Bohomolets Microbiology Lesson #1


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Bohomolets Microbiology Lesson #1

  1. 1. Lesson N1: LABORATORY DIAGNOSIS OF STAPHYLOCOCCAL INFECTIONS1.Scientifically methodical ground of theme There are several types of symbiosis between cocci and the human body. Saprophytic and conditionallypathogenic types of staphylococci and streptococci live on the skin, mucous membranes, and in the respiratorytract. Meningococci may be harboured for long periods in the nasopharynx, and faecal streptococci (enterococci) inthe intestine. When body resistance is lowered or the skin and mucous membranes are injured, these bacteria penetrate thebody tissues and cause infection. The various cocci possess different organotropic ability. This is distinctlymanifest in meningococci, and gonococci but less so in staphylococci and streptococci. Cocci belong to thefamilies Micrococcaceae, Streptococcaceae Peptococcaceae, Neisseriaceae.2.Educational purpose STUDENTS MUST KNOW: 1. Structure, tinctorial and cultural properties of staphylococci. 2. Antigenic structure of staphylococci, their classification. 3. Fermentative properties and toxin production of staphylococci. 4. Differentiation of staphylococci. 5. Main clinical forms of staphylococcal infections. 6. Methods of laboratory diagnosis of staphylococcal infections and main preparations for specificprophylaxis and treatment of staphylococcal infections STUDENTS SHOULD BE ABLE TO: – prepare the smears from pus; – stain the smears by Gram’s technique; – make microscopical examination of the smears; – inoculate pus on solid medium; – inoculate a blood specimen in sugar MPB with the purpose of diagnostics of a sepsis; – inoculate material from a nasopharynx for revealing bacterial carriage.3.Chart of topic content. Staphylococci, being resistent to dry conditions and hight salt concentrations, are well suited to theirecological niche, which is the skin surface of man and animals. Approximately 30% of helthy people are “carriers”of S. aureus, an even higher number carry coagulase-negative staphilocicci. Staphilococci are also commonlypresent on other animals, including farm animals used for milk production, cattle, sheep and gouts; mastitis causedby S. aureus is common and mostly complication of milking. Contamination food can occur from either human oranimal sources; such contamination can result in staphilococcal food poisoning. Staphylococci. The staphylococcus, Staphylococcus aureus, was discovered by R. Koch (18,78), and laterisolated from furuncle pus by L. Pasteur (1880). It has been described as the causative agent of numeroussuppurative processes by A. Ogston (1881), and has been studied in detail by F. Rosenbach (1884) Definition of genusThe chief properties characteristizing the genus Staphylococcus are shown in table N1. Staphylococci resemblemembers of genus Micrococcus morohologically, but they differ in DNA base composition. Micrococcus alsodeffer in having a strictly aerobic metabolism. Table N1:Characteristics of the genus of StaphylococcusGram-positive cocciCatalase positiveDivide in more than one plane to form irregular, grape- lake clusterCapable of aerobic and anaeribic metabolismOccur widly in the surface of man and other vertebrate animalsMorphology. Staphylococci are spherical in shape, 0.8-1 mem in diameter, and form irregular clusters resemblingbunches of grapes. In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci.Large spherical (L-forms) or very small (G-forms) and even filterable forms may be seen in cultures which havebeen subjected to various physical, chemical, and biological (antibiotics) factors. Staphylococci are Gram-positive organisms which possess no flagella and do not form spores. Amacrocapsule can be seen on ultrathin sections of Staphylococci isolated from infected mice. The nucleoid 1
  2. 2. occupies most of the cytoplasm and is filled with DNA fibrils. The G+C content in DNA ranges between 30.7 and39.0 per cent. Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a pHof 7.2-7.4 at a temperature of 37 °C but do not grow at temperatures below 10 °C and above 45 °C. At roomtemperature with adequate aeration and subdued light – the organisms produce golden, white, lemon-yellow, andother pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, benzene,acetone, chloroform, and alcohol. They are most readily formed on milk agar and potatoes at a temperature of20-25° C. On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1-2to 2.5 mm in diameter. Under the microscope the course granular nature of the colonies can be seen, the latter areopaque and have a dense centre. Their colour epends on the pigment produced by the organisms. Besides thetypical colony types, Staphylococci produce R-, G-, and L-forms. Growth of Staphylococci in meat-peptone brothproduces diffuse opacity throughout the medium and, subsequently, a precipitate. In some cases when there issufficient aeration, the organisms form a pellicle on the surface of the broth. Staphylococci