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Microbiology, Virology, and Immunology Department Ecology of microorganisms. The microflora of the human body. Dysbacteriosis. What is Microbial Ecology? What is “Microbial”? – of or referring to a minute life form; a microorganism, especially a bacterium that causes disease. Not in technical use. What is “Ecology”? – the study of the interactions between organisms and their environment Microbial ecology = The study of interactions between microorganisms and their environment (chemical, physical, and biological environment!) In microbial ecology the main concepts are: population - an elementary evolutional unit (structure) of a definite species; biotope - site, habitation of a population, for parasites - place of their localization in an organism; In microbial ecology the main concepts are: microbiocenosis - microbial association, that is collection of populations of different species of microorganisms, which live in the defined biotope (for example, in an oral cavity, skin); ecosystem - system, in which enters a biotope and microbiocenosis. Symbiosis - living together of two dissimilar organisms Types of symbioses: Neutralism - the populations, existing in one biotope do not stimulate and do not oppress each other. Mutalism exists when organisms live in an obligatory but mutually beneficial relationship. The mutualistic association between rhizobia and N-fixing bacteria E. coli synthesizes vitamin K in the intestine in exchange the large intestine provides nutrients necessary for survival of the microorganisms Types of symbioses: Commensalism - such relationship, at which the member called the commensal receives benefits, while its coinhabitant is neither harmed nor benefitted. Bifidobacteria Types of symbioses: Antagonism - oppression of one population another. The microbes – antagonists produce antibiotics, bacteriocines, fatty acids, which cause destruction of bacteria or delay their reproduction. A B Types of symbioses: Parasitism - such kind of symbiosis, at which one population (parasite) brings harm to the host, and for itself has a benefit. parasitic microorganisms Microflora of soil • The soil is the major environment for a habitation of microorganisms. • The amount of bacteria in one gram of soil can be very great - from 200 millions up to 10 billions • The ground mass of bacteria is on depth 10-20 cm. The microflora of soil includes hundreds of species of bacteria, viruses, protozoa, actinomyces and fungi. It is various species of putrefactiving, ammonifying, nitrifying, denitrifying, nitrogen-fixing bacteria, numerous iron bacteria and sulphur-bacteria. The most often inhabitants of soil are the representatives of genus Azotobacter, Nocardia, and Clostridium. producers consumers The role of microorganisms ? decomposers Help in - the decomposition of pollutants and toxic wastes the efficient utilization of limited natural resources transformations of chemical substances that can be used by other organisms • critically important to all form of life closely linked with the flow of energy • the ultimate source of all carbon is CO2 - raw material for photosynthesis - major waste product of respiration and combustion Org.cpd. CO2 fixation Anaerobic respiration and fermentation (anaerobic m.o.) (phototrophic bacteria) Methanogenic Anaerobic procaryotes CO2 Aerobic CH4 CO2 Methane-oxidizing procaryotes Respiration (animals, plants, and m.o.) Org.cpd. CO2 fixation (cyanobacteria, algae, plants, and chemoautotrophic procaryotes) The carbon cycle Beggiatoa sulfate R-SH assimilation (some procaryotes) So Thiobacillus sulfate assimilation Aerobic desulfurylation R-SH Thiothrix SO42- H2 S R-SH Anaerobic Chromatium Chlorobium Dissimilatory sulfate reduction Desulfovibrio So Chromatium S2O32- Chlorobium Higher plant bacteria phytoplankton Dissolved org.ortho-P Dissolved org.-P Sediment zooplankton Precipitated inorg.-P General microbial number (GNM) – quantity (amount) of all saprophytic bacteria in 1 g of soil is examined. More exact rating is manufactured at definition coli - index, that is amount of bacteria E.coli in 1 g of soil. The soil is considered pure, if its coli-index does not exceed 2000, and the quantity of termophilic bacteria is in limits 100 - 1000. Sanitary - indicative bacteria of soil Escherichia coli, Streptococcus faecalis, Clostridium perfringens termophilic bacteria. A degree of fecal contamination of soil is determined on presence and quantity(amount) of first three species. Soil Pathogens Clostridium botulinum, Clostridium tetanus, Clostridium perfringens, Bacillus anthracis Microflora of water Water is a natural medium of a habitation of various microorganisms. In sweet and salty waters representatives of all groups of bacteria are found