Chapter 1 - The Microbial World (General Bacteriology 2024/2025 PDF)

Blida 1 University Institute of Veterinary Sciences General Bacteriology (2nd year) 2024 / 2025 Dr AKLOUL K. CHAPTER 1 THE MICROBIAL WORLD 1. MICROORGANISMS AND MICROBIOTA Microorganisms are living beings characterized by their small size, simple structure and invisibility to th...

Blida 1 University Institute of Veterinary Sciences General Bacteriology (2nd year) 2024 / 2025 Dr AKLOUL K. CHAPTER 1 THE MICROBIAL WORLD 1. MICROORGANISMS AND MICROBIOTA Microorganisms are living beings characterized by their small size, simple structure and invisibility to the naked eye. They can only be seen using an optical or electron microscope. Most microorganisms are beneficial to humans, animals and plants. For example, they play an essential role in the major biological processes of transformation of plant and animal organic matter. They allow the putrefaction of organic matter. The colibacilli existing in the human intestine can provide certain nutrients, especially vitamin K. They allow the digestion of food (in the absence of bacteria, cattle, sheep and goats could not digest the hard fibers of plant cellulose), they protect our skin and mucous membranes. On the industrial level, bacteria play an essential role in the manufacture of cheese, yogurt (lactic acid bacteria), vinegar (acetic acid bacteria), etc. Molds play an important role during the ripening phase: Penicillium roqueforti gives the blue color to Roquefort, Penicillium camemberti the white down of Camembert. Saccharomyces cerevisae yeasts ferment sugars into alcohol and carbon dioxide (making bread, beer). Microalgae are used in industry to produce biogas. There are three categories of pathogenic bacteria: Strict or specific: These bacteria cause disorders regardless of the patient. For example: Salmonella typhi and Vibrio cholerae. Opportunistic: bacteria causing disorders when the host's immune defenses are weakened (immunocompromised subjects). Ex: Pseudomonas aeruginosa. Occasional: which are most often harmless but some strains of which are pathogenic. Ex: Escherichia coli or Staphylococcus aureus The human body hosts a whole community of microorganisms (bacteria, archaea, yeasts and viruses) grouped under the term "Microbiota". We host microorganisms in the skin (cutaneous microbiota), in the mouth (oral microbiota), in the genitals (vaginal microbiota) and in the intestine (intestinal microbiota). The microbiome is the collection of all the genomes of these microorganisms. The holobiont corresponds to the biological unit composed of the host and its microbiota 1 2. CLASSIFICATION OF THE MICROBIAL WORLD In 1868, the German biologist Ernst Haeckel created a third kingdom, that of the protists. This kingdom includes all organisms that are not higher animals or plants. The protists include bacteria, cyanobacteria, protozoa, algae and fungi. Viruses are excluded because they are not cellular organisms. Protists comprise two fundamentally distinct groups: s of higher plants and animals. They have a nucleus surrounded by a nuclear envelope, mitochondria, lysosomes, a Golgi apparatus and chloroplasts in the case of photosynthetic cells. This is the case 2 for algae, protozoa and microscopic fungi. They are called Eukaryotes and form the group of higher protists; -organisms have a more rudimentary cellular organisation. There is no true nucleus and no nuclear membrane. The genetic apparatus consists of a single chromosome. There are no mitochondria or chloroplasts. They have no cytoplasmic partitioning and their membranes do not contain sterols but are lined with a layer of peptidoglycan forming the cell wall. This organisation is characteristic of bacteria, archaea and cyanobacteria. They are Prokaryotes and are called lower protists. 3 In 1969, Robert Whittaker divided organisms on the basis of three main criteria: cell type, mainly based on nuclear structure (prokaryotic or eukaryotic) and cell wall composition, level of organisation (unicellular or multicellular) and type of nutrition. He thus defined the classification of living organisms into five kingdoms: Monera or Procaryotae, Protista, Fungi, Animalia and Plantae. In 1970, Carl Woese proposed the classification of all organisms (based on cellular organization and in particular by comparison of 16 S ribosomal RNA) into three domains: Bacteria, Archaebacteria and Eukaryotes. The first two lineages are made up only of prokaryotic cells, while the third contains exclusively eukaryotes. 