Special Microbiology Handout PDF

Yerevan State Medical University after M. Heratsi Department of Medical Microbiology M.S. Hovhannisyan SPECIAL MICROBIOLOGY Edited by V.A. Shekoyan Handout on Special microbiology for foreign students Yerevan 2016 1 UDC 579 (07) Approved by YSMU Foreign students Education Methodological and Medico-biological committees and was guaranteed for publication by the academic council of the YSMU after M. Heratsi by protocol N 3, 02.12.2015 Reviewers: Hasmik S. Hovhannisyan Vigen A. Asoyan Special Microbiology for foreign students Edited by M.S. Hovhannisyan – Yerevan: M. Heratsi’s Yerevan State Medical University publishing, 2015, 270 pages. Language Editor: N. Nazaretyan M. Bisharyan Computer Design: Manyak Avetisyan ISBN 978-9939-65-134-7 @ M. Heratsi’s Yerevan State Medical University, 2016 2 Brief Description of Special Microbiology Medical microbiology, virology, immunology being basic sciences, studies microbes’ structure, physilogy, lows of deveolopment and belong to such sciences, which is nessesary to each doctor and medworker, because it insures general biological knowleges and at the same time as a propedeutics direction, is used in the practical sphera of medicine. Medical microbiology consists of two parts: General and Special. The studying subject of Special microbiology are pathogenic microorganisms, which are the source of infectious diseases and different pathological processes, their morphology, physiology, biological and antigenic properties and the role of distinct species in different infectious diseases etiology and pathogenesis, as well as elaboration of methods of laboratory diagnosis, prophylaxis and treatment of infectious diseases. 3 MICROBIOLOGICAL LABORATORY DIAGNOSTIC METHODS  Microscopic-preparation of smears from investigating material, staining by Gram method and microscopy. Description of microorganisms by morphology and staining properties.  Bacteriological method-isolation of pure culture from investigating material and identification of them by morphology, staining properties (tinctorial), cultural properties, bio-chemical activities, antigenic structure, toxigenicity, etc.  Serological: revealing of specific antibodies or antigens in blood serum by immunological reactions (agglutination, precipitation, complement fixation, lysis, immune enzyme, RIA).  Skin-allergic tests-revealing of development of infectious allergy (DTH) in some infectious diseases (tuberculosis, brucellosis, anthrax).  Biological: infection of pathogenic material into susceptible animal organism and isolation of pure culture from these organisms. Choosing of pathological material (feces, urine, blood, mucous, sputum, etc.) depends on clinical diagnosis, pathogenesis of infections, stages of infection, and chosen method of examination. ANTIGEN-ANTIBODY REACTIONS IN THE LABORATORY Reactions of antigens and antibodies are highly specific. An antigen will react only with antibodies elicited by itself or by a closely related antigen. Because of the great specificity, reactions between antigens and antibodies are suitable for identifying one by using the other. This is the basis of serologic reactions. Types of diagnostic tests: Many properties of diagnostic tests are performed in the immunological laboratory. Most of these tests can be designed to determine the presence of either antigen or antibody. To do this, one of the components, either antigen or antibody, is known and the other is unknown. Immunoelectrophoresis-immunoelectrophoresis combines electrophoresis immunodiffusion (immune precipitation in gel). This method can be used for analyzing complex antigens in biological fluids. A glass slide is covered with molten agar or agarose. A well for antigen and a trough for antiserum is cut on it. Antigen well is filled with antigen mixture (human serum). The slid is then placed in an electric field for about an hour to allow for the electrophoretic migration of various antigens. Different antigens will migrate at different rates or even in different directions, depending upon their size and charge and the conditions of electrophoresis. After the completion of electrophoresis, antiserum trough is filled with appropriate antiserum (antiserum to whole human serum). Antigens and antibodies diffuse towards each other, resulting in the formation of precipitin bands, for individual antigens and antibodies, whenever they are both in zones of optimal proportions, in 18-24hours. Because immunoelectrophoresis uses electric charge in addition to diffusion, it is more likely to separate antigen than is simple diffusion alone. By this method, over 30 different antigens can be identified in human serum. This technique is useful for detection of normal and abnormal serum proteins. Radioimmine assay (RIA)-This method is used for the quantitation of antigens or haptens that can be radioactively labeled. It is based on the competition for specific antibody between the labeled (known) and unlabeled (unknown) concentration of material. The complexes that form between the antigen and antibody can be separated and amount of radioactivity measured. The more unlabeled antigen is present, the fewer radioactivity there is in the complex. The 4 concentration of the unknown (unlabeled) antigen or hapten is determined by comparison with the effect of standards. RIA is a highly sensitive method and is commonly used to assay hormones or drugs in serum. The radioallergosorbent test (RAST) is a specialized RIA that is used to measure the amount of serum IgE antibody which reacts with a known allergen (antigen). Enzyme-Linked Immunosorbent Assay (ELISA) – the method can be used for the quantitation of either antigens or antibodies in patient specimens. It is based on covalently linking an enzyme to a known antigen or antibody, reacting the enzyme -linked material with the patient’s specimen, and the assaying for enzyme activity by adding the substrate of the enzyme. The method is nearly as sensitive as RIA yet requires no special equipment or radioactive labels. For measurement of antibody, known antigens are fixed to a surface (eg, the bottom of small wells on a plastic plate), incubated with dilutions of the patient’s serum, washed, and then reincubated with antibody to human IgG labeled with an enzyme, eg, horseradish peroxidase. Enzyme activity is measured by adding the substrate for the enzyme and estimating the color reaction in a spectrophotometer. The titer of antibody in the patient’s serum is the highest dilution of serum that gives a positive color reaction. Immunofluorescence (Fluorescent antibody) - Fluorescent dyes eg, fluorescein and rhodamine, can be covalently attached to antibody molecules and made visible by ultraviolet (UV) light in the fluorescence microscope. Such “labeled” antibody can be used to identify antigens, eg, on the surface of bacteria(such as streptococci and treponemas), in cells in histologic section, or in other specimens. The immunofluorescence reaction is direct when known labeled antibody interacts directly with unknown antigen and indirect when a two-stage process is used. For example, known antigen is attached to a slide, the patient’s serum (unlabeled) is added, and the preparation is washed. If the patient’s serum contains antibody against the antigen, it will remain fixed to it on the slide and can be detected on addition of a fluorescent dye-labeled antibody to human IgG and examination by UV microscopy. The indirect test is often more sensitive than direct immunofluorescence, because more labeled antibody adheres per antigenic site. Furthermore, the labeled antiglobulin becomes a “universal reagent”, ie, it is independent of the nature of the antigen used because the antibody to IgG is reactive with all human IgG. Immunoblot (Western Blot) - is a technique that combines electrophoresis with ELISA to separate and identify protein antigens in a sample. It has many research applications, but its main clinical use is to confirm positive ELISA screening tests for antibodies against HIV. The ELISA tests are simpler and cheaper, but they give a small percentage of false positive results; therefore, positive results need to be confirmed by different separated by electrophoresis in a polyacrylamide gel. Smaller proteins migrate faster than larger ones. The resulting bands of separated antigens can react with specific antibodies, but antibodies do not diffuse well into these gels. Therefore, it is necessary to transfer the antigens present in the bands from the gel by blotting them onto a filter. To test for antibodies against HIV, commercial kits are available in which antigens of the HIV virus have been electrophoresed and blotted onto the filter, which is then cut into strips for each test. Each sample to be tested for antibodies to HIV is applied to a strip and incubated to allow specific antibodies to react with the viral antigens. To detect the antigen – antibody combinations formed, a label is used, usually an enzyme – labeled anti-HGG, as in ELISA, or sometimes a radioactive label. The Western blot is a more laborious and expensive technique than the ELISA; however, it offers greater specificity, since antigens are identified by two criteria: their size and their reactivity with antibodies. 5 MICROCOCCACEAE FAMILY GENUS STAPHYLOCOCCUS Staphylococcus aureus The Staphylococcus, S. aureus, was discovered by R. Koch (1878), and later isolated from furuncle pus by L. Pasteur (1880). It was described as the causative agent of numerous suppurative processes and studied in detail by F. Rosenbach (1884). Staphylococci are included in the class Bacteria, family Micrococcaceae, genus Staphylococcus. According to the contemporary classification Staphylococci are classified into 20 species and 15 subspecies, which we classify into 2 groups: coagulase positive, coagulase negative. Three of them ecologically are connected with human organism: S. aureus produces golden pigment (S. aureus) S. epidermidis produces white pigment (S. albus) S. saprophyticus produces yellow pigment (S. citreus) Other species are parasitic on animals. Some are members of the normal flora of the skin and mucous membranes of humans; others cause suppuration, abscess, a variety of pyogenic infections, and even a fatal septicaemia, they can cause 120 nosological forms. Morphology: Staphylococci are spherical in shape, 0.6-1.5µm in diameter. They are arranged characteristically in grape-like clusters (in smears from pure culture). Cluster formation is due to cell division occurring in more than one plane with daughter cells remaining close together. In smears from liquid media and pus, the cocci appear singly or in pairs, short chains, but not clusters. Staphylococci are Gram-positive organisms which are non-motile, non-sporing and, with the exception of rare strains, non-capsulated. Cultivation: Staphylococci are facultative anaerobes. They grow well on ordinary nutrient media with a pH of 7.2-7.4 at a temperature of 37º C. At room temperature with adequate aeration and subdued light the organisms produce golden, white, lemon-yellow, and other pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, alcohol. They are most readily formed on milk agar and potatoes at temperature of 20-25º C. The selective medias for Staphylococcus are salt agars: yolk-salt agar and milk- salt agar: Salt (6-15%) agars are good selective plating media and are useful for staphylococci isolation from food, dust, faeces and pus where mixed bacterial flora is expected. On yolk-salt agar they form smooth (S-form), convex, shiny, opaque colonies. Round the colonies they form pearl zone (lecithinase