grow well on potatoesand coagulated serum. After 24-48 hours of incubation there is usually abundant growth along the inoculation staband liquefaction of gelatin media. On the fourth or fifth day the gelatin medium resembles a funnel filled withfluid. On blood agar pathogenic Staphylococci cause haemolysis of the erythrocytes. Rabbit and sheeperythrocytes are the most sensitive to the staphylococcal haemotoxin. Fermentative properties. Staphylococci produce enzymes which cause the lysis of proteins and sugars (seeTable 2). There is no indole production in young cultures. The organisms liquefy gelatin, coagulate milk andoccasionally serum, reduce nitrates to nitrites, produce urease, catalase, phosphatase, ammonia, and hydrogensulphide. They ferment glucose, levulose, maltose, lactose, saccharose, mannitol, and glycerin, with acid formation.A connection has been revealed between arginase activity and the level of γ-toxin formation. Toxin production. Staphylococci produce α-, β-, δ- and γ-haemolysins which are characterized by lethal,haemolytic, and necrotic activity. Filtrates of staphylococcal broth cultures contain an enterotoxin which causesfood poisoning on entry into the gastro-intestinal tract. Staphylococci excrete exofoliatines which causestaphylococcal impetigo and pemphigus neonatomm in children. Leucocidin, a substance which destroys leucocytes, haematoblasts of the bone marrow and nerve cells, isalso produced by the Staphylococci. The organisms also coagulate blood plasma. Their ability to coagulate, plasmais a stable property and is used for differentiating various strains. Coagulase is thermoresistant. It can be isolatedfrom staphylococcal broth cultures. Staphylococci produce fibrinolysin which when added to a blood clot dissolves the latter within 24-48 hours. The Staphylococci produce hyaluronidase which breaks down hyaluronic acid, a component of connectivetissue. Coagulase, fibrinolysin, lecithinase, hyaluronidasa and phosphatasa all belong to the group of enzymespossessing destructive properties. Lecithinase destroys the lecithin protective membranes of the colloidal particlesof a substance found in human, sheep, and rabbit erythrocytes. An anticoagulant which inhibits blood coagulationhas also been derived from the staphylococcal cultures. This staphylococcal anticoagulant is produced in exudatesof inflamed tissues, occurring during staphylococcal infections. Haemagglutmins which cause the agglutination ofrabbit erythrocytes have also been found in staphylococcal culture filtrates. Virulent Staphylococci inhibit thephagocytic activity of leucocytes. Many microbiologists believe that the Staphylococci isolated from patients produce alpha-haemolysins,while the organisms pathogenic for animals (e. g. itesponsible for mastitis in cows) more often produce beta-haemolysin. Staphylococcal exotoxin, inactivated by treatment with 0.3-0.5 per cent formalin at 37 °C for 7-28 days, andinjected parenterally to humans and animals, stimulates the production of a specific antitoxin capable of reactingwith the toxin. Antigenic structure. Polysaccharides A and B have been obtained from a staphylococcal suspension bytreating the latter alternately with acid and alkali and removing the proteins with trichloracetic acid. Polysaccharide A was extracted from pathogenic strains isolated from patients with septicaemia,furunculosis, osteomyelitis, and acute conjunctivitis, etc. Polysaccharide B is found in avirulent, non-pathogenicstrains. Polysaccharides A and B differ not only in their serological reactions but also in their chemical structures. Antigen C, containing a specific polysaccharide, has been recently isolated. Staphylococcal polysaccharidesdemonstrate a marked type specificity. Even in a 1 :1000 000 dilution they give a distinct precipitin reaction. Theprotein antigen is common to all species and types of staphylococci. 2
  3. 3. Three types (I, II, III) of staphylococci have been revealed by the agglutination test and precipitin reaction.However, a quite number of cultures are unsuitable for serological typing. Recent studies have revealed fifteentype-specific staphylococcal antigens. Classification. Staphylococci are included in the class Bacteria, family Micrococcaceae, genusStaphylococcus.Currently there are some 32 different species of staphylococci. These fall into two main groups onbasis of their ability to clot blood plasma by action enzyme coagulase. S. aureus is far the most importantcoagulase-positive species. The coagulase- negative staphylococci are skin commensals, which are now recognazedas important oportunic pathogens that can cause infections associated with prostheses, catheters and implants (S.epidermidis, and uritary tract (S. saprophyticus) Table 2Differentiation of Staphylococci Species Main characteristics S. aureus S. S. epidermidis; saprophyticus Presence of: Plasmacoagulase + — — Phosphatase + + — Protein A in + — — superficial antigen Oxidation Mannitol + — + Production of alpha- + – – toxin Resistance to S S R novobiocin