4 3. DIVERSITY OF MICROORGANISMS Micro-organisms come in a wide variety of shapes and sizes 3.1. Bacteria Unicellular prokaryotic organisms. They are about 1 to 10 μm long (most of them are only about 1 to 2 μm in diameter), and vary in shape: bacillary, coccoid, spiral. Some constituents are always present such as the wall (except mycoplasmas), the plasma membrane, the cytoplasm, the nuclear apparatus, the ribosomes. Others can be added such as flagella, pili, the capsule, plasmids or the spore. 5 Their multiplication occurs by fission. A mother bacterium thus divides into two daughter bacteria sharing the same genetic material. Bacteria colonise all environments (microbiota, deep sea, atmosphere, etc.). Growth conditions vary from strain to strain in terms of nutrients, oxygen, temperature, humidity, pH, salinity and pressure. Examples: Staphylococcus, Brucella,... 3.2. Archea Archaea (formerly archaeobacteria) are prokaryotes. Their cell wall contains no peptidoglycan (a component of the bacterial wall that maintains the shape of the cells and provides protection against osmotic pressure), but contain a pseudo-peptidoglycan. Archaea essentially comprise only anaerobic species, living in extreme environments: these are known as extremophilic organisms (very acidic or very alkaline saline environment, environment at near-boiling temperature). They are often found in environments where extreme conditions prevail: methanogenic bacteria, which produce methane as a waste product of respiration; extreme halophilic bacteria, which live in extremely salty environments; extreme thermophilic bacteria, which live in hot sulphurous waters. Archaea are not only extremophiles, they are also more common organisms that live in classic living conditions such as marshes or ruminant rumens. Archaea do not appear to cause disease in humans. Archaea reproduce asexually by binary fission, like bacteria. In terms of membrane and chemical structure, archaea cells share characteristics with eukaryotic cells (membrane composed of ether lipids). 3.3. Cyanobacteria A phylum of bacteria (prokaryotes), also known as blue-green algae. They are not algae in the botanical sense. Cyanobacteria are present in the surface layers of seawater as well as in the surface layers of freshwater. They are also present on shaded soils, rocks, mud, wood and even some living organisms. They are photosynthetic, creating energy in the form of carbohydrates from the sun's energy. They fix atmospheric nitrogen into ammonia, enabling them to adapt to any environment, however poor. Some species of cyanobacteria produce toxins that affect humans and animals. These particles present in the water can produce a toxin that is fatal to animals and cause numerous side-effects in humans, such as vomiting, nausea and muscular pain. 6 3.4. Protozoa They are protists (eukaryotes, often unicellular and without specialised tissues) that are heterotrophic (draw their carbon source from different organic compounds), mobile (pseudopods, flagella or cilia) and ingest their food by endocytosis. They range in size from 1 to 700 μm, but amoebae can reach up to 5 mm. As they have no walls, their plasma membrane is in direct contact with the outside environment, from which they must draw their nutrients. They live in water, damp soil or inside an organism (in lung mucus, the intestine, the rumen of certain animals, etc.). They come in a variety of forms and can be free-living entities or parasites. Protozoa reproduce either asexually or sexually, and some use both modes. Examples include -Leishmania donovani (responsible for visceral leishmaniasis) -Entamoeba histolytica (diarrhoea), -Trypanosoma (sleeping sickness) -Trichomonas vaginalis (urogenital infection) -Giardia lamblia (diarrhoea) -Plasmodium falciparum (malaria) -Toxoplasma gondii (toxoplasmosis) 3.5. Microscopic fungi (or mycetes) Fungi are eukaryotes. Their cell walls are mainly composed of chitin. Their general structure is similar to that of an animal cell (plasma membrane, nucleus, cytoplasm, organelles). Unicellular fungi, known as yeasts, are between 10 and 50 μm in size. Their shape can be spherical, ovoid, elongated, cylindrical, etc. The most typical multi-cellular fungi are molds. Fungi reproduce by forming spores, either sexually or asexually. They feed by absorbing solutions of organic matter from their environment, whether soil, seawater, freshwater, a host animal or a host plant. Pathogenic fungi can cause poisoning by producing mycotoxins that affect the liver, kidneys, nerves and skin. They also release allergenic molecules and some species produce lytic enzymes. Examples include -Saccharomyces cerevisiae is a yeast used in bread and beer production. -Candida albicans, responsible for mycosis -Penicillium, used to produce penicillin, - Aspergilus flavus secrete aflatoxins in poorly stored cereals. 