activity). On milk-salt agar pigment production is enhanced. Pigment is not formed in liquid media. On blood agar colonies are similar to those on salt agar, but may be surrounded by a zone of ß- hemolysis (haemolytic activity). Sugar broth (as an enrichment media) is used for examination of blood (sepsis). Fermentative properties: Staphylococci produce enzymes which cause lysis of proteins and sugars. They hydrolyze proteins and form H2S, liquefy gelatin (as a funnel), don't form indole; fermentation of sugars (glucose, maltose, lactose, saccharose) with acid formation, without gas. Fermentation of glucose and mannite in anaerobic condition has differential diagnostic meaning. There is no indole production in cultures. S. aureus is catalase-positive and oxidase –negative. Staphylococcus aureus strains usually exhibit the following characteristics: 1. coagulase positive; 2.greater biochemical activity, (differential diagnostic is fermentation of mannite and glucose in anaerobic condition); 3.produce clear hemolysis on blood agar; 4. produce a golden yellow pigment; 5.liquefy gelatin; 6.produce phosphatase; 7.in a medium containing potassium tellurite, reduce tellurite 6 to form black colonies; 8.produce pearl zone on yolk-salt agar; 9.produce thermostable nucleases which can be demonstrated by the ability of boiled cultures to degrade DNA in an agar diffusion test 10.Urease activity is positive for S. epidermidis and S. saprophyticus. Staphylococci are variably sensitive to many antimicrobial drugs. They are resistant to many types of penicillin (β-lactamates) because they produce beta-lactamase enzyme. Antigenic structure: Antigenic structure of S. aureus is very complex. Staphylococci contain anti- genic polysaccharides and proteins as well as other substances important in cell wall structure. These include: 1. Capsular polysaccharide: A few strains of S. aureus are capsulated and these tend to be more virulent than non-capsulated strains. The capsule is composed of antigenic polysaccharide. It prevents ingestion of the organism by polymorphonuclear leucocytes. The capsule may promote adherence of the organism to host cells and to prosthetic devices. 2. Teichoic acid are polymers, are linked to the peptidoglycan and can be antigenic. Teichoic acid is a major antigenic determinant of all strains of S. aureus. Teichoic acids function in the specific adherence of gram-positive bacteria to mucosal surface. Teichoic acid participates in staining of bacteria also. 3. Peptidoglycan: is a polysaccharide polymer containing linked subunits, provides rigid exoskeleton of the cell wall. It stimulates both humoral and cellular immune responses in the host. The antibodies against peptidoglycan possess opsonizing activity; however, increased levels of antibodies may predispose some patients to immune complex disorders. In addition to their role in providing rigidity and resilience to the staphylococcal cell wall, peptidoglycan and teichoic acid also have several biologic activities that are thought to contribute to virulence. These properties include the ability to activate complement, to inhibit chemotaxis of inflammatory cells, and to stimulate antibody production. 4. Protein A: Protein A is a group - specific antigen and is a cell wall component of many S. aureus strains that binds IgG molecules, non-specifically, through Fc region leaving specific Fab sites free to combine with specific antigen. When suspension of such sensitized cells is treated with homologous (test) antigen, the antigen combines with free Fab sites of IgG attached to staphylococci cells. This is known as co-agglutination. 5. Cross reacting antigens. Pathogenicity: Pathogenicity of Staphylococcus aureus depends on invasive properties, capsule formation, which has antiphagocytic ability; protein A, which inactivate complement. The pathogenic factors are combined with toxin production and enzymes too. They produce several toxins: Leukocidin: Leukocidin act to damage polymorphonuclear leucocytes and especially macrophages (antiphagocytic action), and can produce dermonecrosis. Hemolysins (ά, β, γ and δ): These toxins have haemolytic activity. Alpha toxin is the most important in pathogenicity. This toxin lyses erythrocyte. It is also leucocidal (is toxic for human macrophages and platelets and causes degranulation of polymorphonuclear leucocytes through disruption of their lysosomes), dermonecrotic, cardiotoxic (alfa toxin has a powerful action on the vascular smooth muscle) and neurotoxic. This is lethal toxin. Enterotoxins: An important cause of food poisoning. Enterotoxins (A to F) are soluble, heat- stable (they resist boiling for 30 minutes) and resistant to the action of gut enzymes. Enterotoxins are produced when S. aureus grows in protein food (e.g., creams, creamery products, dairy products). By mechanism of action it is cytotoxin. The usual incubation period ranges from 2-6 hours after the ingestion of food. Patient develops nausea, vomiting and diarrhoea. The duration of acute symptoms is usually less than 24 hours. In addition to the ability to cause nausea and vomiting, enterotoxins are pyrogenic, mitogenic and are capable of producing thrombocytopenia and hypotension. Enterotoxin F is reported to be responsible for the Toxic shock syndrome (TSS): In humans, the toxin is associated with fever, shock, and multi-system involvement, including a desquamative skin rash. Toxic shock syndrome toxin (TSST): TSST is the same as the enterotoxin F. TSST and the enterotoxins are recognized as superantigens, i.e. they are potent activators of T- lymphocytes without relation to their epitope specificity resulting in the liberation of cytokines such as tumour necrosis factor, interleukins 1, 2; interferon gamma and they bind with high affinity to 7 mononuclear cells. These characteristics partly explain the florid and multisystem nature of the clinical conditions associated with these toxins. Exfoliative toxin: This is epidermolytic toxin. This toxin is responsible for scalded skin syndrome in young children, causes impetigo of the new-born (characterized by isolated pustules that become crusted and rupture). Staphylococci produce enzymes: Hyaluronidase, which breaks down hyaluronic acid, a component of connective tissue, which facilitates the spread of bacteria to adjacent areas (known as spreading factor). Coagulase accelerates the formation of a fibrin clot from its precursor, fibrinogen (this clot may protect the bacteria from phagocytosis by walling off the infected area and by coating the organisms with a layer of fibrin). Fibrinolysin (staphylokinase): This enzyme dissolves coagulated plasma and probably aids in the rapid spread of bacteria through tissues. Lecithinase, which is hydrolyses lecithin in the cell membrane resulting in destruction of the membrane and widespread cell death. Lipase,which participates in adhesion and invasion. DNA-ase –plays a role in pathogenesis of diseases. Catalase – suppresses oxidative burst in phagocytic cell. Resistance: Staphylococci are among the more resistant of non-sporing bacteria. They are relatively resistant to drying, heat (they withstand 50º C for 30 minutes), a 3 per cent phenol solution kills the organisms in 15-20 minutes. 1% chloramine kills the staphylococci in 2-5 minutes. Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treating pyogenic skin diseases caused by these organisms. Staphylococci are variably sensitive to many antibacterial drugs, they can develop drug resistance. They produce Beta-lactamase which causes resistance to penicillin and similar drugs (beta- lactamates). Pathogenesis and diseases in man: Staphylococci, particularly S. epidermidis, are members of the normal flora of the human skin and respiratory and gastrointestinal tracts. Nasal carriage of S. aureus occurs in 40-50% of humans. Staphylococci are also found regularly on clothing, bed linen, and other fomites in human environment. Staphylococci enter the body through the skin and mucous membranes. When they overcome the lymphatic barrier and penetrate into the blood, staphylococcal septicaemia sets in. Both the exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms. Staphylococci are responsible for a number of local lesions in humans: abscess, furuncle, carbuncle, osteomyelitis, dermatitis eczema, peritonitis, meningitis, etc, 120 nosologic forms. In some cases, Staphylococci may give rise to a secondary infection in individuals suffering from smallpox, influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumonia in children are particularly severe diseases. Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases of wound infections, diphtheria, and tuberculosis. S. aureus infection can also result from direct contamination of a wound, e.g., postoperative staphylococcal wound infection or infection trauma (chronic osteomyelitis subsequent to an open fracture, meningitis following skull fracture). If S. aureus disseminates and bacteremia ensues, endocarditis, acute hematogenous osteomyelitis, meningitis, or pulmonary infection can develop (table 1). Food poisoning due to staphylococcal enterotoxin is characterized by a short incubation period (1-8 hours); violent nausea, vomiting, and diarrhoea; and rapid convalescence. There is no fever. 8 Table 1 Staphylococcal diseases Skin and soft tissue Folliculitis, furuncle (boil), abscess(particularly breast abscess) wound infection, carbuncle, impetigo, paronychia, less often cellulitis Musculoskeletal Osteomyelitis, arthritis, bursitis, pyomyositis Respiratory Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia Central nervous Abscess, meningitis, intracranial thrombophlebitis system Endovascular Bacteremia, septicaemia, pyemia, endocarditis Urinary Staphylococci are uncommon in routine urinary tract infections, though they do cause infection in association with local instrumentation, implants or diabetes Immunity: Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies (antitoxins, precipitins, agglutinins). The phagocytic and humoral factors act together and supplement each other. Post-infectious immunity following staphylococcal diseases is of low grade and short duration. Treatment: diseases are treated with antibiotics, sulphonamides, and anti-staphylococcal gamma- globulin. During Staphylococcal chronic infections anatoxin, autovaccine are used. Prophylaxis: The general precautionary measures include: hygiene in working and everyday-life conditions, treatment of vitamin deficiency, prevention of traumatism and excess perspiration, observance of rules of hygiene in maternity hospitals, surgical departments, and children’s institutions. Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriological examination of the personnel for carriers is used. Examination of pathogenic staphylococci resistant to antibiotics is important. For prophylaxis staphylococcal anatoxin and bacteriophage are recommended. For the formation of passive response antistaphylococcal immunoglobulin and donor's antistaphylococcal hyperimmune sera is recommended. Laboratory diagnosis: Diagnostic methods are: 1.Microscopic 2.Bacteriological 9 STREPTOCOCACEAE FAMILY GENUS STREPTOCOCCUS Streptococcus pyogenes Streptococci were discovered by T. Billroth (1874) in tissues of patients with erysipelas and wound infections and by Pasteur in patients with sepsis. Streptococci are placed in the family Streptococaceae. They are part of the normal flora of humans and animals. Some of them are human pathogens. The most important of them is Streptococcus pyogenes causing pyogenic infections. Morphology: The streptococci are gram-positive spherical cells measuring 0.5 to 1.0μm in diameter and form chains (they divide in only one plane and tendency of cells to remain united results in the development of the characteristic chains). They are non-motile, do not form spores. Some strains are capsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in short chains, while in smears from broth cultures they form long chains. Cultivation: The majority of streptococci are aerobes and facultative anaerobes. The optimal temperature for growth is 37º C. The organisms show poor growth on ordinary meat-peptone agar, and grow well on selective media - sugar, blood, serum, ascitic agar. On solid media they produce small, translucent colonies. In sugar broth medium growth is in the form of fine-granular precipitates on the walls and at the bottom of the tube. Some streptococcal strains cause haemolysis on blood agar. Based upon their haemolytic properties, streptococci can be classified into three groups: 1.