serotypes).

The first step in identification of bacteria is Gram staining. There after they can be classified according to the above features. A schematic identification scheme is presented in the following flow charts,

Identification scheme for Gram +ve Bacteria

Identification scheme for Gram -ve Bacteria

The mechanism of bacterial Pathogenicity :

Pathogenicity is the ability to produce disease in a host organism. Microbes express their pathogenicity by means of their virulence, a term which refers to the degree of pathogenicity of the microbe. A pathogen can cause disease if it can,

  1. Attach to or enter host tissue.
  2. Evade host responses.
  3. Proliferate.
  4. Damage the host.
  5. Transmit itself to new hosts.

Following are the examples of virulence factors produced by two very important periodontal pathogens,

Virulence factors of A. actinomycemtemcomitans:

  1. Leukotoxin (RTX):  Induce apoptosis
  2.  Cytolethal distending toxin (CDT)
  3.  Chaperonin 60
  4.  LPS: Apoptosis, bone resorption, etc
  5. OMP, vesicles
  6. Fimbriae
  7. Actinobacillin
  8. Collagenase
  9. Immunosuppressive factor

Virulence factors of P. gingivalis:

  1. Involved in colonization and attachment: ™Fimbriae, hemagglutinins, OMPs, and vesicles
  2. Involved in evading (modulating) host responses: ™Ig and complement proteases, LPS, capsule, other  antiphagocytic products
  3. Involved in multiplying:™ Proteinases, hemolysins
  4.  Involved in damaging host tissues and spreading: ™Proteinases (Arg-, Lys-gingipains), Collagenase, trypsin-like activity, fibrinolytic , keratinolytic, and other hydrolytic activities

Know more………………

Bacterial Endotoxin:

Endotoxins are associated with gram –ve bacteria. They belong to part of the outer membrane of the cell wall of Gram-negative bacteria. Although the term “endotoxin” is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly reserved to refer to the lipo-polysaccharide complex associated with the outer membrane of Gram-negative pathogens. The biological activity of endotoxin is associated with the lipopolysaccharide (LPS).

Lipopolysaccharides  and Cytokine induction:

Lipolysaccharides are potent inducers of cytokine production1-3.

Let’s first try and understand the structure of Lipopolysaccharides. LPS is built up of three separate ‘‘building blocks’’:

  • Lipid A.
  • An inner core region.
  • O-specific side chain.

These separate building blocks have widely different compositions and structures, and this is reflected in their biological activities and functions.

Lipid A: It is important part of LPS and the ‘‘endotoxic’’ activities of LPS are largely due to the lipid A region, although activity can be modulated by other regions of the molecule4-5 .

Lipid A is the general term for a family of β(1-6)-linked disaccharides to which are attached medium to long-chain fatty acids (10- to 28-carbon chain length) linked to the sugar residues by ester or amide linkages and containing glycosidic and nonglycosidic phosphoryl groups. The lipid A molecules from bacteria can differ in terms of the sugar residues; the nature, chain length, number, and location of acyl residues; the degree of phosphorylation; and the nature of the substituents on phosphate groups6 .

The structural requirements for proinflammatory cytokine induction are:

  • Disaccharide,
  • Two phosphoryl groups,
  • Six fatty acids in a defined location such as is found in Escherichia coli LPS.

Core region: It consists of an inner and an outer core. The biological activities of the outer core have not been investigated in any detail. Although it is immunogenic and functions as a phage receptor, it does not appear to be directly involved in cytokine induction. The inner core is characterized by the presence of the unusual sugars heptose and 2-keto-3-deoxyoctonic acid (KDO), and these have been reported to exert an effect on the biological activities of lipid A, including its capacity to induce cytokine synthesis.

O-antigenic side chain: Although the O-antigenic side chain has a number of important biological functions, its role in the cytokine-inducing activities of LPS is uncertain. It does not appear to be essential for the induction of IL-1 synthesis by monocytes/macrophages. In one study7  it was demonstrated that the LPS from several of the putative periodontopathogens, including A. Actinomycetemcomitans ATCC 29523, Y4, 67, P. Gingivalis ATCC 33277, E. corrodens ATCC 23834, H. aphrophilus ATCC 19415 and H. segnis spp., were all capable of producing considerable amounts of IL-1 and tumor necrosis factor (TNF) from human peripheral blood monocytes. While it is not clear how the LPS molecule functions to activate the monocyte, it is more than likely that the monocyte “recognizes” specific LPS molecules that emerge from the bacterial surface.

References:

  1. Dinarello, C. A. 1989. Was the original endogenous pyrogen interleukin-1? p. 17–28. In R. Bomford and B. Henderson (ed.), Interleukin-1, inflammation and disease. Elsevier/North-Holland Publishing Co., Amsterdam.
  2. Manthey, C. L., and S. N. Vogel. 1994. Interactions of lipopolysaccharide with macrophages. Immunol. Ser. 60:63–81.
  3. Rietschel, E. T., T. Kirikae, F. Ulrich Schade, U. Mamat, G. Schmidt, H. Loppnow, A. J. Ulmer, U. Zahringer, U. Seydel, F. Di Padova, M. Schreier, and H. Brade. 1994. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8:217–225.
  4. Cavaillon, J.-M., and N. Haeffner-Cavaillon. 1990. Signals involved in interleukin- 1 synthesis and release by lipopolysaccharide-stimulated monocytes/ macrophages. Cytokine 2:313–329.
  5. Rietschel, E. T., L. Brade, B. Lindner, and U. Zahringer. 1992. Biochemistry of lipopolysaccharides, p. 3–41. In D. C. Morrison and J. L. Ryan (ed.), Bacterial endotoxic lipopolysaccharides, vol. 1. Molecular biology and cellular biology. CRC Press, Inc., Boca Raton, Fla.
  6. Rietschel, E. T., L. Brade, B. Lindner, and U. Zahringer. 1992. Biochemistry of lipopolysaccharides, p. 3–41. In D. C. Morrison and J. L. Ryan (ed.), Bacterial endotoxic lipopolysaccharides, vol. 1. Molecular biology and cellular biology. CRC Press, Inc., Boca Raton, Fla.
  7. Lindemann, R. A., Economou, J. S., and Rothermel, H., Production of interleukin-1 and tumor necrosis factor by human peripheral monocytes activated by periodontal bacteria and extracted lipopolysaccharides, J. Dent. Res., 67, 1131, 1988.
  8. Bailey & Scott's Diagnostic Microbiology, 12th Edition.
  9. Medical Microbiology, 4th edition. Edited by Samuel Baron.
  10. Microbiology. Robert Bauman.
  11. Microbiology 5th Edition. Michael Pelczar.

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