Protozoa, Fungi, Viruses, Water - plant. The microflora of reservoirs consists of two groups of microbes: autochtonal (or aqueous) microorganisms / permanently live and are multiplied in water/. It is more often such species: Micrococcus candicans, M. roseus, Sarcina lutea, Bacterium aquatilis communis, Pseudomonas fluorescens. The anaerobes represent by species Clostridium, Bacillus cereus, B. mycoides. allochtonal (brought from the outside). The microbial composition of water is in the large degree mirrors a soil microflora. A major factor, which determines quantity of microbes in water is presence in it of necessary nutritious materials, that are various biological matters. Main path of microbial contamination of water is penetration of unpurified waste from sewage of the populated area. Therefore, the more water is polluted with similar organic matters, there are more in it of microorganisms. Zones of water clearness Polisaprogenic zone is the zone of strong contamination. It contains many organic matters and almost there is no oxygen. The quantity of bacteria in 1 ml of water reaches one million and more. Mesosaprogenic zone is the zone of moderate contamination(pollution)). In it the nitrifying and aerobic bacteria prevails. The total number of microorganisms compounds hundreds of thousand in one ml. Oligosaprogenic zone (zone of pure water). The quantity of bacteria changes from 10 up to 1000 in one ml. Sometimes the pathogenic bacteria in water can get and they will be saved for some time: Salmonella spp., S. typhi, V. cholerae, .S. dysentеriae, L. interrogans. Often various viruses are found: Polioviruses, Rotaviruses, Enteroviruses, Hepatitis A virus and others. The pathogenic microorganism getting in reservoirs, abundant in a zone of strong contamination, gradually die in a zone of moderate contamination. They are almost not found in oligosaprogenic zones. Common microbial number of water is examined by inoculation of 1 ml of water in melted and refrigerated up to 50 C in agar in Petri dishes. Coli-index is determined by a method of membrane filters or fermentation tests. Membrane filters method Membrane filtration for determinging the coliform count in a water sample using vacuum filtration. Membrane filters method Enzymatic assay for coliform • Colilert system system(for P-A test) • β-galactosidase of of coliform -galactosidase coliform substrate →→ nitrophenol (yellow)+Gal substrateONPG ONPG nitrophenol • β-glucuronidase coli -glucuronidaseofofE.E. coli fluorogenic MUG fluorogenicsubstrate substrate MUG Microflora of air Air for microorganisms is less favorable environment, than soil and water. There are almost no the nutritious materials necessary for reproduction of bacteria. Desiccation and the ultraviolet rays operate perniciously on microorganisms. Nevertheless, many microbes in air can be saved more or less long time. Main sources of microbial air pollution is the soil, water and man. Microflora of air The composition of a microflora of air is very various. It depends on cleanness of air, deposits, temperature, humidity, climate and geographical conditions. Than it is more in air of a dust, smoke, soot, the more micro-organisms. Vice-versa, above mountains, seas, oceans and woods, where air is pure, it is not enough of microbes. In open air spaces there are less of microbes, than in the closed rooms. The constant microflora of air is shaped at the expense of soil microorganisms. In its composition many species of Actinomyces, Penicillinum, Aspergillus, Mucor and others go into. There are many Micrococcous roseus, M. candicans, M. luteus, Sarcina lutea, S. alba, S. rosea, Bacillus subtilis, B. mycoides, B. mesentericus. Many microbial diseases are transmitted through the air during sneezing, coughing, or even normal breathing: bacterial - tuberculosis, diphtheria, legionellosis, wooping cough, acute angine, epidemic meningitidis, viral - influenza, measles, mumps, adenoviral infection A sanitary - hygienic rating of air include general microbial number sanitary - indicative microorganisms: Streptococci haеmolyticus, S. viridans, Staphylococcus aureus. Examination of air microflora is made according to Koch’s sedimentation method Krotov’s aspiration method. Where the Normal Flora Are Found • • • • • Skin Conjunctiva Oral cavity Intestinal tract Upper respiratory tract • Urogenital tract Dental plaque consists of a biofilm of bacteria 300500 cells in thickness. The organism of the man is occupied • more than 500 species of bacteria, • about 50 species of viruses, • more than 20 species of protozoa. It is a huge zoological garden. Normal flora Numbers of bacteria that colonize different parts of the body • Human body – 1013 cells – 1014 bacteria • Normal microbial flora – Relatively stable Numbers represent the number of