ß-haemolytic streptococci: these organisms produce a wide (2-4mm in diameter) clear zone of complete haemolysis in which no red blood cell is visible on microscopic examination. 2.α-haemolytic streptococci: The colonies are surrounded by a narrow zone (1-2 mm) of haemolysis; they produce a green zone round the colony, as a result of conversion of haemoglobin into methaemo- globin. The streptococci producing α-haemolysis are also known as streptococci viridans. 3.γ-haemolytic streptococci: These organisms do not produce any haemolysis on blood agar. Enterococcus faecalis is an important organism of this group. 10 Fermentative properties: Streptococci are non-proteolytic, do not liquefy gelatin, and do not reduce nitrates to nitrites. They coagulate milk; dissolve fibrin, ferment glucose, maltose, lactose, saccharose with acid formation. S. pyogenes is soluble in bile (40%) and are not soluble in 10per cent bile, unlike pneumococci. Streptococci are catalase negative (table 1). Antigenic structure: By the group-specific polysaccharide (C substance) the streptococci are classified into 20 serological groups which are designated by capital letters: A, B, C, D…(Lancefield classification). S. pyogenes is in A group. Depending on the type-specific M, T and R protein antigens the serological group A is subdivided into serotypes (about 100 serotypes). The M protein is the most important of these. It acts as a virulence factor by inhibiting phagocytosis (it binds IgG molecules, non-specifically, through Fc region leaving specific Fab sites free to combine with specific antigen). The T and R proteins have no relation to virulence. Various structural components of S. pyogenes exhibit antigenic cross reaction with different tissues of the human body. Antigenic relationships have been demonstrated between capsular hyaluronic acid and human synovial fluid, cell wall protein and myocardium, group A carbohydrate and cardiac valves, cytoplasmic membrane antigens and vascular intima and peptidoglycan and skin. Pathogenicity: The pathogenetic factors are: adherence, colonization, invasion, secretion of toxins and enzymes. Toxin production: Streptococci produce exotoxins with various activities: Haemolysins: a)Streptolysin-S is nucleoprotein with haemolytic and cytotoxic activities, non-antigenic, oxygen stable and sensitive to heat and acid. It is responsible for hemolysis around the surface colonies. In addition to causing ß-hemolysis, it is able to inhibit chemotaxis and phagocytosis. It can cause destruction of cell’s lysosomal membranes. Therefore, this haemolysin appears to be an important factor of group A streptococcal infection. b)Streptolysin-O (oxygen labile) is protein and is strongly antigenic. It is heat labile. SLO induces brisk response, usually within 10-14 days. Anti streptolysin O(ASO) appears in sera following streptococcal infection. Estimation of this antibody (the titre of ASO antibodies can be important in the diagnosis of rheumatic fever) is a standard serological procedure for the retrospective diagnosis of infection with S. pyogenes. It can cause lysis of erythrocytes, destruction of cell’s lysosomal membranes leading to necrosis of tissues. Streptolysin-O is toxic for leucocytes, produces haemolytic, cytotoxic, leukotoxic, cardiotoxic activities. Leukocidin, which destructive to leucocytes (inhibition of phagocytosis); occurs in highly virulent strains. Erythrogenic (Dick, scarlatinal) toxin: It is a protein, antigenic, relatively heat stable. This is superantigen. This toxin is responsible for characteristic skin rash with pharyngitis and tonsillitis in scarlet fever. It is produced only by certain strains of S. pyogenes lysogenized by a bacteriophage carrying the gene for the toxin. This toxin is neutralized by antibodies found in the convalescent sera. This property has been used for developing susceptibility and diagnostic tests for scarlet fever (Dick test gives positive result in persons lacking antitoxin, i.e., susceptible person). This test is known to be only of historical importance as scarlet fever is no longer a common or serious disease. Cytotoxins: Peptides, which destroy kidney cells and cause glomerulonephritis. Cardiohepatic toxin, which causes affection of myocardia and forms granules in liver. Enzymes: Fibrinolysin (streptokinase) activates plasminogen to form plasmin, which dissolves fibrin clots. It can be used to lyse thrombi in the coronary arteries of heart attack patients. Fibrinolysin plays a biological role in streptococcal infections by breaking down the fibrin barrier around the lesions and facilitating the spread of infection. Desoxyribonucleases – D Nase (streptodornase) depolymerizes DNA in exudates or necrotic tissue. Pyogenic exudates contain large amounts of DNA, derived from the nuclei of necrotic cells. Sterptodornase helps to liquefy the thick pus and may be responsible for the thin serous character of streptococcal exudates. 11 Hyaluronidase degrades hyaluronic acid, which is the ground substance of subcutaneous tissue. Hyaluronidase is known as a spreading factor because it facilitates the rapid spread of S. pyogenes in skin infections. Pathogenesis: Streptococci cause a wide variety of infections. Streptococci are mainly spread by the air droplet route. S. pyogenes causes three types of diseases: 1. Pyogenic diseases such as local pyogenic inflammation, abscesses, phlegmona, lymphadenitis, pyelitis, otitis, sinusitis, meningitis, peritonitis, and pharyngitis, tonsillitis (inflammation of the pharyngeal and tonsillar mucosa). S. pyogenes is the most common bacterial cause of sore throat. Invading the blood, streptococci produce a serious septic condition. 2. Toxigenic diseases such as scarlet fever and toxic shock syndrome. 3. Immunologic diseases such as rheumatic fever and acute glomerulonephritis (certain strains of group A streptococci contain cell membrane antigens that cross-react with human heart tissue antigens- autoimmune processes develop). Epidemiology and resistance: Many streptococci are members of the normal flora of the human body. The source of infection is carriers and sick persons. The main ways of spreading are droplet; and endogenous which occur during immunodeficiency. S. pyogenes is a delicate organism. It can be killed by heating at 54º C for 30minutes. It is also killed by usual strengths of disinfectants, but is more resistant to crystal violet than many other bacteria. Streptococci live for a long time at low temperature, are resistant to desiccation, and survive for many months in pus and sputum. When exposed to a temperature of 70ºC, they are destroyed within one hour. A 3-5 per cent phenol solution kills the organisms within 15 minutes. Immunity: Resistance against streptococcal diseases is type-specific. Thus, a host who has recovered from infection by one group A streptococcal is relatively insusceptible to reinfection by the same type but fully susceptible to infection by another type. Immunity against erythrogenic toxin is due to antitoxin in blood. Immunity after scarlet fever is stable, life-long. Antibody to streptolysin O (antistreptolysin O - ASO) develops following infection; it blocks hemolysis by streptolysin O but does not indicate immunity. Immunity after streptococcal infections is of a low grade and short duration. Treatment: Antibiotics. Prophylaxis: There is no specific prophylaxis (vaccines). Streptococcal infections are prevented by the practice of general hygienic measures in everyday life. Streptococcus pneumoniae Morphology: Streptococcus pneumoniae is gram-positive, about 1μm in diameter, lancet (flame) shape, and arranged in pairs (diplococci), capsule forming, non-motile and non-sporing organisms. It is inhabitant of upper respiratory tract of human and some animals, causes infection primarily of the respiratory tract, conjunctivitis, otitis, meningitis. They differ from streptococci chiefly in their morphology, optochin sensitivity and possession of a specific polysaccharide capsule. 12 Cultural characteristics: Pneumococcus is aerobe and facultative anaerobe. Optimum temperature is 37ºC, pH 7.8 and grows only in enriched media (blood, serum, ascitic agar). Pneumococci form a small round colony, primarily dome-shaped and later developing a central plateau with an elevated rim. In broth they produce diffuse turbidity. Pneumococci are ά-hemolytic on blood agar. For growth they need 5-10℅ CO2 for primary isolation. Biochemical reactions: Pneumococci ferment glucose, saccharose, lactose and inulin with the production of acid but no gas. Fermentation of inulin by pneumococci is a useful test for differentiating them from streptococci which do not ferment it. Pneumococci are bile soluble (40%). Bile solubility is a constant property of pneumococci and hence is of diagnostic importance. They are highly sensitive to optochin (ethyl hydrocuprein hydrochloride). Optochin sensitivity test is used for identification of pneumococci and distinguishing them from viridans streptococci, both of which produce α –haemolysis on blood agar. Pneumococci are catalase and oxidase negative. Antigenic structure: The most important antigen of the pneumococci is the capsular polysaccharide which is type specific. By the capsular antigen pneumococci are subdivided into 85 serotypes. The antigenic structure depends on M protein. The somatic portion of the pneumococcus contains an M protein that is characteristic for each type and a group-specific carbohydrate that is common to all pneumococci. The carbohydrate can be precipitated by C-reactive protein, a substance found in the serum of certain patients. Production of disease: Pneumococci produce disease through their ability to multiply in the tissues. The virulence of the organism is a function of its capsule, which prevents or delays ingestion of encapsulated cells by phagocytes. The virulence is connected with C-polysaccharide, M-protein, which is antiphagocytic, hemolysins (S-streptolysin), leukocidin, and enzymes: peptidase, which destroys sIgA, hyaluronidase, which is a spreading factor (degrades hyaluronic acid). Immunity: Immunity to infection with pneumococci is type-specific and depends on both antibodies to capsular polysaccharide and intact phagocytic function. Treatment: Streptococci are sensitive to many antimicrobial drugs. Some species are interesting for medical microbiology: Group A streptococci are among the most important human pathogens. The great majority of haemolytic streptococci that produce human infections belong to group A. Haemolytic streptococcus group A is known as S. pyogenes. Group B (S. agalactiae) streptococci colonize the genital tract of some women and cause neonatal meningitis and sepsis. Group C streptococci colonize the respiratory and urinary tracts. Group D streptococci (Enterococcus faecalis), which are pathogenic for human and animals occur as a part of the normal flora in the gut, are noted for their ability to cause urinary, biliary, and cardiovascular infections. Group H and K is noted during endocarditis. Oral streptococci (S. mutans, S. salivarius) occur in dental caries. Diagnostic laboratory tests for Streptococcal infections: 1.Microscopic: Test material is obtained from the pus of wounds, inflammatory exudate, tonsillar swabs, blood, urine, and foodstuffs. Tests include microscopy of pus smears. 