3.6. Microscopic algae (Microalgae or microphytes) Like higher plants, algae are eukaryotes capable of using light as a source of energy. They are autotrophic (capable of generating their own organic matter from mineral elements) and photosynthesis using their pigments, the most important of which is chlorophyll. Their cell walls, like those of plants, are made up of cellulose. 7 Algae are found in fresh and salt water, in the soil and in association with plants. They play an important role in the carbon cycle and, more generally, in the biogeochemical cycles of lakes and oceans. Some algae are mobile, others are not. Algae have different colors because they contain different combinations of pigments. Reproduction in algae is complex as they exhibit vegetative reproduction as well as sexual reproduction. There are different groups of algae: chlorophytes, including green algae, euglenophytes, pyrrophytes, chrysophytes or golden algae, pheophytes including brown algae and rhodophytes including red algae, Xanthophyta or yellow-green algae, etc. Algal blooms have become recurrent in recent decades, due to climate change (warmer waters) and human activities that encourage faster growth of these micro- organisms, such as nitrogen or phosphate discharges and the reduction in biodiversity. Although only 2% of these compounds are recognized as toxic to animals and humans, such algal blooms represent a threat to the consumption of seafood, particularly bivalve mollusks, which filter and accumulate the toxins, but also fish, which bioaccumulate them via the food chain. 3.7. Viruses Viruses are only visible under an electron microscope and are acellular. The mean size of viruses varies widely depending on the type, but most viruses are extremely small, typically ranging from 20 to 300 nanometers (nm). Very simple in structure, the viral particle consists only of a nucleocapsid. The genetic material, either DNA or RNA, is surrounded by a protein capsid, sometimes covered by a lipid membrane called the envelope. Because they have no cellular structure, metabolism or growth, they cannot be considered living beings. Viruses can replicate, i.e. reproduce, but only if they use the machinery and energy of a living cell. This is why all viruses are obligatory intracellular parasites; outside a living cell, they are inert. Every organism can be infected by one or more viruses. There are even viruses capable of infecting other viruses! Examples: Lyssavirus (rabies virus), Coronavirus,... 3.8. Prions A prion (proteinaceous infectious particle) is a protein-based pathogen which, unlike other types of infectious agents such as viruses, bacteria, fungi and parasites, does not contain nucleic acid (DNA or RNA) as the genetic carrier of its infectious potential. Prions or Non-Conventional Transmissible Agents are classified as biological agents, although they are not micro-organisms. 8 Prions are protein particles naturally present in organisms. Its pathogenicity in spongiform encephalopathies is thought to be linked to a change in its conformation. Prions are responsible for diseases manifested by degeneration of the central nervous system linked to their multiplication in the affected individual. They are responsible for degenerative diseases in animals (scrapie in sheep, bovine spongiform encephalopathy - BSE-); and in humans, Kuru disease, or Creutzfeld- Jakob disease, leading to a certain form of dementia. Since prions are not living organisms, they cannot be cultivated, but can be extracted from cell cultures or organisms maintained for this purpose. 