Classification, nomenclature, taxonomy,identification

1. Classification, nomenclature, taxonomy,identification • Classification is arrangement of bacteria into groups ( the same organisms can be classified differently according to the view: serotype classification, antimicrobial resistance classification…) • Nomenclature (name) is the mean of communicating - it is binominal • Taxonomy - science of classification, identification, nomenclature and making a system • Identification is practical use of classification criteria to distinguish certaine organism from others

2. Graduating • subspecies: serotyping Streptococcus pneumoniae type 8 • species - distinct organism with certain characteristic features, similar organism within genus Streptococcus pneumoniae, S.sp • genera:Streptococcus, • families: Micrococaceae, • orders

3. Approaches to taxonomy • Numerical, • phylogenetic, • genome size, • Guanin+cytosine content, • DNA relatedness, thermal stability of DNA sequences, DNA relatedness under supraoptimal conditions……. • In practice - polyphasic approche - depending on importnace and purpose of nomenclature

4. Bacterial identification in practice • Pure culture • Colony morphology • gram staining - preliminary identificationG+G- • growth characteristics aerobic, anaerobic • Biochemical properties genus + species • antigenic properties type • binominal nomenclature - clinical purposes • Molecular and genetic characteristic • G+C, DNA homology, thermal stability -epidemiological and forensic puroses

5. Principles of diagnosis • Symptomatic patient - manifestation of infecton • Suspection of microbial ethiology - exogenous or endogenous • Specimen selection, collection - • Specimen processing and microbiological examination - specific techniques • Result communication and consultation • Asymptomatic patient

6. Practical taxonomy Miscellanous bacteria Mycoplasma, Ureaplasma, Chlamydia Ricketsiaceae Spirochetales - Spirochetes, Leptospira, Borrelia Mycobacterium Nocardia , Actinomycetes,

7. Lectures 1,2 • Micrococcaceae (Staphylococcus), Streptococcaceae G+cocci • Neisseriaceae: G- cocci • Bacillaceae, Corynebacterium: G+rods Listeria, Erysipelotrix, • Mycobacteria: G- acid fast rods • Actinomyces, Nocardia: G+rods with special characteristic

8. Staphylococcus • Pathogens of man • Divided in 2 groups acc to plasmacoagulase production - artificial PC negative - common comensales of human skin and animals, some of them cause infections in some circumstances (Staphyoloc. epidermidis, warneri, haemolyticus, saprophyticus, hyicus)PC positive - Staphylococcus aureus, St. intermedius

9. G+cocci in clusters - gram staining

10. Cocci in clusters in fluorescein preparation

11. Growth characteristic – growing on salt media – staphylococci Fermenting manitol –yellow – St. aureus vs. St.epidermidis

12. Staphylococcus aureus G+coccus • colonizes nasal passage and axillae • able to grow on salt medium (10%), catalase +, grows in clusters, PC positive - distinguishing factor • Structure: capsule, peptidoglycan,protein A, teichooic acid, clumping factor, cytoplasmic membrane • Toxins: alfa, beta, delta, gama, leukocidin, exfoliating,toxic shock syndrome TSST,enterotoxin • Enzymes: coagulase, catalase, hyaluronidase, staphylokinase, lipases, fibrinolysin, nuclease, penicilinase

13. Virulence factor and patogenesis • Multifactorial • adherence surface proteins (att.to fibronectin), fibrinogen/fibrin binding protein (att. to clots and traumatised cell, the most important part of slime on biomaterial of indwelling devices), fibronectin • avoidance of host defence capsule polysaccharide, protein A - bind IgG - disruption of opsonisation, leukocidin - toxic for PMNL • damage of host tissue - toxins and enzymes

14. Toxíny • alfa - cytotoxický pre bb, rozrušuje bunkovú membránu a hladkú svalovinu ciev, nekrotizujúci • beta - sfingomyelináza C, termolabilný, hydrolýza fosfolipidov, deštrukcia tkaniva, tvorba abscesu • delta - termostabilný, cytolýza, detergentné vlastnosti • gamma - erytrocytolýza • leukocidín-zvyšovanie permeability a tvorba pórov v bb. membráne, odolnosť voči fagocytóze • exfoliatívny toxín - zodpovedný za SSS – skin scaled sydrome, rozrušuje medzibunkové spoje - dezmozómy v stratum granulosum kože, tvorba protektívnych protilátok - len u detí • TSS-1 - vzniká pri raste u niektorých kmeňov St.aureusTSS – toxic shock syndrome – toxický šokový syndróm • enterotoxíny - odolný voči hydrolýze žal. kyselinou, termostabilný, 5 typov A-E, B - pseudomembranózna kolitída, neurotoxín, hnačky a zvracanie

