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 is naming, 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 certain organism from others

2. Graduating • subspecies: serotyping Haemophilus influenzae type b • species - distinct organism with certain characteristic features, similar organism within genus Salmonella typhi • genera:Streptococcus, Salmonella, • families: Micrococaceae, Enterobacteriacae • orders

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

4. Bacterial identification in practice • Pure culture • Colony morphology and gram staining - preliminary identification • Biochemical properties, antigenic properties, growth characteristics - 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 on plasmacoagulase production - artificial PC negative - common comensales of human skin and animals, some cause infections in some circumstances (Staphyoloc. epidermidis, warneri, haemolyticus, saprophyticus, hyicus) PC positive - Staphylococcus aureus, St. intermedius

9. 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

10. 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 - alfa - membrane damaging, necrosis, beta - sfingomyelinase, delta - detergen like action, leucocidin - dermonecrosis, exfoliatin - epidermolytic toxin

11. 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)

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

13. 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

14. 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 - nosocomial strains - meticilin res.

15. 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

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

17. 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

18. 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

19. 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 - ?

20. 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

21. 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

22. 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

23. 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 form 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

24. 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

25. 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

26. 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

27. CRP • CRP - C substance of str. pneumoniae - During bacterial infecions organism syntetises CRP - C reactive protein - protein of acute phase of inflammation, reacting with C substance of Str. pneumoniae

28. Streptococcus pneumoniae - infections • Encapsulated strains - virulent - pneumo, (lobal, crupous -without ATB resolution after 14 days - crisis - production of anticapsular antibodies) meningitis, bakteremia - age predilection -in seniors (superinfection od viral infection of RT) and children up to 3 years ( imunologically defect, encapsulated strains T independent - protectiveantibodies ( 90 serotypes, vaccination, conjugated vaccines) • Nonencapsulated strains - sinusitis, otitis media

29. ATB susceptibility • Str. pyogenes - 100% susceptible for PNC, (in allergies macrolides ERY) • Str. agalactiae - good susceptibility for PNC, some strains tolerate - inhibition, not resistence, resistence to ERY, TTC • Str. salivarius - susceptible for PNC, • Str. pneumoniae - PNC, TTC, CMP, CEF., resistence for PNC - multiresistence - decline of affinity of ATB to PBP

30. Enterococcus • G+ cocci, colonisisng in great quantities intestine and colon, able to grow in the presence of bile - dif. dg. - esculin media • E. faecalis • E. faecium • urine tract infections, intraabdominal abscesses, bakteraemia • ATB nonbactericidal for enterococcie, resistence - synergic therapia: aminoglykosides + cell wall acting • Resistence plasmid transferable - (on staphylococci too - vancomycine)

31. Family NeisseriacaeFamily Moraxellaceae - genera Branhamella, Moraxella, Acinetobacter • Genus Neisseria • Species: N. meningitidis -colonisation of URT or menigitis, setpicaemia- meningococcaemia - fulminant sepsis, pneumonia, arthritis N.gonorrhoeae - uretritis, cervicitis, salpingitis, proctitis, septicaemia, arthritis, conjunctivitis, pharyngitis, Pelvic inflamatory disease other neisseria - colonisation of mucous membranes and skin

32. Neisseria meningitis • Encapsulated (polysaccharide capsule), G- diplococcus, coffee beans, • Serogroups (13) - A - (1 serotype),B,C(several serotypes 20),Y, W135… most frequent, 8 immunotypes • Pathogenicity: colonisation of nasopharynx (pilli) - attachment to noncilliated cell of columnar epitel spread - escape to phagocytosis - antiphagocytic property of capsule toxic effect - endotoxin LPS - vascular damage (endothelial damage, inflamation of vessel wall, DIC, trombosis) - hyperproduction of membrane fragments in growing cells

33. Neisseria meningitis - clinical syndromes • Meningitis - high mortality when not treated 100% • Meningococcemia - life-threatening,, trombosis of small blood vessels, multiorgan invovement, petechiae, overwhelming disseminated intravascular coagulation with shock - destruction of adrenal glands- Waterhouse Friderichsen sy, - chronic septicaemia • Therapy - PNC(genetic alteration of PBP ),CMP, cephalosporin, • Eradication - (spread of strains) sulphonaminds, rifampicin • Vaccination - A,C,Y,W vaccine - not for children under 2, conjugate vaccine - immunogenic for children, B vaccine - polysaccharide is weak immunogene