Deoxyribonuclease + - - Note: S, sensitive; R, resistant Certain strains of the family Micrococcaceae are strict anaerobes. Peptococcus niger, Peptococcusanaerobius, Peptococcus asaccharolyticus and others are usually conditionally pathogenic for human beings. Theylive in the mouth mucosa, in the intestine, urogenital tract, and in other parts of the human body. In weakenedindividuals and in people suffering from chronic diseases the anaerobic micrococci may give rise to variousdiseases and complications. Resistance. Staphylococci are characterized by a relatively strong resistance to desiccation, freezing,sunlight, and chemical substances. After desiccation they can survive for more than 6 months. Repeated freezing.and thawing do not kill the organisms. They survive for many hours under direct sunlight. Staphylococci maintaintheir viability for more than 1 hour at 70 °C. At a temperature of 80 °C they are destroyed within 10-60 minutesand at boiling point, they instantly perish. A 5 per cent phenol solution kills the organisms in 15-30 minutes.Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treatingpyogenic skin diseases caused by these organisms. Staphylococci possess high resistance to antibacterial agents, in70-80 per cent of cases they are resistant simultaneously to 4-5 agents. Cross resistance to antibiotics of themacrolide group (erythromycin, oleandomycin, etc.) is encountered. Pathogenicity for animals. Horses, cattle, sheep, goats, pigs, and, among laboratory animals, rabbits, whitemice, and kittens are susceptible to pathogenic staphylococci. An intracutaneous injection of a culture of pathogenic staphylococci produces inflammation and subsequentnecrosis in the skin of the rabbit. An intravenous injection of a staphylococcal culture filtrate causes a conditionsimilar to acute poisoning in rabbits, which is characterized by motonc excitation, respiratory disorders,convulsions, paralysis of the hind extremities, and sometimes, by diarrhoea and urine discharge. After completefatigue the animal shortly dies. Staphylococci or their toxin will cause vomiting, diarrhoea, and weakness in kittens if introduced per os orintraperitoneally. Functional disorders of the digestive tract arise owing to the effect of tne enterotoxin which isdistinguished from the other fractions of the staphylococcal toxin by its thermoresistance. It withstands atemperature of 100 °C for 30 minutes. The most reliable test for the presence of enterotoxin is an intravenousinjection to adult cats. Pathogenesis and diseases in man. Staphylococci enter the body through the skin and mucous membranes.When they overcome the lymphatic barrier and penetrate the blood, staphylococcal septicaemia sets in. Both the 3
  4. 4. exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms.Consequently, staphylococcal diseases should be regarded as toxinfections. The development of staphylococcal diseases is also influenced by the resulting allergy which in many casesis the cause of severe clinical forms of staphylococcal infections which do not succumb to treatment. Staphylococci are responsible for a number of local lesions in humans: hidradenitis, abscess, paronychia,blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronicpyodermia, peritonitis, meningitis, appendicitis, and cholecystitis. Diabetes mellitus, avitaminosis, alimentary dystrophy, excess perspiration, minor occupational skinabrasions, as well as skin irritation caused by chemical substances, are some examples of the conditions conduciveto the formation of pyogenic lesions of the skin and furunculosis. In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox,influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumoniain children are particularly severe diseases. Ingestion of foodstuffs (cheese, curds, milk, rich cakes and pastry, icecream, etc.) contaminated with pathogenic staphylococci may result in food poisoning. Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases ofwound infections, diphtheria, tuberculosis, actinomycosis, and angina. The wide use of antibacterial agents, antibiotics in particular, lead to considerable changes in the severity anddegree of the spread of staphylococcal lesions. Growth in the incidence of diseases and intrahospital infections mobstetrical, surgical and childrens in-patient institutions, intensive spread of the causative agent, and increase in thenumber of carriers among the medical staff and population have been noted in all countries of the world.Intrauterme and extrauterine contamination of children with staphylococci has been registered, with thedevelopment of vesiculopustular staphyloderma, pemphigus, infiltrates, abscesses, conjunctivitis, nasopharyngitis,otitis, pneumonia, and other diseases. It has been established that staphylococci become adapted rapidly to chemical agents and antibiotics due tothe spread of R-plasmids among these bacteria. The high concentration of drugs in the body of humans and in thebiosphere has resulted in essential disturbance in the microflora and the