15. Enzýmy • Koaguláza - viazaná (fibrinogén - fibrín) a voľná (cez medziprodukty), tvorba fibrínovej vrstvy a abscesu- ochrana pred fagocytózou • Kataláza - premena toxického H2O2 -- H2O + O2 • Fibrinolyzín- stafylokináza - rozpúšťa fibrínový trombus • Lipáza - rôzne typy, prežívanie stafylokokov v seboroických oblastiach - prenikanie do kože a podkožia, tvorba povrchových kožných infekcií • Hyaluronidáza - rozrušuje mukopolysacharidy v spojivovom tkanive, šírenie • Nukleáza - termostabilný E • Penicilináza - beta laktamáza, enzým rozrušujúci betalaktámový kruh PNC antibiotík

16. Klinické manifestácie infekcie spôsobenej St. aureus Hnisavé • Koža– hnisavé ochorenie kože: vredy, abscesy, akné, karbunkul, impetigo, • Systémové - endokarditída, pnuemónia,bakterémia, osteomyelitída, septická artritída, flebitídy, mastitídy, meningitída osteomyelitis, infekcia moč. mechúra • Nozokomiálne infekcie a infekcie kože, katétrov, popálenín • GITinfekcie – enterotoxín - zvracanie, hnačky • TSS- toxic shock syndrom SSS- scalded skin syndrom

18. Superantigen: enterotoxins and TSST • TSST - exotoxin secreted during the growth of some strains, connected with superabsorbent tampons - fever, hypotension, shock, rash, desquamation, multi organ involvement TSST-1: 75% menstrual TSS., TSST-2 enterotoxin B and C: 50% non menstrual TSS • Enterotoxins A-E- resistant to hydrolysing gastric enzymes and to heat 100*C (neurotoxin) • Superantigen: superstimulation of T cells nonspecifically 1 of 5 cells instead of 1 of 10 000 with APC - cytokines released in large amounts - (any T cell with Vb element is stimulated)

19. Clinical manifestation of St.aureus • Skin - stye, soil, carbuncule, impetigo, endocarditis, pnuemonia, • General pyogenic Nosocomial and wound infection, infection of indwelling devices, burns, immunosuppression, phlebitis, mastitis,meningitis • Toxic GIT infections - emesis, diarhea TSS - toxic shock syndrome SSS scalded skin syndrome osteomyelitis,

20. impetigo

21. folikulitis

22. carbuncul

23. Stafylococcal scaled skin syndrom SSSS– Riter´s disease

24. St. epidermidis and other PC negative • Infection of indwelling devices - catheter, shunt, artificial joint • Infection of valve - artificial or damaged - endocarditis - indolent course - 1 year after surgery • Staphylococcus saprophyticus - urinary infection of young sexually active women • Contamination or ethiological agens

25. ATB susceptibility and resistance • Penicillin, staphylococal penicillin - oxacillin, methicilin,TTC,CMP,ERY, KANA, GEN, STM, Fluorochinolony • Quick development of resistance : penicilinase • Now some strains are resistant to convential ATB • Hospital strains resistant to many ATB - including glycopeptides, vancomycin, teicoplanin • MRSA - meticilin resistant St.aureus • VRSA - vancomycin resistant St.aureus - resistance transfered from enterococci • St.epidermidis - - often meticilin resistant.

26. Nosocomial problem • MRSA • multiple resistance • VRSA • resistance to antiseptics and disinfectants • Mechanism of resistance acquiring: 1)extrachromosomal plasmids, transposons or DNA insertion 2)mutation in chromosomal genes

27. G+cocci catalase negat. • Genus: • Streptococcus: • Enterococcus • Aerococcus, Gemella, Lactococcus, Leuconostoc, Pediococcus - very rarely pathogenic in men

29. G+cocci, catalase negat. • genus • Streptococcus: • Enterococcus • Aerococcus, Gemella, Lactococcus, Leuconostoc, Pediococcus - seldom pathogenic for human

30. shapeG+cocci in chains or diplococci

31. Streptococcus - taxonomy and properties • Taxonomy - according to haemolysis on blood agar : alfa - incomplete, beta - complete, gama - without haemolysis - according to serological groupes sc. Lancefield A-H, K-V, not all streptococci have this groupe specific antigen of cell wall • A S. pyogenes bacitracin beta hemolysis • B S. agalactiae CAMP beta hemolysis • C S. anginosus beta, alfa • D S. bovis alfa, gama • - S. pneumoniae optochin + alfa • - S. salivarius optochin - alfa

32. Streptococcus alfa hemolysis • - S. pneumoniaeoptochin + alfa • - S. salivarius optochin - alfa • - Streptococcus beta hemolyticus Streptococcus alfa hemolyticus

33. Streptococcus – beta hemolyticus A S. pyogenes beta hemolysis (bacitracín+) B S. agalactiae beta hemolysis (CAMP)

34. Streptococcus sk A - pyogenes • In bouillon long chains • Antigenic structure - capsule - hyaluronic acid -identical with connective tissue - nonimunogennic • peptidoglycan • groupe and type specific antigens - - most important M antigen - on the surface of fimbriae, virulence - T protein, R protein, F protein, • lipoteichoic acid

35. Factors of patogenity and imunity • Capsule nonimunogennic - inhibits phagocytosis • M protein - protection against phagocytosis and cooperation with C´. Type 1,3,18 - invasive diseases., M3 a M18 - Rheumatic fever • F protein - receptor for fibronectin (matrix of eukaryotic cells - adhesin • Lipoteichoic acid - ?