organisms per gram of homogenized tissue or fluid or per square centimeter of skin surface. Normal microflora of the human body: Constant (obligate, resident, indigenous, autochthonous) microflora is native, no imported one of different biotopes. Microbes become established. Transient (temporary, facultative, allochthonous) microflora is not aboriginal, it is acquired one. Microbes occupy the body for only short periods. Bacterial Flora of the Body Site Total Bacteria (per/ml or gm) Upper Airway Nasal Washings 103-104 Saliva 108-109 Tooth Surface 1010-1011 Gingival Crevice 1011-1012 Gastrointestinal Tract Stomach 102-105 Small Bowel 102-104 Ileum 104-107 Colon 1011-1012 Female Genital Tract Endocervix Vagina 108-109 108-109 Ratio Anaerobes:Aerobes 3-5:1 1:1 1:1 1000:1 1:1 1:1 1:1 1000:1 3-5:1 3-5:1 Initial Colonization of the Newborn • Uterus and contents are normally sterile and remain so until just before birth • Breaking of fetal membrane exposes the infant; all subsequent handling and feeding continue to introduce what will be normal flora 42 Initial Colonization of the Newborn Flora of the Human Skin • Skin is the largest and most accessible organ • Two cutaneous populations – Transients: influenced by hygiene – Resident: stable, predictable, less influenced by hygiene Normal flora of skin: Staphylococcus epidermidis Staphylococcus aureus varius streptococci Corynebacterium Pseudomonas aeruginosa Propionibacterium Yeasts Normal flora of the skin: Benefit • Inhibit fungal growth (athlete’s foot) • Body odor Harm • Body odor • Acne • Opportunistic infections Microflora of an eye The tears perniciously affect on microbes. In 47 % of people the microflora here is completely absent. Only few species have adapted to existence on a conjunctiva: Corynebacterium Neisseria Staphylococcus Mycoplasma Adenovirus Herpervirus Normal flora of the respiratory tract: Staphylococcus aureus Staphylococcus epidermidis Corynebacterium varius Streptococci Neisseria species Haemophilus influenzae Moraxella lacunata In a larynx, the trachea and large bronchuses there are few of microbes. Smaller bronchi and alveoli are normally sterile. Normal flora of respiratory tract – nasal membranes: Benefit • Competition with pathogens for colonization sites Harm •Main carrier site for pathogenic Staph. aureus Normal flora of respiratory tract – pharynx (throat): Benefit • Competition with pathogens for colonization sites • Production of substances that inhibit pathogens Harm •Main carrier site for pathogens transmitted primarily by respiratory contact Flora of the Genitourinary Tract • Sites that harbor microflora – Females – Vagina and outer opening of urethra – Males – Anterior urethra • Changes in physiology influence the composition of the normal flora – Vagina (estrogen, glycogen, pH) 52 Normal flora of the genitourinary tract In an outside part of a urethra Peptococcus Corynebacterium Mycobacterium smaegme Fusobacterium Staphylococcus Mycoplasma are found. The kidney, the ureter, the bladder and upper urethra are sterile. Normal flora of vagina: Lactobacillus Corynebacterium B. fragilis, varius streptococci C. albicans Internal reprodactive organs are normally sterile. Degrees of vaginal secret cleanness of health woman I Epitelhial cells acid-tolerant lactobacilli (Doderllein`s bacilli) many glicogen II the pH is maintained between 4.4 and 4.6 Degrees of vaginal secret cleanness of sick women Epitelhial cells III acid-tolerant lactobacilli (Doderllein`s bacilli) are absent little glicogen the pH is maintained between 6.9 and 7.6 IV many proteins, leucosytes staphylococci and streptococci Normal flora of the vagina Benefit • Competition with pathogens for colonization sites • Production of lactic acid that inhibits pathogenic bacteria and yeasts Harm • none Microflora of the intestinal tract At birth a gastrointestinal path and feces of the child are sterile. In 3 - 4 days there is an intensive invading them by bacteria of genus Escherichia, Proteus, Veilonella, Lactobacterium, Enterococcus, Staphylococcus. However, by the end of the first week putrefacient bacteria are superseded by a lactate microflora. It mainly Bifidobacterium bifidum, B. longum, B. infantis, Lactobacillus acidophilus are. Bifidobacterium and Lactobacterium play the large role in decomposition of mammary milk, help the process of digestion. Microflora of an oral cavity In an oral cavity there is a plenty of microorganisms. More than 100 species of aerobic and anaerobic bacteria are described. In 1 ml of a spit can be found about 100 million of microbial cells. Constant (stationary) temperature (37 ºС), humidity, the oddments of food, alkaline reaction of a spit and wide aeration promote reproduction of microbes. Streptococci Lactobacterium Veilonella Neisseria Corynebacterium Bacteroides