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Practicals 5WT enterobacteriaceae

Practicals 5WT enterobacteriaceae. Enterobacteriaceae – characteristic, structure, types od diseases, sampling – E. coli, Proteus sp., Klebsiella sp Microscopy – E. coli sc Gram, - capsule detection K. pneumoniae sc. Burri

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Practicals 5WT enterobacteriaceae

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  1. Practicals 5WT enterobacteriaceae • Enterobacteriaceae – characteristic, structure, types od diseases, sampling – E. coli, Proteus sp., Klebsiella sp • Microscopy – E. coli sc Gram, - capsule detection K. pneumoniae sc. Burri • Cultivation – E. coli and K.pneumoniae on blood agar and Endo medium, - P. mirabilis – Raussov fenomen - moving • Biochemical properties - Enterotests, - reading of Enterotest E. coli, Kl. Pneumoniae a P. vulgaris., - biochemical diagnostic cultivation medium sc Hajn • Serology – antigenic structure detection of unknown strain polyvalent and monovalent sera – slide agglutination

  2. G- aerobe rods • 1) Facultatively anaerobic fermenters: Enterobacteriaceae - E. coli, Salmonella, Schigella, Enterobacter, Citrobacter, Serratia, Klebsiella, Proteus, Morganella, Providencia Vibrionaceae - Vibrio, Aeromonas, Plesiomonas Campylobacter, Helicobacter • Widest and most heterogenous group of medically important bacteria • 27 genus, 102 species (95% medically important ones belong to 25 species) - differentiation based on DNA homology, biochemical properties, antigen characteristics, ATB susceptibility • Commonly present in nature (soil, water, plants), parts of physiological flora of colon. • Obligatory pathogenic (Yersinia pestis), oprtunistic pathogens (Klebsiella, E. coli, Proteus) • Transmission from annimals (Salmonella), from human carrier (S. typhi), endogenous infection (E.coli)

  3. Sites of infections • CNS - E. coli • URT infections - Klebsiella, Enterobacter, E. coli • Septicaemia - E. coli, Klebsiella, Enterobacter • GIT infections -Salmonella, Shigella, Yersinia, E.coli • Urinary tract infections - E. coli, Proteus, Providencia, Klebsiella

  4. Physiology and structure • G-rods, usually motile (flagella)- besides Klebsiella, non spre forming, facultative anaerobe, poor nutrition requirements, biochemical active, catalase +, oxidase -, cytochromoxidase COX - = dif.dg.from other G - rods • Fermentation of lactose + or – (patogenic Salmonella, Shigella, Yersinia, a Proteus are lactose negative), resistence to bile salt (Salmonella), capsule (Klebsiella) - dif.dg of Enterobacteriaceae • 3 groups of antigens: • - somatic O antigen – most important antigen of cell wall, heat resistant, part of LPS – composed of 3 parts - O polysachride, core polysacharide and lipid A – with endotoxin activity • - capsular K antigen – heat susceptible, cross reacting wiht antibodies against othe bacteria. In Salmonella typhi known as Vi antigen. • - flagellar H antigen - termosusceptible,

  5. Laboratory diagnostic • growing well on non selective media • Selective media for detection of pathogens - lactose negative strains Salmonella, Shigella • Biochemical identification – Hajn medium, Enterotests – group of biochemical tests • Serodiagnosis – detection of antigenic structure – Salmonella, Shigella, E.coli • Interpretation of results – in stool sample – Salmonella, Shigella, Yersinia – pathogens, other enterobacteriaceae – according the the state of eubiosis, when overgrowth - from physiologically sterile materials: urine – UTI, ascendent infection, blood – most improtant etiological agens, wounds, sputum – in monoculture, nose and throat sample - monoculture

  6. E. coli • Present in GIT • Endogennous infection after breaking of immune barieres, - bakterial sepsis( from focuses in urinary tract or GIT) most common cause of G - sepsis, - urinary tract infection – mostly in out patients, transmitted from GIT ascendently, specific serotypes adhering to urinary epitel - gastroenteritis – EPEC, EIEC, ETEC - neonatal meningitis - hemolytic uremic sy – EHEC – O:157, H:7 • Forms complex of numerous O-somatic, H- flagellar and K - capsular antigens

  7. Klebsiella • Encapsulated – mucous colonies, increased virulence, non motile • Klebsiella pneumoniae – pneumonia in pattients with problems of clearing of RT from possible aspirate from mouth. Necrotic destruction of alveolar spaces. Bloody sputum. Infections of wounds, soft tissues and urinary tract. • K. rhinoscleromatis - sklerom • K. ozaenae – atrofic disease of nasal epitelium

  8. Proteus • Motile G- rod, Rauss phenomen • P. mirabilis – infections of urinary tract, production of ureasea ( lysis of urea, alcalinisation, increased possibility for calculli formation, toxicity for epitelium • Proteus vulgaris