36. Toxins • Exotoxins - erytrogenic toxin -termolabil, type A,B,C - hypersensitivity, endotoxic, cytotoxic, non specific mitogen T and imunosupresive B lymfocytes activity, rash at scarlatina • Dick´s, Schultz´s test • Streptolysin S - oxgen stabile, lysis of ery, releasing of lysosyme, nonimunogen • Streptolysin O - reversibily inactivated by oxygen, imunogen, antibodies against streptolysine O - ASLO, killing of leukocytes • Streptokinase - lysis of blood trombus, spread of bacteria • DN-ase - noncytolytic, depolymerisation of free DNA - in pus, declines viscosity of pus, spread

37. Streptococcal infections • Streptococcus pyogenes HSA -pharyngitis - dif dg. from viral - scarlat fever - pharyngitis with exanthem - infectious strain gained by lysogenic conversion ability to produce erytrogenic toxin (rash with desquamation of skin, circumoral whiteness, strawberry thongue) - toxic shock syndrome - cellulitis, necrotising fasciitis, hypotension, erytrodermia, multiorgan failure, bakteraemia, M1, M3, M18 - types - erysipelas, pyodermia - purulent skin diseases

41. Flesh eating streptococcus Toxic shock syndrome – celulitís and necrotising faciitis - superantigen

42. Streptococcal infections- late sequelae • Febris rheumatica - rheumatic fever - nonpurulent streptococcal disease - inflamatory disease of heart, joints, vessels and submucosis - autoimmune disease related to some serotypes of M protein ( types specific antigen, factor of patogenity, numbered M18, M3…) sequelae present only after respiratory infections. Not after skin infection • Poststreptococcal acute glomerulonefritis - specific nefritogen strains, after skin and respiratory infections

43. Diagnosis - laboratory • Microscopy - gram staine, G+ cocci in chains, not colonising skin, in skin swab together with leu - significant for disease. Of any value from URT sample • Detection of antigens - directly from clinical material (also from URT), detection of groupe specific antigens of cell wall, specific, not sensitive - negative tests should be confirmed by cultivation • Cultivation, Identification - hemolysis, bacitracin test • Antibodies - ASLO - confirmation of preliminary streptococcal infection in patients with RF a GNF (+ anti Dnase antibodies

44. Streptococcus agalactiae, HSB • newborne infections (menginitis, pneumonia), puerperal - postpartum sepsis /colonisation of URT, GIT and vagina - danger of contamination of newborne during prolonged and preterm labour, importance of maternal imunity/ • G+ cocci in chains, beta hemolysis, dif dg.from HSA - CAMP test - strenthened hemolysis of Staf. aureus - papillon • Strukture - polysaccharid capsule, cell wall - peptidoglycan with type and groupe specific antigens and lipoteichoic acid, cell membran • Antigenic strukture - antibody against capsule antigens are protective - diseases in newborne • Enzymes - Dnase, hyase,protease, hemolysin

45. Other streptococci • Beta hemolytical group C,F,G - URT and skin infections never late complications • Viridant -alfa a nonhemolytical streptococci - Streptococcus salivarius, viridans - bacteremia, subacute endokarditis, caries, intraabdominal purulent infections • Important condition for development of disease is the preliminary damage of tissu ( tooth, valve) • Carries - formation of dextranu from glucose

46. Streptococcus pneumoniae • G+cocci in pairs, diplococcus, lancet shape - candle and flame,viridant - dif.dg - positivity of optochin test, bile solubility test - colonies are dissolved by powdre bile • encapsulated and nonencapsulated strains- polysaccharide capsule - factor of virulence - more than 90 serotypes based on capsule antigens peptidoglycan of cell wall typical for G+ cocci (N acetylmuramic and N acetyl glukosamin net), teichoic acid and cholin ( unique substance, important for cell division) • 2 forms of teichoicacid in cell wall - surface (C substance) and covalently bound

47. STREPTOCOCCUS PNEUMONIAE • Streptococcus pneumoniae: Gram-positívediplococcus 0.5-1.25 / um

48. STRUCTURE

49. G+ diplococcus in CSF fluorescent microscopy