34. Laboratory diagnosis • CSF - Gram stain - G-diplococci, leucocytes, antigen detection - latex agglutination in CSF, urine • cultivation of CSF and blood culture - chocolatised agar + 5%CO2, !not refrigerator! biochemical properties - dif dg. from other meisseria serogroupe typisation - agglutination, • ATB susceptibility test • from nasopharyngeal swab - dif. dg. from non pathogenic neisseria - biochemical test

35. Neisseria gonorrhoeae • Most common sexually transmitted disease (STD) uretritis, cervicitis, arthritis, conjunctivitis, local complications • G- diplococci, 5 different types of colonies (T1-5), cytochromoxidase positivity, glucose fermentation • Structure: capsule, pilli - in virulent strains(T1,2) - attachment, OMP - outer membrane protein- I.,II.,III, Iron binding protein - removal of iron from host cells - iron essential for metabolism of gonococci, LPS-endotoxin, protease, penicillinase

36. Pathogenesis and immunity • Attachment - pilli, penetration and multiplication and pssage throughthe cells into subepitelial space. Nonpilliated are avirulent. Protein I interfere with neutrophil degranulation. Endotoxin - tissue destruction. • IgG and C´: individuals with C´deficiencis are at increased risk for systemic disease. • Only humans, asymptomatic reservoir • Multiple infections - lack of protective immunity - antibodies against pilin protein - antigenic diversity

37. Neisseria meningitis - immunity • Bactericidal antibodies(serogroup) (only in type B serotype ab are also protective) and complement - protective • polysaccharide capsule - susceptibility of children under 3 years - T independent Ag • susceptibility of infant after decline of maternal antibodies • Acquired immunity - 2 weeks after asymptomatic colonisation • Transmission by droplets, prolonged close contact (millitary, crowded communities) • Sporadic and epidemic spread

38. Clinical signs • Infection in men - uretritis+complication • in women- cervicitis - ascending genital inf. • Disseminated infection with septicaemia, skin, joints infections:(fever, artralgias-migratory,suppurative monoarthritis-wrist, knee, ankles, rash over extremities • Perihepatitis, purulent conjunctivities in newborne, anorectal gonorrhoe in homosexual, pharyngitis • Chemoprophylaxis - - ineffective

39. Cultivation +ATB • Neisseria gonorrhoe - PNC – penicilinase production - chromosome type of resistence - changes in cell surface, - ceftriaxon. TTC, chinolons, makrolides - azitromycin – therapy of chlamydia infection • Microscopy: Vagina swab in susp. gonorrhoe: - Gram staining: G - diplococci, coffee beans, epitelial cells., leukocytes. From culture colonies: G- diplococci • Cultivation: Swab from vagina or discharge – on blood agar, modified blood agar, chocolate agar + ATB – inhibition of contaminating flora, Grey cololnies after application of cytocromoxidase – become black – slide sc. Gram, - biochemical tests for diff.dg. from other Neisseria

40. Genus:Bacillus - G+sporeforming bacilli, aerobic and facultatively anaerobic • Bacillus anthracis,Bacillus cereus,Other Bacillus sp. • Bacillus cereus - 2 enterotoxins - heat stable - emetic form - contaminated rice - heat resistant spores survive initial cooking that kills vegetative cells, germinate, multiply and toxin is not destroyed by reheting - heat labile - diarrheal form - adenylcyclase-cAMP system stimulation in intestinal cells - fluid accumulation -contaminated meat and vegetable - toxin is produced in situ, longer incubaion • Panophthalmitis - traumatic eye infection (soil, penetrating object), complete loss, massive destruction - toxins - necrotic - heat labile enterotoxin -cereolysin -hemolysin - phospholipase C - lecithinase • Ubiquitous, isolation witout symptoms = contamination • Other Bacillus - immunosupressed patients - shunt and catheter inf.