  9. Surface of tube medium – aerobe environment, lactose negative bacteria do not ferment, it is alkaline, red Lower part - in anaerobe environment, enterobacteria ferments glycids – acidic – yellow or black H2S . , nonfermenters do not ferment = red – Ps. aeruginosa 1 negative control 2 Ps.aeruginosa : not fermenting - red 3 Shigella sonnei: H2S - negat.,gas – negat., TSI – alcaline/acidic red/yellow lact.negat 4 Salmonella typhi: H2S – pozit., gas–negat., TSI – alcaline/alcaline red/yellow- black 5 Escherichia coli: H2S – negat.,gas -posit., TSI – acid/acidic yellow/yellow 6 Proteus mirabilis: H2S – posit, gas - negat., TSI – acidic /acidice yellow/yellow-black

  10. Microscopy • E. coli – sc Gram – G- rod • Klebsiella pneumoniae – sc Burri – capsule

  11. Cultivation • E. coli on blood agar –grey colonies without hemolysis • E. coli on Endo medium – prominent colonies eith metal look • K. pneumoniae on blood agar – mucous colonies • K. pneumoniae on Endovej agar – rose • Proteus mirabilis – Raussov fenomen – concentric growth from the central one colony

  12. Slide agglutination • On slide – suspension with FR • 1) Suspension + drop of polyvalent serum • suspension + poly ABC = aggl+ S+ poly DEF = aggl– • 2) Suspenson + drop of monovalent serum • susp + mono A = aggl.+ susp + mono B = aggl – susp + mono C = aggl.– • result – tested strain is type A

  13. Biochemical properties - Enterotest • E.coli – + + + – – + – – – – – + + + + • K.pneumoniae – + – – + – + + + + + + + + + + • P.vulgaris + – + – + + + – – – – – + – + – • H2S LYS IND ORN URE PHE ESL SCI + black blue red blue red green black blue • – no col. green yellow green yellow yellow no col. yellow • MAL INO ADO CEL SUC SOR TRE MAN + blue yellow yellow yellow yellow yellow yellow yellow yellow green greeen green green green green green

  14. Group of biochemical tests aligned so that they allow numeric identification based on statistical probability of the result of one test In the positive result the well is attributed the chifer according to the position in the triplet.( 1 2 or 4) Addition of chifers in triplet gives the number and each result of the triplet gives a subsequent one position of the code that is the combination of numbers of tested triplets. This code is corresponding to one bacteria - - - 0 - + + 6 + - - 1 + + + 7 - + - 2 - - + 4 + - + 5 + + -3

  15. G-rods Hajn glucose succrose lactose maltose manit indol urea/motility H2S Proteus vulgaris + + - + -* - + +/+ Proteus mirabilis + + - - - - - +/+ Salmonella typhi + + - - + + - -/+ Escherichia coli - + + + + + + -/+ Shigella - - - - - + + -/+

  16. MikroLaTest Identification of Klebsiella pneumoniae – Enterotest + /- + - - +- + + + + + + + + + + /+

  17. Praktiká 6 - Salmonella, shigella, • Mikroskopický dôkaz - Gramovo farbenie Salmonella sp. - Kultivácia - Salmonella, Shigella, E.coli na Mackonckey, dezoxycholát, krvný agar -Biochemické vlastnosti - fermentácia cukrov - Hajnova pôda a Enterotesty: Salmonella, Shigella, E.coli • Určenie antigénnej štruktúry Shigella flexneri Widalova reakcia - diagnostické možnosti brušného týfusu

  18. Mikroskopický dôkaz • Mikroskopický dôkaz - Gramovo farbenie Salmonella sp. - G - paličky

  19. Kultivácia G- paličiek - Salmonella, Shigella, E.coli • Kultivácia - Salmonella, Shigella, E.coli • krvnom agare - G - palička, šedavé kolónie neodlíšiteľné, • na Mc agare - E.coli laktózu skvasuje - ružové kolónie, Salmonella laktózu neskvasuje - žlté, priesvitné - s tvorbou sirovodíka, Shigella - laktózu neskvasuje, bez sirovodíka, • na DC - dezoxycholátovom agare - selektívna pôda - rastie Salmonella (laktóza negat. so sírovodíkom) a Shigella (laktóza negat. bez sírovodíka). E. coli nerastie

  20. Biochemické vlastnosti Salmonella • Biochemické vlastnosti - fermentácia cukrov - Hajnova pôda (skvasovanie cukrov, tvorba plynu, tvorba sírovodíka, skvasovanie urey, pohyb, skvasovanie simonscitrátu) - Enterotesty

  21. Biochemické vlastnosti Shigella • Biochemické vlastnosti - fermentácia cukrov - Hajnova pôda a Enterotesty:Shigella

  22. Biochemické vlastnosti E.coli • Biochemické vlastnosti - fermentácia cukrov - Hajnova pôda a Enterotesty: E.coli

  23. Enterotesty • Biochemické vlastnosti - fermentácia cukrov - • H Sírovodík čierna bezfarebná • G Lyzín modrá zelená • F Indol červená žltá • E Ornitín modrá zelená • D Ureáza červená žltá • C fFnylalanín tmavozelená žltá • B Eskulín čierna bezfarebná • A Simonscitrát modrá žltá • H Malonát modrá žltá • G Inositol žltá zelená • F Adenitol “ “ • E Celobioza “ “ • D Sacharóza “ “ • C Sorbitol “ “ • B Trehalozs “ “ • A Mannitol “ “