H. Influenzae Treponema Leptospira C. albicans Actinimycetes Mycoplasma Protozoa. Normal flora of the oral cavity: Benefit • Compete with pathogens for colonization sites • Produce substances that inhibit pathogens • Stimulate local immunity Harm • Plaque formation and dental disease Microflora of the stomach Yeast Sarcina Fungi Lactobacterium Staphylococcus Campylobacter Helicobacter pylori (all up to 30 species). Normal flora of GI tract - stomach Helicbacter pylori Harm • Causes gastric ulcers • Probable association with duodenal ulcers Microflora of the small intestine Bifidobacterium Lactobacillus Bifidobacterium bifidum Enterococcus E. coli Lactobacillus Normal flora of GI tract: small intestine Benefit • Production of vitamins and nutrients • Competition with pathogens for colonization sites • Production of substances that inhibit pathogens Harm • Possible relationship with inflammatory conditions • Transfer antibiotic resistance to pathogens The microflora of a large intestine is most abundant and is diverse. Here there are most favourable conditions for reproduction of many microorganisms. Now microbiologists have detected and have described more than 270 species of bacteria. Their common biomass compounds about 1,5 kg. During day the man with feces discharges of 17-18 billions microbes. Normal flora of the colon: Bacteria: Bacteroides Bifidobacterium Eubacterium Fusobacterium Lactobacillus coliforms (Escherichia coli, Enterobacter) fecal Steptococci Clostridium Fungi: Candida Protozoa: Entamoeba coli, Trichomonas hominis Normal flora of GI tract : large intestine Benefit • Competition with pathogens for colonization sites • Production of substances that inhibit pathogens • Stimulate development and activity of immune system Harm • Relationship with inflammatory bowel diseases • Production of carcinogens and relationship with colon cancer • Methanogenesis Importence of normal flora: 1. Colonization resistance 2. Antagonistic role 3. Stimulate immunity 4. Take part in all kinds of metabolism 5. Produce vitamines, enzymes, hormons 6. Digestive role Normal flora - Benefits Prevent colonization by competing for attachment sites Prevent colonization by competing for essential nutrients Antagonize bacterial growth: Fatty acids, peroxides, bacteriocins kill or inhibit nonindigenous species Normal flora - Benefits Synthesize and excrete vitamins: vitamin K, vitamin B12 Stimulate development of certain tissues: Caecum and lymphatic tissues (Peyer’s patches) in GI tract Production of cross-reactive (“natural”) antibodies Influence immunology of gut-associated lymphatics Normal flora - Risks Not so beneficial: “side effects” • Dental plaque – Biofilm on hard, smooth enamel surface • 300-500 bacterial cells thick Streptococcus sanguis, Streptococcus mutans Normal flora - Risks • Dental caries: destruction of enamel, dentin or cementum of teeth – bacteria in plaque produce lactic acid Streptococcus mutans – Lactic acid demineralizes enamel Normal flora - Risks • Periodontal disease – Gingiva, cementum, periodontal membrane, alveolar bone – Rich in anaerobes Opportunistic flora Some normal flora become opportunistic pathogens Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, Streptococcus pneumoniae, Pseudomonas aeruginosa, etc. Opportunistic flora Breach of skin/mucosal barrier: trauma, surgery, burns •Surgery to mouth leads to abscesses of bone, lung, brain •Dental manipulation: wounds seed with oral streptococci might invade via blood and adhere to heart valve previously damaged due to rheumatic fever •Intra-abdominal abscesses: anaerobes from intestinal tract Opportunistic flora Bacterium at one site may be commensal, but might be pathogenic at another site – Commensal in gastrointestinal tract (E. coli) might be pathogenic in lung or urinary tract The dysbacteriosis is change of a quantitative relation and composition of a normal microflora of an organism, mainly of it intestine. Thus there is a decrease or disappearance of some bacteria and sharp increase others, especially infrequent or not characteristic for the given biotope of microorganisms. As a rule, the quantity of an facultative- anaerobic microflora is enlarged. It can be the Gram-negative rods, staphylococci, Candida albicans and species Clostridium. Candidosis Probiotics –Oral administration of living organisms to promote health –They contain the dried up clouds of alive bacteria having strong antagonistic properties –Mechanism speculative: competition with other bacteria; stimulation of nonspecific immunity –Species specific: adherence and growth Prebiotics –Non-digestible food that stimulates growth or activity of GI microbiota, especially bifidobacteria and lactobacillus bacteria (both of which are noninflammatory) –Typically a carbohydrate: soluble fiber