3.9. Viroids In 1971, Theodor Diener discovered an acellular particle that he named viroid, meaning ‘virus-like’. Viroids, which are smaller than viruses (about 50 nm long), consist only of circular single-stranded RNA capable of self-replication. Their genome represents only 1/10th of the genetic information of the smallest known viruses. They only affect plants. The first viroid to be discovered turned out to be the cause of potato tuber spindle disease, which slows germination and causes various deformities in potato plants. They replicate autonomously. Like viruses, viroids control host machinery to replicate their RNA genome. Unlike viruses, viroids do not have a protein coat to protect their genetic information, and do not code for any proteins. There is no treatment for viroid infections, other than destroying contaminated plants. 3.10. Virusoïds These are sub-viral particles described as non-self-replicating RNAs. Virusoid RNA replication requires that the cell is also infected by a specific ‘helper’ virus. An example of a helper virus is the subterranean clover mottle virus, which has an associated virusoid encapsulated inside the viral capsid. Once the helper virus enters the host cell, the virusoids are released and can be found free in the cytoplasm of plant cells, where they possess ribozyme activity. The helper virus undergoes typical viral replication independently of the activity of the virusoid. 9 4. TAXONOMY 4.1.Classification of Bacteria The kingdom Procaryotae is the first level of classification. This is followed by the domain Bacteria, the phylum, the class, the order, the family, the genus and the species. The species is the unit of classification. However, it is often necessary to subdivide a species into different subspecies. Example: Classification of Escherichia coli: - kingdom: Procaryotae - domain: Bacteria - phylum: Proteobacteria - class: Gammaproteobacteria - order: Enterobacteriales - family: Enterobacteriaceae - genus: Escherichia - species: Escherichia coli 10 4.2 Nomenclature The nomenclature of bacteria is binomial. The full name includes a genus name and a species name; the genus name begins with a capital letter, the species name does not; the genus name precedes the species name. The names of micro-organisms are written in Latin and italics (e.g. Escherichia coli, Staphylococcus aureus) 11 The subspecies rank is noted subsp. (subspecies) followed by the Latin name in italics (or underlined). To designate all the species in a genus, the name of the genus is followed by spp. (‘speciei pluralia’ meaning ‘several species’). Membership of a genus, without specifying the species, is noted by the name of the genus followed by sp. (species) as long as the isolate is not identified with a species. The term ‘Candidatus’ can be added before the binomial name of a species that lacks information for an official description (information on lifestyle, culture, etc.). ‘Candidatus’ is written in italics, the rest of the name is written straight without italics. For example ‘Candidatus Midichloria mitochondrii’. Bacteria can also be given vernacular (or common) names. These names are part of everyday language. For example, Escherichia coli is also called colibacillus, while Staphylococcus aureus is called “golden staph”. 4.3. Identification 4.3.1. Phenotypic or phenetic taxonomy The classification of bacteria is based on several types of observation and study. Bacteria can be classified according to : - microscopic morphology (cocci, bacilli, vibrion; isolated, in pairs, in chains,.. ) - macroscopic morphology (size, shape, color of colonies on agar culture media) - mobility (mobility or immobility at a given temperature) - presence of spores - result of Gram staining (positive or negative Gram staining) - growth temperature (4° C, 20° C, 30° C, 37° C...) - respiratory type (aerobic, anaerobic, facultative aero-anaerobic, microaerophilic…) - nutritional requirements (specific substances for development) - ability to use certain sources of carbon or nitrogen Bacteria can be classified according to their characteristics - biochemical (classification into biotypes or biovars) - morphological (morphovar) - antigenic (serotypes or serovars) - pathogenic (pathotypes or pathovars) - enzymatic (zymotypes or zymovars) - sensitivity to antibiotics (antibiotypes) - sensitivity to bacteriophages (phageotypes, phageovars or phagovars) 12 4.3.2. Numerical or Adansonian taxonomy Approach based on the comparison of characters of different natures: morphological, physiological, genetic belonging to strains taken two by two. The selected characters are quantified numerically. Binary quantification (0 or 1, i.e. absence or presence) of similarities and differences then allows taxa to be characterized