extensive spread of resistant strainspossessing more manifest virulence. The L-forms of staphylococci are especially marked by increased degree ofresistance to antibiotics. Table N3 Virulence factors of S. aureusVirulence factor ActivityCell wall polymersPeptidoglican Inhibitor of inflammatory responseTeichoic acid Phage adsorbtion;Cell surface priteinsProtein A Binds to Fc portion of antibody, inhibiting phagocytosis (blocks attachment to Fc receptors on white blood cells). Inhibit phagocytosis of nonopsonized bacteriaCaosules (microcapsules)ExoproteinsLysin (alpha,beta, gamma, delta) Impearent of membrane permeability; cytotoxic effects on phagocytic and tissue cellsLeucocidin Kills white blood cells by producing holes in their cytoplasmic membrane Convert fibrinogen to fibrin, may impede progress ofCoagulase leucocytes into infected area by producing clots in the surrounding capillariesEnterotoxin Inducing vomiting and diarrheaEpixdermolic toxin (exfoliation) Separates layers of epidermis, causeng scalded skin syndromeToxic shock syndrom toxin Causes rash, diarrhea, toxic shock (multisystem effect) 4
  5. 5. Lipase Break down fats by hydrolyzing the bond between glycerol and fatty acidProtease Degrade collagen and other tissue proteinsHyaluronidase Break down hyaluronic acid component of tissue, thereby promoting extension of infection Immunity. The tendency to run a chronic flaccid course or relapse is regarded as a characteristic symptomof staphylococcal infections. This peculiarity gives a basis for concluding that postmfectional immunity followingstaphylococcal diseases is of low grade and short duration. Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies(antitoxins, precipitins, opsonins, and agglutinins). The inflammation restricts the staphylococci to the site of penetration and obstructs their spreadingthroughout the body. At the centre of inflammation the organisms undergo phagocytosis. Neutralization of thestaphylococcal toxin by the antitoxin is an important stage of the immunity complex. As a result of capillarypermeability, the antitoxin penetrates from the blood into the inflammation zone and renders harmless the toxinproduced by staphylococci. Thus, the phagocytic and humoral factors act together and supplement each other. The presence in the human organs and tissues of antigens which are also common in the staphylococci(mimicry antigens) is among the causes of low immunity. This causes a state of immunological tolerance tostaphylococci and their toxins, which provides favourable conditions for uninhibited reproduction of the causativeagent in the patients body. The wide use of antibacterial agents promotes intensive selection of staphylococcalstrains resistant to the natural inhibitors of the microorganism. Laboratory diagnosis. Test material may be obtained from pus, mucous membrane discharge, sputum,urine, blood, foodstuffs (cheese, curds, milk, pastry, cakes, cream, etc.), vomit, lavage fluids, and faeces. The material is examined for the presence of pathogenic staphylococci. Special rules are observed whencollecting the material since non-pathogenic strains are widespread in nature. Laboratory studies comprise the determination of the main properties of the isolated staphylococci (i. e theirmorphologic, cultural and biochemical characteristics), as well as their virulence. For this purpose the followingprocedures are carried out. Smears are made from pus and stained by the Gram method. Pus is inoculated ontoblood agar and meat-peptone agar containing crystal violet. In cases of septicaemia blood is inoculated into glucosebroth.The isolated pure culture is tested for its haemolytic (by inoculation onto blood agar plates),plasmacoagulative (by inoculation into citrated rabbit plasma), and hyaluronidase activities. Virulemce isdetermined in rabbits by intracutaneous injection of 400 million microbial cells. Necrosis develops at the site ofinjection within 24-48 hours. Pigment production of the isolated culture is also taken into account. For revealingsources of infection, particularly food poisoning and outbreaks of sepsis in maternity hospitals, serological typingand phage typing are carried out. To ensure effective therapy the isolated cultures are examined for sensitivity toantibiotics. In cases of food poisoning presence of the enterotoxin in th isolated staphylococcal culture is tested for byintravenous injection of the culture filtrate to adult cats. In cases when intoxication is due to ingestion of the milkof a cow suffering from mastitis, the culture grown on starch medium is tested directly for toxin production as ameans of detecting staphylococci of animal origin. When the causative agent cannot be detected (osteomyelitis andother diseases), the patients serum is tested for agglutinins. Treatment. Staphylococcal diseases are treated with antibiotics (penicillin, phenoxymethyl penicillin,tetracycline, gramicidin, etc.), sulphonamides (norsulphazol, sulphazol, etc.), and antistaphylococcal gamma-globulin. When treating patients