2.Bacteriological: Isolation of the pure culture and its identification (Table1). 3.Biological: White mice are sensitive to pneumococcus. 13 14 GRAM–NEGATIVE COCCI NEISSERIACEAE FAMILY The genus Neisseria consists of gram negative, aerobic, non-sporulating, nonmotile, oxidase- positive cocci, typically arranged in pairs (diplococcus). N. meningitidis and N. gonorrhoeae are the primary human pathogens of the genus. Besides the two important pathogens, N. meningitidis and N. gonorrhoeae, the family contains many other genera: Moraxella, Acinetobacter, Kingella, which occur as commensals (conditionally pathogenic) and saprophytes. N. meningitidis and N. gonorrhoeae are pathogenic for human and typically are found associated with or inside polymorphonuclear cells. Most importantly, the two species are differentiated by the usual clinical presentations of the diseases they cause: meningococci typically are found in the upper respiratory tract and cause meningitis, while gonococci cause genital infections. GENUS NEISSERIA Neisseria gonorrhoeae (Gonococcus) Morphology: N. gonorrhoeae is Gram-negative, non-motile diplococcus, approximately 0.8 µm in diameter, non-capsulated. They are kidney (coffee-bean) shaped. Gonococci possess pili on their surface. Pili facilitate adhesion of the cocci to mucosal surfaces and promote virulence by inhibiting phagocytosis. Under the action of chemotherapeutic preparations they can transfer into L-forms. Cultural characteristics: Gonococci grow best on media containing complex organic substances such as heated blood, hemin, and animal proteins and in an atmosphere containing 5-10% CO2. They are aerobic. Optimal temperature for growth is 37ºC; pH-7.2-7.4. On ascites agar they form small, round, transparent, convex colonies (S-type). Biochemical reactions: Gonococci ferment glucose with producing acid only. They are catalase and cytochrome oxidase positive. They lack protein lysing activity. Antigenic structure: Gonococci are antigenically heterogeneous. They are capable of changing their surface structures and antigenic structure. The antigenicity is connected with: 1. Pili: which are hairlike structures, act as virulence factors by promoting attachment to host cells. Pili undergo antigenic variations (16serotypes). 2. Cell membrane proteins: Outer membrane proteins show antigenic diversity, which helps in typing gonococcal strains. These proteins act as ligands attaching the coccus to the host cells. They also form transmembrane channels (proteins) which play a role in the exchange of molecules across the outer membrane. 3. Lipopolysaccharides of the cell wall. 4. Superficial polysaccharide K antigen. Toxicity in gonococcal infections is largely due to the endotoxic effect of LPS. Virulence factors are: fimbria, capsule, LPS of the cell wall which possesses endotoxic and antiphagocytic activities, sIgA- protease, β-lactamase enzyme, production of which depends on R- plasmids. Resistance: The gonococcus is a very delicate organism, readily killed by heat, drying and antiseptics. Pathogenicity: Gonorrhoea is a venereal disease which has been known since ancient times. The name gonorrhoea (meaning flow of seed) was first employed by Galen in150AD. The disease is acquired by sexual contact. Gonococci require cylindric epithelium. They attack mucous membrane of the genitourinary tract, eye, rectum, and throat producing acute suppuration that may lead to tissue invasion; this is followed by chronic inflammation and fibrosis. The incubation period is 2-8days. In males the disease starts as an acute urethritis with yellow, creamery pus containing gonococci in a large number, and painful urination. The process may extend to the epididymis. In females, the primary infection (acute gonorrhoea) is in the endocervix and extends to the urethra and vagina, giving rise to mucopurulent discharge. It may then progress to the uterine tubes, causing salpingitis, fibrosis and obliteration of the tubes. Infertility occurs in 20℅ of women with gonococcal salpingitis. Chronic gonococcal cervicitis or proctitis is often asymptomatic. Blennorrhoea, gonococcal ophthalmia neonatorum, an infection of eye of the newborn, is acquired during passage through an infected birth canal. Initial conjunctivitis rapidly progresses and, if untreated, results in blindness. For prevention of gonococcal ophthalmia neonatorum, instillation of tetracycline, erythromycin or silver nitrate into the conjunctival sac of the newborn is used. 15 Immunity: Repeated gonococcal infections are common. Protective immunity to reinfection does not appear to develop as part of the disease process, because of the antigenic variety of gonococci. Treatment: Antibacterial drugs (-lactamates-penicillin, cephalosporins and other antibiotics). Epidemiology, prevention, control: It is transmitted by sexual contact, often by women and men with asymptomatic infections. The infection rate can be reduced by avoiding multiple sexual partners, rapidly eradicating gonococci from infected individuals, by means of early diagnosis and treatment, and finding cases and contacts through education and screening of populations at high risk. Vaccination has no place in prophylaxis. Diagnostic laboratory tests: 1.Acute gonorrhoea: Microscopic method- direct examination of gram-stained smear of pus. This can be used to demonstrate the characteristic intracellular gram negative diplococci in symptomatic urethritis (phenomenon of incomplete phagocytosis). 2.Chronic gonorrhoea: a)bacteriological method b)serological method: Bordet-Gengou CFR Neisseria meningitidis (Meningococcus) Morphology: Meningococci (N. meningitidis) are gram-negative cocci that resemble paired coffee bean, non-motile diplococcus, and 0.6-1m in diameter. They form prominent polysaccharide capsule. They are non-sporing and non-motile. Cultural characteristics: Meningococci do not grow on ordinary media. The organisms grow best on blood agar (chocolate agar), serum agar incubated at 37C in an atmosphere of 5-8 CO2. They are strict aerobes. On solid media, after incubation for 24hours, the colonies are small (about 1mm in diameter), translucent, round, convex, bluish grey, with a smooth surface(S-type). Growth is poor in liquid media, producing a granular turbidity with little or no surface growth. The biochemical activity of meningococci is feebly marked: they ferment glucose and maltose with acid formation. Indole and hydrogen sulphide are not produced and nitrates are not reduced. They are catalase and oxidase positive. Antigenic structure: N. meningitidis possesses a polysaccharide capsule and on the basis of this it has been subdivided into 13 serogroups. The most important serogroups associated with disease in humans are A, B, C, D, X, Y, Z, 29E, W-135, H, I, K, L. Serogroups H, I, K and L have been isolated from carriers and have not been associated with disease. Meningococcal antigens are found in blood and cerebrospinal fluid of patients with active disease. By the cell wall proteins they are subdivided into serotypes, which are designated by Arabic numerals (1,2,3, ….). Ecology: Meningococci are very delicate organisms, being highly susceptible to heat, desiccation, alterations in pH and disinfectants. Epidemiology: The human nasopharynx is the only reservoir of the meningococcus. Asymptomatic nasopharyngeal carriers rarely contract the illness but serve to infect their contacts. Transmission is essentially by airborne droplets or less often by fomites. During interepidemic periods, the carrier rate is about 5-10 per cent. An increase in carrier rate heralds the onset of an epidemic. During epidemics the carrier rates in closed communities may go up to 90 per cent. Meningitis is common in children between 3 months and 5 years of age. Epidemics usually occurs in semi-closed communities living in crowded conditions, as in jails and ships formerly, and in army camps in recent times. Meningococci colonize the membranes of the nasopharynx and become part of the transient flora of the upper respiratory tract without producing symptoms. The organisms attach to epithelial cells with the aid of pili. From nasopharynx meningococci can enter the bloodstream producing bacteremia and spread to specific sites, such as the meninges or joints, or be disseminated throughout the body. The most important manifestations of disease are nasopharyngitis, meningococcemia and meningitis. Fulminant meningococcemia (Waterhouse-Frederichsen syndrome) is more severe, with high fever, shock and hemorrhagic rash; there may be disseminated intravascular coagulation, and adrenal insufficiency. Cerebrospinal meningitis is the most common complication of meningococcemia. It usually begins suddenly with fever, headache, vomiting, stiff neck and progresses to coma within a few hours. 16 The important virulence factors of meningococci are: 1.A polysaccharide capsule that enables the organism to resist phagocytosis by polymorphonuclear leukocytes. 2.Endotoxin-LPS of the cell wall, which causes fever, shock and other pathophysiologic changes (in purified form, endotoxin can reproduce many of the clinical manifestations of meningococcemia). 3.An immunoglobulin A protease, which by cleaving secretory Ig A helps the bacteria to attach to the membranes of the upper respiratory tract. 4.Pili: Meningococci possess pili on their surface which allow intimate contact with host cell and organisms release endotoxin. 5.Outer membrane proteins. 6.They produce enzymes hyaluronidase, neuraminidase, fibrinolysin, which promote their invasion in tissues, sIgA- protease and plasmocoagulase. Immunity: Immunity to meningococcal infection is associated with the presence of specific, complement-dependent, bactericidal antibodies in the serum. Immunity is tense, lifelong and relapse occurs rarely. Prophylaxis: Monovalent and polyvalent vaccines containing the capsular polysaccharides of groups A, C, W-135 and Y are available. Meningococcal chemical vaccine is used. Laboratory diagnosis: Methods: 1. Microscopic (RIF) 2. Bacteriological -isolation of pure culture (in ristomycin containing media (ristomycin inhibits gram positiv microbes in investigated material) and identification (table 1). 3.Serological(PHAR). Table 1. Differences between pathogenic and non-pathogenic meningococci Non-pathogenic Pathogenic 1.Growth on ordinary + - nutrient media 2.Growth temperature the borders are high 22-39 oC 3.Biochemical activity high low (only fermentation of glucose and maltose) 4. Pigment formation + - Sample tests Staphylococcus 1. Which of the following enzymes are typical of Staphylococcus aureus: 1. lecithinase 2. hyaluronidase 3.coagulase 4. mucinase a)1.2.3. b)1.2.4 c)1.3. d)3.4. 2. Which toxins are produced by Staphylococcus aureus: 1. hemolysin 2. enterotoxin 3. erythrogenic toxin 4. leukocidin a)1.2.3. b)1.2.4. c)1.3. d)2.3.. 3. Which medias are used for Staphylococci cultivation: 1. milk-salt agar 2. egg- yolk agar 3. peptone water 4. sugar broth a)1.2.3. b)1.2.4. c)1.3. d)1.4. 