by a similarity coefficient. For a precise and reliable classification, compare many characters (at least 50). For each pair of organisms in the group, an association coefficient is calculated which measures the agreement between the characteristics of the two organisms. The coefficient most used in microbiology is that of Jaccard, given by the following relation : SAB = nS+ /nS+ + nd SAB: coefficient of similarity between strain A and strain B nS+: number of similar characters nd: number of different characters to give a significant value to the study. The results are expressed in the form of a dendrogram, in which the organisms with the greatest similarity are grouped into clusters called phenons. It is estimated that phenons with more than 80% similarity can be assimilated to the same species. 13 4.3.3. Molecular taxonomy The advent of molecular biology in the 1990s brought new ways of studying ‘non- cultivable’ organisms. Enzymatic DNA amplification (PCR) provided access to bacterial gene sequences without the need for culture. Identification is based on the comparison of nucleic acid sequences (DNA, RNA) or protein profiles of a microorganism with documented data from known organisms. 4.3.3.1. Chargaff’s rules (GC%) DNA contains as much purine as pyrimidine, i.e. (A+G) / (C+T) = 1, as much thymine as adenine A/T = 1, and as much guanine as cytosine G/C = 1. However, the ratio (A+T)/(C+G) varies greatly and is characteristic of the species. This coefficient, known as the Chargaff’s rules, can be calculated following sequencing using the formula: GC% = [(G+C)/G+C+A+T] X 100 14 -Two bacteria belonging to the same species have identical GC% (within 2.5%). -Two bacteria with different GC% do not belong to the same genetic community. -Two bacteria with identical GC% do not necessarily have the same nucleotide sequences, and can therefore be genetically distant. - Two microbial species with the same nucleotide sequences necessarily have the same GC%. 4.3.3.2 DNA/DNA hybridization This method enables the entire genome of two bacteria to be compared by measuring the degree of homology between the two DNAs. In vitro renaturation of two heterologous DNA strands (each strand from two bacteria being compared) results in the formation of a heteroduplex. The degree of homology is the percentage of complementary sequences relative to the total sequences Two strains belong to the same species only if the hybridization rate (renaturation) between the two DNAs is greater than or equal to 70%. 4.3.3.4. Nucleic acid sequencing Ribosomal RNAs (rRNAs) are present in prokaryotic and eukaryotic cells and have a well-conserved structure in all living organisms. Portions of rRNA have an identical sequence in all living organisms. They are abundant in the cell and easy to purify and sequence (using a reverse transcriptase). Work on 16S rRNA has made it possible to distinguish Eubacteria from Archaea. For 16S rRNA, levels of nucleotide identity greater than or equal to 95% and greater than or equal to 98.7% are required at the genus and species levels, respectively. 15 4.3.4. Methods based on proteomic techniques Proteomic tools offer an excellent complement to classical genomics-based techniques for bacterial identification MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry allows the identification of bacteria by analysis of their total proteins. This is a physical process that produces mass spectra specific to the proteome of the bacteria detected. Comparison with a database of spectra enables the type of bacteria to be deduced. 16 4.3.5. Fatty acid profiling The fatty acid profile of bacteria refers to the specific types and quantities of fatty acids present in the bacterial cell membrane. These fatty acids are a critical part of the cell’s phospholipid bilayer, determining membrane properties such as fluidity and permeability. The composition of fatty acids can vary greatly among bacterial species, making the fatty acid profile a valuable tool for bacterial identification and classification. A common method is gas chromatography (GC), which separates and quantifies the different fatty acids in a bacterial cell membrane. A bacterium like Bacillus subtilis may have a high concentration of branched-chain fatty acids, while Pseudomonas aeruginosa is rich in unsaturated fatty acids. These differences in fatty acid composition are used in labs to differentiate between the two species. *** 17