suffering from staphylococcal infections one should bear in mind that it is necessaryto relieve intoxication and improve the immunological defence forces of the body (infusion of glucose, plasma,blood transfusion, injection of cardiac stimulants). In cases of chronic staphylococcal lesions specific therapy is recommended: autovaccines, staphylococcalanatoxin, antitoxic serum, and diphage containing staphylococcal and streptococcal phages. Staphylococcal produce strains resistant to sulphonamides, antibiotics, and bacteriophage, which advancesthe wide distribution of staphylococcal infection. This variability is of particular importance in the therapy ofstaphylococcal pyogenic diseases. The medical services produce semisynthetic preparations of penicillin andtetracycline which are effectively used for the treatment of these diseases. 5
  6. 6. 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 hygienein maternity hospitals, surgical departments, childrens institutions, industrial plants and enterprises, particularlycanneries, observance of personal hygiene and frequent washing of hands in warm water with soap. Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriologicalexamination of the personnel for carriers of pathogenic staphylococci resistant to antibiotics are also necessary.In some cases specific prophylaxis by means of immunization with the staphylococcal anatoxin may berecommended for individuals subject to injury or infection with antibiotic-resistant staphyloc4. Student’s independent study program 1. General characteristics of gram-positive cocci. Structure, staining properties of staphylococci. Cultivation. 2. Antigenic structure of staphylococci. Classification of staphylococci. 3. Fermentative properties and toxin production of staphylococci. Virulence factors of staphylococcus(toxins, plasmocoagulase, fibrinolysin, hyaluronidase, lecithinase, DNase, proteinase etc). 5. Differentiation of staphylococci. 6. Main clinical forms of staphylococcal infections. 7. Peculiarities of obtaining of tested material and bacteriological examination stages of sepsis, enteritis,furunculosis and other pyo-inflammatory processes, which are caused by staphylococci. 8. Staphylococcal carriage and hospital infections. 9. Value of staphylococcal phagotyping, procedure’s technique. 10. Active and passive prophylaxis of staphylococcal infection 5. Students’ practical activities: 1. To prepare the smears from pus, to stain them by Gram’s technique. 2. To familiarize with cultivation methods of staphylococci and streptococci (blood MPA, yolk-salt agar,MPB etc.). 3. To familiarize with the tests for determination of virulence factors of bacteria (plasmocoagulase, DNase,hyaluronidase etc.).Coagulase test plasma is clotted by Staphilococcus aureusStaphilococcus epidermidis Staphilococcus aureus Plasma Incubate 370 C Clot 4. To inoculate a specimen of pus onto yolk-salt agar. Using your loop, streak your unknown bacteria (pus) for isolation on a plate of yolk-salt agar. 5. To inoculate patient’s blood with suspicion on a sepsis in sugar MPB. Take a tube with patients‘ blood, open its and flame the edges of a tube. Take a flask with sugar MPB and dothe same actions. Pour a blood into a flask. Sterile the cotton wool plugs and edges of a tube and a flask in theflame and close them. Sign a flask (type of material, date, no of group). 6. Control questions and tests Choose the correct statements: 6
  7. 7. 1. To staphylococci have next properties: a – in smears they occur in pairs; b – Gram-positive; c– form spores; d – form irregular clusters resembling bunches of grapes; e – Gram-negative. 2. Growth onto nutrient media: a – might grow onto ordinary media; b – there are haemolysis zones on blood agar around colonies; c – they produce pellicle on the surface of sugar broth; d – grow on media with bile; e – on blood agar don’t cause hemolysis. 3. Toxin production of staphylococci: a – produce an alpha-, beta- delta-hemolysins; b – don’t produce enterotoxins; c – produce Hyaluronidase, plasmocoagulase; d – don’t produce an exfoliative toxin; e – have plasmids. 4. Choose disease, which are caused by staphylococci: a – Rheumatic Fever; b – sepsis; c – phlegmon; d – Scarlet Fever; e – pyodermia. 5. The bacteriological preparations, which are used for creation active and passive immunity: a – staphylococcal anatoxin; b – antistaphylococcal human immunoglobulin; c – staphylococcal toxin; d – antistaphylococcal antitoxic serum; e – staphylococcal autovaccine. 6. Main method of laboratory diagnosis of staphylococcal infections: a – serological method (precipitation test); b – bacteriological method; c – express method (ELISA); d – allergic method; e – serological method ( CFT). 7. For revealing a staphylococcal carriage it is necessary: a – to take material from a nasopharynx; b–to isolate of a pure culture; c-to carry out CFT; d- to carry out phagotyping of Staphylococcus aureus.7. List of literature: 1. I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004, chapter15, p.3-11. 7