  24. Určenie antigénnej štruktúry Shigella flexneri • Polyvalentné séra A - Sh sonnei, C - Sh dysenteriae, B - Sh. flexneri (1a,1b,2a,2b,3a,3b,3c,4a,4b,5,6,x,y,) • Monovalentné séra I II III IV V VI

  25. Salmonella • 1500 sérotypov, rôzne názvy podľa miesta izolácie • DNA analýza: 1 druh (S. enterica) a jej 7 podskupín • Prítomná u všetkých zvierat, kontaminovaná potrava - primárne od kontaminovaných zvierat, vajíčka - salmonelové ochorenie sliepok, sekundárne - od infikovaného človeka - nosiča. Potrebné veľké inokulum 106-8 - šírenie potravinami, v ktorých sa môže pomnožiť. Osoby so zvýšeným rizikom - imunokompromitovaný, deti, redukovaná acidita žalúdočnej tekutiny • Enteritída - postihnutie HČ, teplota, nauzea, zvracanie, kŕče, bolesť hlavy a svalov. Dg.: tampón stolice, 3 x za sebou. Lactóza negatívne kolónie, biochemická identifikácia. Stanovenie séroskupiny sklíčkovou aglutináciou podľa Kauffmann Whitovej schémy - epidemiologický význam ( S. enteritidis, S. infantis, S. agona, S. kentucky…………………….)

  26. Salmonella typhi • Týfus - horúčka s dezorientáciou - celkové závažné ochorenie - penetrácia do lamina propria, lymfatických uzlín, RES (množenie v pečeni -šírenie cez žlčník a žlčové cesty do črev - , slezine, kostnej dreni), krvi - bakterémia, exantém - roseola, teplota, bolesti hlavy. GIT príznaky menej, perforácia čreva. Časté nosičstvo a vylučovanie. • Dg. - výter stolice, opakovaný, po stimulácia MgSO4. Typický vzhľad. Aglutinácia. Dôkaz aj nepriamo - sérologicky - Vidalova reakcia. Vi aglutinácia • Th. - CMP • Salmonella paratyphi A, B, C - miernejšia podoba týfusu • Septikémia - pri bakterémii, - osteomyelitída, endokarditída

  27. Shigella • 4 druhy - Sh. dysenteriae, Sh. flexneri, Sh. boydii, Sh. sonnei a 38 sérotypov • Dyzentéria - vodnaté hnačka s prímesou krvi, ochorenie často epidemické, kontaminovanými rukami. Prežíva vo vode až 6 mesiacov. Malá infekčná dávka - asi 200 baktérií. Ochorenie tenkého čreva, najskôr bez invázie epitelu. Produkcia enterotoxínu - invázia mukózy, deštrukcia sliznice a tvorba vredov - hnis a krv v stolici • Dg. Na základe biochemických vlastností ( charakteristický zápach, netvorí plyn, laktózu neskvasuje)., odlíšenie druhov - sklíčkovou aglutináciou

  28. Yersinia • 7 druhov - Y.pestis, Y. pseudotuberculosis, Y. enterocolitica, + oportunistické Yersínie • Y. pestis - spôsobuje mor - mestský a lesný typ, nie GIT ochorenie. Adaptovaná na i.c. parazitizmus, v prírode neprežíva. Faktory virulencie - i.c., polysacharidové púzdro, endotoxin, koaguláza, fibrinolyzín, vychytávanie Fe. Mestský mor - udržiavaný potkanmi, prenesený na človeka hmyzom infikovaným počas bakterémie hlodavcov. Replikácia v čreve hmyzu a náhodný prenos na hlodavcov alebo človeka. Lesný mor - prériový psy a hmyz. Nekontrolovateľný. • 2 klinické formy - bubonická - 7 dní po poštípní bolestivé zdurenie lymf. uzlín, bakterémia,75% úmrtnosť. - pneumonická - 2 dni po inhalácii - teplota, pneumónia, šírenie kvapôčkovou nákazou, úmrtnosť 90%

  29. Widalova reakcia - diagnosis of typhoid fever • Cultivation (direct diagnosis) blood (7th-10th day), stool (from 2nd week, bone marrow, urine(from 3rd week) a detection of agglutinins in serum - Widal reaction (indirect diagnosis) – antibodies against O a H antigens of S. typhí – fourfold rise of titer - acute disease or for carriere detection • Knowledge of clinical signs and patogenesis – enable the cultivation from right material – blood, stool, urine or detection of antibodies

  30. Widal reaction – diagnosis of typhoid fever (fig) Urine Stool Serum Blood week

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