17 Gonorrhoea: 1. Name diseases caused by gonococci: 1. enterities 2. blenorea 3. rheumatic fever 4. gonorrhea a)1.2.3. b)2.4. c)1.2. d)1.4. 2. Which of the following are typical of Gonorrhea: 1. gram (+) 2. motile 3. gram(-) 4. non-motile a)3.4. b)1.2.4. c)1.3. d)2.3.4. 3. Which are the characteristic features of N. gonorrhea: 1.arranged in pairs 2.arranged in chains 3.resemble coffee bean 4.like grape clusters a)1.2.3. b)1.2.4. c)1.3. d)3.4. Meningococcus: 1. Which of the following cultural properties are typical of N. meningitis: 1. don’t require nutrient media 2. form fine, colorless colonies on serum agar surface 3. biochemical activity is weak 4. growth in anaerobic conditions a)2.3. b)1.2.4. c)1.3. d)2.4. 2.The clinical forms of meningococcal infections are: 1. Nasopharyngitis 2. Epidemic cerebrospinal meningitis 3. Meningococcemia 1,2,3 4. Endocarditis a)1.2.3. b)1.2.4. c)1.3. d)3.4. 3. Specific prophylaxis of meningococcal infections is carried out by: a)meningococcal chemical vaccine b)antimicrobial serum c)antitoxic serum d)auto-vaccine Streptococcus: 1. Which of the following are typical of S. pyogenes: 1. growth on ordinary nutrient media 2. formation of small colonies on agar 3. formation of precipitate on the wall and at the bottom of the tube 4. formation of turbidity in liquid media a)2.3. b)1.2.4. c)1.3. d)1.4. 2. Virulence of S. pneumonia is determined by: 1. peptidase 2. O-streptolysin 3. hyaluronidase 4. capsule a)1.2.3. b)1.3.4. c)1.3. d)3.4. 18 19 20 21 ENTEROBACTERIACEAE FAMILY The Enterobacteriaceae form a large family of gram-negative rods found primarily in the colon of humans and other animals, many as part of the normal flora. The family Enterobacteriaceae includes 30 genera: of all of these the most pathogenic for human organism are: Escherichia, Salmonella, Shigella, Proteus, Klebsiella, Yersinia, etc. Morphology: they are short gram-negative rods with rounded ends and measure 1.5-5.0μm in length, 0.3-0.8μm in breadth. They do not form spores, they are facultative anaerobes. They differ in fermen- tative properties (they ferment a wide range of carbohydrates) and antigenic structure. Antigenic structure: They possess a complex antigenic structure. There are 3 main types of antigens: 1. The cell wall somatic O antigen is the lipopolysaccharide of the cell wall. Somatic O antigen is the basis for the serologic typing of many enteric rods. 2. The H antigen is the flagellar protein. Only flagellated organisms have H antigen (Escherichia, Salmonella). 3. The capsular or polysaccharide K antigen, which contain the thermolabile L and B antigens and thermostable A antigen. Differentiation of the members of Enterobacteriaceae family species Gluc. lactose mannite sacchrose indol urease motility H2S MR VP NO3    E.coli    -  --  --  --  K.pneumoniae     --  -- -- --   Sh.dysenteriae  -- -- --  -- -- --  --  Sh.sonnei    -- -- -- -- --  --  S.typhi  --  -- -- --    --  S.paratyphi A  --  -- -- --  --  --  S.paratyphi B  --  -- -- --    --  S.typhimurium  --  -- -- --    --  P.vulgaris  -- -- --      --  Y.enterocolitica  --     -- --    - acid and gas formation GENUS ESCHERICHIA Escherichia coli This genus is named after T. Escherich who was the first to describe the colon bacillus in 1885. Morphology: E. coli are rods 0.4-0.7 in breadth and 1-3 in length. The ends are rounded. They are motile-peritrichous. Non-sporing, do not form capsule. Capsule and fimbriae are found in some strains. Cultural characteristics: E. coli is a facultative anaerobe. The optimum temperature for growth is 37 C and the optimum pH is 7.2-7.5. Good growth occurs on ordinary media (meat-peptone agar), on which colonies are large, slightly convex, semitransparent, greyish (S-type colonies). They can give S- R dissociation. The S-R variation occurs as a result of repeated subcultures and is associated with the loss of surface antigens and usually of virulence. Some strains of E. coli can produce polysaccharide capsule. The colonies of these strains are “mucoid”. Many strains, especially those isolated from pathogenic conditions, are haemolytic on blood agar. On differential diagnostic media - Endo’s medium (MacConkey’s medium) the organism produce red colonies with metallic hue due to lactose fermentation. On Levin’s media they produce blue colonies. On Ploskirev’s media they form red colonies. 22 Fermentative properties: E. coli possesses sugar lysing and protein lysing activities. Glucose, levulose, lactose, maltose and many other sugars are fermented with the production of acid and gas. Typical strains do not ferment saccharose. The fermentation of lactose with acid and gas formation is differential diagnostic property for E. coli. It produces indole (indole is produced by bacteria that have tryptophanase-enzyme. They metabolised tryptophane from meat-peptone broth into indole). Gelatin is not liquefied, hydrogen sulphide is not formed. They haven’t urease activity. E. coli reduces nitrates to nitrites; Voges-Proskauer (VP) reaction is negative. Methyl red (MR) test is positive. Antigenic structure: It has three antigens that are used to identify the organism in epidemiological investigations: Somatic “O” antigen or cell wall antigen. This is thermostable, lipopolysaccharide antigen of the cell wall. By O antigen they are subdivided into 173 serological groups. The normal colon strains of E. coli belong to early O groups (1,2,3,4,5, etc.) and enteropathogenic strains belong to the latter O groups (26,55,111, 112, etc.). “H” or flagellar antigen –H antigen is type specific. There are 75 H antigens. This antigen is thermolabile protein. Capsular “K” antigen: there are 100 K antigens. (K antigen consists of three components A thermostable; B and L thermolabile). K antigen masks O antigen (for revealing O it is necessary to boil the culture and destroy the K antigen). K antigen is type specific too. On the basis of O antigens, E. coli is subdivided into a number of O groups. Each O group is then divided into subgroups on the basis of K antigen. Each of these subgroups includes strains with different H antigens. Thus, on the basis of antigenic structure an antigenic formula is derived which fully reflects the antigenic properties of the strain. For example, E. coli O26:K60:H12. Specific serotypes are associated with certain diseases; eg, O55 and O111 cause outbreaks of neonatal diarrhoea. Virulence factors: Virulence factors depend on: 1. Adhesive factors (fimbriae-which are chromosomally determined) and colonization 2. Invasive factors which depend on surface proteins, by which microbes penetrate into intestinal epithelial cells, multiply and destroy them. 3. Exotoxins: a)enterotoxins: Enterotoxins: heat labile protein toxin, it activates adenylate cyclase in the enterocysts to form cyclic AMP. The accumulation of cAMP in the intestinal mucosa initiates the hypersecretion of electrolytes and fluids into the lumen, resulting in watery diarrhoea. Heat stable enterotoxin is a low molecular weight polypeptide and poorly immunogenic toxin. It activates guanylate cyclase causing the increased production of cyclic guanosine monophosphate (cGMP) and subsequent hypersecretion of electrolytes resulting in diarrhoea. b)cytotoxin: is phage encoded cytotoxin identical to Shiga toxin produced by Shigella dysentery, which can cause destroying of vessel’s endothelium and intestinal wall. 4. Hemolysins: many strains of E. coli produce hemolysin. A larger proportion of E. coli strains recovered from extra-intestinal lesions of a man are haemolytic than are those isolated from human faeces. 5. Endotoxin - the somatic lipopolysaccharide surface O antigen, besides exerting endotoxin activity, also protects the bacillus from phagocytosis and the bactericidal effects of complement. Diseases caused by E. coli subdivided into: Endogenous: Causes diseases outside the intestinal tract. It causes urinary tract infection-cystitis, pyelonephritis. It can cause meningitis in association with the B streptococci; can cause sepsis (by the immunodeficiency). These infections are caused by conditionally pathogenic E. coli (the member of normal microflora of our organism) and named coli-bacteriosis. Exogenous: is an acute infection of the intestinal tract and is named esherichiosis (diarrhoea). E. coli causing diarrheal diseases is subdivided into 5 groups. They produce diarrhoea with different pathogenic mechanisms: 1.Enteropathogenic E. coli (EPEC): They cause coli-enteritis in infants and children (serogroups O26, O55, O111) usually occurring as institutional outbreaks but they can also cause sporadic diarrhoea in children and less often in adults. The pathogenesis of EPEC diarrhoea is not fully understood. EPEC do not ordinarily produce enterotoxins, and are not invasive. They are seen to be adherent to the mucosa of the upper small intestine by superficial proteins, intimately attached to cup- like projections of the enterocyte membrane, causing disruption of the brush border microvilli (they 23 produce inflammatory process and erosive surfaces). The pathogenesis is limited by endotoxin which causes inflammatory reaction. 2.Enterotoxigenic E. coli (ETEC) causative agent of cholera-like diarrhoea infection. The first step in pathogenesis is adherence of the bacteria to the cells of the jejunum and ileum by pili that protrude from the bacterial surface. ETEC is not invasive but produce enterotoxin, heat-labile toxin (functional blockater), which acts by stimulating adenylate cyclase, the resultant increase in intracellular cyclic AMP (cAMP) concentration stimulates cAMP-dependent protein kinase, causing an outpouring of fluid, potassium, and chloride from the enterocytes. Watery diarrhoea occurs (resembles a mild form of cholera). Its severity varies from mild watery diarrhoea to fatal diseases indistinguishable from cholera. Persons from developed countries visiting endemic areas often suffer from ETEC diarrhoea, a condition known as “travellers diarrhoea”. Though plasmids with enterotoxin genes may be present in any strain of E. coli, in practice only a small number of serotypes become enterotoxigenic (e.g. O6, O8, O15, O25, O27, O167). 3. Enteroinvasive E. coli (EIEC): Causative agent of dysentery-like disease. These resemble shigella in many respects. Many of these strains are non-motile, do not ferment lactose or ferment it late with acid, but without producing gas. Many of these show O antigen cross reaction with shigella. Clinically EIEC infection resembles shigellosis, ranging from mild diarrhoea to frank dysentery: bloody diarrhoea accompanied with inflammation. EIEC usually belong to serogroups O25, O114, O124, O144, O152, O154. They penetrate into enterocytes and produce heat-stable toxin (Shigella-like toxin) which inhibits protein synthesis by removing adenine from rRNA of human ribosomes. 