41. Bacillus anthracis • Spore and capsule not seen in clinical specimens • Antrax toxin - 3 antigenically distinct components: -protective Ag, lethal factor, edema factor - alone not active, • Pathogenesis: capsule - antiphagocytic, Ab are not protective, toxins - protective + lethal or edema f. • Human diseases - cutaneous - inoculation - painless papule, ulcer, necrosis - inhalation - rapid progresive diffuse pulmonary involvement - respiratory failure - 95% mortality - gastrointestinal - ingestion - rare - mesenteric adenopathy, hemorrhage, ascites, 90% mortality • Cultivation - on nonselective media, rapidly growing adherent colonies, no hemolysis, Non motility,liquifaction of agar Microscopy: caput medusae - serpentine chains, • Therapy - PNC (TTC, CMP)., control of animal antrax, vaccination.

42. Non sporeforming G+ bacilli - heterogenous group • Corynebacterium - coryneforms, diphtheroids- C. diphteriae (diphtheria), C. jeikeium (oportunistic). C. urealyticum(urinary tranct inf.), C. pseudodiphthericum (endocarditis), C. ulcerans (pharyngitis) • Arcanobacterium haemolyticum - pharyngitis • Actinomyces - granulomatous ulcerative inf. • Rhodococcus - suppurative pneumonia, opportunistic • Listeria - meningitis, septicemia, granulomatosis infantiseptica • Erysipelothrix - erysipeloid, septicemia, endocarditis

43. Corynebacterium • Pleiomorphic G+ rod, arranging to short chains, forming china letters • Metachromatic granules – the final colour is defferent from original stain and from other parts of cell • Special cell wall structure – mesodiaminopimel acid, mycolic acid – taxonomically near Mycobacteria • C. diphtheriae – preventable disease – inhalation – asymptomatic carriage or disease • C. JL – jeikeium – opportunistic pathogen hematological disorders

44. Pathogenesis • Diphtheric toxin – tox gen – lysogenic conversion • AB toxin – blacking protosynthesis – prolongation of peptid chain on ribosomes • Schick skin test – detection of neutralisation antibodies when i-d appôicated diphtheria toxin

45. Lysogenic conversion • Strain of C. diphtheriae gains his toxigenicity by lysogenic conversion - thanks to bacterophage. Bacterial virus - bactrophage is able to transfere genetic information about the production of toxin - tox gen - fom one cell ( toxigenic) to the other (non toxigenic) C. diphtheriae cell.

46. Enzymes • Phospholipasa D – dermonecrotic toxin – spread – increasing of vascular permeability – C. ulcerans • Urease – alkalinisation of urine calculi C. urealyticum • ATB resistance – selection of resistent strains – C. urealyticum, C.JK – resistent to ATB used for urinary infections

47. Clinical syndromes • Diphtheria – depend on immunity and place of infection – asymptomatic colonisastion. Mild infection or fulminant diphtheria • Pseudomembrane on tonsils, pharynx, nose and general symptomes and complication (myocarditis) • Skin diphtheria • Therapy –ATB .PNC, ERY – elimination of bacteria, not threating intoxication, antitoxin, immunisation • Debacilisation of carrierers – • Protective immunity in skin forme

48. Lab. dg. of corynebacterium • Diagnosis of the disease is based on clinical picture and epidemiological history. Lab. dg. is long and complicated Appropriate sampling • Microscopy - smear with Gram staining and Albert´s staining does not distinguish between C. diphtheriae and other Corynebacteria. • Cultivation: Blood agar - 3 typs od colonies - mitis, gravis, intermedius, Loffler´s medium, tellurit medium brown colonies with halo • Biochemical identification- differentiation from other corynebacteria present in throat • toxigenicity of the strain - ELEK, • ATB susceptibility

49. Cultivation of Corynebacteria • C. diphtheriae - blood agar • C. pseudodiphthericum - blood agar, inverse CAMP • C. diphtheriae on tellurit medium - brown colonies with brouwn ring - halo

50. Detection of toxigenicity of C. diphtheriae • ELEK´s test of toxigenicity - imunodiffusion of suspension of tested strain and antidiphtheric serum in agar - zone of precipitation • Annimal mode: application of diphtheria toxin i.d. ………………..necrosis • antidiphtheric serum (i.p. or i.d.) + toxin …no necrosis