4.Enterohemorhagic E. coli (EHEC): EHEC strains have become well known as the cause of several outbreaks of the disease associated with ingestion on undercooked hamburger or raw milk. Cattle are suspected as the reservoir. These organisms produce verotoxin or shigella-like toxin, which is cytotoxin. This toxin is responsible for hemorrhagic colitis (an inflammation of the colon with bleeding). It penetrates into the blood and can defeat kidney. Many patients, especially children, infected by this organism produce stools combined with copious amounts of blood, but without fever. Sometimes it can have fatal result. The typical EHEC is serotype O157:H7 5. Enteroaggregative E. coli (EAEC): These strains are so named because they appear aggregated in a “stacked brick” formation on Hep-2cells or glass. They have been associated with persistent diarrhoea, especially in developing countries. They produce a low molecular weight heat stable enterotoxin called EAST1(enteroaggregative heat stable enterotoxin-1). In animal experiments they cause shortening of villi, hemorrhagic necrosis and mild oedema with mononuclear infiltration of the submucosa. Most of them are O-untypable, but many are H-typable. Resistance: E. coli is more resistant to physical and chemical factors of the external environment than the other members of Enterobacteriaceae family. At 55 C the organism perishes in 1 hour, and at 60 C in 15 minutes. E. coli is sensitive to brilliant green and other disinfectants. E. coli is a common inhabitant of the large intestine of humans and mammals. The bacteria are excreted in great numbers with the faeces and are always present in the external environment (soil, water, foodstuffs, and other objects). Detection of E. coli in drink water is used as the indicator of faecal contamination (coli titer, coli index) Coli-titer is the minimal quantity of the water which contains one E. coli. It equals 300. Coli-index is the quantity of E. coli in one litre water. It equals 3. As a member of normal flora E. coli (with bifidum bacteria, lactobacilli) participates in nutritional function by producing several vitamins B, K, D. E. coli produce colicins (antibiotic like substances) which suppress exogenous and endogenous toxic products, and suppress the pathogenic flora (fungi, strepto-staphylococci, other intestinal pathogens). Antibiotic therapy inhibits the predominant normal flora and some diseases can occur (e.g., mycoses can occur and during antibiotic therapy we use anti-fungal drugs). E. coli takes parts in stimulation of formation of immune system (due to presence of muramil peptide in their cell wall). Immunity: During endogenous infection there is humoral immune response, but in this case immunoglobulins haven’t protective property. Immunodeficiency assists depressing phagocytosis. In the time of exogenous infection humoral immune response takes place too. IgG cross the placenta and from the blood it penetrates into the intestine. Here participate secretory IgA too. Treatment and prophylaxis: Antibiotics-penicillin, cephalosporins. Eubiotics: bifidobacteria, lactobacteria. 24 There is no specific prophylaxis. These infections are prevented by the practice of general hygienic measures in everyday life. The role and functions of normal microflora are numerous: 1.The normal microflora is non- specific defense factor of the organism. 2.The normal microflora has antagonistic role against pathogenic and putrefactive (saprogenous) microflora due to production of lactic acid, acetic acid, antibiotics, bacteriocines; at powerful biological potential’s expense can compete with foreign microflora. 3.The normal microflora participates in water - salt metabolism, regulation of gas composition of intestine, in metabolism of proteins, carbohydrates, fatty acids, cholesterin, nucleic acids, and also in production of biologically active compounds: antibiotics, vitamins (k, B group and others), toxins, etc. 4.The normal microflora participates in digestion and detoxication of exogenous substrates and metabolites, which is close to liver’s function. 5.The normal microflora participates in regulation of steroid hormones and bile salts as a result of excretion of metabolites from liver into intestine and following restitution in it. 6.The normal microflora plays morphokynetic role in development of different organs and systems of the organism, participates in physiological inflammation of mucous membrane and in replacing the epithelium. 7.The normal microflora plays anti-mutagenous function due to the destruction of cancerogenic substances in the intestine. At the same time some bacteria can produce powerful mutagens. So, enzymes of intestinal bacteria transform artificial cyclomate into active cancerogene (cyclohexamine) for urinary bladder. 8.Exopolysaccharides (glycocalyx) of microorganisms, which is the part of biological membrane, prevent microbial cells from different physical-chemical influence. Intestinal mucous membrane is also under the protection of biological membrane. 9.The normal microflora possesses a significant influence on formation and support of immune system. There are approximately 1,5kg of microorganisms in intestine, which antigens stimulate immune system. Natural non-specific stimulator of immunogeneses is muramyldipeptide, which is formed from bacterial peptidoglycane under the influence of intestinal lysozyme and other lytic enzymes. It results in abundant saturation of intestinal tissue with lymphocytes and macrophages, so, in norm the intestine is in chronic inflammation -`like conditions 10. The important function of normal microflora is participation in colonizative resistance. Laboratory diagnosis: 1. Bacteriological method-isolation of pure culture and identification 2. Serological method: slide agglutination, tube agglutination. 25 GENUS SALMONELLA Genus Salmonella has been named after American microbiologist, D.E Salmon. The genus Salmonella consists of bacilli that parasitize the intestine of large number of vertebrate species and which infect man, they cause enterocolitis, enteric fevers such as typhoid fever, septicemia and the carrier state. The morphology of salmonella corresponds with general characteristics of the Enterobacteriaceae family: gram-negative, middle-sized rods (2-5μm in size), they are motile (peritrichous), do not form capsule and spore. Antigenic structure: Salmonella possess three main types of antigens on the basis of which they are serologically typed. These are: cell wall O, flagellar H, capsular Vi (virulence) which are important for taxonomic and epidemiologic purpose. F. Kauffman and P. White classified the Salmonella into a number of groups according to antigenic structure (table 1). By the somatic O antigen, which is LPS of the cell wall Salmonella are subdivided into 65 serological groups (which are designated by capital letters A, B, C, D, ….) based on the presence of distinctive O antigen, in which O antigen factors were designated by Arabic numerals (1, 2, 3, etc.). H antigen: This antigen present on the flagella is a heat labile protein. It is composed of two phases: phase I is specific, and the phase II is non-specific. Within each group this bacteria is identified by the specific phase of H antigen (S. paratyphi A constitutes group A, paratyphi B belongs to group B (table). Vi antigen -surface antigen envelops the O antigen. It is heat – labile acidic polysaccharide. It is destroyed by heating the bacteria at 100ºC for one hour. When fully expressed, it renders the bacterium inagglutinable by O antiserum but agglutinable by Vi antiserum. 26 Salmonella typhi, S. paratyphi A and S. paratyphi B The morphology of the typhoid salmonella corresponds with the general characteristic of the Enterobacteriaceae family. Cultivation: Salmonella are facultative anaerobes. The optimum temperature for growth is 37º C. They grow on ordinary media at pH 6.8-7.2. On meat-peptone agar S. typhi forms semitransparent, large colonies (2-4 mm in diameter). Colonies of S. paratyphi B (schottmulleri) have a rougher appearance and mucus swelling round the colonies. This is a characteristic differential cultural property. On Ploskirev’s and Endo’s media S. typhi and S. paratyphi form colourless colonies due to the absence of lactose fermentation. On Wilson and Blair’s brilliant-green bismuth sulphite medium black colonies with metallic sheen are formed due to production of H2S. Salmonella paratyphi A and other species that do not form H2S produce green colonies. Selenite broth and bile broth are employed as enrichment media. In Rappaport media they grow and produce turbidity. Fermentative properties: S. typhi does not liquefy gelatin; they ferment proteins and liberate hydrogen sulphide (H2S); does not produce indole and reduces nitrates to nitrites. They ferment glucose, mannite, and maltose with acid formation. S. paratyphi ferments carbohydrates with acid and gas formation. Lactose, saccharose are not fermented. They are MR positive, VP negative, urea is not hydrolysed. S. paratyphi A and S. paratyphi B differentiated by the hydrogen sulphide production. S. paratyphi B formed H2S, S. paratyphi A not (table 2). By antigenic structure S. typhi in the D group, S. paratyphi A in the A group, S. paratyphi B in the B group and S. paratyphi B can cause infection in animal organism too (zoo-anthroponose). Pathogenicity: The main virulent factors are endotoxin, Vi antigen. Epidemiology and Pathogenesis: The source of infection is a patient, or far more frequently, a carrier. Man acquires infection by ingestion of contaminated water or food. Typhoid fever occurs in two epidemiological types. The first is endemic or residual typhoid that occurs throughout the year though seasonal variations may sometimes be apparent. The second is epidemic typhoid, which may occur in endemic or non-endemic areas. Typhoid epidemics are water, milk or foodborne. On reaching the gut, the bacilli attach themselves to microvilli of the ileal mucosa and penetrate to the lamina propria and submucosa. They are phagocytized there by polymorphs and macrophages. The ability to resist intracellular killing and to multiply in these cells is a measure of their virulence. For laboratory diagnosis of this infection it is necessary the pathogenesis of disease, which depends on different stages: 1.Digestive stage –penetration of microorganism into stomach. 2.Mesenterial lymphadenitis stage: On reaching the gut, the bacilli attach themselves to the epithelial cells of the intestinal villi and penetrate to the lamina propria and submucosa (invasive stage). After it they enter into the mesenteric lymph nodes where they multiply. This is incubation 27 period which is usually 10-14 days. After it they enter the bloodstream and the next stage is developed (bacteremeia). 3.Bacteremia period(prodrome period) during which the bacilli are seeded in all the organs and tissues (liver, gall bladder, spleen, bone marrow, lymph nodes, lungs, kidney where further multiplication takes place). Prodromal period is usually 2-3 days during which non-specific symptoms such as fever, malaise and loss of appetite occur (The first week of the diseases). 4.Parenchymatouse dissemination period (specific-illness period) As bile is a good culture medium for the Salmonella, it multiplies abundantly in the gall bladder and specific-illness period occurs during which the overt characteristic signs and symptoms of diseases occur: headache, malaise, anorexia, a coated tongue and abdominal discomfort with either constipation or diarrhoea. Red rash (rose spots) appears on the skin during the second and or third week. Some develop psychoses, deafness or meningitis. Cholecystitis, arthritis, abscesses, periostitis, nephritis, haemolytic anaemia, venous thrombosis and peripheral neuritis are other complications found 5.Allergic-secreted period when the bacteria are discharged into the intestine where involves the Peyer’s patches and lymphoid follicles of the ileum which had been previously sensitized by the Salmonellae in the initial stage. These become inflamed; undergo necrosis and slough off, leaving behind the characteristic typhoid ulcers and may be followed by perforation of the intestine and peritonitis and circulatory collapse. Ulceration of the bowel leads to the two major complications of the disease-intestinal perforation and hemorrhage. 6.Convalescence (recovery period) is slow. The typhoid-paratyphoid salmonellae together with pro- ducts of their metabolism induce antibody production and promote phagocytosis. These processes reach their peak on the fifth-sixth week of the disease and eventually lead to recovery from the disease. In about 5-10% cases, relapse occurs during convalescence. The relapse rate is higher in patients treated early with chloramphenicol. Clinical recovery does not coincide with the elimination of the pathogenic bacteria from the body. The majority of convalescents become carriers during the first weeks following recovery. Patients who continue to shed typhoid bacilli in faeces for three weeks to three months after clinical cure are called convalescent carriers. Those who shed the bacilli for more than three months but less than a year are called “temporary carriers” (acute carriers), and 3-5 per cent of the cases continue to excrete the organisms for many months and years after the attack and, for life (chronic carriers). Inflammatory processes in the gall bladder (cholecystitis) and liver are the main causes of a carrier state since these organs serve as favorable media for the bacteria, where the latter multiply and live for long periods. Besides, this typhoid-paratyphoid salmonella may affect the kidneys and urinary bladder, giving rise to pyelitis and cystitis. In such lesions the organisms are excreted in the urine. Immunity: Post infections immunity is tense, stable, lifelong, which depends on humoral and cellular immune response. Prophylaxis: General measures amount to rendering harmless the sources of infection. This is achieved by timely diagnosis, hospitalization of patients, disinfection of sources, and identification and treatment of carriers. Of great importance in prevention of typhoid fever and paratyphoids are such measures as disinfection of water, safeguarding water supplies from pollution, systematic and thorough cleaning of inhabited areas, fly control, and protection of foodstuffs and water from flies. Regular examination of personnel in food-processes factories for identification of carriers is also extremely important. In the presence of epidemiological indications specific prophylaxis of typhoid infections is accomplished by vaccination. The TABte vaccine was used which contains O and Vi-antigens of typhoid, paratyphoid A; B, and a concentrated purified and sorbed tetanus anatoxin. A new areactogenic vaccine (chemical vaccine) consisting of the Vi-antigen of Salmonella typhi has been produced. It is marked by high efficacy and used in immunization of adults and children under seven years of age. Specific bacteriophage can be used too. Treatment: Patients with typhoid fever and paratyphoids are prescribed chloraminphenicol and the other antibiotics which act on gram-negative bacteria. Laboratory diagnosis: 1.Bacteriological: Isolation of haemo-culture (bacteremic phase). For this examination Rappaport media is used (MPB, 10% bile, glucose, indicator, float for indication of gas formation), in which differentiation of S. typhi and S. paratyphi A and B is possible. If S. typhi is present fermentation of 28 glucose with acid formation is occurs; for S. paratyphi A and B – acid and gas formation. A pure culture is isolated from faeces and urine copro-culture or urino-culture (recovery period). The test material is inoculated into bile broth; Ploskirev’s, Endo’s media or bismuth sulphite agar. 2.Serological method –Widal tube agglutination reaction. This is a test for the measurement of O and H agglutinins for typhoid and paratyphoid bacilli in the patient’s sera (sufficient number of agglutinins accumulate in blood on the second week of the disease -specific illness period). 3.Passive hemagglutination test. 4.Skin-allergic test. 5.Diagnosis of carriers: The detection of carriers is important for epidemiological and public health purpose. The demonstration of Vi agglutinins has been claimed to indicate the carrier state. SALMONELLA-GASTROENTERITIS (Causative agents of food toxinfection) The genus salmonella comprises many species and types of bacteria which cause food toxinfection (S. typhimurium; S. enteritidis; S. anatum; S.heidelberg, S. derby, S. haifa, S. infantis). It may be caused by any salmonella except S. typhi. Salmonella gastroenteritis or food poisoning as distinct from typhoid fever and paratyphoids A and B is generally is zoo-anthroponose disease, the source of infection being animal products. Morphology: Morphologically Salmonella organisms possess the general characteristics of the family Enterobacteriaceae. Fermentative properties: They do not liquefy gelatin and do not produce indole. The majority of species produce hydrogen sulphide and ferment glucose, maltose with acid and gas formation. Resistance: Salmonella are relatively stable to high temperatures (60-75ºC), high salt concentrations, and to acids. They withstand 8-10 per cent solution of acetic acid for 18 hours, and survive for 75-80 days at room temperature. A characteristic feature of foodstuffs contaminated by salmonella is that they show no changes which can be detected organoleptically. Virulence factors are: 1.adhesion and colonization. 2.enterotoxin which acts by adenylate cyclase mechanism It is necessary to note that salmonella's protein toxins have intracellular location and pass internal medium of macroorganism in the case of microbe's structure destruction. 3.cytotoxin, which is similar to shigella exotoxin 4.different enzymes-protease, mucinase, decarboxylase, etc. 5.endotoxin Pathogenesis: Human infection result from the ingestion of contaminated food. The most frequent sources of salmonella food poisoning are poultry, meat, milk, and milk products. Of great concern are eggs and egg products. Meat may be infected while the animal is alive or after its death. Depend on virulence, infectious dose of bacteria and immune state of macroorganism, there are following clinical forms of infection: 1.food toxinfection 2.salmonella diarrhoea 3.generalized (typhoid) Intoxication develops in a few hours following infection. Masses of microbes ingested with the food are destroyed in the gastrointestinal tract and in the blood (bacteremia is infrequent). This results in the production of large amount of endotoxin which, together with the endotoxin entering the body with the ingested food, gives rise to intoxication. Salmonella produce exotoxin-enterotoxin too, which is similar to E. coli thermolabile toxin. The mechanism of enterotoxin is disturbances of water-salt metabolism, which cause diarrhoea. Clinically, the disease develops after short incubation period (18-48 hours or less), with diarrhoea (with or without blood), which can vary from mild to severe, vomiting, abdominal pain and fever. It is self-limited, causes non-bloody diarrhoea, and does not require medical care except in the very young and very old. By the generalization of the processes the diseases are of long duration or become chronic. 29  It may vary in severity from the passage of one or two loose stools to an acute cholera- like disease  It usually subsides in 3-5 days, but in some cases more prolonged enteritis develops, with passage of mucus and pus in faeces resembling dysentery.  In a few, typhoid or septicemic type of fever may develop. Immunity: Immunity acquired after salmonellosis is tense and type specific. Treatment: Treatment of uncomplicated, non-invasive salmonellosis is symptomatic. Antibiotics should not be used. But for the serious invasive cases antibiotic treatment is needed. Prophylaxis: Veterinary-sanitary and other anti-epidemiological measures. Control of salmonella food poisoning requires the prevention of food contamination. Food may become contaminated at various levels, from natural infection in the animal or bird, to contamination of the prepared food. Proper cooking of food destroys salmonellae. Laboratory diagnosis: Bacteriological method: Isolation of pure culture from the faeces of patients and food. In outbreaks of food poisoning, the causative article of food can often be identified by taking a proper history. 30 GENUS KLEBSIELLA They are in family Enterobacteriaceae, genus Klebsiella (the discoverer was Klebs) which contain two species: Klebsiella pneumoniae and Enterobacter. For human organism the most important is Klebsiella pneumoniae, which includes three subtypes: K. pneumoniae, K. ozaenae, K. rhinoscleromatis. Morphology and physiology: The Klebsiella are gram-negative thick short bacilli 0.6-6.0μm in length and 0.3-1.5 µm in breadth. They have rounded ends, are nonmotile. They occur mainly in pairs but may be seen frequently as single organisms, and are normally surrounded by polysaccharide capsule, non spore forming. Cultivation: The Klebsiella are facultative anaerobes, which grow readily on common nutrient media at pH 7.2-7.4 and at temperature of 35-37º C. They form turbid mucilaginous colonies on agar and produce intense turbidity in broth. On differential diagnostic media (Endo, Ploskirev) they form red colonies due to fermentation of lactose (table 3). Antigenic structure: Klebsiella contain O and K antigens. There are 11 “O” antigens and 80 and more “K” antigens. Ecology: Klebsiella are widely distributed in nature. They are contained in normal intestinal biocenosis. They may be found on the skin and mucous membranes. They are as commensals in the intestine and as saprophytes in soil and water. The source of infection is patient. K. pneumoniae is excreted from human throat (pharynx), gastrointestinal tract in 5% healthy people. Pathogenesis: The virulence factors are: adhesion (fimbria, superficial proteins), capsular polysaccha- ride (antiphagocytic factor), colonization, endotoxin, enterotoxin and cytotoxin. K. pneumoniae produces enterotoxin (which is very similar to the heat-stable toxin of E. coli) which stimulates secretion of liquids and development of diarrhoea. Three species of bacteria play most important role in human pathology: the causative agents of pneumonia, ozaenae and rhinoscleromatis. K. pneumoniae (Friedlander rod): is responsible for pneumonia and hospital infections (bronchitis and bronchopneumonia), which involves one or several lung lobes, sometimes producing fused foci and lung abscesses. The death rate is quite high. In some cases the organisms may be responsible for meningitis, appendicitis, cystitis. They may also cause inflammation in cases of mixed infections. As they produce enterotoxin they are causative agents of enteric infection in new-borns. K. ozaenae: morphological characteristics are given above. They are responsible for rhinitis which is characterized by an offensive nasal discharge. K. ozaenae affects the mucous membranes of the nose, nasal sinuses, and conchae. This results in production of a viscid discharge which dries up and forms thick scabs with an offensive odour. These scabs make breathing difficult. Ozaenae is transmitted by the air-droplet route. It is possible that other factors (trophic and endocrine disturbances, etc.) also contribute to its development. K. rhinoscleromatis: These organisms are responsible for chronic granulomatosis of skin and mucous membranes of the nose, pharynx, larynx, trachea, and bronchi, with the formation of granulomas. Rhinoscleroma is mildly contagious disease. Treatment is a matter of great difficulty and involves complex therapeutic measures which must be carried out over a long period of time. Immunity: Diseases caused humoral and cell immune response. The formed antibodies don’t have protective properties. Immunity is low and in short duration. Treatment: Antibiotics; cephalosporins. Prophylaxis: is ensured by recognition of the early stages of ozaenae and rhinoscleroma, active antibiotic therapy, and prevention of healthy individuals from being infected by the sick. There is not specific vaccino-prophylaxis. Laboratory diagnosis: Includes the following methods: 1. Microscopic: examination of smears made from sputum (from patients with pneumonia) nasal mucus discharge (from patients with pneumonia), and tissue specimens (from patients with rhinoscleroma). 2. Bacteriological method: Isolation of the pure culture and identification by cultural, biochemical, and serological properties (table1). 3. Serological reaction: Complement fixation reaction. 31 32 GENUS PROTEUS Proteus was discovered in 1885 by G. Hauser. Genus Proteus contains the following species P. vulgaris, P. mirabilis, P. morgani, P. rettgeri, P. inconstans. P. vulgaris and P. mirabilis cause toxinfections and pyo-inflammatory processes in human organism frequently. The species are differentiated by studying their fermentative properties. Morphology: They are polymorphous (in young cultures most of them are long curved and filamentous), gram–negative rods (1-3x0.6μm), motile (peritrichous). Non-flagellar, non-motile variants are also encountered. The organism does not form either spores or capsules. Cultural properties: Facultative anaerobes, and grows readily on common media. They are characterized by creeping growth (H-forms – hauch in German means breathing. These are motile forms). Some strains are non-motile and form large, S-form colonies (O-forms – in German one hauch - non-breathing). Cultures of Proteus have a characteristic putrid odour described as “fishy”. Proteus liquefies gelatin and coagulates serum. They produce indole, ammonia, hydrogen sulphide, ornithindecarboxilase, reduce nitrates to nitrites and ferment levulose, glucose, galactose, maltose with acid and gas formation (table 1). Antigenic structure: Proteus contains O, H and K antigens. “O” antigen is cell wall LPS by which they are subdivided into 49 serogroups; by “H” antigen – into 19 serovars. Weil and Felix observed that certain non-motile strains of P. vulgaris, called “X” strains, were agglutinated by sera from typhus fever patients. This can be used in agglutination reaction for revealing Rickettsia (Weil and Felix agglutination reaction-three non-motile Proteus strains OX2, OX19, and OXK are used in this test) Pathogeneses and pathogenicity: Proteus is conditionally pathogenic bacteria (common members of the indigenous microflora of the colon; opportunistic pathogens; a fairy common cause of cystitis). The virulence factors are the following:  Fimbriae  Endotoxin-LPS of the cell wall  Protease, increases permeability of the vessels and affects immunoglobulins  Urease  Hemolysin  Hemagglutinins In gastrointestinal tract they can cause toxinfections and acute gastroenteritis. P. mirabilis is the most frequently associated bacterium with urinary tract infection (especially dangerous infection in newborns: Proteus transferred from umbilicus which often leads to bacteraemia and meningitis). In association with other conditionally pathogenic microorganisms they can cause local pyo- inflammatory processes and septic infections: cystitis, pyelitis, pleuritis, abscesses, pneumonia, meningitis, sepsis. Specific prophylaxis is absent; treatment is by antibiotics, Proteus phage can also be used. 33 GENUS YERSINIA Yersinia are short, pleomorphic, gram-negative rod that can exhibit bipolar staining. They do not form spores and are catalase-positive, oxidase-negative, and microaerophilic or facultative anaerobes. The natural hosts for them are animals but they can produce a serious disease in humans. Genus Yersinia contains 7 species. The causative agents of human infections are: Y. pestis - causative agent of plague. Y. pseudotuberculosis -causes fatal typhoid-like illness with hepatoslenomegaly. Y. enterocolitica - important causes of human diarrheal diseases. Yersinia enterocolitica These are non-lactose-fermenting, gram-negative ovoid coccobacilli showing polymorphism in older cultures, motile (peritrichous) rods. In 37ºC they lose motility, they are active motile in 18-22ºC. 1-3x0.5-0,8μm, non-capsulated (some virulent strains can form capsule), non-spore forming organisms. They are urease-positive and oxidase-negative. Cultivation: These organisms grow readily on common nutrient media at temperature 28-30 C. In liquid media they produce diffuse turbidity, on solid media they form small (0.1-0.2mm), shinny, light blue, S-type colonies. They are found in the intestinal tract of a variety of animals, in which they may cause a disease, and are transmissible to humans, in whom they can produce a variety of clinical syndromes. Antigenic structure: Y. enterocolitica possesses “H”-flagellar and somatic “O” antigens. According to somatic “O” antigen they are subdivided into 34 serovariants. Causative agents of infections in human organism belong to O3, O5 - O9 serovariants. Ecology: Y. enterocolitica has been isolated from rodents and domestic animals (eg, sheep, cattle, swine, dogs, and cats) and waters contaminated by them. Transmission to humans probably occurs by contamination of food, drink, or fomites. Person to person transmission with either of these organisms is probably rare. Pathogenicity and pathogenesis: Y. enterocolitica is facultative intracellular parasite. Pathogenicity depends on adhesins (fimbria, microcapsule), cytotoxins and invasive properties (invasion depends on superficial proteins, which can inhibit activities of bactericidal factors-phagocytosis, complement), enzymes (phosphatase, proteinkinase), endotoxin, thermostable enterotoxin (guanilate cyclase mechanism of action). An inoculum of 108-109 Yersinia must enter the alimentary tract to produce infection. During the incubation period of 5-10 days, Yersinia multiplies in the gut mucosa (enterocytes, Peyer’s patches), particularly in the ileum. This leads to inflammation and ulceration, and leukocytes appear in faeces. The process may extend to mesenteric lymph nodes and rarely, to bacteremia. Early symptoms include fever, abdominal pain, and diarrhoea. Diarrhoea may be due to an enterotoxin or to the invasion of the mucosa, and it ranges from watery to bloody. At times, the abdominal pain is severe and located in the right lower quadrant, suggesting appendicitis. One to 2 weeks after the onset some patients develop arthralgia, arthritis. Very rarely, Yersinia infection pro- duces pneumonia, meningitis, or sepsis. The clinical forms are gastroenteritis, lymphadenopathy, and acute appendicitis. Treatment: Y. enterocolitica are generally susceptible to aminoglycosides, chloramphenicol, tetracyc- line, third-generation cephalosporins. Prevention and control: Contact with farm and domestic animals, their faeces, or materials contami- nated by them probably accounts for most human infections. Meat and dairy products have occasional- ly been indicated as sources of infection, and group outbreaks have been traced to contaminated food or drink. Conventional sanitary precautions are probably helpful. There are no specific preventive measures. Laboratory diagnosis: Bacteriological method: Isolation of pure culture and identification by biochemical activity (table 1). Serological method: agglutination reaction. 34 35 GENUS SHIGELLA Shigella are the causative agents of bacterial dysentery. In 1898 this organism was studied in detail by K. Shiga in Japan and according to the current International Nomenclature, all dysentery bacilli are grouped together in one genus known as Shigella. Morphology: Morphologically dysentery bacilli correspond to the organism of the family Enterobacteriaceae. They are slender gram-negative rods, non-sporing, non-capsulated, non-motile, the difference from other members of Enterobacteriaceae is dysentery bacteria have no flagella. They possess microvilli, fimbriae, pili like other members of Enterobacteriaceae. Cultural characteristics: Shigella are facultative anaerobes and grow readily on common media (but less readily than other enterobacteria) at pH 6.7-7.2, optimal temperature for growth - 37º C. On solid media they form small (1-1.5 mm), fragile, semitransparent colonies. In meat broth dysentery bacilli produce a diffuse turbidity. Colonies on Endo’s (MacConkey agar), Levin’s, Ploskirev’s media are colorless due to the absence of lactose fermentation. An exception is S. sonnei which ferments lactose late (after 48-72 hours) with acid formation (without gas) and forms pale pink colonies. Antigenic structure: The antigenic structure of Shigella is associated with somatic O-antigen and surface K-antigens. By somatic O antigen Shigella are subdivided into four groups (A, B, C, D) and 40serotyps (table 1). 1. Group A - S. dysenteriae which subdivided into 1-10 serovars; 2. Group B - S. flexneri ( 1-6 serovars); 3. Group C – S. boydii (1-15 serovars) 4. Group D – S. sonnei (-). Dysentery bacilli are differentiated on the basis of the whole complex of antigenic structure and biochemical properties. Dysentery bacilli ferment glucose with acid formation, don’t ferment lactose except S. sonnei, which ferment lactose during 48-72 hours (in this case they differ from E. coli-fermentation of lactose during 18-24 hours). None of species of dysentery bacilli liquefy gelatin nor produce hydrogen sulphide. Fermentation of mannite is of importance in classification and Shigella has traditionally been divided into mannite positive, mannite negative. S. dysenteriae is mannite negative, does not ferment mannite, others are mannite positive, they ferment mannite. Shigella are MR positive and reduce nitrates to nitrites (table 2). Ecology and spreading:. They are killed at 56ºC in one hour and 1% phenol in 30 minutes. In water and ice they remain viable for 1-6 months, boiling or chlorination of water and pasteurisation of milk destroy the bacilli. In faeces they die within a few hours due to the acidity produced by the growth of coliforms. S. sonnei is, in general, more resistant than other species. Shigellosis are highly communicable; the infective dose is 102 organisms (105 for salmonella and 1011 for vibrio). 36 Dysentery is anthroponose infection; human beings are the only natural hosts for shigella. The modes of transmission are: 1. direct, through contaminated fingers and hand (infection of dirty hands) 2. through fomites such as door handles, water taps, lavatory seats. 3. through water 4. through contaminated food and drink. 5. through flies, which may transmit the infection as mechanical vectors. Ecology and spreading:. They are killed at 56ºC in one hour and 1% phenol in 30 minutes. In water and ice they remain viable for 1-6 months, boiling or chlorination of water and pasteurization of milk destroy the bacilli. In faeces they die within a few hours due to the acidity produced by the growth of coliforms. S. sonnei is, in general, more resistant than other species. Virulent factors are the following:  adhesion on epithelium of large intestine, which depends on fimbriae, superficial proteins of external membrane  colonization  invasion into the cells, which depends on superficial proteins and enzymes (neuraminidase, hyaluronidase, mucinase)  endotoxin: all shigella release an endotoxin after autolysis. It is thermostable lipopolysaccharide of the cell wall.  exotoxins:  Shiga toxin: Shigella dysentery produces a powerful exotoxin. It is heat-labile protein and acts as a cytotoxin (suppresses protein synthesis in target cells), which has enterotoxic, cardiotoxic and neurotoxic action. As a neurotoxin it damages endothelial cells of small blood vessels of the cen

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